microsoft word d3000_vol1no1_erratum(2) errata for “lack of association between irf6 polymorphisms and nonsyndromic oral clefts in south indian population,” volume 1, no 1 dentistry 3000 issn: 2167-8677 (online) this page reflects errors that were found in the article “lack of association between irf6 polymorphisms and nonsyndromic oral clefts in south indian population," volume 1, no 1 of dentistry 3000. 1. gurramkonda, venkatesh, jyotsna murthy, altaf hussain syed, & bhaskar vks lakkakula. "lack of association between irf6 polymorphisms and nonsyndromic oral clefts in south indian population." dentistry 3000 [online], 1.1 (2013): n. pag. web. 3 sep. 2013 the article title should be “evidence of association between irf6 polymorphisms and nonsyndromic oral clefts in south indian population.” last updated wednesday, september 4, 2013 microsoft word vieira editorial 2017.docx   vol  5,  no  1  (2017)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2017.81           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     five  years  of  dentistry  3000   alexandre  rezende  vieira     editor  in  chief   department  of  oral  biology,  university  of  pi4sburgh  school  of  dental  medicine,  pi%sburgh  pa,  usa                 cita%on:  vieira  ar.  (2017)  five  years  of  den.stry   3000.  den!stry  3000.  1:a001   doi:10.5195/d3000.2017.81   received:  december  5,  2017   accepted:    december  12,  2017   published:    december  15,  2017   copyright:  ©2017  vieira  ar.  this   is  an  open  ac-­‐ cess  ar!cle   licensed  under  a  crea!ve  commons   a"ribu%on  work  4.0  united  states  license.   email:  arv11@pi(.edu      dentistry  3000  is  a  journal  pub-­‐ lished  by  the  university  library   system  (uls),  university  of  pitts-­‐ burgh,  as  part  of  its  d-­‐scribe  digital   publishing  program  and  is  cospon-­‐ sored  by  the  university  of  pitts-­‐ burgh  press.    in  that  capacity,  we   have  created  a  journal  that  is  free   from  the  pressures  of  publishers,   authors,  funding  agencies,  and  other   interest  groups.    dentistry  3000  has   no  publication  fees  for  authors  and   is  readily  accessible  to  all  via  web.     the  main  goal  of  the  journal  contin-­‐ ues  to  be  disseminating  scientific   information  in  dentistry,  which  is  a   field  that  suffers  due  to  the  limited   number  of  journals  interested  in   publishing  a  wide  range  of  topics  of   dental  research.          we  have  been  encouraging   young  authors  to  submit  their  first   scholarly  works  as  a  way  to  incen-­‐ tivize  the  development  of  and  inter-­‐ est  in  academic  and  scholarly  activi-­‐ ties.    we  have  also  been  supporting   authors  from  smaller  centers,  facili-­‐ tating  the  publication  of  their  work.     we  provide  help  with  manuscript   preparation  and  improving  lan-­‐ guage  and  style  during  the  peer-­‐ review  process.    we  believe  these   practices  can  only  benefit  the  scien-­‐ tific  community,  and  we  have  no   concerns  regarding  specific  individ-­‐ ual  agendas  and  misrepresented   perceptions  of  quality.    we  do  not   measure  dentistry  3000  by  impact   factor,  which  in  reality  is  a  measure   of  the  impact  of  the  authors  that   publish  in  a  particular  journal.    we   understand  that  a  small  fraction  of   papers  recieves  many  citations,   whereas  the  vast  majority  of  papers   get  few  or  none  at  all.            in  the  last  five  years,  we  have   published  over  40  papers.    our  goal   moving  forward  is  to  continue  the   practices  we  have  established:  fair   and  unbiased  peer-­‐reviews,  with   emphasis  on  content  and  findings   rather  than  looking  for  reports  with   positive  results.    we  aim  to  continue   supporting  young  authors  in  pub-­‐ lishing  their  first  works  and  valuing   all  disciplines  of  dentistry  equally.          we  are  looking  forward  to  your   future  contributions  to  dentistry   3000.       microsoft word cetnar 2016.doc   vol  4,  no  1  (2016)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2016.52           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     observing  anterior  crowding  in  a  population  living  a  century  ago   christopher  cetnar1     1  university  of  pittsburgh  school  of  dental  medicine,  pittsburgh  pa,  usa abstract   purpose:  to  observe  the  frequency  of  mandibular  anterior  crowding  in  a  population  who   lived  during   the  nineteenth  century  and  compare   the   rate  of  occurrence   in   the  current   population,  which   is   claimed   to  be   two-­‐thirds   of   adults   experiencing   crowding  by   early   adulthood.  materials  and  methods:  the  school  of  dental  medicine  at   the  university  of   pittsburgh   possesses   a   collection   of   skulls   of   individual’s   likely   living   in   the   northeast   united  states  during  the  late  19th  century  and  early  20th  century.  of  this  skull  collection,   29  skulls  were  evaluated  for  the  presence  of  mandibular  anterior  crowding.  results:  55%   of   the   evaluated   mandibular   anterior   arches   were   positive   for   mandibular   anterior   crowding.  57%  of  the  male  specimens  were  positive  for  anterior  crowding,  while  50%  of   the  females  were  recorded  to  have  anterior  crowding.  these  frequencies  are  lower  than   the  one  suggested  for  the  current  live  population  in  the  united  states.  conclusion:  from   this  sample  of  the  population  living  a  century  ago,  there  was  a  lower  prevalence  of  ante-­‐ rior  crowding  (55%)  than  the  research  suggested  two-­‐thirds  of  adults.     citation:  cetnar,  c.  (2016)  observing  anterior   crowding  in  a  population  living  a  century  ago.   dentistry  3000.  1:a001   doi:10.5195/d3000.2016.52   received:  may  31,  2016   accepted:    june  13,  2016   published:    september  28,  2016   copyright:  ©2016  cetnar,  c.  this   is  an  open  ac-­‐ cess  article   licensed  under  a  creative  commons   attribution  work  4.0  united  states  license.   email:  cwc23@pitt.edu   introduction   orthodontic  treatment  has   become  very  common  in  the   modern  model  of  dental  treat-­‐ ment.  it  has  been  recorded  that   80%  of  u.s.  teenagers  have  com-­‐ pleted  or  are  currently  undergoing   orthodontic  treatment  according   to  the  american  association  of   orthodontists  (aao;   https://www.aaoinfo.org/).  it  can   be  contemplated  that  in  the  near   future  orthodontic  treatment  may   eventually  become  the  standard  of   care,  if  it  has  not  already.   one  key  to  understanding   orthodontic  treatment  is  the  idea   that  the  oral  cavity  is  dynamic,   consisting  of  many  interacting   components  including  pressures   from  soft  tissue,  musculature,   function,  and  normal  growth.  this   dynamic  environment  is  under-­‐ stood  by  orthodontics  and  is  com-­‐ bated  with  the  use  of  removable   or  permanent  retainers  after   treatment  to  prevent  the  altera-­‐ tion  of  the  established  occlusion   (orthodontic  regression).     mandibular  incisor  crowd-­‐ ing  is  a  phenomenon  that  is  ob-­‐ served  in  both  patients  that  have   received  orthodontic  treatment   and  those  who  have  not.  as  the   aging  process  occurs,  the  smile   line  tends  to  shift  exposing  more   mandibular  teeth  when  a  patient   smiles  or  opens  their  mouth  in   normal  conversation.  patients  who   had  previous  orthodontic  treat-­‐ ment  notice  that  their  once   straight  teeth  become  “crooked”   or  “crowded”  and  point  towards   their  mandibular  incisors.  re-­‐ search  suggests  that,  without  or-­‐ thodontic  treatment,  approxi-­‐ mately  2/3  of  adolescents  with   good  alignment  and  “normal”  oc-­‐ clusions  will  develop  incisor  ir-­‐ regularity  by  early  adulthood  [1].     the  school  of  dental  medi-­‐ cine  at  the  university  of  pittsburgh   possesses  a  collection  of  skulls   that  is  made  up  of  individuals  liv-­‐ ing  in  the  northeastern  united   states  during  the  late  19th  and   early  20th  century  [2].  this  popu-­‐ lation  living  a  century  ago  would   not  have  undergone  modern  day   orthodontic  treatment  and  from   this,  anterior  crowding  can  be  ob-­‐ served  whether  or  not  two-­‐thirds   of  adolescents  have  crowding  in   their  mandibular  anterior  teeth.   this  information  would  yield  use-­‐    observing  anterior  crowding  in  a  population  living  a  century  ago   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.52    http://dentistry3000.pitt.edu   2   ful  data  in  understanding  the   complex  process  of  anterior   crowding  and  the  prevalence  in  a   population  that  was  not  exposed   to  modern  dentistry  and  ortho-­‐ dontic  practices.   methods   at  the  university  of  pitts-­‐ burgh  school  of  dental  medicine   there  is  a  collection  of  skulls.  most   of  them  were  acquired  from  a   seller  in  new  york  city  around  the   1930’s  indicating  that  these  indi-­‐ viduals  lived  in  the  east  coast  of   the  united  states  [2].  the  skulls   have  previous  tagged  displaying   gender,  estimated  age  (>40  years,   <40  years;  unless  a  known  age  was   indicated),  as  well  as  ethnicity.     in  evaluation  of  anterior   crowding,  a  set  of  criteria  was  es-­‐ tablished  to  determine  if  the  skull   was  eligible.  the  first  condition   was  that  the  skull  had  to  have  full   adult  dentition,  which  excluded   the  six  pediatric  skulls  found  in  the   museum.  the  second  condition   was  that  the  specimen  had  to   have  present  all  six  of  the  man-­‐ dibular  anterior  teeth.  the  last  cri-­‐ teria  was  evaluating  the  condition   of  the  crowns  on  the  anterior   teeth  which  eliminated  specimens   that  had  more  than  one  full  crown   missing  or  that  had  crowns  that   were  unable  to  be  evaluated  for   crowding  (missing  contacts,  frac-­‐ tured  crowns  etc).    after  the  selec-­‐ tion  process  was  completed  there   were  29  qualified  mandibular   arches  evaluated,  23  males  and  six   females.   since  the  intent  is  not  to   measure  the  severity  of  crowding,   a  different  method  of  evaluating   crowding  was  applied  in  determin-­‐ ing  whether  or  not  the  subject  has   crowding  present.  crowding  was   evaluated  by  proximal  tooth  con-­‐ tact-­‐point  displacement,  which   was  observed  by  visualizing  irregu-­‐ larity,  flaring,  or  protrusion  of  the   mandibular  incisors  [4].    for  an   arch  to  qualified  as  a    “yes  or  posi-­‐ tive”  response  to  crowding,  two  or   more  teeth  in  the  anterior  arch   must  have  improper  bilateral   proximal  contact  or  must  display   excessive  flaring,  retrusion  or  pro-­‐ trusion  due  to  insufficient  interca-­‐ nine  distance.  for  example,  figure   2a  would  have  recorded  as  posi-­‐ tive  for  anterior  crowding,  while   figure  2b  would  have  been  nega-­‐ tive  for  anterior  crowding  since   there  is  ideal  proximal  tooth  con-­‐ tact  and  no  excessive  malposition   of  the  anterior  teeth  due  to  lack  of   arch  space.   there  are  multiple  meth-­‐ ods  to  determining  whether  ante-­‐ rior  crowding  exists  besides  the   mere  clinical  presentation  of  misa-­‐ ligned  teeth.  one  method  to  de-­‐ termine  the  severity  of  anterior   crowding  to  determine  appropri-­‐ ate  treatment  is  the  widths  of  all   the  teeth  anterior  to  the  molars   are  measured  and  subtracted  from   the  sum  of  two  measurements   (mesial  of  the  lower  incisor  to  the   distal  of  the  lower  canine,  plus  dis-­‐ tal  of  lower  canine  to  the  mesial  of      observing  anterior  crowding  in  a  population  living  a  century  ago   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.52    http://dentistry3000.pitt.edu   3   the  first  molar)  to  give  the  degree   of  crowding  [3].  this  method  is   described  in  figure  1.   results   from  table  1,  it  can  be  ob-­‐ served  that  out  of  the  29  subjects   that  were  eligible  for  evaluation,   16  (55%)  presented  with  anterior   crowding,  while  13  of  the  speci-­‐ mens  had  normal  spacing  and   proximal  contacts  between  all  six   of  the  mandibular  anterior  teeth.       table  2  shows  a  compari-­‐ son  between  male  and  female   museum  specimens.  these  data   show  that  males  were  at  a  slightly   higher  rate  of  56.5%  occurrence  of   anterior  crowding  in  comparison   with  50%  of  the  female  subjects   being  positive  for  anterior  crowd-­‐ ing.   table  3  shows  a  compari-­‐ son  between  age  groups  and  sexes   that  were  recorded  in  the  muse-­‐ um  records.  these  data  show  that   males  had  nearly  the  same  preva-­‐ lence  for  anterior  crowding  in   both  age  groups,  57%  for  males   below  the  age  of  40  and  56%  for   males  above  the  age  of  40.  while   the  females  below  the  age  of  40   only  showed  25%  positive  for  an-­‐ terior  crowding  and  both  of  the   two  specimens  above  the  age  of   40  being  positive  for  anterior   crowding.   discussion   the   physiology  be-­‐ hind  anterior   crowding  is   understood  as   combination   between  me-­‐ sial  movement   of  posterior  teeth  and  lingual   movement  of  anterior  teeth.  es-­‐ sentially,  both  of  these  processes   shorten  the  arch  length,  which   leads  to  the  crowding  process.  the   mesial  movement  of  posterior   teeth  theory  has  been  more  or   less  discounted  as  the  source  of   crowding  and  has  been  used  as   justification  for  surgical  removal  of   third  mo-­‐ lars.  studies   have  shown   that  there  is   no  clinically   reduced   level  of   crowding   due  to  third   molar  ex-­‐ traction  as   well  as  crowding  affecting  people   with  congenitally  missing  third   molars  the  same  as  those  who   have  retained  molars  [5]..  anterior   lingual  movement  is  discussed   through  the  equilibrium  theory  of   tooth  position,  with  evidence  from   the  differential  mandibular  growth   theory.    anterior  crowding  is  most   definitely  a  multifactorial  process   and  evidence  seems  to  lean  to-­‐ wards  a  combination  of  the  equi-­‐ librium  theory  and  relapse  from   orthodontic  treatment.     there  are  a  number  of   theories  attempting  to  explain   mesial  acting  force;  pressure  from   erupting  third  molars,  inherent   mesial  migration  or  drifting,  matu-­‐ ration  and  contraction  of  perio-­‐ dontal  soft  tissue  and  fibers,  oc-­‐ clusal  forces  on  anterior  teeth,   and  loss  of  arch  length  due  to  inci-­‐ sor  overbite.  it  is  illogical  to  as-­‐ sume  that  there  is  a  single  cause   of  late  mandibular  anterior  crowd-­‐ ing  and  more  realistically  under-­‐ stood  as  a  complex  and  multifac-­‐ torial  process.  like  other  multifac-­‐ torial  processes,  there  is  always  an   interaction  between  genetics  and   the  environment.  most  of  the  re-­‐ searched  processes  tend  to  be   considered  environmental  instead   of  making  links  to  genetic  suscep-­‐ tibility.  from  a  genetic  approach,   there  may  be  certain  susceptibili-­‐ ties  that  can  be  observed  to  help   predict  the  severity  of  future  ante-­‐ rior  crowding  besides  observing   familial  trends.  this  could  lead  to   creating  personalized  treatment   plants  to  prevent  relapse  in  pa-­‐ tients  who  have  undergone  ortho-­‐ dontic  treatment  and  are  interest-­‐ ed  in  persevering  their  inter-­‐arch   length.     table 1. prevalence of anterior crowding found in the observed population specimens positive for anterior crowding 16 specimens negative for anterior crowding 13 percent of specimens positive for anterior crowding 55% table 2. comparison of crowding by sex. specimens positive for anterior crowding specimens negative for anterior crowding percent of specimens positive for anterior crowding males 13 10 56.5% female 3 3 50%    observing  anterior  crowding  in  a  population  living  a  century  ago   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.52    http://dentistry3000.pitt.edu   4   the  common  understand-­‐ ing  with  mandibular  incisor  crowd-­‐ ing  is  that  without  orthodontic   treatment,  approximately  two-­‐ thirds  of  adolescents  will  develop   incisor  irregularity  by  early  adult-­‐ hood.  this  hypothesis  might  be   observable  in  modern  day  adults   in  which  have  had  a  mosaic  of   modern  orthodontic,  surgical,  and   restorative  treatment,  all  of  which   may  affect  late  mandibular  incisor   movement  ultimately  resulting  in   crowding.  observing  a  population   almost  a  century  apart  in  which   these  more  modern  approaches  to   occlusion  and  comprehensive  or-­‐ thodontic  treatment  were  not   available  could  contest  this  dog-­‐ ma.  with  the  understanding  that   orthodontics  is  in  place  to  correct   original  malocclusion  and  does  not   play  a  role  in  later  life  develop-­‐ mental  processes  that  lead  to   crowding,  it  is  very  interesting  to   observe  see  if  we  are  better  off   now  that  we  were  a  century  ago  in   relation  to  ante-­‐ rior  crowding  in   adults.   from  the   data  present  in   table  1,  it  was   observed  that  in   the  population   living  on  the   eastern  coast  of   the  united   states  nearly  a   century  ago   (1930s)  repre-­‐ sented  in  the   museum,  there   was  observable   anterior  crowd-­‐ ing  in  55%  of  the  subjects.  this  is   an  interesting  number  because  it   is  actually  lower  than  the  expected   two-­‐thirds  (66%)  that  research   suggests.           the  data  presented   in  table  3  compares  age  and  gen-­‐ der  and  shows  that  the  male  spec-­‐ imens  that  were  eligible  for  analy-­‐ sis  showed  a  relatively  consistent   percentage  of  anterior  crowding,   but  the  data  collected  from  the   few  female  specimens  told  a  dif-­‐ ferent  story.  only  25%  of  the  fe-­‐ males  under  the  age  of  40  showed   anterior  crowding  while  both  of   the  specimens  aged  above  40   were  positive  for  anterior  crowd-­‐ ing.  though  the  sample  size  was   very  small  for  the  females,  it  is   consistent  with  the  modern  ob-­‐ servation  in  which  correlates  inci-­‐ sor  crowding  and  thickness  of  the   mandibular  symphysis,  cancellous   bone  thickness  [6].   one  drawback  of  this  ob-­‐ servation  is  the  lack  of  sample   size;  of  the  100  specimens  found   in  the  museum  only  29  were   found  to  be  qualified  for  the  data   collection,  which  means  that  this   small  sample  size  might  not  be   representative  of  the  whole  popu-­‐ lation.  thus,  it  must  be  taken  into   consideration  that  if  a  larger  sam-­‐ ple  size  were  analyzed,  then  the   percentage  of  anterior  crowding   might  be  closer  to  the  suggested   two-­‐thirds.  it  must  also  be  taken   into  consideration  that  a  skull  col-­‐ lection  sold  as  a  museum  might   have  been  selective  and  not  an   accurate  representation  of  the   population.   in  conclusion,  from  the   small  sample  size  of  a  population   living  a  century  ago  it  was  deter-­‐ mined  that  anterior  crowding  was   present  in  55%  of  the  population   (56%  in  males  and  50%  in  fe-­‐ males),  which  is  comparable  to  the   research  suggested  two-­‐thirds   (66%).   references   1.  late  mandibular  incisor   crowding.  american  association  of   orthodontics:  orthodontic  dia-­‐ logue.  1999;11(1).   2.  caries  and  periodontal   disease:  insight  from  two  us  popu-­‐ lations  living  a  century  apart.  rose   e,  vieira  ar.  oral  health  prev   dent.  2008;6(1):23–8.   pmid:18399304   3.  orthodontics.  part  3:  pa-­‐ tient  assessment  and  examination   ii.  roberts-­‐harry  d,  sandy  j.  br   table 3. comparison of crowding by age and sex. specimens positive for anterior crowding specimens negative for anterior crowding percent of specimens positive for anterior crowding males <40 4 3 57.1 males >40 9 7 56.3 females <40 1 3 25 females >40 2 0    observing  anterior  crowding  in  a  population  living  a  century  ago   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.52    http://dentistry3000.pitt.edu   5   dent  j.  2003  nov  22;195(10):563– 5.  pmid:14631426   4.  goldberg  ai.  an  evalua-­‐ tion  of  mandibular  anterior  crowd-­‐ ing  as  it  relates  to  facial  diver-­‐ gence  in  treated  and  untreated   subjects.  2012;  a  thesis  presented   to  the  graduate  faculty  of  saint   louis  university  in  partial  fulfill-­‐ ment  of  the  requirements  for  the   degree  of  master  of  science  in   dentistry.   5.  the  effect  of  extraction   of  third  molars  on  late  lower  inci-­‐ sor  crowding:  a  randomized  con-­‐ trolled  trial.  harradine  nw,  pear-­‐ son  mh,  toth  b.  br  j  orthod.  1998   may;25(2):117-­‐22.  pmid:9668994   6.  mandibular  anterior   bony  support  and  incisor  crowd-­‐ ing:  is  there  a  relationship?  uysal   t,  yagci  a,  ozer  t,  veli  i,  ozturk  a.   am  j  orthod  dentofacial  orthop.   2012  nov;142(5):645–53.   pmid:23116505                   microsoft word 68 2017.docx   vol  5,  no  1  (2017)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2017.68           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     a  case  of  molar-­‐incisor  hypomineralization  with  genetic  and     environmental  influences   erin  kello1      1university  of  pi.sburgh,  department  of  anthropology abstract   background:  molar-­‐incisor  hypomineraliza1on  (mih)  is  a  common  childhood  dental  pathology.  this  paper  describes  a   case  with  familial  involvement  and  environmental  risk  factors.  case  descrip+on:  a  35-­‐year  old  female  has  yellowish-­‐ brown  opaci+es  present  on  central  and  lateral  maxillary  incisors,  right  and  le)  maxillary  canines,  right  and  le)  maxil-­‐ lary  first  molars,  and  right  and  le2  maxillary  second  molars.  a  33-­‐year  old  male  sibling  has  yellowish-­‐brown  opaci+es   on  the  right  and  le  central  and  lateral  maxillary  incisors.  the  male  sibling’s  fraternal  twin  does  not  have  evidence  of   mih.  a  maternal  grandmother  also  had  evidence  of  mih,  though  involved  teeth  are  not  known.  prac%cal  implica%ons:   mih   is  a  condi,on  with  both  gene,c  and  environmental   components.  prac,,oners   should  consider  both  e,ologies   when  pa(ents  present  with  the  condi(on.       cita%on:  kello,  e.  (2017)  a  case  of  molar-­‐incisor   hypomineraliza-on  with  gene-c  and  environmental   influences.  den!stry  3000.  1:a001   doi:10.5195/d3000.2017.68   received:    may  2,  2017   accepted:    may  9,  2017   published:    june  8,  2017   copyright:  ©2017  kello.  this   is   an   open  access   ar!cle   licensed   under   a   crea!ve   commons   a!ribu%on  work  4.0  united  states  license.   email:  erinkello@gmail.com   introduction   molar-­‐incisor  hypominer-­‐ alization  (mih)  is  diagnosed  by  the   presence  of  yellowish-­‐brown  or   white/cream  enamel  opacities  [1].   prior  to  2001,  the  nomenclature  of   mih  was  not  standardized  [1].   other  terms  for  this  condition  in-­‐ cluded:  hypomineralized  first   permanent  molars  (fpm),  idio-­‐ pathic  enamel  hypomineralisation   in  non-­‐fluoride,  hypomineraliza-­‐ tion  in  fpm,  and  cheese  molars   [1].  these  names  were  eventually   subsumed  under  molar-­‐incisor   hypomineralization  (mih).  we-­‐ erheijm  et  al.  suggested  the  term   “mih”  and  the  attendant  defini-­‐ tion:  “hypomineralization  of  sys-­‐ temic  origin  of  1-­‐4  fpm,  frequent-­‐ ly  associated  with  affected  inci-­‐ sors”[2].  weerheijm  et  al.  further   defined  mih  by  noting  enamel  de-­‐ fects  are  occasionally  present  on   the  permanent  canines  and  se-­‐ cond  molars  in  addition  to  the  first   molars  and  incisors.       the  prevalence  rate  for   mih  is  estimated  at  2.8%  to  25%   depending  on  the  study  and  the   geographical  region.  some  evi-­‐ dence  for  differing  rates  of  mih  in   the  mandible  and  maxilla  has  been   found,  but  studies  do  not  agree  on   which  location  is  more  common   [1].  there  also  does  not  appear  to   be  a  significant  difference  in  mih   instances  between  the  sexes  [1].   environmental  factors   several  environmental  fac-­‐ tors  have  been  linked  to  mih.  ala-­‐ luusua  et  al.  linked  exposure  to   dioxin  in  breast  milk  to  enamel   hypomineralization  in  children   [3,4].  the  authors  found  a  correla-­‐ tion  between  the  “frequency  and   severity  of  lesion  and  total  expo-­‐ sure.”      jalevik  et  al.  found  a  signif-­‐ icant  correlation  between  early   life  respiratory  illnesses  and  mih   [5].    beentjes  et  al.  found  children   with  mih  became  ill  more  fre-­‐ quently  in  the  first  four  years  of   life  than  those  without  mih.  the   authors  assert  there  “appears  to   be  an  association  with  pneumonia,   otitis  media  and  high  fevers,  spe-­‐ cifically”  [6].  muratbegovic  et  al.   also  found  an  association  between   mih  and  childhood  illnesses  with  a   high  fever  [7].  in  another  study,   the  development  of  mih  has  also   been  associated  with  the  use  of   antibiotics  in  early  life  [8].       genetic  associations   genetic  association  studies  in  mih   are  few.  a  2013  genome-­‐wide  as-­‐ sociation  study  identified  single   nucleotide  polymorphisms  on   chromosomes  4,  9,  16,  20,  and  22   associated  with  mih  [9].  the  low    a  case  of  molar-­‐incisor  hypomineraliza,on  with  gene$c  and  environmental  influences   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.68    http://dentistry3000.pitt.edu   est  p-­‐value  was  found  in  the  re-­‐ gion  that  encodes  the  scube1   gene.  21.8  %  of  children  who  car-­‐ ried  the  minor  allele  of   rs13058467  had  mih.  of  children   who  were  homozygous  for  the   major  allele,  10.6  %  had  mih.   scube1  is  a  bmp  inhibitor  in-­‐ volved  in  the  development  of  the   tooth  germ.  the  scube  gene  fami-­‐ ly  is  involved  in  epithelial  to  mes-­‐ enchymal  signaling  during  tooth   development,  although  it’s  role  is   not  yet  well  understood.  the  au-­‐ thors  hypothesize  that  the  scube1   polymorphism  may  “disturb  regu-­‐ lar  tooth  development  and  miner-­‐ alization...or  distinct  systemic  etio-­‐ logical  factors  may  influence  the   protein  functionality  [9].”     more  broadly,  the  devel-­‐ opment  of  enamel  has  been  linked   to  certain  genes.  some  evidence   can  be  found  in  the  regulation  of   the  genes  linked  to  amelogenesis   imperfecta  (ai)  [10].    most  ai  fol-­‐ lows  an  autosomal  dominant  ex-­‐ pression  pattern.  bailleul-­‐forestier   et  al.  linked  several  genes  includ-­‐ ing  tuft1,  ambn  and  amtn  to   non-­‐syndromic  deficient  enamel   formation[10].   a  2016  family-­‐ based  associa-­‐ tion  study  inves-­‐ tigated  63  snps   and  21candidate   genes  related  to   ai  [11].    this   study  built  on   earlier  work  [12].   the  study  cohort   was  65  individu-­‐ als  without  mih,   96  of  unknown  mih  status  and   130  individuals  with  mih.    signifi-­‐ cant  results  were  obtained  for  11   snps  associated  with  genes  in-­‐ volved  in  amelogenesis  (table  1).   the  authors  conclude  these  varia-­‐ tions  confer  underlying  suscepti-­‐ bility  to  mih.   vieira  and  kup  argue  mih   is  a  genetic  disease  because  the   disease  affects  teeth  in  different   morphogenic  fields  [13].  the  au-­‐ thors  assert  that  additional  gene   variants  may  influence  the  presen-­‐ tation  of  mih  on  teeth  adjacent  to   the  first  molars  and  on  incisors.     the  authors  argue  there  is  a  ge-­‐ netic  basis  of  mih  based  on  di-­‐ verse  geographic  prevalence  rates   and  lack  of  clearly  associated  envi-­‐ ronmental  factors.  based  on  the   large  number  of  associated  genes   and  possible  environmental  asso-­‐ ciations,  mih  is  likely  a  multifacto-­‐ rial  condition.     case  report   a  35-­‐year  old  female  with   yellowish-­‐brown  opacities  present   on  central  and  lateral  maxillary   incisors,  right  and  left  maxillary   canines,  right  and  left  maxillary   first  molars,  and  right  and  left   maxillary  second  molars.  the  pa-­‐ tient  had  veneers  on  the  right  and   left  central  and  lateral  incisors  and   right  and  left  canines.  the  pa-­‐ tient’s  first  dental  restoration  was   performed  at  16  when  resin  com-­‐ posite  veneers  were  installed.   these  were  replaced  with  the   same  resin  composite  material  at   age  25.  the  current  veneers  were   installed  at  age  35.     the  patient’s  early  medical   history  includes  a  high  fever  treat-­‐ ed  with  amoxicillin  at  age  3.  also   at  age  4,  the  patient  had  facial  re-­‐ construction  surgery  following  a   fall  from  a  bed.     an  evaluation  of  familial  history   identified  a  33-­‐year  old  male  sib-­‐ ling  with  yellowish-­‐brown  opaci-­‐ ties  on  the  right  and  left  central   and  lateral  maxillary  incisors.  the   patient’s  fraternal  twin  was  unaf-­‐ fected  by  mih,  but  did  have  more   than  13  carious  lesions  before  age   30.  composite  resin  restorations   were  performed  on  this  patient   several  times.  the  patient  smoked   for  11  years.  interviews  with  fami-­‐ ly  members  revealed  that  the  ma-­‐ ternal  grandmother  also  had  yel-­‐ lowish  brown  opacities  on  her  in-­‐ cisors.     discussion   the  history  of  mih  is  ob-­‐ fuscated  by  the  lack  of  standard-­‐ ized  naming  before  2001  [1,6].   this  had  led  some  authors  to   question  whether  the  disease  is  of   modern  origin  [14].  the  small  por-­‐ tion  of  the  archeological  record   that  has  been  assessed  for  the   presence  of  mih  offers  differing   conclusions.  additionally,  the  dif-­‐ table  1.  snps  and  genes  involved  in  amelogenesis   snp   gene   snp   gene   rs7821494   fam83h   rs7664896   enam  gene   rs34367704     ambn  gene   rs1711399   mmp20   rs3789334   bmp2   rs1711423     mmp20   rs6099486     bmp7   rs2278163   dlx3   rs762642     bmp4  gene   rs6996321     fgfr1    a  case  of  molar-­‐incisor  hypomineraliza,on  with  gene$c  and  environmental  influences   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.68    http://dentistry3000.pitt.edu   ferential  preservation  of  carious   teeth  associated  with  mih  may   skew  any  evaluation  of  an  archeo-­‐ logical  population.  ogden  et  al.   found  an  mih  prevalence  rate  of   93.2%  in  a  london  cemetery  popu-­‐ lation  dating  to  1559  [15].  howev-­‐ er,  the  study’s  sample  size  was   small  and  focused  on  sub  adults.   kühnisch  et  al.  found  a  prevalence   rate  of  3.1%  among  a  late  medie-­‐ val  period  german  cemetery   population,  leading  the  authors  to   conclude  mih  is  linked  to  contem-­‐ porary  living  conditions  [14].     another  archeological   population,  dating  to  the  11th-­‐16th   centuries  from  thuringia,  germa-­‐ ny,  had  an  mih  prevalence  rate  of   12.2%  [16].  the  authors  postulate   the  prevalence  of  mih  may  have   been  higher  than  this,  but  factors   such  as  more  prevalent  ante-­‐ mortem  tooth  loss,  heavy  wear,   and  extended  carious  lesions  may   have  masked  the  true  prevalence.   hence,  mih  is  likely  not  a  modern   disease,  but  the  prevalence  rate   may  have  changed  in  modern   times  with  the  introduction  of  new   environmental  stressors  that  af-­‐ fected  underlying  genetic  varia-­‐ tion.     the  cases  reported  in  this   paper  present  an  interesting  prob-­‐ lem.  the  35-­‐year  old  female  pa-­‐ tient  was  exposed  to  a  high  fever,   amoxicillin  and  an  additional   stressful  injury.  a  high  fever  and   amoxicillin  have  both  been  linked   to  mih  [5-­‐8].  the  presence  of  mih   in  the  male  sibling  suggests  a  ge-­‐ netic  component.  this  male  was   not  yet  born  at  the  time  the  fe-­‐ male  was  exposed  to  the  fever,  so   had  no  exposure  himself.  the   male’s  fraternal  twin  did  not  de-­‐ velop  mih,  suggesting  environ-­‐ mental  or  epigenetic  factors  may   have  been  contributory.  addition-­‐ ally,  the  maternal  grandmother   likely  had  mih,  further  arguing  for   a  genetic  component.     it  is  possible  the  family  has   an  underlying  genetic  susceptibil-­‐ ity  to  mih  that  was  acted  upon  by   environmental  factors.  the  female   had  more  affected  teeth  than  the   male  and  was  also  exposed  to   more  environmental  stressors.   this  argues  for  underlying  suscep-­‐ tibility  exasperated  by  environ-­‐ mental  stressors,  or  multifactorial   inheritance.  to  determine  the  role   of  genetic  variations  in  the  devel-­‐ opment  of  mih  in  this  family,  gen-­‐ otyping  of  both  male  twins  and   the  female  would  be  advised.   references   1.  willmott  ns,  bryan  ra,  duggal   ms.  molar-­‐incisor-­‐ hypomineralisation:  a  literature   review.  eur  arch  paediatr  dent.   2008  dec;9(4):172-­‐9.  review.   pmid:  19054470.   2.  weerheijm  kl,  jälevik  b,  ala-­‐ luusua  s.  molar-­‐incisor  hypo-­‐ mineralisation.  caries  res.  2001   sep-­‐oct;35(5):390-­‐1.  pmid:   11641576.   3.  alaluusua  s.  aetiology  of  molar-­‐ incisor  hypomineralisation:  a  sys-­‐ tematic  review.  eur  arch  paediatr   dent.  2010  apr;11(2):53-­‐8.  re-­‐ view.  pmid:20403298.   4.  alaluusua  s,  lukinmaa  pl,  var-­‐ tiainen  t,  partanen  m,  torppa  j,   tuomisto  j.  polychlorinated  diben-­‐ zo-­‐p-­‐dioxins  and  dibenzofurans  via   mother's  milk  may  cause  devel-­‐ opmental  defects  in  the  child's   teeth.  environ  toxicol  pharmacol.   1996  may  15;1(3):193-­‐7.  pmid:   21781681.   5.  jälevik  b,  norén  jg,  klingberg  g,   barregård  l.  etiologic  factors  in-­‐ fluencing  the  prevalence  of  de-­‐ marcated  opacities  in  permanent   first  molars  in  a  group  of  swedish   children.  eur  j  oral  sci.  2001   aug;109(4):230-­‐4.  pmid:   11531068.   6.  beentjes  ve,  weerheijm  kl,   groen  hj.  factors  involved  in  the   aetiology  of  molar-­‐incisor  hypo-­‐ mineralisation  (mih).  eur  j  paedi-­‐ atr  dent.  2002  mar;3(1):9-­‐13.   pmid:  12871011   7.  muratbegovic  a,  markovic  n,   ganibegovic  selimovic  m.  molar   incisor  hypomineralisation  in  bos-­‐ nia  and  herzegovina:  aetiology   and  clinical  consequences  in  me-­‐ dium  caries  activity  population.   eur  arch  paediatr  dent.  2007   dec;8(4):189-­‐94.  pmid:  18076849   8:  laisi  s,  ess  a,  sahlberg  c,  arvio   p,  lukinmaa  pl,  alaluusua  s.   amoxicillin  may  cause  molar  inci-­‐ sor  hypomineralization.  j  dent   res.  2009  feb;88(2):132-­‐6.  doi:   10.1177/0022034508328334.   pmid:  19278983.    a  case  of  molar-­‐incisor  hypomineraliza,on  with  gene$c  and  environmental  influences   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.68    http://dentistry3000.pitt.edu   9.  kühnisch  j,  thiering  e,  heitmül-­‐ ler  d,  tiesler  cm,  grallert  h,  hein-­‐ rich-­‐weltzien  r,  hickel  r,  heinrich   j;  gini-­‐10  plus  study  group.;  lisa-­‐ 10  plus  study  group  genome-­‐wide   association  study  (gwas)  for  mo-­‐ lar-­‐incisor  hypomineralization   (mih).  clin  oral  investig.   2014;18(2):677-­‐82.  doi:   10.1007/s00784-­‐013-­‐1054-­‐8.   pmid:  23918034.   10.  bailleul-­‐forestier  i,  molla  m,   verloes  a,  berdal  a.  the  genetic   basis  of  inherited  anomalies  of  the   teeth.  part  1:  clinical  and  molecu-­‐ lar  aspects  of  non-­‐syndromic  den-­‐ tal  disorders.  eur  j  med  genet.   2008  jul-­‐aug;51(4):273-­‐91.  doi:   10.1016/j.ejmg.2008.02.009.  re-­‐ view.  pmid:  18499550.   11.  jeremias  f,  koruyucu  m,  küch-­‐ ler  ec,  bayram  m,  tuna  eb,  deeley   k,  pierri  ra,  souza  jf,  fragelli  cm,   paschoal  ma,  gencay  k,  seymen  f,   caminaga  rm,  dos  santos-­‐pinto  l,   vieira  ar.  genes  expressed  in  den-­‐ tal  enamel  development  are  asso-­‐ ciated  with  molar-­‐incisor  hypo-­‐ mineralization.  arch  oral  biol.   2013  oct;58(10):1434-­‐42.  doi:   10.1016/   j.archoralbio.2013.05.005.   pmid:23790503   12.  jeremias  f,  pierri  ra,  souza  jf,   fragelli  cm,  restrepo  m,  finoti  ls,   bussaneli  dg,  cordeiro  rc,  secolin   r,  maurer-­‐morelli  cv,  scarel-­‐ caminaga  rm,  santos-­‐pinto  l.   family-­‐based  genetic  association   for  molar-­‐incisor  hypomineraliza-­‐ tion.  caries  res.  2016;50(3):310-­‐8.   doi:  10.1159/000445726.  pmid:   27179118.   13.  vieira  ar,  kup  e.  on  the  etiol-­‐ ogy  of  molar-­‐incisor  hypomineral-­‐ ization.  caries  res.   2016;50(2):166-­‐9.  doi:   10.1159/000445128.  pmid:   27111773.   14.  kühnisch  j,  lauenstein  a,   pitchika  v,  mcglynn  g,  staskiewicz   a,  hickel  r,  grupe  g.  was  molar   incisor  hypomineralisation  (mih)   present  in  archaeological  case  se-­‐ ries?  clin  oral  investig.  2016   dec;20(9):2387-­‐2393.  pmid:   26780019.   15.  ogden  ar,  pinhasi  r,  white   wj.  nothing  new  under  the  heav-­‐ ens:  mih  in  the  past?  eur  arch   paediatr  dent.  2008  dec;9(4):166-­‐ 71.  pmid:  19054469.   16.  lang  j,  birkenbeil  s,  bock  s,   heinrich-­‐weltzien  r,  kromeyer-­‐ hauschild  k.  dental  enamel  de-­‐ fects  in  german  medieval  and  ear-­‐ ly-­‐modern-­‐age  populations.  an-­‐ thropol  anz.  2016  nov   1;73(4):343-­‐354.  pmid:  27643784.     wettability of two different artificial saliva substitutes on injection moulded heat polymerized acrylic resin and cad-cam acrylic resin: an in vitro study. vol 11 no 1 (2023) doi 10.5195/d3000.2023.184 http://dentistry3000.pitt.edu wettability of two different artificial saliva substitutes on injection moulded heat polymerized acrylic resin and cadcam acrylic resin: an in vitro study. quraish lal¹, aamir godil², mosin shaikh¹, smita musani², ramandeep dugal², ajinkya kirad² ¹p.g. student (m.a. rangoonwala college of dental sciences and research centre, pune), india ²mds prosthodontics; (professor, m.a. rangoonwala college of dental sciences and research centre, pune), india abstract aim: the purpose of this in vitro study was to evaluate the wettability of two different artificial saliva substitutes on injection moulded heat-polymerized acrylic resin and cad-cam acrylic resin and to compare these properties with natural saliva and distilled water. materials and method: forty heat polymerized (injection moulded) acrylic resin specimens and forty cad-cam acrylic resin specimens were fabricated with each specimen measuring 10x10x2mm. four groups of liquids namely distilled water, wet mouth™, mouth kote™ and human saliva were used. a contact angle goniometer was used to measure the contact angles. two contact angle readings were obtained on the right side and on the left side of the test specimen. the observations were recorded and a mean of the two readings was calculated. the same procedure was repeated for all specimens (heat cure resin and cad-cam) with all the four test groups of liquids. results: contact angle measurements were significantly higher in wet mouth™ and mouth kote™ solution of heat cure resin group compared to the cad-cam group (p-value<0.05 for both). mouth kote™ had lower mean contact angle (62.63 ± 4.59) than wet mouth™ (77.46 ± 9.16) on cad/cam denture base resin. mouth kote also had lower mean contact angle (70.80 ± 4.79) than wet mouth™ (97.77 ± 7.79) on injection moulded denture base resin. conclusion: mouth kote had better wettability than wet mouth on both cad-cam and injection moulded acrylic resin. both the saliva substitutes had better wettability on cadcam than on injection moulded acrylic resin. citation: quraish lal, et al. (2023) wettability of two different artificial saliva substitutes on injection moulded heat polymerized acrylic resin and cad-cam acrylic resin: an in vitro study dentistry 3000. 1:a001 doi:10.5195/d3000.2023.184 received: may 11, 2021 accepted: july 18, 2021 published: february 14, 2023 copyright: ©2023 quraish lal, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: quraishlal786@gmail.com introduction xerostomia is a condition characterized by qualitative and/or quantitative alteration in salivary secretion with/without increased dehydration of the oral mucosa.[1] it is a commonly reported condition, with prevalence around 22%-26% in general population and upto 82%-83% in patients seeking palliative care.[2] dry mouth predisposes to oral irritation and epithelial atrophy, which may progress into inflammation, fissuring, and ulceration. furthermore, lack of saliva significantly hampers denture retention and causes dysphagia. [3] the physical forces involved in the retention of a denture are adhesion, cohesion, capillarity, atmospheric pressure, surface tension, and viscosity, which are directly or indirectly dependant on saliva.[4] moreover, there is an increased incidence of oral candidiasis in xerostomia patients. artificial saliva substitutes were introduced as a replacement for natural saliva in individuals presenting with hyposalivation. [5] in such patients, for a denture to have sufficient adhesion to the supporting mucosa; the artificial saliva substitutes must flow over the denture surfaces with ease to ensure adequate wetting. [4] wettability is defined as the tendency of an adhesive to spread or wet the adherent. [6] the propensity of a liquid to spread enhances, when the contact angle decreases. therefore, the contact angle is a reliable inverse measure of wettability.[4] contact angle can be defined as the equilibrium angle of contact of a liquid http://dentistry3000.pitt.edu/ wettability of two different artificial saliva substitutes on injection moulded heat polymerized acrylic resin and cad-cam acrylic resin: an in vitro study. vol 11 no 1 (2023) doi 10.5195/d3000.2023.184 http://dentistry3000.pitt.edu on a rigid surface, measured within the liquid at the contact line where three phases (liquid, solid, gas) meet.[7] heat polymerized acrylic resin is the most preferred and popular choice of material among clinicians and patients. the contact angles of heat polymerized acrylic resin on artificial saliva and distilled water have been found to be 66.6 ± 5.5 and 64.1 ± 3.6 respectively. [4] on human saliva, heat polymerized acrylic resin has shown mean contact angle of 58.42 ±1.68. [6] however, the denture fabrication process with this material is a timeconsuming procedure and involves a lot of manual labour. certain other disadvantages include residual monomer content, polymerisation shrinkage and processing errors like porosities. to overcome these drawbacks, computer-aided design/ computer-aided manufacturing (cad/cam) based materials have shown to provide more predictable outcomes. although sufficient evidence exists in literature regarding the superior mechanical and handling properties of cad-cam poly-methymethacrylate (pmma), there is insufficient evidence on the wettability of these polymers with salivary substitutes. the best choice of material in patients suffering from xerostomia still remains debatable and inconclusive. therefore, this in vitro study was undertaken to evaluate the wettability of two different artificial saliva substitutes on heat-polymerized acrylic resin and cad-cam acrylic resin and to compare these properties with natural saliva and distilled water. the null hypothesis of the study was that there is no difference in wettability of the two test resins with the different artificial salivary substitutes. materials and methods two types of denture base materials (specimens) were used including injection moulded heat polymerized acrylic denture base material (sr, ivocap high impact, ivoclar vivadent, liechtenstein) and cad-cam denture base material (bloomdentm, china). four groups of liquids were used for contact angle measurements. these were: distilled water, wet mouth ™ (sodium carboxy methyl cellulose 0.5% w/v, glycerine 30%w/v; icpa health products ltd), mouth kote™ (xylitol, sorbitol, yerba santa; parnell pharmaceuticals) and human saliva. each of these liquids were tested on 20 acrylic denture base specimens each (heat polymerized n=10 and cad-cam n=10). fabrication of specimens forty heat polymerized injection moulded acrylic resin specimens and forty cad-cam acrylic resin specimens (in accordance with the power analysis) were fabricated with each specimen measuring 10x10x2mm. injection moulded specimens: preproportioned capsules of resin and monomer (sr, ivocap high impact, ivoclar vivadent, liechtenstein) were combined and loaded in the cap vibrator (ivoclar vivadent) for 5 minutes. the flask halves were clamped in the frame under 6,000 lbs of pressure and the mixed capsule was inserted. this assembly was connected to a compressed air supply (6 bar/85 psi) to enable the plunger to descend and push the material into the mold. total injection time was programmed to ten minutes, at room temperature. specimens measuring 10x10x2mm were obtained after processing. cad/cam specimens: cad/cam pmma discs (bloomden™, china) of 98 mm diameter and 25 mm height were scanned in the dc5 milling system (dental concept systems gmbh, ulm, germany). a layout of a square block measuring 10x10x2mm was designed on the cad file. the 5-axis milling system was used to mill these blocks. the specimens were finished as per manufacturer’s instructions to obtain an even thickness of 2 mm using acrylic finishing burs. however, no polishing was done for the surface to be tested (tissue surface) to simulate the intaglio surfaces of denture bases. all the specimens were subjected to thermocycling for 10,000 cycles while they were immersed in alternating water baths at 5 ± 1°c and 55 ± 1°c with a 60-second dwell time. [8] for decontamination of the surface of the specimens to be tested, the specimens were first cleaned with http://dentistry3000.pitt.edu/ wettability of two different artificial saliva substitutes on injection moulded heat polymerized acrylic resin and cad-cam acrylic resin: an in vitro study. vol 11 no 1 (2023) doi 10.5195/d3000.2023.184 http://dentistry3000.pitt.edu soap water and then wiped clean with universal solvent (100% ethanol). the specimens were then immersed in an ultrasonic cleaner for 15 minutes.[9] after the cleaning procedure, the test specimens were dried in an oven at 44°c for half an hour and then brought down to the room temperature.[6] the heat polymerized specimens were numbered sequentially and randomly divided into four groups of ten specimens each. similarly, cad-cam specimens were also numbered and randomly divided into four groups. a morning sample of human saliva was collected from a medically healthy male donor. unstimulated whole saliva was collected by requesting the donor to relax and tilt his head forward in order to drool the saliva from the lower lip into a test tube. contact angle measurement a contact angle goniometer (fig. 1), consisting of a photography area and computer software (sca 20 dataphysics, germany), was used to measure the contact angles. using a pipette, 10 microlitres was collected from the test-tube. the test resin specimen was placed in the center of the table of the goniometer and human saliva was dropped on it from the pipette (fig. 2). contact angle measurements were done on the software (sca 20 dataphysics, germany). two contact angle readings were thus obtained on the right side and on the left side of the test specimen (fig. 3). the observations were recorded and a mean of the two readings was calculated. the same procedure was repeated for all specimens (heat cure resin and cad-cam) with all the four test groups of liquids. fig. 1. contact angle goniometer. fig. 2. denture base specimen with 10 microlitre drop of human saliva. fig. 3. contact angle measurements shown on the software (sca 20 dataphysics, germany) results the mean contact angles of the four test solutions on cad-cam and injection moulded heat polymerized acrylic resin are summarized in table 1. the inter-group statistical comparison of distribution of means of continuous variable is done using independent sample t test for two groups and by one-way analysis of variance (anova) with post-hoc bonferroni’s correction for multiple group comparisons for more than two groups. the underlying normality assumption was tested before subjecting the study variable to t test and anova. contact angle measurements were significantly higher in wet mouth™ and mouth kote™ solution of heat cure resin group compared to the cad-cam group (p-value<0.05 for both) indicating that both the artificial saliva substitutes had better wettability on cad-cam denture base (table 1). mouth kote™ had lower mean contact angle (62.63° ± 4.59) than wet mouth™ (77.46° ± 9.16) on cad/cam denture base resin. mouth kote also had lower mean contact angle (70.80° ± 4.79) than wet mouth™ (97.77° ± 7.79) on injection moulded denture base resin (table 2). among the four solutions used, mouth kote™ had best wettability (least contact angle) followed by human saliva, wet mouth™ and distilled water in both the groups of specimens (cad-cam and heat polymerized). http://dentistry3000.pitt.edu/ wettability of two different artificial saliva substitutes on injection moulded heat polymerized acrylic resin and cad-cam acrylic resin: an in vitro study. vol 11 no 1 (2023) doi 10.5195/d3000.2023.184 http://dentistry3000.pitt.edu table 1. inter-material group comparison (cad-cam vs injection moulded) of mean angular measurement according to type of solution. angle (deg) cad-cam (n=40) injection moulded (n=40) p-value solution mean sd mean sd distil (n=10) 95.31 4.67 88.17 4.34 0.037* wet mouth (n=10) 77.46 9.16 97.77 7.79 0.005** mouth kote (n=10) 62.63 4.59 70.80 4.79 0.025* human saliva (n=10) 73.94 6.26 75.92 5.77 0.617ns p-value by independent sample t test. p-value <0.05 is considered to be statistically significant. *p-value<0.05, **p-value<0.01, ns-statistically non-significant. table 2. inter-solution group comparison of mean angular measurement. angle (deg) cad-cam (n=40) injection moulded (n=40) solution mean sd mean sd distil (n=10) 95.31 4.67 88.17 4.34 wet mouth (n=10) 77.46 9.16 97.77 7.79 mouth kote (n=10) 62.63 4.59 70.80 4.79 human saliva (n=10) 73.94 6.26 75.92 5.77 p-value (inter-solution) distil v wet mouth 0.003** 0.115ns distil v mouth kote 0.001*** 0.001*** distil v human saliva 0.001*** 0.026* wet mouth v mouth kote 0.013* 0.001*** wet mouth v human saliva 0.999ns 0.001*** mouth kote v human saliva 0.081ns 0.999ns p-value by anova with post-hoc bonferroni’s correction for multiple group comparisons. p-value <0.05 is considered to be statistically significant. *p-value<0.05, **p-value<0.01, ns-statistically non-significant. http://dentistry3000.pitt.edu/ wettability of two different artificial saliva substitutes on injection moulded heat polymerized acrylic resin and cad-cam acrylic resin: an in vitro study. vol 11 no 1 (2023) doi 10.5195/d3000.2023.184 http://dentistry3000.pitt.edu discussion the null hypothesis was rejected since a statistically significant difference was found in wettability of the two test resins with the different artificial salivary substitutes. heat polymerized acrylic resin dentures are the most commonly used type of material by clinicians. however, its fabrication is a time consuming procedure and involves a lot of manual labour. one of the fastest evolving aspects of modern prosthodontics is cad/cam technology. dentures fabricated using this technique involve minimal time and manual labour. other disadvantages of heat polymerized dentures such as polymerization shrinkage and the possibility of porosities in the denture are also addressed by the cad/cam technique. for a denture to show adequate adhesion to the supporting tissues, the saliva must wet and flow easily over both the contact surfaces i.e. intaglio surface of the denture and the denture bearing mucosa. the flow properties seem to have a huge impact on the clinical efficacy of saliva or its substitutes in addition to viscosity of the liquid; since saliva helps enhance the retention of complete dentures through multiple factors. wettability on oral mucosa and denture base resin is indispensable for the maintenance of lubrication and denture retention. patients with hyposalivation have problems with mastication, swallowing, and speech. dry mouth makes the mucosa prone to irritation and epithelial atrophy; leading to possible inflammation, fissuring, and ulceration. wettability can be measured in terms of the contact angle that a liquid forms with the surface being tested. contact angle has been found to be inversely proportional to wettability. current treatment options for patients with xerostomia include intrinsic and extrinsic approaches. intrinsic approach involves using sialogogues such as pilocarpine and cevimeline. the extrinsic approach is to administer salivary substitutes. the aim of developing salivary substitutes is to attain a viscoelastic configuration comparable to that of human whole saliva. a variety of materials have been used as artificial saliva substitutes over the years. these substitutes have proved to be excellent agents for the palliative treatment of hyposalivation at present. they provide increased lubrication and moisturizing effect of the oral cavity and are available in a range of formulations such as gels, sprays, solutions and oils. the commonly used substitutes are mucin based or carboxymethyl cellulose (cmc) based or herbal saliva substitutes. however, mucin-based saliva substitutes are obtained from porcine gastric tissues which makes it unacceptable for people who keep away from pork. carboxymethyl cellulose based substitute such as wet mouth™, on the other hand, is derived from natural cellulose. mouth kote™, the saliva substitute used in the study, contains yerba santa which is a natural herb and sorbitol which is a natural sweetener. aydin et al, in their study concluded that the mucin, carboxymethylcellulose, and concentrated ion materials all had superior wetting properties than human saliva on the denture base resin.[1] vissink et al[10] had similar results in their study. however, ramanna pk [11] did not find any significant difference between the contact angles of human saliva, distilled water and a cmc based saliva substitute. the current study indicates that the herbal saliva substitute has better wettability than cmc based saliva substitute. ziad al-dwairi [12] et al measured the contact angles made by cad-cam and pmma with distilled water and found no significant difference between the contact angles in the two groups. the current study shows that there is a significant difference between the contact angles of the saliva substitutes on cad/cam and injection moulded pmma. reijden et al [13] believed that new saliva substitutes should not only possess suitable rheological properties but also need more muco-adhesive activity so that the retention of a saliva substitute may be enhanced by acrylic resins binding onto the mucous layer of the oral mucosa. the limitations of the study include that in vitro investigations cannot duplicate the clinical situation. http://dentistry3000.pitt.edu/ wettability of two different artificial saliva substitutes on injection moulded heat polymerized acrylic resin and cad-cam acrylic resin: an in vitro study. vol 11 no 1 (2023) doi 10.5195/d3000.2023.184 http://dentistry3000.pitt.edu clinically, the tissue surface of the denture is irregular, whereas the surface of the test specimens was flat. a better understanding of the flow properties of human saliva and salivary proteins may be a rational approach for the development of a more effective artificial saliva substitute. dynamic contact angle measurements will give a better insight into the rheological properties of the saliva substitutes. within the limitations of this study, it is recommended that cad/cam denture bases should be given along with mouth kote™ in patients having hyposalivation. conclusion mouth kote had better wettability than wet mouth on both cad-cam and injection moulded acrylic resin. both the saliva substitutes had better wettability on cad-cam than on injection moulded acrylic resin. references 1. aydin ak, terzioglu h, ulubaram k, hasirci n. wetting properties of saliva substitutes on acrylic resin. int j prosthodont 1997;10:473-7. 2. davies a, finlay ig. oral care in advanced disease. oxford: oxford university press; 2005. p. 100-3. 3. van der reijdenwa, veerman eci, nieuw amerongen av. treatment of oral dryness related complaints (xerostomia) in sjögren’s syndrome. ann rheum dis 1999;58:465-73. 4. kilani bhz, retief dh, guldag mv, castleberry dj, fischer te. wettability of selected denture base materials. j prosthet dent 1984;52:288-91. 5. park ms, kim yk. viscosity and wettability of carboxymethylcellulose (cmc) solution and artificial saliva. j oral med 2007;32:365-73. 6. jaiswal n, patil pg, gangurde a, parkhedkar rd. wettability of 3 different artificial saliva substitutes on heat-polymerized acrylic resin. the journal of prosthetic dentistry. 2019 mar 1;121(3):517-22. 7. glickman ts. glossary of meteorology. 2nd ed. american meteorological society (2000). 8. lira af, consani rl, mesquita mf, nóbilo ma, henriques ge. effect of toothbrushing, chemical disinfection and thermocycling procedures on the surface microroughness of denture base acrylic resins. gerodontology. 2012 jun;29(2):e891-7. 9. monsenego p, baszkin am, de lourdes costa m, lejoyeux j. complete denture retention. part ii: wettability studies on various acrylic resin denture base materials. j prosthet dent 1989;62:308-12. 10. vissink a, de jong hp, busscher hj, arends js, 'sgravenmade ej. wetting properties ofhuman saliva and saliva substitutes. journal of dental research. 1986 sep;65(9):1121-4. 11. ramanna pk. wettability of three denture base materials to human saliva, saliva substitute, and distilled water: a comparative in vitro study. j indian prosthodont soc 2018;18:248-56. 12. al-dwairi zn, tahboub ky, baba nz, goodacre cj, özcan m. a comparison of the surface properties of cad/cam and conventional polymethylmethacrylate (pmma). journal of prosthodontics. 2019 apr;28(4):452-7. 13. van der reijden wa, veerman ec, nieuw amerongen av. rheological properties of commercially available polysaccharides with potential use in saliva substitutes. biorheology. 1994 jan 1;31(6):631-42. http://dentistry3000.pitt.edu/ vol 7, no 1 (2019) issn 2167-8677 (online) doi 10.5195/d3000.2019.86 http://dentistry3000.pitt.edu new articles in this journal are licensed under a creative commons attribution 4.0 united states license. this journal is published by the university library system, university of pittsburgh as part of its d-scribe digital publishing program and is cosponored by the university of pittsburgh press. applicability of moyer’s prediction tables to estimate the mesio-distal diameter of canines and premolars amanda de sousa galdino1, tânia braga ramos1, vitor marques filgueiras2, rosa helena wanderley lacerda 2 1. brazilian dentistry association/ paraiba, brazil 2. paraiba federal university, university hospital, cleft center, brazil abstract objective: the aim of this study was to verify the applicability of moyer’s prediction tables to estimate the mesio-distal diameter of canines and pre-molars in a population of north-eastern brazil. materials and methods: a sample of 100 plaster moulds of 12-18-year-old patients was selected from the dental archives of the brazilian association of orthodontists dental school clinic in the city of joão pessoa, state of paraiba. analyses were performed with r software (version 3.1.3). the upper and lower canines and premolars on both sides of the arch were measured. data analysis: linear regression analysis was used to assess the closeness of the estimated values in each percentile of moyer’s prediction table. analyses were performed at a significance level of 5%. results: gender dimorphism (p < 0.001) was found in both upper and lower arches. in the upper arch, the best regression models for data analysis of female, male and both were, respectively, p5 and p6”, p25 and p35, and p35 and 75, whereas in the lower arch were p15, p35 and p75, with “p75 being the best-fitted percentile following application of correction equations for both genders. conclusions: the moyer’s prediction table can be applied for estimating the mesio-distal diameters of canines and pre-molars, provided that a correction factor is used for the population under study. citation: galdino as, et al. (2019) applicability of moyer’s prediction tables to estimate the mesio-distal diameter of canines and premolars. dentistry 3000. 1:a001 doi:10.5195/d3000.2019.86 received: october 7, 2018 accepted: december 6, 2018 published: june 26, 2019 copyright: ©2019 galdino as, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email:rhelenawanderley@msn.com introduction the majority of malocclusions are related to an imbalance between mesio-distal diameter of the teeth and their bone support (1). this statement was observed by angle (1899), who classified the malocclusions of the teeth. antero-posterior malocclusions were classified as follows: class i normal relationship between maxilla and mandible; class ii the maxilla is more anteriorly positioned than the mandible; and class iii – the mandible is more anteriorly positioned than the maxilla (2, 3). as there are several changes within the alveoli due to bone growth during the phase of mixed dentition, the majority of malocclusions occur in these sites. also, because some teeth (e.g. canines and pre-molars) are still unerupted, it is complicated to evaluate any discrepancy between teeth and bone supports. in this phase, it is important to estimate the size of the unerupted permanent teeth in order to evaluate whether the dental sockets will correspond to the dental volume after eruption (4). there are some methods to evaluate discrepancies and the moyer’s method is one of the most used as it has some advantages, such as simplicity in usage, quickness and no need of periapical radiography (5). the moyer’s analysis is based on a statistical method in which one can predict the sum of mesiodistal measurements of unerupted canines and pre-molars through its relationship with the sum of the mesio-distal measurements of the already erupted lower incisors (6). however, the method proposed by the author was based on a north-american population, and thus its accuracy may vary for populations from other countries (7). the objective of this work was to assess the applicability of the moyer’s prediction tables to http://dentistry3000.pitt.edu/ http://www.library.pitt.edu/ http://www.pitt.edu/ http://www.library.pitt.edu/articles/digpubtype/index.html http://upress.pitt.edu/ http://creativecommons.org/licenses/by/4.0/ applicability of moyer’s prediction tables to estimate the mesio-distal diameter of canines and premolars vol 7 no 1 (2019) doi 10.5195/d3000.2019.86 http://dentistry3000.pitt.edu estimate the mesio-distal diameter of canines and premolars in a population of northeastern brazil. materials and methods samples • all the plaster moulds of 12-18-year-old patients who attended the brazilian association of orthodontists dental school clinic in the city of joão pessoa, state of paraiba, between 2010 and 2015 were evaluated for selection, resulting in a sample of 100 moulds according to inclusion and exclusion criteria. were included plaster moulds in good conditions, that is without fractures or distortions, with all the teeth should be erupted (i.e. dentition from first molar to first molar); in patients aged between 12 and 18 years old. were excluded teeth with anomalies in shape or size and evidence of caries or proximal restorations in the teeth to be measured. the present study was approved by the local human research ethics committee according to protocol number 44133515.6.0000.5180 and resolution number 196 of 1996 by the national health committee and national ethics committee of the ministry of health. pilot study a pilot study was performed to calibrate the examiner, with kappa test being 0.835 at significance level of 5% and confidence interval of 95%. in the pilot study, a total corresponding 10% of the total sample, and therefore 10 models, evaluated twice, by the same examiner with an interval of 10 days and only the research was initiated after obtaining the concordance index intra-examiner above 0.7, considered good agreement and therefore means the examiner was calibrated and fit for research development. these models did not part of the survey sample. measurement the mesio-distal diameter of the lower incisors of each plaster mould was measured to obtain the sum of this space. next, the measurements of upper and lower canines and pre-molars were performed on both sides based on the largest distances between the contact points of the proximal surfaces. a digital calliper (digimess, são paulo, brasil) was used for the measurements ( calibration 0,005mm/0,002”), being positioned in parallel to the occlusal surface and perpendicular to the axis of the tooth. the sample was divided into groups of 10 moulds each and they were individually measured, avoid the vies of the fatigue that could compromise the quality of the measurement. next, the sum of the measurements of upper and lower canines and premolars were transferred to excel spreadsheets, which also included gender and age as variables. statistical analysis normality distribution test was applied to the mean values obtained from the total measurements of the mesio-distal diameter of canines and premolars, resulting in non-normal distribution for both females (p = 0.031; shapiro-wilk) and males (p < 0.001; kolmogorov-smirnov) as well as in complete distribution for both genders (p < 0.001; kolmogorov-smirnov). in view of the results of the normality distribution test, the mann-whitney test was performed to assess the existence of gender dimorphism between females and males regarding the mean measurements of canines and pre-molars, showing presence of dimorphism (p < 0.001) in both upper and lower arches. next, the spearman’s correlation coefficients were calculated between mean values and moyer’s percentiles for genders separately. in view of the significant results for correlation analysis, a linear regression analysis was http://dentistry3000.pitt.edu/ applicability of moyer’s prediction tables to estimate the mesio-distal diameter of canines and premolars vol 7 no 1 (2019) doi 10.5195/d3000.2019.86 http://dentistry3000.pitt.edu conducted to assess the prediction of measurements by using the moyer’s percentiles, including generation of determination coefficient, which was the parameter adopted to chose the percentiles closer to the actual values found in the population of the city of joão pessoa in both males and females as well as in the general population. for each mould in each analysis, the following was verified: error normality, homoscedasticity of residuals, error independence, linearity of predicted mean values and absence of aberrant, leverage or influence values. all the models proposed met the requisites of a fitted linear regression model. all the analyses were conducted by using the statistical software r (version 3.1.3) at significance level of 5%. results table i show the measurements of canines and pre-molars regarding gender dimorphism, respectively, in the upper and lower arches. tables i upper arch the results listed in table ii show that there are significant correlations between actual values and those estimated by the moyer’s percentiles for males and females separately and for both genders, with stronger correlations being observed in males only, followed by both genders. for assessment of a correlation, values above 0.8 are considered as having excellent internal consistency. prieto & muniz (2000) suggest that, for samples smaller than 200 subjects, values above 0.6 are considered adequate enough so that the spearman’s ranking can be the following: 0.70-0.79 (adequate); 0.80-0.84 (good) and above 0.85 (excellent) (8). table ii in addition, the correlation values are positive, meaning that the values observed follow the actual ones so that the higher the actual values, the higher the estimated values. linear regression analysis the best models of linear regression obtained for analysis of data regarding females, males and both genders were, respectively, p5 and p65, p25, and p35 and p75. with regard to equal models regarding the determination coefficient for assessment of females and both genders, the models were diagnosed to decide which the best one was. in this way, standardised residuals, cook’s distance and residual square sum were assessed to verify the existence of differences between the models for these parameters. table iii the smaller the standardised errors, the cook’s distance and the residual square sum, the better the models because of the smaller distances between actual values and estimated ones. therefore, there was no difference between the models generated for females, with the 75th percentile model being the best to predict space for unerupted teeth in both genders due to the lowest residual square sum. in this way, one can observe that the 5th percentile (r² = 0.624) and 65th percentile (r² = 0.750) for female gender had the same parameters; the 25th and 75th percentiles were the best to predict measurements, respectively, for male gender and both genders. however, in view of the low rate of adjustment of the models for the female gender (62.4%), it is suggested that correction factors should be used for the best models for male gender and both genders, and in the case of females, it is suggested the use of parameters for estimation in both genders. the correction factors for these models are described below in table iv, with respective rates of adjustment. table iv lower arch assessment of correlation between mean values of premolars and each percentile by gender and regarding the whole set of data. these results indicate that there are significant correlations between actual values and estimated ones by using the http://dentistry3000.pitt.edu/ applicability of moyer’s prediction tables to estimate the mesio-distal diameter of canines and premolars vol 7 no 1 (2019) doi 10.5195/d3000.2019.86 http://dentistry3000.pitt.edu moyer’s percentiles for males and females separately as well as for both genders, with stronger correlations being observed for data on males followed by data on both genders. in addition, the positive correlation values mean that the values observed correspond with the actual values so that the higher the actual values, the higher the estimated ones ( table v). linear regression analysis the best models of linear regression obtained for analysis of data regarding females, males and both genders involving the predictor variable were, respectively, p15 and p35, p75. by observing the values obtained for the different parameters of each model in each analysis (i.e. female, male and both), one can realise that they are equal. in this way, standardised residuals, cook’s distance and residual square sum were assessed to verify the existence of differences between the models for these parameters. however, according to table vi, one can observe that there are differences neither in the mean values of residuals, nor in the mean values of cook’s distance and residual square sum and respective standard deviations between the models of percentiles p15, p35 and p75 in all analyses. table v and vi the smaller the standardised errors and the cook’s distance, the better the models because of the smaller distances between actual values and estimated ones. therefore, there was no difference between the models generated for females, with the 75th percentile model being the best to predict space for unerupted teeth in both genders due to the lowest residual square sum. therefore, table vi shows that there was no difference between the models generated for each assessment (i.e. female, male and both). in this way, one can realise that any percentile (i.e. p15, p35 and 075) can be used to estimate the space for pre-molars, provided that the following correction factors are used for the population under study. table vii discussion the success of the orthodontic therapy depends mainly on the correct diagnosis (9), thus the treatment planning should include key elements of an early diagnosis. the methods to predict the mesiodistal diameter of permanent teeth and the discrepancy between moulds play an important role in this context, since space problems can be diagnosed and treated even during the mixed dentition in a less invasive manner (10). in view of this, the present study was aimed at assessing one of the most used methods by surgeondentists and its applicability in a study population. the presence of gender dimorphism (tables i) in the findings of our study is corroborated by several works in the literature, that is, showing that men have larger upper and lower dental arches than women (11,12,13). yamaguti & vasconcelos (2005) also reported that there is gender dimorphism as all the mesio-distal dental dimensions of male individuals are greater than those of females (14). by applying the moyer’s prediction table to our population, the correlation coefficients revealed that the percentiles corresponding to the best regression models in the upper arch for males and females separately as well as for both genders were p5 and p65, p25, p35, and p75, with the 75th percentile being the best model for both genders. in the lower arch, the percentiles p15, p35 and p75 had the best regression models. some studies (15,16,17) showed that the moyer’s table could not be used in their populations, since the actual values did not correspond to the estimated ones, by either overestimating or under-estimating the values, as in the studies by farret et al. and cabral & pessoa (16,17), respectively. however, by means of statistical tests, we found that the moyer’s tables can be applied to different populations other than that used in the original study, provided that they are adjusted by using correction factors. this was corroborated by a similar study performed in the north-eastern brazil, with the probability tables being shown to be reliable for use http://dentistry3000.pitt.edu/ applicability of moyer’s prediction tables to estimate the mesio-distal diameter of canines and premolars vol 7 no 1 (2019) doi 10.5195/d3000.2019.86 http://dentistry3000.pitt.edu in caucasian individuals (12), but it is necessary to use correction factors with an equation (table iv), as for lower arch: p75 25.094 + 2.019 x 20.4 = 16.09. fida & bherwani (18) (2011) compared the use of the linear equations developed for the north-american population to that developed by them for a population of pakistani children and also compared the results to those found by moyers and tanaka & johnstone, reporting no significant differences. however, the authors concluded that the methods used for the northamerican population did not fit to the pakistani one, leading to the need to develop other method (18, 19). in a study on caucasian brazilians, melgaço, araujo & ruelas (2006) also used linear regression equations and reported that the prediction method was good in determining the width of canines and pre-molars. the moyer’s prediction tables have probabilities ranging from 5 to 95 percent, but the author suggests that a 75-percent level should be used to estimate the values in order to prevent crowding (21). in the study by cabral (2002), the values of the moyer’s table were used at a 50percent level, since they are mean values and t-test compares them (16,17). in this study, such a percentile was also chosen because it represents a high adjustment rate, that is, r2 = 76.3 percent. however, the adjustment rate was low for females and for this reason we suggest its use to standardise the use of the equation for both genders. table viii and iv from these results, we propose that the tables v and vi should be used in patients of north-eastern brazil in order to facilitate the treatment. based on the results found, we have concluded that: 1) there was gender dimorphism between the statistical models for females and males, with the sum of the mesio-distal diameters of incisors, canines and pre-molars in men being greater than that in women regarding upper and lower arches; 2) the correlation tests and linear regression analyses showed that the best regression models to assess data from both genders was those using the 75th percentile; 3) the moyer’s table can be used to predict the mesiodistal diameter of canines and pre-molars in the population of northeastern brazil, provided that it is adjusted by means of a correction factor. references 1. patterns of dental crowding in the lower arch and contributing factors. a statistical study. shigenobu n, hisano m, shima s et al. angle orthod; 2007;15:303310. https://doi.org/10.2319/00033219(2007)077[0303:podcit]2.0. co;2 2.classification of malocclusion. angle eh. dent cosmos; 1899; 41: 248-64. 3. avaliação comparativa de três métodos para cálculo do espaço requerido na dentição mista . oliveira mvp, ruellas mm, oliveira ac. . revista odonto ciência – fac. odonto/pucrs; 2007:148-153. http://scholar.google.com.br/scho lar_url?url=http%3a%2f%2fwww. matheuspithon.com.br%2fv2%2f wpcontent%2fuploads%2fodonto2007-226.pdf&hl 4. análise da dentição mista: tomografia versus predição e medida radiográfica. felício lg, ruellas aco, bolognese am, santana ef, araújo mtz. dental press j orthod; 2010; 15: 159-165. 5. new, fast, and accurate procedure to calibrate a 2dimensional digital measurement method. paredes v, gandia jl, cibrian r. am j orthod dentofacial orthop; 2005; 127: 518-519. 6. handbook of orthodontics for the student and general practioner. moyers re.. 2ª ed. chicago; year book medical, 1963. 7. orthodontic probability tables forblack patients of africa descent: mixed dentition analysis. schirmer ur, wiltshire, wa. am j orthoddentofacial orthop.;1997; 112: 545-551. http://dentistry3000.pitt.edu/ applicability of moyer’s prediction tables to estimate the mesio-distal diameter of canines and premolars vol 7 no 1 (2019) doi 10.5195/d3000.2019.86 http://dentistry3000.pitt.edu 8. un modelo para evaluar la calidad de los tests utilizados en españa. prieto g; muñiz, j.; disponível em: <http//www.cop.es/tests/modelo. htm>. 9. prediction of the mesiodistal width of unerupted permanent canines and premolars: a statistical approach. boboc a, dibbets j. am j orthod dentofacial orthop; 2010; 137: 503-507. 10. comparação de diferentes incidências radiográficas para a predição de tamanho dentário na dentição mista. gonçalves ts, menezes lm, lima, ems. revista ortodontia gaúcha; 2011;15: 1-12. 11. determinação do diâmetro mésio-distal de dentes caninos e pré-molares em indivíduos brasileiros da região de araraquara. paixão rf, cordeiro, rcl, gandini junior, l. r dental press ortodon ortop facial; 2002; 7:45-43. 12. applicability of moyers' probability table in the population of the northeast of brazil. pithon mm , lacerda-santos r, araujo jts, coqueiro rs. biosci. j. 2015; 31: 311-318. 13. mixed dentition space analysis in kodava population: a comparison of two methods. ramesh n, reddy ms, palukunnu b, shetty b, puthalat u. journal of clinical and diagnostic research; 2014; 8: 01-06. 14. determinação das medidas dentárias mésio-distais em indivíduos brasileiros leucodermas com oclusão normal. yamaguto ot, vasconcelos mhf. dental press ortodon ortop facial; 2005; 10: 99-107. 15. aplicabilidade da tabela de moyers na predição do tamanho dos caninos e pré-molares em brasileiros leucodermas descendentes de europeus. farret mmb, jurach em, lopes lfd, porto ss, porto vs.ortodontia; 2005; 38: 163-168. 16. análise de dentição mista – avaliação das tabelas de moyers em campina grande, paraíba. cabral ed, guedes ap. j bras ortodon ortop facial; 2002; 7: 235-237. 17. evaluation of moyers' tables to estimate the width of canine and premolars: a study in natal-rn. cabral ed, caldas ja , frança a, cabral filho h,caldas sgfr. j. bras. ortodon. ortop. facial; 2004; 9:3740. 18. development of a prediction equation for the mixed dentition in a pakistani sample. bherwani ak, fida m. american journal of orthodontics and dentofacial orthopedics; 2011;140: 626-632. 19. the prediction of the size of unerupted canines and premolars in a contemporary orthodontic population. tanaka mm, johnston le. j am dent assoc;1974; 88: 798801. 20. applicability of three tooth size prediction methods for white brazilians. melgaço ca, araújo mt, ruellas aco. angle orthodontist; 2006; 76: 644-649. 21. applicability of moyers analysis in mixed dentition: a systematic review. galvão mab, dominguez gc, tormin st, akamine a, tortamano a, fantini sm. dental press j orthod; 2013; 18:100-105. http://dentistry3000.pitt.edu/ sodium hypochlorite for primary teeth pulpotomy vol 9 no 1 (2021) doi 10.5195/d3000.2021.124 http://dentistry3000.pitt.edu a clinical and radiographic assessment of sodium hypochlorite versus formocresol pulpotomy in primary molar teeth: 12-month follow-up zahra bahrololoomi1, fatemeh zarebidoki 2, atefeh shakib3* 1 associated professor, social determinants of oral health research center, department of pediatric dentistry, shahid sadoughi university of medical sciences, yazd, iran. 2 assistant professor, department of pediatric dentistry, shahid sadoughi university of medical sciences, faculty of dentistry, yazd, iran. 3 pedodontist abstract objective: the aim of this study was to compare the clinical and radiographic success rates of pulpotomy in primary molars using formocresol versus sodium hypochlorite. methods: twenty-three children aged 4-9 years with at least two primary molars requiring pulpotomy were randomly allocated into two groups. all teeth received stainless steel crown after conventional pulpotomy procedure with either naocl or formocresol. clinical and radiographic signs/symptoms were recorded at six and 12 months. outcomes were statistically analyzed using fisher’s exact test and chisquare test. results: clinical success rates at 6 and 12-month follow-up in both groups was 100%. at 6-month followup, radiographic success rate for naocl and formocresol groups was 100%. at 12-month recalls, in naocl group, 20 teeth (87%) and in formocresol group, 21 teeth (91.3%) had radiographic success. no significant difference was found in the radiographic success rates at 12 months (p=1.00). internal root resorption was the most common radiographic pathologic finding in both groups. conclusion: clinical and radiographic success rates in naocl group was comparable with formocresol group, so naocl can be suggested as an alternative for primary teeth pulpotomies. however further clinical studies with long-term follow-ups are needed. keywords: formocresol; pulpotomy; sodium hypochlorite; tooth; deciduous citation: bahrololoomi z, et al. (2021) a clinical and radiographic assessment of sodium hypochlorite versus formocresol pulpotomy in primary molar teeth: 12-month follow-up. dentistry 3000. 1:a001 doi:10.5195/d3000.2021.124 received: december 4, 2020 accepted: march 6, 2021 published: july 19, 2021 copyright: ©2021 bahrololoomi z, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: dr.1370@yahoo.com introduction pulpotomy is a common therapeutic method for treatment of primary teeth in which teeth had deep carious lesions approaching the pulp and some cases also had a history of pain. this pain lasted less than 20 minutes, was provoked by chewing foods, especially sweets, did not wake the child from sleep [1]. various medicaments are used in pulpotomy of primary teeth among which formocresol (fc) is the commonest. the reported success rate of fc pulpotomy is 70-98% [2, 3], but some studies showed the possibility of such local or systemic side effects as pulpal reactions with inflammation, necrosis, cytotoxicity, and potential mutagenic or carcinogenic effects and immunologic responses after fc use. there are also concerns http://dentistry3000.pitt.edu/ sodium hypochlorite for primary teeth pulpotomy vol 9 no 1 (2021) doi 10.5195/d3000.2021.124 http://dentistry3000.pitt.edu about the effect of fc on the enamel of the succedaneums permanent teeth after pulpotomy of primary teeth [4]. considering the side effects of fc, its substitution with a safer substance seems necessary. therefore, in different studies, medicament and non-medicament methods have been proposed to replace fc in pulpotomy of primary teeth [5]. sodium hypochlorite (naocl) is a substance used for canal irrigation, hemostasis and removing biofilms and debris [6]. it has strong biological properties that include antimicrobial activity and organic tissue dissolution capacity [7]. it presents a wellrecognized in vitro antimicrobial activity although in some cases this solution fails to achieve a bacteria-free root canal under clinical coditions [7-9] the antimicrobial effect of naocl solutions depends on their free available chlorine, which consists of hypochlorous acid and the hypochlorite ion [8, 9] clinical studies in which naocl was used for pulpotomy of primary molars, showed that its clinical and radiographic success rate was comparable with fc and ferric sulfate [4, 10]; although there are few studies on this issue. therefore, this study was designed to assess the clinical and radiographic success rate of primary molar pulpotomy using fc and naocl. material and methods this was a double-blind randomized controlled trial approved by the ethics committee of shahid sadoughi university of medical sciences (code: ir.ssu.rec.1394.107) and registered in iranian center for registration of clinical trials (code: irct20150927935n5). totally, 25 children 4-9 years old referred to the pediatric department of dental school in shahd sadoughi university of medical sciences with at least two primary molars requiring pulpotomy entered the study. two children (4 teeth) didn't continue the study during 12-months follow-up. a parallel design was used, because only some of the children had at least two primary molars requiring pulpotomy. the tooth was considered the unit for randomization. on the day of the procedure before the pulpotomy was performed, the operator was given a sealed envelope that contained a letter denoting the medicament group. the patients were not informed about their group allocation. inclusion criteria were: in clinical examination: primary molars with deep caries and pulp exposure; lack of pulp degeneration in the affected teeth (e.g. spontaneous and nocturnal pain, swelling, mobility, abscess, fistula and tenderness to percussion); in radiographic examination: lack of internal or external root resorption and furcation radiolucency; being restorable by stainless steel crowns. before examinations, the objectives of the study were explained for the parents and an informed consent was obtained from them. at first, the tooth was anesthetized using lidocain 2% and epinephrine 1/80000 (daroupaksh co., tehran, iran). after isolation of selected teeth with a rubber dam, caries were removed and then pulpal chamber accessed by a fissure diamond bur n≠330 (tizkavan, tahran, iran) in a watercooled high-speed hand-piece. for removing pulp tissue, a spoon excavator was used and after saline irrigation, cotton pellets were inserted in place for 5 minutes to induce hemostasis. if bleeding continued, the patient was excluded from the study, and if hemostasis was induced, treatment was continued in both groups. in first group, a 1:5 dilution of buckley's fc (ssa, produits dentaires, switzerland) was used for 5 minutes and in the http://dentistry3000.pitt.edu/ sodium hypochlorite for primary teeth pulpotomy vol 9 no 1 (2021) doi 10.5195/d3000.2021.124 http://dentistry3000.pitt.edu second group, naocl 5% (sehhat, tehran, iran) was applied by a cotton applicator in the pulp chamber for 30 seconds. then, in both groups, reinforced zinc oxide eugenol (kemdent, uk) was inserted in the access cavity and in the same session, teeth were restored by stainless steel crowns (3m/espe, dental products, st. paul, mn, usa). all procedures were performed by postgraduate pedodontist. clinical and radiographic outcome assessments were made by one independent experienced pedodontist who was blind to the treatment at follow-up visits. the success of treatment was determined as the absence of spontaneous pain, mobility, swelling, fistula and tenderness to percussion. radiographic success was determined as lack of internal or external resorption, and interradicular and periapical radiolucency. all radiographs were taken by plammeca oy (helsinki, finland) using e films (skydent, slovakia). and the images were reevaluated by the same specialists after 7 days (intra-examiner agreement>90%). data were analyzed by spss (ver. 20) using fisher's exact test and ci square test. the p value <0.05 was considered significant. results this study was started with 25 children 4-9 years old (13 females and 12 males) with mean age of 6.5 years. fifty primary molars were randomly allocated into two treatment groups. two children (4 teeth) didn't continue the study during 12-months follow-up. therefore, 23 patients were evaluated. table 1 shows the distribution of the teeth in two groups. there was not a statistically significant difference between two groups regarding gender, age, tooth type and tooth location (p>0.05) clinical findings: from 46 pulpotomized teeth at 6 and 12-month recalls, 100% of the cases in both fc and naocl groups showed clinical success. radiographic findings: at 6-month recall, 100% of cases in both groups showed radiographic success (fig. 1). after 12 months, in naocl group, three teeth (13%) showed internal resorption, among which two teeth showed external resorption and involvement of inter-radicular bone as well; and in fc group, failure was observed in two teeth (8.7%), among which one tooth showed internal resorption (fig 2) and another tooth showed both internal resorption and involvement of inter-radicular bone. the most frequent radiographic failure was internal resorption without insignificant difference between groups (p>0.05). figure 1. successful treatment of naocl pulpotomy of lower left second primary molar after 12 months follow-up. figure 2. radiographic failure of fc pulpotomy on lower left first primary molar with internal root resorption after 12 months follow-up. the 12-month radiographic success rate was 87% and 91.3% in naocl and fc groups, respectively, and the difference was not statistically significant (p = 1.00) (table 2). table 1. distribution of pulpotomied primary molars according to the type of teeth. http://dentistry3000.pitt.edu/ sodium hypochlorite for primary teeth pulpotomy vol 9 no 1 (2021) doi 10.5195/d3000.2021.124 http://dentistry3000.pitt.edu treatment group number of teeth first primary molar at 6 months second primary molar at 6 months first primary molar at 12 months second primary molar at 12 months maxillary mandibular total maxillary mandibular total maxillary mandibular total maxillary mandibular total fc 9 5 14 4 7 11 8 5 13 3 7 10 naocl 6 10 16 9 0 9 5 10 15 8 0 8 table 2. comparison of the clinical and radiographic success rate in primary molars pulpotomied by fc and naocl at 6 and 12-month follow-ups. success rate, n (percent) treatment group clinical radiographic 6 month 12 month 6 month 12 month success failure success failure success failure success failure fc 23 (100) 0 23 (100) 0 23 (100) 0 21 (91.3) 2 (8.7) naocl 23 (100) 0 23 (100) 0 23 (100) 0 20 (87) 3 (13) discussion the first objective of pulp therapy is maintaining the integrity and health of oral tissue. it is necessary to try to maintain pulp vitality [11]. pulp therapy has been successfully performed for severely destructed primary molars by buckley's fc since 1904. controversy to use this substance was due to its potential mutagenic and carcinogenic effect; so other drugs have been introduced with different success rates and costs [12]. previous studies used naocl 2.5 to 5.25 percent. in the current study, similar to al-mutairi et al.[13], vargas et al.[4], vostak et al.[10], and tzu-ying li studies[14], naocl 5% was used. only in the studies conducted by ruby et al.[12] and shabzendehdar et al.[15] naocl 3% was used. akay et al.[16] who assessed the effect of naocl on disinfection before application of the substance covering pulp, used naocl 5% [16]. according to the studies and considering the difficulties in preparing naocl 3%, concentration of 5% was used in the present study. in the current study, clinical success rate of naocl and fc at 6 and 12-month recalls was 100%. radiographic success rate at 6month recall was 100% in both groups which was consistent with the results of farsi et al. [17], although other studies reported success rate to be between 8699% [4, 10, 12-15, 18]. radiographic success rate at 12month recall was 91.3% and 87% in fc and naocl groups, http://dentistry3000.pitt.edu/ sodium hypochlorite for primary teeth pulpotomy vol 9 no 1 (2021) doi 10.5195/d3000.2021.124 http://dentistry3000.pitt.edu respectively, which was in agreement with some other studies [12, 13, 15]. al_mutairi et al. found 12-month clinical and radiographic success rate of naocl to be 94.6 and 86.5%, respectively [13]. they used a method similar to our study. in the study conducted by ruby et al. the success rate of naocl was 100%, though with a lower radiographic success rate, i.e. 90%. they didn't find a significant difference between the success rate of naocl and fc which was consistent with the results of the current study; although they used naocl 3% [12]. in the study of shabzendehdar et al. in agreement with the current study, there was not a significant difference between two substances. they used naocl 3%, but duration of substance application was 30 s, similar to the current study. another difference between the studies was that they performed pulpotomy only on mandibular first molars and their participants had at least one tooth requiring pulpotomy [15]. tzu-ying li et al. conducted a retrospective study with a larger sample size and a longer duration of recall (24 months). the most frequent radiographic finding in the present study was internal resorption which was observed in 21.7% of teeth at 12month recall. although no clinical signs were seen in these teeth which was consistent with the results of the previous studies [4, 10, 14, 15, 18]. we couldn't find a satisfactory explanation for the internal resorption occuring after pulpotomy; even though during pulpotomy it is possible that abnormal pulpal tissue remains in the place, and if the inflammation extends to the entrance of the pulp canal, odontoclasts will be attracted to the area [19]. internal resorption may occur due to zinc oxide eugenol use, because eugenol is irritant for pulp and may induce pulp inflammation. the fixing effect of fc can probably prevent the irritating effect of eugenol, and the hemostatic properties of naocl may prevent the necrosis layer to be formed [20]. bony changes may lead to the formation of abscess, fistula, pain and mobility of the tooth, while internal resorption is limited to the tooth and is from dental origin which was explained earlier. internal resorption is not so important clinically, and it is possible that the problem occurs after pulpotomy without any alarming signs for the patient and the dentists, and it may be accidentally discovered in radiography[21]. other radiographic failures include external resorption and bone lucency which are among bony changes which are more important than internal resorption in their effect on the development of permanent teeth. these changes were fortunately less frequent than internal resorption in the current study. the failures after treatment of vital pulp and eventual failure in the formation of a calcified bridge on the vital pulp depends on the patient's age, the degree of surgical trauma, pressure during sealing, selection of inappropriate covering substance, low patient's resistance threshold and the presence of microorganisms and the eventual infection. in summary, the reason for difference between studies is probably due to different methods, age range of the participants, difference in tooth type, design of the study and sample size. it is recommended that clinical studies with larger sample size and longer duration of recalls be performed. conclusion http://dentistry3000.pitt.edu/ sodium hypochlorite for primary teeth pulpotomy vol 9 no 1 (2021) doi 10.5195/d3000.2021.124 http://dentistry3000.pitt.edu the findings of the current study showed that clinical and radiographic success rate of naocl is comparable to fc. a substance covering the pulp should have bacteriocidal and biocompatibility properties with low cost. naocl has all these properties and is inexpensive and easily available. conflicts of interest the authors deny conflicts of interest to this study. acknowledgment: the authors wish to thank the vice chancellor of research of shahid sadoughi university of medical sciences. references 1. farooq ns, coll j, kuwabara a, shelton p. success rates of formocresol pulpotomy and indirect pulp therapy in the treatment of deep dentinal caries in primary teeth. pediatric dentistry. 2000;22(4):278-86 . 2. haghgoo r, abbasi f. a histopathological comparison of pulpotomy with sodium hypochlorite and formocresol. iran endod j. 2012;7(2):60-2 . 3. bahrololoomi z, fekrazad r, zamaninejad s. antibacterial effect of diode laser in pulpectomy of primary teeth. journal of lasers in medical sciences. 2017;8(4):197. 4. vargas kg, packham b, lowman d. preliminary evaluation of sodium hypochlorite for pulpotomies in primary molars. pediatr dent. 2006;28(6):511-7 . 5. bahrololoomi z, moeintaghavi a, emtiazi m, hosseini g. clinical and radiographic comparison of primary molars after formocresol and electrosurgical pulpotomy: a randomized clinical trial. indian j dent res. 2008;19(3):219-23 . 6. poggio c, arciola cr, dagna a, chiesa m, sforza d, visai l. antimicrobial activity of sodium hypochlorite-based irrigating solutions. int j artif organs. 2010;33(9):654-9 . 7. tejada s, baca p, ferrerluque cm, ruiz-linares m, valderrama mj, arias-moliz mt. influence of dentine debris and organic tissue on the properties of sodium hypochlorite solutions. int endod j. 2018 . 8. arias-moliz mt, morago a, ordinola-zapata r, ferrer-luque cm, ruiz-linares m, baca p. effects of dentin debris on the antimicrobial properties of sodium hypochlorite and etidronic acid. j endod. 2016;42(5):771-5 . 9. bystrom a, sundqvist g. the antibacterial action of sodium hypochlorite and edta in 60 cases of endodontic therapy. int endod j. 1985;18(1):35-40 . 10. vostatek sf, kanellis mj, weber-gasparoni k, gregorsok rl. sodium hypochlorite pulpotomies in primary teeth: a retrospective assessment. pediatr dent. 2011;33(4):327-32 . 11. shayegan a, atash r, petein m, abbeele av. nanohydroxyapatite used as a pulpotomy and direct pulp capping agent in primary pig teeth. j dent child (chic). 2010;77(2):77-83 . 12. ruby jd, cox cf, mitchell sc, makhija s, chompu-inwai p, jackson j. a randomized study of sodium hypochlorite versus formocresol pulpotomy in primary molar teeth. int j paediatr dent. 2013;23(2):14552 . 13. al-mutairi ma, bawazir oa. sodium hypochlorite versus formocresol in primary molars pulpotomies :a randomized clinical trial. eur j paediatr dent. 2013;14(1):33-6 . 14. li t-y, chuang l-c, tsai ai. a retrospective study of sodium hypochlorite pulpotomies in primary molars. journal of dental sciences. 2016;11(3):261-5 . 15. shabzendedar m, mazhari f ,alami m, talebi m. sodium hypochlorite vs formocresol as pulpotomy medicaments in primary molars: 1-year follow-up. pediatr dent. 2013;35(4):329-32 . http://dentistry3000.pitt.edu/ sodium hypochlorite for primary teeth pulpotomy vol 9 no 1 (2021) doi 10.5195/d3000.2021.124 http://dentistry3000.pitt.edu 16. akcay m, sari s, duruturk l, gunhan o. effects of sodium hypoclorite as disinfectant material previous to pulpotomies in primary teeth. clin oral investig. 2015;19(4):803-11 . 17. farsi dj, el-khodary hm, farsi nm, el ashiry ea, yagmoor ma, alzain sm. sodium hypochlorite versus formocresol and ferric sulfate pulpotomies in primary molars: 18-month follow-up. pediatr dent. 2015;37(7):535-40 . 18. ghasemi d, dehghan z. one year comparison of clinical and radiographic success rate of ferric sulfate and sodium hypochlorite for primary molar teeth pulpotomy. journal of mashhad dental school. 2014;38(1):37-44 . 19. chin jr, kowolik je, stookey gk. dental caries in the child and adolescent. mcdonald and avery's dentistry for the child and adolescent (tenth edition): elsevier; 2016. p. 155-76 . 20. smith nl, seale ns, nunn me. ferric sulfate pulpotomy in primary molars: a retrospective study. pediatr dent. 2000;22(3):192-9 . 21. fuks ab, bimstein e. clinical evaluation of diluted formocresol pulpotomies in primary teeth of school children. pediatr dent. 1981;3(4):321-4 . http://dentistry3000.pitt.edu/ vol 7, no 1 (2019) issn 2167-8677 (online) doi 10.5195/d3000.2019.89 http://dentistry3000.pitt.edu investigation of surface topography of different root-end filling materials: an in vitro study mine koruyucu1, hazal özcan1, merve bayram2, abdullah burak çankaya3, nurullah keklikoglu4, figen seymen1 1istanbul university, faculty of dentistry, department of pedodontics, istanbul, turkey 2istanbul medipol university, faculty of dentistry, department of pedodontics, istanbul, turkey 3istanbul university, faculty of dentistry, department of oral and maxillofacial surgery, istanbul, turkey 4 university, faculty of dentistry, department of histology and embriology, istanbul, turkey abstract aim: although there are many materials that can be used for retrograde filling in surgical endodontics, none of them can be regarded as an ideal material yet. the purpose of this study was to compare the surface topography of three different root-end filling materials. methods: 36 extracted single rooted human incisor teeth were cleaned and decoronated to standardized 10 mm root lengths. the root segments were prepared and 2 mm apical resection were performed. the samples were randomly separeted to three groups (group a: ca(oh)2, group b: mta angelus, group c: proroot mta), each one of them was comprised of 12 roots. materials were placed as 2 mm apical barriers and obturated with guttapercha and ah-plus sealer. each group was dimidiated into two subgroups (a1,a2,b1,b2,c1,c2). groups a1,b1,c1 were stored in normal saline (ns), groups a2,b2,c2 were stored in neutral phosphate buffer saline (npbs) solution and samples were incubated at 370c for 2 weeks. stereomicroscope (32x) was used to photograph the root-end filling. results: all specimens demonstrated white crystals formation and sediment over the root-end filling materials and on the superficial border of the root-end cavities’ wall as a white plaque. a2,b2,c2 samples had more crystal sediment on root-end fillings than samples a1,b1,c1. dissolution and corrosion were observed in groups a1, a2. conclusions: the results of this study revealed that calcium hydroxide is more resorbable than mta angelus and proroot mta. the crystals formation and precipitation were observed in neutral phosphate buffer saline solution which were more than normal saline solution for all groups as a hydroxiapatite crystals. key words: root canal filling materials, root canal medicaments, mineral trioxide aggregate citation: kuruyucu m, et al. (2019) investigation of surface topography of different root-end filling materials: an in vitro study. dentistry 3000. 1:a001 doi:10.5195/d3000.2019.89 received: august 7, 2018 accepted: september 18, 2018 published: july 25, 2019 copyright: ©2019 kuruyucu m, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email:mine.yildirim@istanbul.edu.tr introduction endodontic surgery is performed in cases when treatment with orthograde root canal is treatment-resistant to periradicular inflammation or nonsurgical root canal treatment is unsuccessful [1,2]. in periradicular surgery, a root-end cavity preparation is performed then a retrograde filling material is used for filling. the periradicular surgery aims to block the ways in which transmission can occur between the root canal system and surrounding tissues [3]. surgical endodontic treatment technique is a feasible treatment option, and the type of root-end filling material can affect the end result [4]. root-end filling materials get in touch with periapical bone tissue after apex resection surgery. various materials are invariably used for instance amalgam, bonding systems, zinc oxide eugenol cements, glass ionomer cements, and calcium-silicate cements, all of which are commonly named as mineral trioxide aggregate based cements [5,6]. an ideal root-end filling material should demonstrate particular characteristics. the material http://dentistry3000.pitt.edu/ investigation of surface topography of different root-end filling materials: an in vitro study vol 7 no 1 (2019) doi 10.5195/d3000.2019.89 http://dentistry3000.pitt.edu should adjust to the living tissue with its biocompatibility and therefore be able to start tissue repairing of periodontal ligament complex and most importantly its own cementogenesis. it should also be radiopaque like a natural tooth while being antibacterial, non-corrosive, non-resorbable and moisture indifferent. while being easy to handle and able to adapt to the dentinal walls, they should still be dimensionally stable and cost effective as well as safe to use with its non-toxic characteristics [7]. the problems encountered during pulpal healing have been treated with calcium hydroxide for many decades. calcium hydroxide does not lead to periapical reactions, it has predictable results. another advantage of calcium hydroxide is its mixability with certain liquids. however, calcium hydroxide may have got some disadvantages. these are resorpsion, variable treatment time, unpredictable treatment period, apexification situation, increased risk of tooth fracture, and poor patient compliance due to the long treatment time. disadvantages of calcium hydroxide can affect treatment results [8,9]. calcium silicate based materials have received great interest due to their high sealing ability , biocompatibility, regenerative capacity, and antibacterial properties. there are calcium silicate–based root repair materials developed, such as; proroot mineral triokside aggregate (mta), mta plus and mta angelus [7,10]. due to their excellent biocompatibility, low solubility and impermeability, these materials are widely used to repair perforations in root canals and to form apical barriers [11]. these are hydraulic materials that provide better clinical results when compared to other root filler materials [7,10]. in terms of sealing ability and biocompatibility, mta cements are better than amalgam, superseal, intermediate restorative materials (irm), and glass ionomer cement (gic), which are all conventional root materials. studies demonstrate that mta induce the proliferation of periodontal fibroblasts, dental pulp cells, osteoblasts and osteoblast-like cells, and mesenchymal stem cells [10,12]. there are different root-end filling materials that can be used for endodontic surgery, however they all lack some characteristcs to be the ideal material yet [13]. the purpose of this study was to compare the surface topography of three materials: calcium hydroxide (sultan chemists inc., englewood, nj, usa), mta angelus (angelus soluções odontológicas, londrina, brazil) and proroot mta (dentsply endodontics, tulsa, ok, usa). material and methods ethical approval of the study was obtained from istanbul university faculty of dentistry clinical research ethics committee (no: 2012/1738-1298). in this study, three commercial root end filling materials were tested. these were calcium hydroxide (sultan chemists inc., table 1: contents of materials composition liquid calcium hydroxide calcium hydroxide powder sterile water mta angelus tricalcium silicate, dicalcium silicate, bismuth oxide, tricalcium aluminate, calcium oxide, aluminium oxide, silicon dioxide distilled water proroot mta tricalcium silicate, dicalcium silicate, bismuth oxide, tricalcium aluminate, calcium sulphate dihydrate or gypsum distilled water http://dentistry3000.pitt.edu/ investigation of surface topography of different root-end filling materials: an in vitro study vol 7 no 1 (2019) doi 10.5195/d3000.2019.89 http://dentistry3000.pitt.edu englewood, nj, usa), mta angelus (angelus soluções odontológicas, londrina, brazil) and proroot mta (dentsply endodontics, tulsa, ok, usa). (table 1) thirty-six extracted single-rooted human incisor teeth were used in this study. the teeth with caries, resorption or root fracture were eliminated from study. teeth were cleaned from soft issues, bone residues and debris, and kept in 5.25% sodium hypochlorite for one week and then placed in normal saline solution until to be used in the current study. teeth were decoronated from the cement enamel junction using micromotor handpiece (w&h dentalwerk bürmoos gmbh, austria) and diamond discs (diamant 0.15mm, dentaurum, germany) for standardized 10 mm root lenghts. the root segments were prepared with ultrasonic tips and 2 mm apical resection were performed from vertical to the long axis of the root for each tooth, using a cylindrical carbide bur under water cooling in a high-speed cycle. the canal length was measured by #15 k-file. k-file were used for canal preparation with step-back technique. apical widening was performed up to #40 k-file. the preparing root canal method was completed with 1,2 and 3 gatesglidden drills for the coronal shaping. irrigation was made with 2 ml of 2.5% sodium hypochlorite. 2 ml of 17% edta was applied for three minutes to remove smear layer, then 2 ml distilled water was used as the last irrigation. the prepared canals were dried up with paper points. the samples were randomly divided into three groups (group a: ca(oh)2, group b: mta angelus, group c: proroot mta), each one of them comprised of 12 roots. ca(oh)2, mta angelus and pro root mta were prepared according to the manufacturer’s instructions. materials were placed as 2 mm apical barriers and obturated with guttapercha and ah-plus sealer. each group was dimidiated into two subgroups (a1, a2, b1, b2, c1, c2) (table2). groups a1, b1, c1 were stored in normal saline (ns), groups (a2, b2, c2) were stored in neutral phosphate buffer saline (npbs) solution and samples were incubated at 370c for 2 weeks. a stereomicroscope (leica mz 7.5) at x32 magnification was used to photograph the root-end filling both before placing the samples in their solutions and afterwards. results all specimens demonstrated white crystals formation and sediment over the root-end filling materials and a white plaque was seen on the superficial border of the rootend cavities’ wall. the crystal sediments on root-end fillings in the samples of calcium hydroxide, mta angelus and proroot mta which were stored in normal saline solution (a1, b1, c1) were less than calcium hydroxide, mta angelus and proroot mta which were stored in neutral phosphate buffer saline solution (a2, b2, c2). white crystal formation were observed the same in both proroot mta groups (c1, c2). white crystal formation were observed in npbs group (b2) was more than ns group (b1) in mta angelus group. white crystal table 2: subgroups of the study calcium hydroxide mta angelus proroot mta normal saline a1 b1 c1 neutral phosphate buffer saline a2 b2 c2 http://dentistry3000.pitt.edu/ investigation of surface topography of different root-end filling materials: an in vitro study vol 7 no 1 (2019) doi 10.5195/d3000.2019.89 http://dentistry3000.pitt.edu formation were observed the same in both ca(oh)2 groups. dissolution and corrosion were observed in groups a1, a2 (ca(oh)2) (figure 1). discussion this study intended to evaluate the surface topography of three retrograde filling materials; calcium hydroxide, mta angelus and proroot mta in different solutions. healthy periradicular complex includes multiple tissues like cementum, periodontal ligament and bone. the ability to increase the regeneration of the functional periradicular complex is a feature on demand for root-end filling materials [15]. mta has ability of promoting hard tissue accumulation, especially cement formation [2]. the ability of hydroxyapatite formation on its interfaces when in contact with physiological or simulated body fluids such as phosphate buffer saline indicates that this material is bioactive. the fact that a material is bioactive makes it more figure 1. stereo-microscope images http://dentistry3000.pitt.edu/ investigation of surface topography of different root-end filling materials: an in vitro study vol 7 no 1 (2019) doi 10.5195/d3000.2019.89 http://dentistry3000.pitt.edu preferable in use. the surface topography of the materials which used in this study was examined thus their bioactivity was also identified [3]. sarkar et al. [15] suggest that the physico-chemical reactions of the mta changes according to rootend filling material’s ability of good sealing and bio-compatibility. samples which stored in phosphate buffered saline (pbs) solution were photographed with scanning electron microscopy (sem), examined energy dispersive x-ray analysis (edxa), and x-ray diffraction (xrd) techniques after 2 weeks. sem examination of the surface of mta which were predisposed to the stf showed to be covered with sediments of similar morphology and chemical composition. [15] asgary et al. [3] used extracted teeth. samples prepared with mta or new experimental cement (nec) and subgroups were stored in normal saline solution or neutral phospate buffered solution. samples were photographed with sem before and after placement in solution. the samples which prepared with mta and stored with normal saline did not show any change but other samples showed white crystal formation and sedimentation over the retrograde filling materials, the edges of the materials and rootend cavities’, and around the dentin surfaces as a white plaque. this situation is similar to our study. [3]. saghiri et al. [16] studied how storage medium affects mta and biodentine cement in terms of their surface porosity. the specimens were utilized under sem x1000 magnifications. they concluded the least surface crystallization surveyed in mta samples were stored in stf. the most surface crystallization surveyed in biodentine samples were stored in distilled water (dw). biodentine samples demonstrated higher surface porosities when compared to the mta samples which were stored in dw and stf with significantly lower surface porosities [16]. endosequence root repair material (errm) putty and paste were evaluated in terms of biocompatibility and compared to gray mta by ma et al. [17] each material evaluated by sem.. irm and cavit g showed inadequate crystallized superficial structure. errm putty, errm paste, and mta displayed similar crystalline surface structures. these crystals contained calcium, carbon, and oxygen and low level of phosphorus [17]. vajja et al. [18] investigated in a vitro study that whether the thickness of three root-end filling materials have an effect on sealing ability. cavities were filled with mta, rmgic and irm. teeth were examined under stereomicroscope at 30x magnification. the mta had better sealing properties in proportion to other materials at periapical area [18]. gandolfi et al.[19] tested apatiteforming ability on proroot mta cement after placement in pbs. samples were photographed with an esem-edx analysis and the analysis showed different surface morphologies related on the absorbing time. result of this study proved that proroot mta surface morphology is rapidly modified by the phosphate solution. similar results were obtained in our study [19]. asgary et al. [20] compared the properties of nec and mineral trioxide aggregate (mta). all specimens were photographed and analyzed with scanning electron microscope and electron probe microanalysis (epma). white mta and nec specimens both showed the existence of crystalline particles [20]. all these studies demonstrated that mta specimens’ surface shows crystalline particles under sem examination. these results are similar to properties of our study. in the present study, higher crystallization and porosities occurred in proroot mta, mta angelus than calcium hydroxide, which is in agreement with previous studies. this study was performed in vitro conditions; which may not imitate the exact situation of the oral cavity as blood or moisture contamination may affect the properties of sealant which are sensitive to moisture. longitudinal in vivo studies are required to http://dentistry3000.pitt.edu/ investigation of surface topography of different root-end filling materials: an in vitro study vol 7 no 1 (2019) doi 10.5195/d3000.2019.89 http://dentistry3000.pitt.edu check the surface topography and crystal precipitation for establishment of protocols for routine clinical usage. conclusion the consequences of this study revealed that ca(oh)2 is more resorbable than mta angelus and proroot mta as expected. the crystals formation and precipitation observed in npbs solution was more than ns solution for all groups as hydroxiapatite crystals. references 1. modern endodontic surgery concepts and practice: a review. kim s, kratchman s. j. endod 2006;32:601–23. pmid: 16793466 2. histologic assessment of mineral trioxide aggregate as a root-end filling in monkeys. torabinejad m, pitt ford tr, mckendry dj, abedi hr, miller da, kariyawasam sp. j endod 1997; 23: 225–8. pmid: 9594770 3. effect of two storage solutions on surface topography of two rootend fillings. asgary s, eghbal mj, parirokh m, ghoddusi j. australian endodontic journal. 2009; 35: 147–152. pmid: 19961453 4. comparison of mineral trioxide aggregate and calcium hydroxide for apexification of immature permanent teeth: a systematic review and meta-analysis. lin jc, lu jx, zeng q, zhao w, li wq, ling jq. journal of the formosan medical association 2016 jul; 115(7): 523-30. pmid: 26911724 5. biointeractivity-related versus chemi/physisorptionrelated apatite precursorforming ability of current root end filling materials. gandolfi mg, taddei p, modena e, siboni f, prati c. 2013. j biomed mater res part b 2013;101b:1107– 1123. pmid: 23559495 6. mineral trioxide aggregate: a comprehensive literature review—part iii: clinical applications, drawbacks, and mechanism of action. parirokh m, torabinejad m. j endod 2010;36:400–413. pmid: 20171353 7. properties of a new rootend filling material. chong hk, islam i, yap au, tong yw, koh et. j endod 2005;31:665–8. pmid: 16123702 8. treatment options: biological basis of regenerative endodontic procedures. hargreaves km, diogenes a, teixeira fb. pediatr dent 2013;35:129-40. pmid: 23635981 9. will mineral trioxide aggregate replace calcium hydroxide in treating pulpal and periodontal healing complications subsequent to dental trauma? a review. bakland lk, andreasen jo. dental traumatology. 2012; 28: 25–32. pmid: 21895969 10. apatite formation on bioactive calciumsilicate cements for dentistry affects surface topography and human marrow stromal cells proliferation. gandolfi mg, ciapetti g, taddei p, perut f, tinti a, cardoso m, van meerbek b, prati c. dent mater 2010;26: 974–992. pmid: 20655582 11. biyoseramik esaslı kök kanal patları: derleme. bilgiç a, bodrumlu e. atatürk üniv. diş hek. fak. derg. 2016; 14:114-17. 12. mineral trioxide aggregate: a comprehensive literature review—part ii: leakage and biocompatibility investigations. torabinejad m, parirokh m. j endod 2010;36:190–202. pmid: 20113774 13. physical and chemical properties of a new rootend filling material. torabinejad m, hong cu, mcdonald f, pitt ford tr. j endod 1995; 21: 349–53. pmid: 7499973 14. cementoblasts maintain expression of osteocalcin in the presence of mineral trioxide aggregate. thomson ts, berry je, somerman mj, kirkwood http://dentistry3000.pitt.edu/ investigation of surface topography of different root-end filling materials: an in vitro study vol 7 no 1 (2019) doi 10.5195/d3000.2019.89 http://dentistry3000.pitt.edu kl. j endod 2003; 29: 407– 12. pmid: 12814226 15. physicochemical basis of the biologic properties of mineral trioxide aggregate. sarkar nk, caicedo r, ritwik p, moiseyeva r, kawashima i. j endod 2005;31: 97–100. pmid: 15671817 16. storage medium affects the surface porosity of dental cements. saghiri ma, shabani a, asatourian a, sheibani n. journal of clinical and diagnostic research: jcdr. 2017; 11(8), zc116. pmid: 28969288 17. biocompatibility of two novel root repair materials. ma j, shen y, stojicic s, haapasalo m. j endod. 2011; 37(6), 793-798. pmid: 21787491 18. influence of different thickness of mineral trioxide aggregate, resin modified glass ionomer cement and intermediate restorative material on sealing ability of root end fillings: an in vitro study. vajja s, naik bd, vummidisetti sv, yarlagadda v. journal of clinical & diagnostic research: jcdr 2018; 12(1):zc10-13. pmid: 29899639 19. apatite-forming ability (bioactivity) of proroot mta. gandolfi mg, taddei p, tinti a, prati c. int endod j. 2010; 43(10), 917-929. pmid: 20646080 20. the properties of a new endodontic material. asgary s, shahabi s, jafarzadeh t, amini s, kheirieh s. j endod 2008; 34(8), 990-993. pmid: 18634932 http://dentistry3000.pitt.edu/ microsoft word 94 2020.docx vol 8, no 1 (2020) issn 2167-8677 (online) doi 10.5195/d3000.2020.94 http://dentistry3000.pitt.edu new articles in this journal are licensed under a creative commons attribution 4.0 united states license. comparative evaluation of retentive strength of polyvinyl siloxane impression materials to custom tray using different tray adhesives: an in vitro study jinsa p devassy1, nishna pradeep2, a v sreekumar2, jimmy george1, jittin james3, jacob george1 1. department of prosthodontics, mar baselios dental college, kerala, india 2. department of prosthodontics, kannur dental college, kerala, india 3. department of prosthodontics, indira gandhi dental college, kerala, india abstract the reten(ve strength of the impression material to the impression tray is an important factor that can affect the quality of the final impression. separa(on of the impression from the tray or tearing of the impression while removing from the mouth could be one of the reasons for distor(on. it is impera(ve to know the most effec(ve tray adhesive which can be used with different brands of rubber-based impression material. so, this study was directed to compare the different tray adhesives that can be effec(vely used in our day-to-day clinical prac(ce. keywords: tray adhesive, polyvinyl siloxane impression material, reten(ve strength. cita/on: devassy jp, et al. (2020) compara've evalua'on of reten've strength of polyvinyl siloxane impression materials to custom tray using different tray adhesives: an in vitro study. den(stry 3000. 1:a001 doi:10.5195/d3000.2020.94 received: march 25, 2020 accepted: april 25, 2020 published: may 4, 2020 copyright: ©2020 devassy jp, et al. this is an open access ar(cle licensed under a crea(ve commons avribu(on work 4.0 united states license. email: jinsa.pd@gmail.com introduction the bond strength of an impression material to tray is essential and in case of rubber-based impression materials the retention is mainly achieved via chemical adhesion. to obtain an accurate impression, the impression material must be securely attached to the tray. if the material is not secured to tray firmly while retrieving from the mouth, it will result in improper impression and therefore cause distorted die, wax pattern and casting. [1,2] the polyvinyl siloxane impression materials are addition reaction silicone elastomers which were first introduced in the 1970s. [3] in the past decade, these materials have occupied a large share of the impression material market. they possess good physical properties and handling characteristics. they have achieved a high level of dentist and patient acceptance as they are clean, odorless and tasteless. [3] an effective adhesive is especially indicated when the impression material has a high tear resistance, so that it can effectively be removed from undercuts. the chemistry of the adhesives usually are polydimethyl siloxane and ethylsilicate. the adhesive reacts with the surface of the tray material and forms a chemical bond to the tray and to the impression material. it is usually recommended to wait compara(ve evalua(on of reten(ve strength of polyvinyl siloxane impression materials to custom tray using different tray adhesives: an in vitro study vol 8 no 1 (2020) doi 10.5195/d3000.2020.94 http://dentistry3000.pitt.edu for ten to fifteen minutes after application of the adhesive before making the impression. this allows time for the solvent to react with the tray material. sulong and setchell [4] demonstrated that roughening the surface of the impression tray will significantly improve the effectiveness of polyvinyl siloxane adhesives. impression tray adhesives are applied as a spray, or are manually applied using a brush. prior to tray loading, the adhesive requires a lengthy drying time. suggested drying times for adhesives vary widely among manufacturers. also, adhesive strength of different tray adhesive varies considerably and hence in our clinical practice the knowledge of adhesive strength of various impression materials with certain adhesive is of utmost important for the success of the impression and final result. permanent distortion occurs when the impression material does not adhere to the tray. ideally, the impression material tears instead of undergoing considerable distortion before it is released from the undercut. if the material is not adequately retained in the impression tray when it is removed from the mouth, it may be a potential source of error. the most consistently accurate impression is obtained with the adhesive lined resin custom tray. each class of elastomeric impression material has its own specific adhesive. metal and plastic stock trays are used routinely for dental impressions, especially with the puttywash systems. however, heatacrylic cured trays with an adhesive is recommended for additional silicones [4]. the purpose of this study is to compare the retentive bond strength of the three commonly available brands of poly vinyl siloxane impression materials (aquasil, virtual and expresstm) to acrylic tray using universal tray adhesive (zhermarck) and adhesives supplied by the respective manufactures using the universal testing machine. materials figure 1. armamentarium. armamentarium (fig.1): • acrylic discs impression material: • virtual heavy body impression material-regular set (vivadent) • aquasil ultra heavy (dentsply) • express tm xt pentatm h (3m espe) tray adhesives: brand specific tray adhesives: • virtual • aquasil • 3m espe • universal tray adhesives (zhermarck) methods the polyvinyl siloxane heavy body impression materials selected were aquasil, virtual, expresstm and these formed the groups i, ii and iii. group i: aquasil impression material. group ii: virtual impression material. group iii: express tm impression material. the tray adhesives selected were universal tray adhesives (zhermarck) & adhesives of respective impression materials. the main groups were further subdivided based on the adhesives used. total sample size used were 135. the procedures were divided into five steps: step 1: preparation of the acrylic discs. step 2: application of adhesives. step 3: placement of disc on universal testing machine. step 4: injection of heavy body material to acrylic disc. step 5: checking the retentive strength of impression material. compara(ve evalua(on of reten(ve strength of polyvinyl siloxane impression materials to custom tray using different tray adhesives: an in vitro study vol 8 no 1 (2020) doi 10.5195/d3000.2020.94 http://dentistry3000.pitt.edu step 1: preparation of acrylic disc for preparing the acrylic disc a wax pattern of diameter of 64mm and height 9mm was made using modeling wax. simultaneously another cylindrical wax pattern of diameter 28mm height and 12mm diameter was made for perpendicular shaft. the wax pattern was invested and acrylisation was done using compression molding technique. then heat cured acrylic disc and shaft thus obtained were finished and smoothed using 320-grit silicone-carbide paper to standardize surface roughness. the heat cured acrylic cylinder shaft was attached to center of the heat cure acrylic disc using self-cure acrylic. three pairs of same kind were fabricated for convenience purpose. for each time after the loading, ethanol was used for cleaning the acrylic disc as recommended by the manufacturers of tray adhesives. step 2: application of adhesives on disc based on the adhesives used the main group was divided into subdivisions a, b and c [table 1]. subdivision a of main groups i, ii and iii was kept as control group. the adhesive was not applied to all the samples tested in this subdivision. in subdivision b of group i, caulk tray adhesives [brand specific] was applied on the disc. the adhesive was applied using a brush as thin as possible [fig.2]. only a single coat was applied and it allowed to dry for 5 minutes*. in group ii subdivision b, virtual tray adhesives was applied using brush as thin as possible and allowed to dry for 3 minutes*. in group iii subdivison b, 3m espe tray adhesive was applied as thin as possible and allowed to dry for 5 minutes*. in subdivision c of all groups, the universal tray adhesive (zhermarck) was applied on the acrylic disc in the same manner. it was allowed to dry for 2 minutes*. *time as recommended by the manufacturer. figure 2. applica3on of adhesives. placement of disc on universal testing machine universal testing machine universal testing machine (model lr5k plus) consists of upper compartment and lower compartment. it is an automatic computerized machine for high accuracy load measurements with lcd display [fig. 3]. it is also connected to a personal computer for programming and data collection. it is operated using software called nexygen. once loaded this machine itself will automatically align the disc for measurements. after the application of tray adhesives, the acrylic disc was placed on lower compartment and another acrylic disc was placed on upper compartment. then ensured the acrylic disc aligned properly. figure 3. universal tes3ng machine [lr5k plus]. injection of heavy body material heavy body material was injected [fig. 4] on the lower disc and then upper disc lowered until a space of 4mm [fig. 5]. care was taken to ensure that the heavy body material completely occupied in this 4mm space between the compara(ve evalua(on of reten(ve strength of polyvinyl siloxane impression materials to custom tray using different tray adhesives: an in vitro study vol 8 no 1 (2020) doi 10.5195/d3000.2020.94 http://dentistry3000.pitt.edu acrylic discs [fig. 6]. impression material used was different for each group [table 1]. figure 4. injec3on of heavy body material. figure 6. penta mix dispenser. in group i the aquasil impression material injected using mixing gun and the material was allowed to polymerize for 5 minutes*. in group ii, virtual impression material dispensed using mixing gun and allowed to set for 4.30 minutes*. table 1. grouping of selected impression materials with its subdivisions based on tray adhesives used. in case of group iii for expresstm, pentamix dispenser [fig. 6] was used and allowed to set for 3.30 minutes* * time as recommended by the manufacturer. checking the retentive strength the test specimens were tested in tensile mode at a crosshead speed of 6mm/min until separation failure [fig. 7] occurred and maximum force of separation is automatically recorded and saved to the computer both numerically and graphically [fig.8]. the study was conducted on remaining samples in a similar manner and results were statistically analyzed using one way anova and tukey post-hoc test. figure 7. impression material separa3on at maximum load. group i 1. aquasil [45 samples] 2. sub div a 3. without tray adhesive 4. 15 samples 5. sub div b 6. caulk tray adhesive 7. 15 samples 8. sub div c 9. zhermarck tray adhesive 10. 15 samples group ii virtual [45 samples] 11. 12. sub div a 13. without tray adhesive 14. 15 samples 15. sub div b 16. virtual tray adhesive 17. 15 samples 18. sub div c 19. zhermarck tray adhesive 20. 15 samples 21. group iii 22. express t m [45 samples] 23. sub div a 24. without tray adhesive 25. 15 samples 26. sub div b 27. 3m espe tray adhesive 28. 15 samples 29. sub div c 30. zhermarck tray adhesive 31. 15 samples figure 5. heavy body material between the acrylic discs. figure 8. computer generated graph according to the maximum force of separa3on failure. compara(ve evalua(on of reten(ve strength of polyvinyl siloxane impression materials to custom tray using different tray adhesives: an in vitro study vol 8 no 1 (2020) doi 10.5195/d3000.2020.94 http://dentistry3000.pitt.edu results on comparison aquasil with universal tray adhesive showed greater retentive strength than aquasil with brand specific tray adhesive and aquasil without tray adhesive [table 2]. on comparison virtual with universal tray adhesive showed greater retentive strength than virtual with brand specific tray adhesive and virtual without tray adhesive [table 3]. on comparison expresstm with universal tray adhesive showed greater retentive strength than expresstm with brand specific tray adhesive and expresstm without tray adhesive [table 4]. on applying one way anova, it was found that there was significant difference [p-value<0.001] in mean retentive strength between the study subgroups. the mean value of the retentive strength is highest for virtual with universal tray adhesive and lowest for aquasil without tray adhesive [table 5]. table 6 shows the summary of interactions of sub groups with each other. comparing subdivisions a, b and c of group i, ii and iii, the subdivision c showed the greatest retentive values than subdivision b and subdivision a. when comparing group i, ii, and iii, the group ii showed the greatest retentive values than group i and group iii. table 2. maximum force of separa6on failure in group i in newton [n] table 3. maximum force of separa6on failure in group ii in newton [n] table 4. maximum force of separa6on failure in group iii in newton [n] compara(ve evalua(on of reten(ve strength of polyvinyl siloxane impression materials to custom tray using different tray adhesives: an in vitro study vol 8 no 1 (2020) doi 10.5195/d3000.2020.94 http://dentistry3000.pitt.edu table 5. one way anova test to compare the reten6ve strength of the subdivisions. discussion the impression adhesives used for silicone impression materials contain polydimethyl-siloxane or a similar reactive silicone, and ethyl silicate. polymethylsiloxane of adhesives bonds to the silicone impression material whereas ethylsilicate forms a hydrated silica that bonds to the impression tray material physically. the volatile solvent in the form of ethyl acetate reacts with the acrylic tray material to create microporosities on the tray material so that the adhesive physically and mechanically bonds with it. sulong and setchell [3] studied the properties of tray adhesives of addition polymerizing silicones to impression tray materials. they number of values mean std. deviation lower 95% ci upper 95% ci f p value grp i subdiv a aquasil without tray adhesive 15 250.6 15.85 241.9 259.4 1166 <0.001 subdiv b aquasil brand specific tray adhesive 15 704.2 26.41 689.6 718.9 subdiv c aquasil universal tray adhesive 15 887.5 12 880.9 894.2 grp ii subdiv a virtual without tray adhesive 15 501.6 48.34 474.8 528.3 subdiv b virtual brand tray adhesive 15 805 21.43 793.2 816.9 subdiv c virtual universal tray adhesive 15 891.3 8.801 886.4 896.2 grp iii subdiv a express tm without tray adhesive 15 351.7 20.47 340.3 363 subdiv b express tm brand tray adhesive 15 511.3 11.74 504.8 517.8 subdiv c express tm universal tray adhesive 15 710.4 41.03 687.7 733.1 compara(ve evalua(on of reten(ve strength of polyvinyl siloxane impression materials to custom tray using different tray adhesives: an in vitro study vol 8 no 1 (2020) doi 10.5195/d3000.2020.94 http://dentistry3000.pitt.edu reported that acrylic resin trays roughened with eighty grit silicone-carbide created highest strength and they also concluded that adhesives did not adhere well to chromium plated metal or plastic stock tray material. samman [5] studied about impression tray adhesives and reported that tray adhesives definitely improve the bonding of impression material to the tray. table 6. a cross table showing interaction of subgroups with each other by post-hoc tukey test p value. (*significant p values). vs group i group ii group iii sub div a sub div b sub div c sub div a sub div b sub div c sub div a sub div b sub div c group i sub div a <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* sub div b <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* 0.999 sub div c <0.001* <0.001* <0.001* <0.001* 0.999 <0.001* <0.001* <0.001* group ii sub div a <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* 0.984 <0.001* sub div b <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* sub div c <0.001* <0.001* 0.999 <0.001* <0.001* <0.001* <0.001* <0.001* group iii sub div a <0.001* <0.001* <0.001* 0.984 <0.001* <0.001* <0.001* <0.001* sub div b <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* sub div c <0.001* 0.999 <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* compara(ve evalua(on of reten(ve strength of polyvinyl siloxane impression materials to custom tray using different tray adhesives: an in vitro study vol 8 no 1 (2020) doi 10.5195/d3000.2020.94 http://dentistry3000.pitt.edu tjan and sung [6] reported the significance of tray adhesives in case where the impressions were poured repeatedly, to minimize accidental separation of the impression from the tray. though manufacturers have come out with its own tray adhesives for particular rubber-based impression materials, previous literature [1,7] has reported that it might not be the best. paint-on adhesive on medium body vps is found to be effective. [5,7] considering the above facts, the study was done to compare the effectiveness of the universal tray adhesive, zhermarck with the three commonly available medium consistency vps impression materials, aquasil, virtual and expresstm with their respective tray adhesives, caulk, virtual and 3m espe. among the different groups studied, heavy body impression material with universal tray adhesives [subdivision c] showed the highest value. in subdivision c, the tray adhesive used was zhermarck tray adhesive. the results of the study agree with the findings of study done by land7 et al. and ashiwini et al. [1] in their study, they evaluated the bond strength of the three polyvinyl siloxane materials with a methacyrlate autopolymerizing and light polymerizing tray material, using the adhesives recommended by the manufactures of the impression materials and two universal adhesives (paint on and spray on). they reached the conclusion that paint on universal tray adhesives offers the advantage of providing equal or superior adhesive bond strength for the three vps impression materials to the autopolymerizing and light polymerizing tray materials tested, compared to the manufactures recommended adhesives. when comparing subdivision a [without tray adhesives] of all groups, group ii [virtual heavy body impression material] showed the greatest bond strength and it is statistically significant [table 6, graph 1]. without using any tray adhesives retentive bond strength of virtual heavy body impression material to acrylic resin tray material which was roughened by 320 grit silicon carbide was comparatively higher than group i [aquasil heavy body impression material] and group iii [expresstm heavy body impression material]. when comparing the subdivision b [brand specific] of all groups, group ii [virtual heavy body impression material] showed the greatest bond strength than group i aquasil heavy body impression material and group iii expresstm heavy body impression material [table 6, graph 1 ]. when comparing the subdivision c of all groups, there is no significant difference observed [table 6]. as already stated, on comparison of the subdivisions a, b and c, the subdivision c [with universal tray adhesives] showed the greatest bond strength and is statistically significant [table 6]. variations in bond strength of impression material, adhesive agent group i aquasil group ii virtual group iii expresstm 25 0. 6 50 1. 6 35 1. 7 70 4. 2 80 5 51 1. 3 88 7. 5 89 1. 3 71 0. 4 without tray adhesive brand specific tray adhesive universal tray adhesive graph 1. bar diagram showing comparison of groups and subdivisions. compara(ve evalua(on of reten(ve strength of polyvinyl siloxane impression materials to custom tray using different tray adhesives: an in vitro study vol 8 no 1 (2020) doi 10.5195/d3000.2020.94 http://dentistry3000.pitt.edu and custom tray material combination is related to 1. the chemistry of the adhesive agents 2. the surface chemistry of the resin tray material. [8] the significantly greater bond strengths of zhermarck universal tray adhesive were probably attributable to the higher adhesive capability of these adhesives. the adhesion of the tray adhesives is achieved because of chemical adhesion between the impression material and methyl methacrylate tray material. upon application of the tray adhesive, the carrier solvent present in the adhesive causes swelling of the outermost surface of the tray, thereby allowing the adhesive to penetrate and interact intimately with the impression material. the solvent then evaporates, leaving the entire tray surface covered with the adhesive, which is retained within the molecular network of the impression material. the retention of impression materials to acrylic resin tray material ultimately depends on the ability of the solvent in the adhesive to dissolve the resin tray material. thus solvent evaporation is considered as the “setting” of the adhesive and is dependent on time, temperature and relative humidity [9]. it is generally recommended to wait for a few minutes after application of the adhesive before making the impression. this allows time for the solvent to react with the tray material. therefore, in this present study, the better adhesive bond strength of zhermarck universal tray adhesive in comparison with brand specific tray adhesives may be attributed to the difference in the solubility of the tray material by the solvent present in the tray adhesive. from the results obtained in this study, zhermarck universal tray adhesive with addition silicone impression material could be recommended as an ideal tray adhesiveimpression material combination using acrylic resin tray material. when comparing among the heavy body impression materials selected, aquasil, virtual and expresstm, the virtual heavy body impression material showed the greatest retentive strength to custom tray with both brand specific tray adhesive and universal tray adhesive. clinical implications from the results of this study, it can be concluded that the universal tray adhesive can be used in our clinical practice with most of the elastomeric impression materials to attain maximum results without the need of the adhesive supplied by the respective manufacturer. acknowledgements mr. binish jacob, mr. arun, assistant managers and mr. abilash, engineer in us based amphenol fci-oen connentors ernakulam, india. dr. sreekanth, research investigator, manipal institute of medical science, india. dr. imad muhammed ismail, assistant professor in kannur dental college, india. dr. shahanaz hamza, dr. harsha gangadharan, dr. navaz, house surgeons anjarakandy dental college, india. mr. satheesh, lab assistant in kannur dental college, india. references 1. the bond strength of different tray adhesives on vinyl polysiloxane to two tray materials: an in vitro study. ashwini bl, manjunath s, mathew kx. j indian prosthodont soc. 2014 mar;14(1):29-37. doi: 10.1007/s13191-012-0184z. 2. considerations for adhesion of impression materials to impression trays. bomberg tj, goldfogel mh, hoffman w jr, bomberg se. j prosthet dent. 1988 dec;60(6):681-4. 3. polyvinyl siloxane impression materials: an update on clinical use. mandikos mn. aust dent j. 1998 dec;43(6):428-34. 4. properties of the tray adhesive of an addition polymerizing silicone to impression tray materials. sulong mz, setchell dj. j prosthet dent. 1991 dec;66(6):7437. compara(ve evalua(on of reten(ve strength of polyvinyl siloxane impression materials to custom tray using different tray adhesives: an in vitro study vol 8 no 1 (2020) doi 10.5195/d3000.2020.94 http://dentistry3000.pitt.edu 5. a study of impression tray adhesives. samman jm, fletcher a. quintessence int. 1985 apr;16(4):305-9. 6. comparing effects of tray treatment on the accuracy of dies. tjan ah, whang sb. j prosthet dent. 1987 aug;58(2):175-8. 7. the effect of different adhesives on vinyl polysiloxane bond strength to two tray materials. peregrina a, land mf, wandling c, johnston wm. j prosthet dent. 2005 sep;94(3):20913. 8. bond strength of two nonaqueous elastomeric impression materials bonded to two thermoplastic resin tray materials. payne ja, pereira bp. j prosthet dent. 1995 dec;74(6):563-8. 9. retention strength of impression materials to a tray material using different adhesive methods: an in vitro study. marafie y, looney s, nelson s, chan d, browning w, rueggeberg f. j prosthet dent. 2008 dec;100(6):432-40. doi: 10.1016/s00223913(08)60260-7. salvizan and triamcinolone gel on oral lichenoid vol 10 no 1 (2022) doi 10.5195/d3000.2022.157 http://dentistry3000.pitt.edu comparative effects of salvizan gel (containing salvia officinalis extract) and triamcinolone acetonide gel on the duration and symptoms of oral lichenoid reactions parichehr ghalayani1, fahimeh pakravan2, fatemeh abbasi2 1 dental research center, department of oral medicine, dental research institute, isfahan university of medical sciences, isfahan, iran 2 dental implant research center, department of oral medicine, dental research institute, isfahan university of medical sciences, isfahan, iran abstract objectives: lichenoid reactions are disorders with similar clinical presentations. the current study was conducted to compare the improvement indices in the patients’ symptoms by salvizan and triamcinolone gels. therefore, regardless of the cause, eliminating the pain and symptoms is one of the objectives of the treatment. methods: this study was designed as a randomized double-blind clinical trial. diagnosis of patients was performed by clinical examinations and confirmed by histological evaluation. one group of patients was administered salvizan and another group was administered triamcinolone. after washing their mouth, the patients administered the gel (1 cm) on the inflammatory mucosa three times a day for two weeks. the amelioration rate of the lesions in each visit was measured from according to visual analog scale (vas) on a scale of 0-10. results: the results of repeated measures ancova showed a significant difference between groups in terms of the pain intensity before treatment (p<0.001). moreover, there was a significant difference between different times with regard to pain intensity (p=0.004). to evaluate the clinical indices in the study groups, mann-whitney test was used and the results showed no significant difference for the extent of lesions before treatment (p=0.874), after one week (p=0.503) and after two weeks (p=0.669). conclusions: salvizan could better reduce pain intensity than triamcinolone, but clinically no significant difference was reported between the drugs in terms of the extent of lesion. keywords: lichenoid reaction, gel, scarlet sage, triamcinolone citation: ghalayani p, et al. (2022) comparative effects of salvizan gel (containing salvia officinalis extract) and triamcinolone acetonide gel on the duration and symptoms of oral lichenoid reactions. dentistry 3000. 1:a001 doi:10.5195/d3000.2022.157 received: march 3, 2021 accepted: august 31, 2021 published: march 2, 2022 copyright: ©2022 ghalayani p, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: f.abbasi@dnt.mui.ac.ir introduction lichenoid reactions are a set of disorders with similar clinical presentations, but are created due to various reasons. these reactions are divided into such subtypes as lichen planus, druginduced lichen planus, contact lichenoid reactions and lichenoid reactions due to graft-versus-host disease (gvhd). oral lichen planus (olp) is a rather common and chronic oral disease that mostly affects the oral mucosa [1]. oral lichen planus and lichenoid reactions are common oral mucosal diseases that are frequently encountered in the health centers. this mucocutaneous disease is characterized as desquamative gingivitis without symptoms and with wickham striae or plaque and painful ulcers in oral mucosa. although the primary cause of this disease is unknown, recent studies have shown different immune complexes along with responding cells for inflammatory structures and chronic lesions. lichen planus is generally an immunologic disease that is microscopically similar to a hypersensitive reaction with t-cell interference http://dentistry3000.pitt.edu/ salvizan and triamcinolone gel on oral lichenoid vol 10 no 1 (2022) doi 10.5195/d3000.2022.157 http://dentistry3000.pitt.edu [2]. factors such as diabetes, hepatitis c, trauma and hypersensitivity to medications and metals can cause lichen planus [3]. most of the patients with lichen planus are middleaged people. women are also more affected by lichen planus than men. the prevalence of lichen planus has been reported to be 0.1-2.2%. olp has different clinical features, but is mainly seen as reticular, erosive and bullous forms; the most common of which is reticular form [4]. microscopically, olp is characterized by hyperkeratosis, basal cell degeneration, basement membrane thickening and accumulated submucosal lymphocytes. the characteristics of oral lichenoid reactions include unilateral clinical presentation along with a significant number of plasma cells in lymphocytic community, accumulation of materials or cells in the tissues around the vessels, increase of colloid bodies and presence of acute inflammatory cells such as eosinophils and neutrophils [5, 6]. olp is induced as a result of such drug agents as thiazides, penicillamine, systemic diseases like chronic liver diseases, hepatitis c virus, diabetes, and allergic reactions to metals such as amalgam, food supplies, flowers and viral hemorrhagic disease (vhd) [3, 7]. the microscopic aspect of lichenoid reactions may have a similar presentation to olp [1, 8]. clinical manifestations of lichenoid reactions vary from white painless keratotic lesions to erosions and painful ulcers [9]. although these lesions rarely threaten the patients’ lives, the discomfort and pain associated with them can affect the normal function of mouth, increase the risk of infection and to some extent reduce the patients’ quality of life. the main treatment for lichenoid reaction is administration of systemic and local corticosteroid [10]. its local and potent type such as triamcinolone has increasingly been used for the treatment of lichenoid reaction [11]. another treatment for lichen planus is medicinal plants [12-14]. salvia officinalis is one of these plants whose extract is used in salvizan gel. each 15 gr of salvizan gel contains 28% hydroalcoholic extract. the leaves of salvia officinalis are known to have antioxidant properties. these plants are used for local treatment of gingival inflammatory, microbial and painful complications and oral lesions [15]. this plant enjoys antimicrobial, antifungal and antivirus properties and is able to reduce salivary and sweat secretion. the leaves of this plant contain 3-8% catechin tannins, phenolic acids, monoterpenes, beta-thujone, thymol, cineol and water-soluble tannin [16]. this study was aimed to analyze the treatment effect of salvizan and triamcinolone gels among the patients with lichenoid reactions, provided that they would have a significant impact in the amelioration of mucosal ulcer and their complications would not be statistically significant. materials and methods the study sample in this randomized double-blind clinical trial comprised of the patients with lichenoid reactions referring to the department of oral diseases at school of dentistry, isfahan university of medical sciences in 2014. all the patients participated in the study voluntarily. this clinical trial study was registered in iranian registry of clinical trails (irtc). the inclusion criteria for the patients with lichenoid reaction included minimum age of 18 years, non-pregnant and nonbreastfeeding women and ability to remain during the study period. the patients with allergy to plant products and drug abuse and those who were reluctant to cooperate were excluded from the study. according to the declaration of helsinki, informed http://dentistry3000.pitt.edu/ salvizan and triamcinolone gel on oral lichenoid vol 10 no 1 (2022) doi 10.5195/d3000.2022.157 http://dentistry3000.pitt.edu consent was taken from the patients. in this study, the samples were selected through simple random sampling. the clinical criteria for diagnosis were based on the presence of ulcerative and bilateral lesions that were basically located in the cheeks and in some cases similar bilateral lesions that were at ventral surface, sides of the tongue and gums as desquamate gingivitis. moreover, papules or reticular lines along the ulcerative strips were helpful in diagnosing most patients with lichenoid reactions. finally, sampling was carried out for histopathological analysis for accurate diagnosis. a total number of 30 samples in each group might yield a difference equal to d=0.5 between the mean scores at α=0.05 up to 80%. after taking the informed consent from the patients, they were divided into two groups; the first group underwent treatment with salvizan gel and the second group was treated with triamcinolone gel. the treatment was performed weekly and lasted for two weeks or until the lesions were treated (either of them). one group was administered salvizan gel and the other group was administered triamcinolone gel. the drug was administered three times a day after eating the meal. the patients were provided with required trainings. after eating food and washing their mouth, the patients rubbed the gel (1 cm) on the mucosa and massaged it. the recovery rate of lesions was measured in each visit according to the criteria presented in the study of buajeeb et al, and the pain level was determined according to the visual analog scale (vas) from 0 to 10. the pain level in patients was evaluated by a scaled ruler and vas technique as well as the pain level perceived by the patient on the scale of 0-10. in fact, vas is a 10-cm horizontal line on one end of which the term “no pain” and on the other end the term “severe pain” are printed. the patients were asked to draw a vertical line on the ruler based on the pain level they experienced at that moment. the extent of lesions is shown in table 1 from 0 to 5 according to the parameters mentioned by buajeeb [17]. ulcer or burning, pain during eating and drinking, dry mouth, numbness after taking the studied drugs and use of products like caffeine, tobacco and alcohol were recorded in the patients’ questionnaires (table 2). the obtained data were analyzed by spss-21 software. wilcoxon test was used to analyze the collected data in one group and compare it with another at different intervals and mann-whitney test was administered to compare two groups with each other. survival analysis was performed as well. table 1 . the extent of lesions from 0 to 5 according to buajeeb’s criteria. results among the 30 patients under treatment with salvizan gel, 8 patients were male and 22 patients were female, and among the 30 patients under treatment with triamcinolone gel, 10 patients were male and 20 of them were female. the age range of the patients was 20-60. the results of repeated measures ancova showed a statistically significant difference between salvia officinalis and triamcinolone gels in terms of gender by the extent of lesions according to buajeeb’s criteria grade normal mucosa 0 white poor and mild striae with no erythematosus 1 white striae with atrophy less than one square cm 2 white striae with atrophy more than one square cm 3 white striae with erosion less than one square cm 4 white striae with erosion more than one square cm 5 http://dentistry3000.pitt.edu/ salvizan and triamcinolone gel on oral lichenoid vol 10 no 1 (2022) doi 10.5195/d3000.2022.157 http://dentistry3000.pitt.edu controlling the effect of pain before treatment (p=0.05). the pain intensity before treatment was reported to be significantly different in both groups (p<0.001); however, no significant difference was observed between genders (p=0.786). also, vas levels indicated a significant difference between different times (p=0.004) (figure 1). figure 1. standard deviation of pain level for both drugs at different times. following the ancova test, paired sample t-test was run to compare vas values at different times. the findings revealed a significant difference for salvia officinalis between different times; before treatment and one week after treatment, before treatment and two weeks after treatment, and after one week and two weeks after treatment (p<0.001). for triamcinolone, the same results were reported based on the paired sample t-test. to compare the clinical index of lichenoid reaction in the study groups, mann-whitney test was used for different times and the findings showed no significant difference between groups in terms of the extent of lesions before treatment (p=0.874). this index was also not reported to be significantly different between groups one week after treatment (p=0.503). for two weeks after treatment too, this index was not significantly different between groups (p=0.669). to compare the clinical index of salvia officinalis and triamcinolone gels at different times, friedman test was applied and the results indicated a significant difference between the three times in terms of the clinical index of both drugs (p=0.001). following friedman test, wilcoxon signed-rank test was used and the findings showed a significant difference between the three different times with regard to the clinical index of salvia officinalis and triamcinolone gels (p<0.001). discussion lichenoid reactions are caused by numerous factors. therefore, it seems necessary to make an effort to treat and eliminate the factors associated with the disease in order to prevent the complications resulting from it such as tooth loss due to health problems and possible malignant changes. the treatment plan for lichenoid reactions is as follows: first, local corticosteroid is administered; second, the lesion is conservatively removed and the inducing factors are found using patch test, oral prophylaxis, restoration and regular polishing and using mouth wash. for more severe cases, chemotherapy and surgery are carried out; third, the patient undergoes control every three months for one year and checked again every two years. in sd number mean time drug 2.31 30 6.00 before treatment salvia officinalis 1.75 30 2.56 one week after treatment 1.04 30 1.06 two weeks after treatment 2.17 30 5.13 before treatment triamcinolone 1.75 30 2.53 one week after treatment 0.97 30 1.16 two weeks after treatment table 2 . mean and standard deviation of pain intensity for both drugs at different times. http://dentistry3000.pitt.edu/ salvizan and triamcinolone gel on oral lichenoid vol 10 no 1 (2022) doi 10.5195/d3000.2022.157 http://dentistry3000.pitt.edu the case of changes and recurrence of the disease, biopsy is performed again [18]. this study was carried out to evaluate the effect of salvizan and triamcinolone gels on 60 patients with lichenoid reactions. the findings indicated salvizan gel had a better effect than triamcinolone in terms of reducing the pain, although no significant difference was reported between them with regard to the extent of lesions. based on the obtained results, it can be argued that salvizan, which is a medicinal plant, can be a substitute for triamcinolone, which is a chemical drug and has more side effects, or can be administered along with triamcinolone. medicinal plants have been used successfully to treat oral diseases [19-21]. as an example, sookto et al showed that saliva officinalis had anti-candida properties and prevented the connection of this fungus [22]. moreover, martin et al investigated the effect of oral patches containing licorice extract in the treatment of aphthous ulcers and reported positive effects in the size of the lesion and patient’s pain [9]. another nonpharmacologic and non-steroidal therapy used for the treatment of lichenoid diseases was the use of topical retinoic acid in comparison with topical fluocinolone acetonide which was investigated by buajee et al. the authors concluded that topical fluocinolone acetonide was more effective to reduce the severity of lichen planus [17]. moreover, azizi et al compared the efficacy of adcortyl lotion and topical tacrolimus and showed that topical tacrolimus could be an efficient and safe replacement for the treatment of lichen planus [23]. furthermore, bendas et al introduced hydroxychloroquine as a new and promising drug for the topical treatment of lichen planus for a short-term with few side effects [24]. the limitations of this study included lack of comprehensive control over the use of drugs and small sample size. future studies are recommended to recruit larger study sample, compare different plant compounds, use plant complexes, and make use of topical cortones and plant medicines alternatively in order to obtain more accurate results. conclusion salvizan gel performed better than triamcinolone in decreasing pain intensity but clinically, no difference was reported between the two drugs with regard to the extent of lesion. references 1. marx re, stern d. oral and maxillofacial pathology. chicago: quintessence 2003;789-91. 2. damm dd, bouquot je, neville bw, allen c. oral and maxillofacial pathology. chap. 2002;1:66. 3. austyn jm, wood k. principles of cellular and molecular immunology revista do instituto de medicina tropical de são paulo 1994;36(3):224-. 4. edwards pc, kelsch r. oral lichen planus:clinical presentation and management. j can dent assoc 2002;68(8):494-9 5. boyd as, neldner kh. lichen planus. j am acad dermatol 1991;25(4):593-619. 6. al-hashimi i, schifter m, lockhart pb, wray d, brennan m, migliorati ca, axéll t, bruce aj, carpenter w, eisenberg e, epstein jb. oral lichen planus and oral lichenoid lesions: diagnostic and therapeutic considerations. oral surg oral med oral pathol oral radiol endod 2007;103:25-e. 7. mccreary ce, mccartan be. clinical management of oral lichen planus. br j oral maxillofac surg 1999;37(5):338-43. 8. carrozzo m, gandolfo s. the management of oral lichen planus. oral dis 1999;5(3):196-205. 9. martin md, sherman j, van der ven p, burgess j. a controlled trial of a dissolving oral patch http://dentistry3000.pitt.edu/ salvizan and triamcinolone gel on oral lichenoid vol 10 no 1 (2022) doi 10.5195/d3000.2022.157 http://dentistry3000.pitt.edu containing glycyrrhiza (licorice) herbal extract for the treatment of aphthous ulcers. gen dent 2008;56:206-210. 10. willershausen b, gruber i, hamm g. the influence of herbal ingredients on the plaque index and bleeding tendency of the gingiva. j clin. dent 1991;2(3):75-8. 11. cherevatyi vs, vashchenko tn, shishkov gz. comparative evaluation of the antibacterial action of different extracts from salvia officinalis. rastitel'nye resursy 1980;16(1):137-9. 12. taheri e, ghorbani s, safi m, sani ns, amoodizaj ff, heidari m, chavoshi r, hajazimian s, isazadeh a, heidari m. inhibition of colorectal cancer cell line caco-2 by essential oil of eucalyptus camaldulensis through induction of apoptosis. acta med iran. 2020;58(6):260-265. 13. isazadeh a, hajazimian s, mashayekhi m, shadman b, bedoustani ab, safaei s, chavoshi r, shanehbandi d, baradaran b, nahaei m. anti-cancer effects of probiotic lactobacillus acidophilus for colorectal cancer cell line caco-2 through apoptosis induction. pharm sci. 2021;27(2):368-373. 14. jadid mf, shademan b, chavoshi r, seyyedsani n, aghaei e, taheri e, goleij p, hajazimian s, karamad v, behroozi j, sabet mn. enhanced anticancer potency of hydroxytyrosol and curcumin by plga-paa nano-encapsulation on panc-1 pancreatic cancer cell line. environ toxicol. 2021;36(6):10431051. 15. willershausen b, gruber i, hamm g. the influence of herbal ingredients on the plaque index and bleeding tendency of the gingiva. j clin dent 1991;2(3):75-8. 16. cherevatyî vs, vashchenko tn, shishkov gz. comparative evaluation of the antibacterial action of different extracts from salvia officinalis. rastitel'nye resursy 1980;16(1):137-9. 17. buajeeb w, kraivaphan p, pobrurksa c. efficacy of topical retinoic acid compared with topical fluocinolone acetonide in the treatment of oral lichen planus. oral surg med oral patho oral radiol endod 1997;83(1):21-5. 18. kamath vv, setlur k, yerlagudda k. oral lichenoid lesions-a review and update. indian j dermatol 2015;60(1):102. 19. firouzi amoodizaj f, baghaeifar s, taheri e, farhoudi sefidan jadid m, safi m, seyyed sani n, hajazimian s, isazadeh a, shanehbandi d. enhanced anticancer potency of doxorubicin in combination with curcumin in gastric adenocarcinoma. j biochem mol toxicol. 2020;34(6):e22486. 20. mahdavi s, kheyrollahi m, sheikhloei h, isazadeh a. antibacterial and antioxidant activities of essential oil on food borne bacteria. open microbiol j. 2019;13(1):81-85. 21. mahdavi s, haj azimian s, isa zadeh ar, babash pour m, shishehgar r. study of the antioxidant and antimicrobial effects of the ethanolic extract of eucalyptus camaldulensis dehnh against infectious bacteria isolated from clinical and animal sources. j comparative pathobiol. 2017;13(4):2063-70. 22. sookto t, srithavaj t, thaweboon s, thaweboon b, shrestha b. in vitro effects of salvia officinalis l. essential oil on candida albicans. asian pac j trop biomed 2013;3(5):376-80. 23. azizi a, lawaf s. the comparison of efficacy of adcortyl ointment and topical tacrolimus in treatment of erosive oral lichen planus. j dent res dent clin dent prospects 2007;1(3):99-102. 24. bendas er, abdullah h, elkomy mh, kassem ma. hydroxychloroquine niosomes: a new trend in topical management of oral lichen planus. int j pharm 2013;458(2):287-95. http://dentistry3000.pitt.edu/ microsoft word winnie zhang 2016.docx   vol  4,  no  1  (2016)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2016.49           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     third  molar  eruption  mechanisms  and  patterns   winnie  zhang1     1  university  of  pi.sburgh  school  of  dental  medicine,  pi.sburgh  pa,  usa abstract   third  molars  are  highly  variable  in  their  presence  and  form.  this  report  focuses  on   a  horizontally  impacted  third  molar  and  analyzes  the  poten.al  e.ology  of  this  sit-­‐ ua#on.  upon  a  clinical  and  radiographic  examina#on,  it  was  noted  that  the  pa#ent   had   four   third  molars  present.  the  pa3ent’s   third  molars  began  erup3ng  around   the  age  of  19.  currently,  they  are  asymptoma!c  with  incipient  caries  on  the  occlu-­‐ sal  surfaces.  while  three  of  the  third  molars  erupted  in  a  normal  orienta4on,  one   of  the  third  molars  (mandibular  le2)  erupted  in  an  orienta5on  that  would  be  clas-­‐ sified  as  horizontal  and/or  mesio-­‐angular.  no   treatment  has  been  undertaken  at   the  moment,  however  surgical  extrac4on  can  be  recommended  with  the  progno-­‐ sis   being   very   good.   for   the   clinician   that   has   to   treat   dental   complica6ons   that   arise   from   abnormal   tooth   erup1on,   as   seen   in   numerous   gene1c   and   acquired   disorders,   knowledge   about   the   basic   molecular   mechanisms   involved   may   be-­‐ come  extremely  important.     cita%on:  zhang,  w.  (2016)  third  molar  erup.on   mechanisms  and  pa-erns.  den$stry  3000.   1:a001  doi:10.5195/d3000.2016.49   received:  may  21,  2016   accepted:    june  13,  2016   published:    september  28,  2016   copyright:  ©2016  zhang,  w.  this  is  an  open  ac-­‐ cess  ar!cle   licensed  under  a  crea!ve  commons   a"ribu%on  work  4.0  united  states  license.   email:  wiz5@pi'.edu   introduction   third  molars,  often  re-­‐ ferred  to  as  wisdom  teeth,  are  the   most  distal  (posterior)  teeth  of  the   three  molars  in  each  quadrant  of   human  dentition.  they  generally   erupt  between  the  ages  of  17and   23.  ideally,  the  third  molars  should   erupt  just  like  all  the  other  teeth   and  be  properly  aligned.  however,   often  these  third  molars  are  misa-­‐ ligned  which  can  on  occasion  lead   to  crowding  of  the  teeth  or  dam-­‐ age  to  adjacent  teeth,  and  rarely   to  cystic  pathology  or  root  resorp-­‐ tion  [1].     third  molars  can  also  be   impacted  which  means  they  are   enclosed  within  the  soft  tissue   and/or  jawbone  or  they  only  par-­‐ tially  break  through/erupt  through   the  gum.  partial  eruption  of  third   molars  provides  an  opening  for   bacteria  to  enter  around  the   tooth.  this  could  potentially  cause   an  infection,  which  results  in  pain,   swelling,  jaw  stiffness,  and  other   complications.  partial  eruption   also  makes  the  third  molar  more   susceptible  to  caries  and  perio-­‐ dontal  disease  because  they  are  in   hard  to  reach  areas,  thus  making  it   hard  to  brush  and  floss  those  are-­‐ as  [1].     there  are  multiple  ways  to   classify  impacted  third  molars.   one  way  is  based  on  the  nature  of   the  overlying  tissues.  based  on  the   nature  of  the  overlying  tissue  im-­‐ paction,     impacted  lower  wisdom  teeth  can   be  classified  into  [2]:     1.   soft  tissue  impaction.     when  the  height  of  the  tooth’s   contour  is  above  the  level  of  the   surrounding  alveolar  bone  and  the   superficial  portion  of  the  tooth  is   covered  only  by  soft  (though  this   can  be  dense  and  fibrous)  tissue.     soft  tissue  impaction  is  usually  the   easiest  type  of  impacted  tooth  to   remove.   2.   hard  tissue  ('bony')  impac-­‐ tion.    this  is  where  the  wisdom   tooth  fails  to  erupt  due  to  being   obstructed  by  the  overlying  bone.     this  can  be  sub-­‐divided  into  (a)   partial  and  (b)  complete  bony  im-­‐ pactions.      third  molar  erup.on  mechanisms  and  pa5erns   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.49    http://dentistry3000.pitt.edu   2   a.   partial  bony.    the  superfi-­‐ cial  portion  of  the  tooth  is  covered   only  by  soft  tissue  but  the  height   of  the  tooth's  contour  is  below  the   level  of  the  surrounding  alveolar   bone.    apart  from  cutting  the  gin-­‐ giva  (gum)  and  possible  bone  re-­‐ moval  from  behind  the  tooth,  the   tooth's  roots  may  need  to  be  di-­‐ vided.   b.   complete  bony.    the  tooth   is  completely  encased  in  bone  so   that  when  the  gingiva  is  cut  and   reflected  back,  the  tooth  is  not   seen.    bone  removal  (large   amounts)  together  with  root  sec-­‐ tioning  will  be  needed  to  remove   the  tooth.    these  are  often  the   most  difficult  teeth  to  remove.   another  way  to  classify   impacted  third  molars  is  using   winter’s  classification  which  is   based  on  the  inclination  of  the  im-­‐ pacted  third  molar  to  the  long  axis   of  the  second  molar  [2].  each  type   of  impaction  has  some  definite   path  of  withdrawal  of  the  teeth.   mesially  impacted  teeth  are  (can   be)  easier  to  remove  whereas  dis-­‐ tally  impacted  teeth  are  (can  be)   the  hardest  to  remove.  bucally   positioned  maxillary  teeth  are  eas-­‐ ier  to  remove  as  the  bone  covering   the  tooth  is  thinner  whereas  the   palatally  positioned  tooth  requires   bone  removal  and  hence  makes   the  extraction  difficult  [2].   1.   mesio-­‐angular  (figure  1):   the  impacted  tooth  is  tilted  to-­‐ ward  the  2nd  molar  in  a  mesial   direction.   2.   disto-­‐angular  (figure  2):   the  long  axis  of  the  3rd  molar  is   angled  distally  or  posteriorly  away   from  the  second  molar.     3.   horizontal  (figure  3):  the   long  axis  of  the  third  molar  is  hori-­‐ zontal.   4.   vertical  (figure  4):  the  long   axis  of  the  third  molar  is  par-­‐ allel  to  the  long  axis  of  the   second  molar.   5.   buccal  /  lingual   obliquity  (figure  5):    the   tooth  can  be  buccally  (tilted   towards  the  cheek)  or  lin-­‐ gually  (tilted  towards  the   tongue)  impacted.   6.    transverse  (figure  6):     this  type  is  where  the  tooth  is  in   effect  horizontally  impacted  but   in  the  cheek  or  tongue  direction.   7.   inverse  (figure  7):  orienta-­‐ tion  of  the  impacted  tooth  in   completely  inverted  in  reference   to  the  occlusaly  immediate  oppo-­‐ site  tooth.   familial  aggregation   observations  of  a  patient  and  fam-­‐ ily  members:   a  23  year-­‐old  asian  fe-­‐ male  with  no  significant  medical   history  has  received  regular   dental  check-­‐ups  every  six   months  throughout  her  life  and   had  orthodontic  treatment  from   age  14  to16.  the  patient  has   class  i  occlusion.  upon  a  clinical   and  radiographic  examination,   it  was  discovered  that  the  pa-­‐ tient  had  four  third  molars  pre-­‐ sent  (see  figure  8).  the  pa-­‐ tient’s  third  molars  began  erupting   around  the  age  of  19.  currently,   they  are  asymptomatic  with  incip-­‐ ient  discoloration  on  the  occlusal   surfaces.  while  three  of  the  third   molars  erupted  in  a  normal  orien-­‐ tation,  one  of  the  third  molars   (mandibular  left)  erupted  in  an   orientation  that  would  be  classi-­‐ fied  as  horizontal  and/or  mesio-­‐ angular.          third  molar  erup.on  mechanisms  and  pa5erns   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.49    http://dentistry3000.pitt.edu   3   the  patient’s  father  had   four  erupted  third  molars,  all  with   normal  eruption  orientation.  the   patient’s  mother  has  congenitally   missing  third  molars.  the  patient   has  a  younger  sibling  who  is  19   and  third  molars  have  not  erupted   but  one  of  them  is  slightly  mesially   tipped,  and  impacted  (figure  9).       if  one  considers  third  mo-­‐ lar  impaction  and  agenesis  as  vari-­‐ ation  of  the  same  clinical  presen-­‐ tation,  this  family  would  fit  an  au-­‐ tosomal  dominant  mode  of  inher-­‐ itance,  suggesting  a  major  gene   effect.   mechanisms  controlling  tooth   eruption   the  etiology  of  the  mecha-­‐ nisms  behind  eruption  patterns  of   third  molars  is  currently  not  very   well  understood.  tooth  eruption  is   a  complex  and  tightly  regulated   process  that  involves  cells  of  the   tooth  organ  and  the  surrounding   alveolus.  the  genes   that  are  involved  in  the   process  of  tooth  erup-­‐ tion  are  found  all  over   the  human  genome.     mononuclear   cells  (osteoclast  precur-­‐ sors)  must  be  recruited   into  the  dental  follicle   prior  to  the  onset  of   eruption.  these  cells,  in  turn,  fuse   to  form  osteoclasts  that  resorb   alveolar  bone,  forming  an  eruption   pathway  for  the  tooth  to  exit  its   bony  crypt  [3].  there  are  many   different  genes  that  are  involved   in  tooth  eruption.  some  of  the   molecules  possibly  involved  in  the   signaling  cascades  of  eruption   have  been  proposed  in  studies   from  null  mice,  osteopetrotic  ro-­‐ dents,  injections  of  putative  erup-­‐ tion  molecules,  and  cultured  den-­‐ tal  follicle  cells.  in  particular,  re-­‐ cruitment  of  the  mononuclear   cells  to  the  follicle  may  require   colony-­‐stimulating  factor-­‐one   (csf-­‐1)  and/or   monocyte  chemo-­‐ tactic  protein-­‐1   (mcp-­‐1)  [3].  if  the   recruitment  of   mononuclear  cells   to  the  follicle  were   misguided  and  the   dental  follicle  of  a   third  molar  were  disoriented  to   begin  with,  it  could  potentially  re-­‐ sult  in  the  tooth  erupting  in  a  hori-­‐ zontal  orientation.     paracrine  signaling  by  par-­‐ athyroid-­‐hormone-­‐related  protein   and  interleukin-­‐1α,  produced  in   the  stellate  reticulum  adjacent  to   the  follicle,  has  also  been  found  to   potentially  play  a  role  in  regulating   eruption  [3].  this  is  an  example  of   how  a  gene  seemingly  unrelated   to  tooth  development  can  influ-­‐ ence  a  very  crucial  step  in  denti-­‐ tion  development.  therefore,  it  is   possible  that  a  mutation  anywhere   along  the  genome  can  easily  influ-­‐ ence  a  tooth  to  develop  and  erupt   in  an  abnormal  pattern  such  as   horizontal  eruption.     osteoblasts  might  also  in-­‐ fluence  the  process  of  eruption,   the  most  important  physiologic   role  likely  being  at  the  eruptive   site,  in  the  formation  of  osteo-­‐ clasts  through  signaling  via  the   rankl/opg  pathway.  if  this  sig-­‐ naling  pathway  were  interrupted   by  another  protein,  osteoblasts   function  could  potentially  be  al-­‐ tered  and  interfered  with.  this   could  subsequently  affect  how   bone  was  deposited.  if  bone  was   deposited  only  underneath  the   distal  portion  of  the  third  molar,  it   could  potentially  cause  the  tooth   to  tip  over,  giving  it  the  horizontal   orientation  it  is  in  now.  evidence   thus  far  supports  a  role  for  an  os-­‐ teoblast-­‐specific  transcription  fac-­‐ tor,  cbfa1  (runx2),  in  molecular   events  that  regulate  tooth  erup-­‐ tion  [3].  in  the  case  presented      third  molar  erup.on  mechanisms  and  pa5erns   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.49    http://dentistry3000.pitt.edu   4   above,  one  possibility  is  that  since   neither  of  the  patient’s  parents   had  any  third  molar  eruption  ab-­‐ normalities,  the  genetic  factors   that  caused  this  horizontal  orien-­‐ tation  was  probably  a  polygenic   manifestation  that  resulted  from  a   unique  combination  of  the  par-­‐ ents’  genetic  make-­‐up.  the  pa-­‐ tient’s  mother  had  congenitally   missing  third  molars,  so  it  is  possi-­‐ ble  that  some  of  the  genes  which   contribute  to  missing  third  molars   also  play  a  role  in  tooth  eruption   and  influenced  the  eruption  of  the   horizontal  third  molars  in  her  off-­‐ spring.  the  etiology  of  third  molar   impaction  in  the  population  is   most  likely  complex  with  multiple   genes  influencing  the  final  out-­‐ come  of  a  horizontally  oriented   third  molar,  whereas  the  case   presented  here  exemplifies  one   of  the  instances  a  single  gene   form  of  inheritance  is  in  play.       hence,  an  alternative   hypothesis  for  the  etiology  of   this  case  could  be  that  the  hori-­‐ zontal  impaction  of  the  third   molar  is  a  variation  of  tooth   agenesis.  the  mother  of  the   patient  described  in  this  case  ex-­‐ hibits  agenesis  of  the  third  molars.   the  agenesis  alleles  could  have   been  passed  on  to  the  patient,   which  then  exhibited  a  variable   phenotype  of  agenesis:  horizontal   impaction  of  the  third  molar.  this   hypothesis  is  further  supported  by   the  fact  that  the  patient’s  sister   also  has  a  mesially  tipped  and  im-­‐ pacted  third  molar,  which  could   likely  be  another  variable  pheno-­‐ type  of  the  tooth  agenesis  allele   passed  down  from  the  mother.    if   this  were  the  case,  a  pedigree   drawn  of  this  particular  case  study   would  strongly  suggest  an  auto-­‐ somal  dominant  mode  of  inher-­‐ itance  (figure  10).     in  a  research  study  done   on  patterns  of  third-­‐molar  agene-­‐ sis  and  associated  dental  anoma-­‐ lies,  it  was  found  that  permanent   tooth  agenesis,  microdontia  of   maxillary  lateral  incisors,  and  total   dental  anomalies  are  more  fre-­‐ quently  associated  with  agenesis   of  all  four  third  molars  than  with   their  presence  [4].  horizontal  im-­‐ paction  of  the  third  molar  could  be   categorized  as  a  dental  anomaly.   this  association  could  suggest  a   variable  phenotypic  expression  of   the  same  genes  or  set  of  genes  for   tooth  agenesis.     in  addition,  third  molar   agenesis  seems  to  predispose  for   reduced  size  of  the  remaining   teeth  and  the  delayed  develop-­‐ ment  of  certain  teeth  and  has  also   been  linked  to  diminished  stability   of  specific  molar  cuspal  patterns   [5,6].  a  relationship  between   tooth  agenesis  and  abnormal   morphology  of  remaining  teeth   has  been  observed  within  other   types  of  agenesis.  this  goes  to  fur-­‐ ther  support  the  fact  that  these   dental  anomalies  are  all  interre-­‐ lated  and  thus  likely  to  have  a  sim-­‐ ilar  genetic  pathway  or  mode  of   inheritance.  it  is  very  possible  that   third  molar  agenesis  is  a  trait  that   shows  varying  phenotypes,  such   as  the  anomalies  listed  above  and   perhaps  horizontal  impaction  of   third  molars.     management  of  the  condition   the  optimal  treatment   plan  for  this  case  would  be  extrac-­‐    third  molar  erup.on  mechanisms  and  pa5erns   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.49    http://dentistry3000.pitt.edu   5   tion  of  the  horizontal  third  molar.   third  molars  often  cause  crowding   in  the  mouth  and  therefore  are   recommended  for  extraction.  indi-­‐ viduals  with  impaction  showed   more  moderate  to  extreme   crowding  than  those  with  agenesis   [7].  in  addition,  the  horizontal   eruption  of  the  mandibular  left   third  molar  has  caused  the  mesial   marginal  ridge  of  the  tooth  to   come  in  contact  with  the  distal   cervical  portion  of  the  crown  on   the  mandibular  left  second  molar.   the  orientation  of  the  second  and   third  left  mandibular  molars  cre-­‐ ates  a  food  trap  that  could  poten-­‐ tially  lead  to  caries  on  the  distal  of   the  second  molar  or  periodontal   inflammation.  therefore,  a  possi-­‐ ble  treatment  for  this  case  is  sur-­‐ gical  extraction  of  the  third  molar.     discussion  and  practical   implications   while  the  mecha-­‐ nisms  and  etiology  of  third   molar  eruption  are  not  well   understood  currently,  con-­‐ ducting  more  research  in   this  aspect  can  yield  bene-­‐ fits  for  both  the  field  of   dentistry  as  well  as  medi-­‐ cine  as  a  whole.  skeletal   biologists  view  the  process   of  tooth  eruption  as  a  valu-­‐ able  model  to  study  bone  remod-­‐ eling,  since  the  emergence  of  a   tooth  into  the  oral  cavity  involves   both  coupled  and  uncoupled  bone   turnover  events  [3].  for  cell  and   molecular  biologists,  tooth  erup-­‐ tion  provokes  several  questions   concerning  the  tightly  pro-­‐ grammed  series  of  signaling  inter-­‐ actions  between  cells  of  the  con-­‐ nective  tissue  sac  surrounding  the   tooth  (dental  follicle)  and  the  sur-­‐ rounding  alveolus  [3].     for  a  clinician  that  has  to   treat  dental  complications  that   arise  from  abnormal  tooth  erup-­‐ tion,  as  seen  in  numerous  genetic   and  acquired  dis-­‐ orders,   knowledge  about   the  basic  molecu-­‐ lar  mechanisms   involved  is  ex-­‐ tremely  im-­‐ portant.  knowing   the  etiology  can   not  only  help  the   clinician  formu-­‐ late  a  better  treatment  plan  for   the  patient,  but  it  can  help  den-­‐ tists  better  educate  the  patient  in   their  dental  health.   references   1.  prevalence  of  impacted  teeth   and  associated  pathologies-­‐-­‐a  ra-­‐ diographic  study  of  the  hong  kong   chinese  population.  chu  fc,  li  tk,   lui  vk,  newsome  pr,  chow  rl,   cheung  lk.  hong  kong  med  j.   2003  jun;9(3):158-­‐63.   pmid:12777649.   2.  mandibular  third  molar  impac-­‐ tion:  review  of  literature  and  a   proposal  of  a  classification.   juodzbalys  g,  daugela  p.  j  oral   maxillofac  res.  2013  jul  1;4(2):e1.   pmid:24422029.   3.    cellular,  molecular,  and  genetic   determinants  of  tooth  eruption.   wise  ge,  frazier-­‐bowers  s,  d'sou-­‐ za  rn.  crit  rev  oral  biol  med.   2002;13(4):323-­‐34.   pmid:12191959.   4.  patterns  of  third-­‐molar  agenesis   and  associated  dental  anomalies   in  an  orthodontic  population.  ce-­‐ likoglu  m,  bayram  m,  nur  m.  am  j   orthod  dentofacial  orthop.  2011   dec;140(6):856-­‐60.   pmid:22133951.   5.  third  molar  agenesis  and  size   reduction  of  the  remaining  teeth.   garn  sm,  lewis  ab,  kerewsky  rs.   nature.  1963  nov  2;200:488-­‐9.   pmid:14076752.   6.  third  molar  agenesis  and  varia-­‐ tion  in  size  of  the  remaining  teeth.   garn  sm,  lewis  ab,  kerewsky  rs.   nature.  1964  feb  22;201:839.   pmid:14161232.   7.  third  molar  impaction  and   agenesis:  influence  on  anterior   crowding.  esan  t,  schepartz  la.      third  molar  erup.on  mechanisms  and  pa5erns   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.49    http://dentistry3000.pitt.edu   6   ann  hum  biol.  2016  feb  8:1-­‐29.   pmid:26856343.         vol 7, no 1 (2019) issn 2167-8677 (online) doi 10.5195/d3000.2019.90 http://dentistry3000.pitt.edu aesthetic perception and psychological impact of molar-incisor hypomineralisation among patients and parents fernanda mafei felix da silva1, christiane vasconcelos cruz1, luise leal1, marcelo de castro costa1 1 department of pediatric dentistry and orthodontics, school of dentistry, universidade federal do rio de janeiro, rio de janeiro, rj, brazil abstract the aim this study was investigate`s patient and parent’s aesthetic perception and psychological impact of molar-incisor hypomineralisation (mih) and to assess the correlation of mih with caries experience. the sample comprised 56 children, 28 with mih and 28 without mih (comparison group), and their gardens (n=56). for the perception data we used the child and parent’s questionnaire of teeth appearance. we used the european academic of paediatric dentistry (eapd) criteria to define mih. caries experience was assessed by decayed, missing and filled teeth (dmft) index. chi-square, student’s t-test and correlation tests were used with significance level set at 5%. most children were male (n=35; 62.5%) and patients with mih perceived their affected teeth as stainned (p=0.01). mih was considered by parents (6.96±1.7) to enhance psychosocial condition (social, physical, and psychological social) of their children (p<0.01). patients with severe mih showed the worst perception about the color of the teeth (p=0.07). there was no correlation between dmft scores and presence of mih (p=0.80). patients and parents perceived mih. groups with severe mih (children/ parents) showed the worse aesthetic perception. citation: silva fmf, et al. (2019) aesthetic perception and psychological impact of molarincisor hypomineralisation among patients and parents dentistry 3000. 1:a001 doi:10.5195/d3000.2019.90 received: november 26, 2018 accepted: march 28, 2018 published: july 25, 2019 copyright: ©2019 silva fmf, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: pttpo2009@yahoo.com.br introduction molar incisor hypomineralisation (mih) is defined as a qualitative enamel defect that occurs during amelogenesis [1,2]. its aetiology has been linked to both environmental factors (disturbances during pregnancy, severe infections, frequent use of antibiotics in childhood) and genetic factors (genetic variations in tuft1, tuft11, amelx and enam) [3, 4-6]. the prevalence of this condition varies considerably in different parts of the world ranging from 2.4 to 40% in europe [7,8] and from 12.3% to 40.2% in brazil [9,10]. one of the main characteristics of mih is enamel porosity and discoloration, which may vary from white to yellowish and brown [11]. it usually affects one or more permanent molars and/ or may the permanent incisors [10,12]. in most of the, mih may cause loss of tooth structure, resulting in functional and aesthetic problems for the individual, which may affect their quality of life [13,14]. there are few literature reports on the social impact of mih, especially in cases of changes in color and dental caries susceptibility [14, 15, 16]. over the years, aesthetic demands of the smile has been increasing worldwide, especially among children and adolescents [17, 18, 19]. however, it is difficult to measure the impact caused by changes in tooth enamel, since the http://dentistry3000.pitt.edu/ aesthetic perception and psychological impact of molar-incisor hypomineralisation among patients and parents vol 7 no 1 (2019) doi 10.5195/d3000.2019.90 http://dentistry3000.pitt.edu aesthetic evaluation is subjective. thus, the aims of this study were to determine children, parents’ perceptions, and psychological impact of mih before and after treatment to add to the relatively sparse literature. we also aimed to assess possible correlation between mih and caries experience. methods study population this study was performed after approval from the local ethics committee for research (hospital universitário clementino fraga filho – hucff/ufrj – number: 44598514.7.00005257). all subjects/guardians read and signed a written informed consent before their participation in the study. a convenience sample consisting in invidious of both sexes divided by children between 7 and 14 years old treated at paediatric dentistry clinic of the federal university of rio de janeiro and their parents. the sample was divided into children with mih (n=28) and children without mih (n=28) in treated at paediatric dentistry clinic of the federal university of rio de janeiro, and their parents (n=56). the inclusion criteria for both groups were having permanent incisors, first permanent molars erupted, and in the mih group, the permanent incisors had to be affected. the exclusion criteria for both groups included children with syndromes, dental trauma or the use of a fixed appliance. the clinical examination was performed by one calibrated dentist (fmf) and was carried out with a mirror and a probe in the dental chair after dental prophylaxis using artificial light. caries experience was assessed by the dmft index [20, 21]. mih was evaluated using the eapd criteria (european academic of paediatric dentistry) [11,12]. mild mih cases was considered when the tooth presented demarcated opacity, without any loss of structure, while severe cases were considered when the teeth showed loss of structure past or current lesion requiring treatment or presence of atypical restorations were defined as having severe mih [11]. to assess the intra examinerreliability, an exercise was previously applied by the examiner (s.fm) and in the gold standard (c.mc) evaluator with 20 clinical pictures of dental enamel defects (eight with mih). two weeks after the first assessment, a new assessment was carried out by the examiner and the resulting kappa was 0.88. at the end of the clinical examination, each patient and parents received oral hygiene instructions. in addition, the patients received a dental prophylaxis and a topical fluoride application questionnaires a perception survey was carried out using the child and parent’s questionnaire of teeth appearance [22]. this instrument has been validated and adapted to the brazilian context [23] and represents a standard tool for estimating the impact of aesthetic perceptions of enamel opacities. this instrument, has a version for children and adults, includes physical, psychological and social issues. in addition, questions about the dental color aspects, dental appearance, alignment and oral health. the response options for the five questions and sub-items were presented in the form of multiplechoice items. responses are grade ranging from best condition (0) to worst possible condition (4). the first three questions investigate how the child or her/his parent has felt over the past two months and whether he or she has felt uncomfortable, concerned or has been prevented from smiling because of the appearance of his or her teeth, according to the views of his or her parents/guardians and himself or herself. an additional question with four subratings for the perception of children and their parents about appearance, position, colour and health of their teeth (or their children) was asked. the last item asked the interviewee about his/her opinion on satisfaction or dissatisfaction with the colour of the teeth, according to the following sentence: ‘the colour of http://dentistry3000.pitt.edu/ aesthetic perception and psychological impact of molar-incisor hypomineralisation among patients and parents vol 7 no 1 (2019) doi 10.5195/d3000.2019.90 http://dentistry3000.pitt.edu my teeth (or the tooth of my son) are nice and beautiful’. statistical analysis data were analysed using spss version 20.0 (statistical package for social sciences, spss inc., chicago, iii). descriptive statistics was performed. a shapiro-wilks test was carried out to evaluate the pattern of sample distribution. student’s t-test and binary logistic regression were performed to assess the difference between groups (patients and parents). chisquare and correlation tests, with significance level set at 5%, were applied to disclose the associations and correlation between mih and caries experience. results a total of 112 individuals (parents and children) completed questionnaires. the majority of the parents were female (87.5%; n=49), with an average age of 39.3±7.4 and most of children were male (62.5% n=35) (table 1). in the groups with mih (n=28) and control (n=28) there was no difference (p>0.05) between sex, ethnicity, socio-economic condition or dental caries experience (table1). patients with mih perceived their stained teeth (p=0.01) (table 2). there was no statistically significant difference between the two groups when comparing individuals with mih (4.18±1.3) and the comparison group (4.82±2.1) regarding physical, psychological and social conditions (table 2). in addition, in the mih parent group, a worse perception (6.96±1.7) than the one in the comparison group (6.50±2.9) was seen, which was statistically significant with regards to staining perception of teeth and to opinion about oral health (p=0.01 and p=0.03, respectively) (table 2). perception between parents and children showed the former had a worse perception in both psychosocial conditions (6.96±1.7) and appearance (21,81±3.9) (table 3). regarding the severity of the mih, 64.3% (n=18) showed mild and 35.7% (n=10) showed severe mih. patients with severe mih (4.80±1.2) and their parents (7.5±2.0) showed the worst perception on physical, psychological and social conditions. in addition, children with severe mih showed greater table 1. characteristics of the sample children mhi group control group p value mean age (sd) 9.39(2.04) 9.43(1.91) 9.36(2.19) sex n (%) 0.58 female 35(62.5) 19(67.9) 16(57.1) male 21(37.5) 9(32.1) 12(42.9) a cceb n (%) 0.77 0.80 b2 18(32.1) 10(35.7) 8(28.6) c1 27(48.2) 14(40) 13(46.4) c2 8(14.3) 3(10.7) 5(17.9) d2 3(5.4) 1(3.6) 2(7.1) b dmft x mih 0.95(± 1.6) 1.00(±1.54) 0.89(±1.68) a brazilian economic classification criteria cceb b2< r$ 3.118, c1< r$1.865 c2<r$ 1.277 d2<r$ 895 bcorrelation test performed between mih group and controls. note: p value was based on chi square (p<0.05). http://dentistry3000.pitt.edu/ aesthetic perception and psychological impact of molar-incisor hypomineralisation among patients and parents vol 7 no 1 (2019) doi 10.5195/d3000.2019.90 http://dentistry3000.pitt.edu dissatisfaction with tooth coloration. (table 4). discussion aesthetic alterations in dental enamel may have a significant impact on the physical, social and psychological well-being of individuals and also affect their families [24,25]. mih may be considered as an impacting enamel disturbance because it highly affects aesthetic and function. moreover, in the most severe cases, it may lead to loss of structure and dental sensitivity as well as an increase in caries experience [26,27]. physical, psychological and social conditions related to children and their parents were perceived in the present study and may affect their quality of life. furthermore, the appearance of mih teeth (displayed as the perception of teeth being stained) was perceived by and influenced both children and parents. according to a study, the most severe enamel defects were related to aesthetic complaints by the patients and sometimes required aesthetic restorative treatment [14]. in relation to tooth alignment (tooth position), this was perceived by both children and parents in the control group. this leads us to support that the position of the teeth can also be related to a negative impact on perception of the smile aesthetics. this study showed that parents were more concerned than children with regard to dental appearance, colour of teeth and tooth alignment [19]. regarding dental caries in the present study, there was no statistically significant association between mih and dental caries, although other studies related to mih showed increased caries susceptibility in mih patients [26,27]. in this context, we can table 2. evaluation of agreement between parents/children (mih and controls) for aesthetic perception and psychosocial conditions. subjects (children) p value parents p value mih group controls mih group controls *physical, psychological, and social conditions 4.18(±1.3) 4.82(±2.1) 0.17 6.97(±1.7) 6.50(±2.9) ** concerns with tooth position 2.75(±1.37) 2.96(±1.73) 0.61 3.29(±1.4) 2.93(±1.5) 0.73 tooth condition 2.54(±1.20) 2.24(±1.73) 0.22 2.68(±1.0) 2.75(±1.4) 0.04 staining perception teeth 2.75(±1.04) 2.07(±0.85) 0.01 3.21(±0.83) 2.64(±1) 0.01 oral health opinion 2.0(±1.12) 1.71(±1.08) 0.33 2.71(±1.0) 2.04(±1.2) 0.03 satisfaction with the colour of the tooth 2.64 (±1.2) 2.24 (±1.0) 1.00 3.61(±0.95) 3.14(±1.1) 0.10 *the sum of the first three questions: discomfort with appearance of their teeth, concern about the appearance of the teeth and discomfort with smile, was assed by student’s t-test (p<0.05). http://dentistry3000.pitt.edu/ aesthetic perception and psychological impact of molar-incisor hypomineralisation among patients and parents vol 7 no 1 (2019) doi 10.5195/d3000.2019.90 http://dentistry3000.pitt.edu suppose that the severity of mih may be related to caries experience, since in this study, mild mih was predominant. it is noteworthy that some studies reported that even mih patients with lower dental caries activity needed to be carefully monitored. this is because the teeth affected with mih present more porous enamel, leading to fractures, facilitating accumulation of the biofilm [28]. in our study, all patients received oral hygiene instructions, fluoride application and restorative treatment, if necessary. children with mih received treatments ranging from preventive care (application of sealants and fluoride varnish) to restorations. the majority of treated teeth were at the severe level and most of them were first permanent molars. some studies have shown that regardless of the affected tooth or locality, patients with enamel defects after treatment have a better perception of their quality of life [12]. as limitations, we can mention the child’s age. this may have an influence on perception, because the sample was patients less than 14 years old. age could hamper the understanding of the questions because the older the children, the higher the aesthetic concerns. furthermore, caries experience (dmft scores) tends to increase with age, but we do not think that age influenced our results since the mih group and controls presented a similar mean age. this study demonstrated that children and their parents had the knowledge about the existence of the mih, in which may affected their quality of life, supposing that color alteration can influence the negative perception in the patient. we suggest that other studies about the aesthetic perception among individuals with mih will be performed by group of teenagers or adults who have a higher level of aesthetic requirements. also, assess the actual need for aesthetic treatment in patients with mih affecting incisors without a loss of structure. conclusion mih was perceived by both patients and parents as affecting physical, psychological or social table 3. comparison of aesthetic perceptions between children and parents with mih and controls. mih controls pvalue children n=28 parents n=28 children n=28 parents n=28 *physical, psychologi cal, and social conditions 4.18(±1.3) 6.96(±1.7) 4.82(±2.1) 6.50(±2.9) 0.03 **aesthetic perception of position, staining, oral, health and satisfactio n with the colour of the teeth 20.25(±3.7) 25.07(3.7) 20.11(±5.1) 22.86(2.0) 0.03 note: p-value was assessed by student’s t-test (p<0.05) comparing children and parents. *the sum of the first three questions: discomfort with appearance of their teeth, concern about the appearance of the teeth and discomfort with smile. **the sum of additional question with four sub-ratings of the perception and last question of satisfaction with the colour of the tooth. http://dentistry3000.pitt.edu/ aesthetic perception and psychological impact of molar-incisor hypomineralisation among patients and parents vol 7 no 1 (2019) doi 10.5195/d3000.2019.90 http://dentistry3000.pitt.edu features, and parents showed worse perception scores than children. caries experience was not correlated to mih in this sample. acknowledgements this study was financed in part by the coordenação de aperfeiçonamento pessoal de nivel superior brasil(capes) this study is part of the masters dissertation of the first author. references 1. developmental defects of enamel-historical and present-day perspectives of their pathogenesis. suckiling gw. advances in dental research 1989; 3(2): 87-94. 2. clinical diagnosis of enamel defects: pitfalls and practical guidelines. seow wk. international dental journal 1997; 47(3): 173– 82. 3. amoxicilin may cause molar incisor hypomineralization. laisi s, ess a, sahlberg c, arvio p, lukinma pl, alaluusua s. j dent res 2009; 88: 132-136. table 4. evaluation of agreement between parents/children (mih group) for aesthetic perception and psychosocial conditions accord into level of severity. groups with mih children parents mild severe p value mild severe p value *physical, psychological, and social conditions 3.94(±1.24) 4.55(±1.30) 0.23 6.9(±1.32) 7.5(±2.30) 0.90 concerns with tooth position 2.35(±1.27) 3.36(±1.36) 0.60 3.29(±1.36) 3.27(±1.68) 0.97 tooth condition 2.65(±1.17) 2.36(±1.36) 0.56 2.88(±1.11) 2.36(±0.81) 0.19 staining perception teeth 2.71(±0.85) 2.82(±1.33) 0.79 3.29(±0.84) 3.09(±8.3) 0.54 oral health opinion 2.06(±0.97) 1.91(±1.38) 0.74 2.82(±1.19) 2.55(±0.93) 0.52 satisfaction with the colour of the tooth 2.09(±1.30) 3.0 (±1.17) 0.07 3.65(±0.10) 3.55(±0.93) 0.80 *the sum of the first three questions: discomfort with appearance of their teeth, concern about the appearance of the teeth and discomfort with smile". p -value assessed by student’s t-test (p<0.05) http://dentistry3000.pitt.edu/ aesthetic perception and psychological impact of molar-incisor hypomineralisation among patients and parents vol 7 no 1 (2019) doi 10.5195/d3000.2019.90 http://dentistry3000.pitt.edu 4. clinical e. molecular analysis of the enamelin gene enam in colombian families with autosomal dominant amelogenesis imperfecta. gutierrez s, torres d, bricen ˜ o i, go ´mez am, baquero. genetics and molecular biology 2012; 35(3): 557–66. 5. genes expressed in dental enamel development are associated with molar-incisor hypomineralization. jeremias f, koruyucu m, kuchler ce, bayram m, tuna be, deeley k, pierri a, souza j, fragelli mb, paschoal ab, koray gf, seymen ms, caminaga lp, vieira ar. archives of oral biology 2013; 58: 1434–1442 a. 6. aetilogy of molar-incisor hypomineralization: a systematic review. alaluusua s. european archives of paediatric dentistry 2010; 11(2): 53-8. 7. prevalence and diagnosis of molar-incisor hypomineralisation (mih): a systematic review. jälevik b. eur arch paediatr dent 2010; 11: 59-6. 8. prevalence and distribution of demarcated opacities in permanent 1st molars and incisors in 6 to 8 year-olddanish children. wogelius p, haubek d, poulsen s. acta odontol scand 2008; 66: 5864. 9. prevalence and distribution of demarcated opacities and their squeals in permanent first molars and incisor in 7 to 13-year-old brazilian children. soviero v, haubek d, trindade c, matta t, poulsen s. acta odontol scand 2009; 66: 170-175. 10. molar incisor hypomineralization: prevalence severity and clinical consequences in brazilian children da costa silva cm, jeremias f, souza jf, cordeiro rcl, santos pinto l, zuanon acc. int j paediatr dent 2010; 20(6): 426-34. 11. best clinical practice guidance for clinicians dealing with children presenting with molar-incisorhypomineralisation (mih): an eapd policy document. lygidakis na, wong f, jälevik b, vierrou am, alaluusua s, espelid i. eur arch paediatr dent 2010; 11(2): 75-81. 12. judgement criteria for molar incisor hypomineralisation (mih) in epidemiologic studies: a summary of the european meeting on mih held in athens, 2003 weerheieijm kl, duggal m, majáre i, papagiannoulis l, koch g, martens hc, et al.. eur j paediatr dent 2003; 4: 110-3. 13. dental enamel formation and its impact on clinical dentistry. simmer jp, hu jcc. journal of dental education 2001; 65(9): 896– 905 14. meneghim mc, kozlowskl fc, antonio c,pereira a v. assaf, tagliaferro ps. international journal of paediatric dentistry 2007; 17: 205–210. 15. perception of dental fluorosis and other oral health disorders by 12-year-old brazilian children. jeremias f, souza jfd, costa silva cmd, cordeiro rdcl, zuanon acc, santos-pinto l. dental carie experience and molar-incisor hypomineralization. acta odontol scand 2013; 71: 17 b. 16. clinical studies on molar– incisor hypomineralisation part 2: development of a severity index. chawla n, messer lb, silva m. eur arch paediatr dent 2008; 9: 191–9. 17. clinical evaluation of desensitizing treatment for incisor teeth affected by molar-incisor hypomineralization ozgül bm, saat s, sönmez h, oz ft.. j clin pediatr dent 2013; 38(2): 101-5. 18. aesthetic concerns of children and parents in relation to different classifications of the tooth surface index of fluorosis. clark dc, hann hj, williamson mf, berkowitz j. community dent oral epidemiol 1993; 21: 360–364. 19.comparison of dental esthetic perception of young adolescents and their parents. kavand g, broffitt b, levy sm, warren jj j public health dent 2012; 72(2): 164-71. 20. w.h.o. world health organization. oral health surveys. basic methods. geneva: world health organization; 1997. 21. sex differences in dental caries experience of elementary school children. klein, h., palmer, c. e. studies on dental caries. vii. publ hlth rep 1938; 53(38):1685-1690. http://dentistry3000.pitt.edu/ aesthetic perception and psychological impact of molar-incisor hypomineralisation among patients and parents vol 7 no 1 (2019) doi 10.5195/d3000.2019.90 http://dentistry3000.pitt.edu 22. development of a questionnaire to measure perceptions of, and concerns derived from, dental fluorosis. martínez-mier ea, maupomé g, soto-rojas ae, ureña-cirett jl, katz bp, stookey gk. community dent health 2004; 21(4): 299-305. 23. percepção da fluorose dentária e avaliação da concordância entre relatos de pais e filhos: validação de um instrumento. furtado ges, sousa mlr, barbosa ts, wada rs, martínez-mier ea, almeida mel. cad saude publica. 2012 ago;28 (8):1493-505. 24. esthetic perception and psychosocial impact of developmental and enamel defects among malaysian adolescents. sujak sl, abdul karim a, dom tn. j oral sci 2004; 46(4): 221-6. 25. seeking children’s perspectives in the management of visible enamel defects. abdul-karim a, yesudian g, o’mahony j, marshman z. int paediatr dent 2011: 21(2): 89-95. 26. increase in severity of molarincisor hypomineralization and its relationship with the colour of enamel opacity: prospective cohort study. da costa-silva cm, ambrosano gm, jeremias f, de souza jf, mialhe fl. int j paediatr dent 2011; 21(5): 333-41. 27. molar incisor hypomineralization in bosnia and herzegovina: aetiology and clinical consequences in medium caries activity population. muratbegovic a, markovic n, gannibegovic selimovic m. eur arch paediatr dent 2007; 8(4): 189-94. 28. nonfluoride hypomineralizations in the permanent first molars and their impact on treatment need. lepäniemi a, lukinmaa pl, alaluusua s. caries res. 2001; 35(1): 36-40.1 http://dentistry3000.pitt.edu/ erratum to: incorporation of fluconazole and ocimum sanctum oil in soft denture liners to treat biofilms of candida albicans associated with denture stomatitis vol 9 no 1 (2021) doi 10.5195/d3000.2021.150 http://dentistry3000.pitt.edu erratum to: incorporation of fluconazole and ocimum sanctum oil in soft denture liners to treat biofilms of candida albicans associated with denture stomatitis. godil, a., bhagat, d., das, p., kazi, a., dugal, r., & satpute, s. dentistry 3000 2021; 9(1). doi: https://doi.org/10.5195/d3000.2021.120 abstract in incorporation of fluconazole and ocimum sanctum oil in soft denture liners to treat biofilms of candida albicans associated with denture stomatitis. godil, a., bhagat, d., das, p., kazi, a., dugal, r., & satpute, s. dentistry 3000 2021; 9(1). doi: https://doi.org/10.5195/d3000.2021.120 , we apologize that the name of one of the authors was misspelled by the managing editor when creating the pdf. ashi ilyas kazi was corrected to arshi ilyas kazi. the figure legends were also not in line with the respective figures and were fixed in the new version. the original manuscript has been updated to reflect the aforementioned changes. http://dentistry3000.pitt.edu/ https://doi.org/10.5195/d3000.2021.120 https://doi.org/10.5195/d3000.2021.120 https://doi.org/10.5195/d3000.2021.120 effects of a novel dye containing toothpaste versus regular fluoridated toothpaste for supragingival biofilm reduction in patients with chronic gingivitis: a randomized controlled trial vol 9 no 1 (2021) doi 10.5195/d3000.2021.117 http://dentistry3000.pitt.edu effects of a novel dye containing toothpaste versus regular fluoridated toothpaste for supragingival biofilm reduction in patients with chronic gingivitis: a randomized controlled trial avita rath, chong jun ning, priyadarshini hr, brandon nicholas teo, naik hsian zhen, ong wey yin segi university, malaysia abstract aim: to compare the effects of a plaque disclosing toothpaste with a regular fluoridated toothpaste for supragingival biofilm reduction in patients with chronic gingivitis. materials and methods: this study was a randomized controlled trial with a parallel design. forty patients, both males and females aged 18-25 years with chronic generalized gingivitis (n=40) were randomly allocated to two groups of twenty each (n=20), using lottery method. participants in the experimental group brushed with a plaque disclosing toothpaste, while participants in the control group brushed with regular fluoridated toothpaste. clinically, biofilm was measured using silness & loe plaque index, both at baseline and at follow up visits by two assessors who were blinded to group allocation. adjunctly, intraoral photographs were taken for digital plaque image analysis of the remaining dental biofilm in the upper and lower anterior labial tooth surfaces, and calculated based on the area of stained pixels. data was analyzed using spss version 22. mean plaque scores between the groups were compared using independent sample t-test and p<0.05 was considered significant. results: data of all the 40 participants were analyzed as they were no drop outs. no significant differences were found in the mean plaque scores between the two groups (p=0.059) at the end of the study. similarly, digital plaque image analysis showed no significant difference in the mean plaque scores between the groups (p= 0.93). conclusion: this study showed that there are no additional benefits of using a plaque disclosing toothpaste, in enhancing the removal of the supragingival dental biofilm during routine oral hygiene maintenance. keywords: dental biofilm; supragingival; gingivitis; oral hygiene; dentifrice citation: rath, a et al. (2021) effects of a novel dye containing toothpaste versus regular fluoridated toothpaste for supragingival biofilm reduction in patients with chronic gingivitis: a randomized controlled trial dentistry 3000. 1:a001 doi:10.5195/d3000.2021.117 received: september 10, 2020 accepted: january 16, 2021 published: june 21, 2021 copyright: ©2021 rath, a et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: reachdocpriya@gmail.com introduction dental biofilm is the primary cause for many oral diseases and conditions including periodontitis [1]. currently, periodontal disease is estimated to be the 11th most prevalent disease globally [2]. periodontitis can result in tooth loss and worsening of certain systemic illness, leading to a compromised quality of life [2]. thus, adequate plaque control should be the primary goal of daily oral hygiene, as it prevents the occurrence of oral diseases and future tooth loss [3,4]. even though tooth brushing is the cornerstone of home oral hygiene practices, the high incidence and prevalence of periodontal problems globally reveal that these home hygiene practices are not adequate to maintain good oral hygiene [4-6]. http://dentistry3000.pitt.edu/ effects of a novel dye containing toothpaste versus regular fluoridated toothpaste for supragingival biofilm reduction in patients with chronic gingivitis: a randomized controlled trial vol 9 no 1 (2021) doi 10.5195/d3000.2021.117 http://dentistry3000.pitt.edu while brushing is the most widespread and universally suitable measure for the mechanical removal of the dental biofilm, it should be associated with constant motivation [7-9]. incomplete biofilm removal may be partly attributed to the invisible nature of dental biofilm. this can be overcome by adding a dye, thereby disclosing the biofilm, especially in the posterior retentive areas. increasing the visibility of the pathological source to patients can be a promising and reliable way to improvise their home oral hygiene practices, and thus can motivate them to achieve ideal brushing technique [10]. studies suggest that the use of disclosing agents such as erythrosine, will help to remove dental biofilm more easily when used prior to brushing [11]. however, there is still a wide gap in knowledge regarding the effectiveness of such products in home-use. various indices have been used to quantify dental biofilm in different clinical and research settings. though these indices quantify biofilm in an objective manner, they are not devoid of certain limitations such as subjective assessment of the operator [12-14]. several authors have proposed other methods such as the use of grids and planimetric systems including the use of fluorescent light to visualize and measure biofilm [12,15]. the assessment of the outcome of oral hygiene practices needs tools with higher accuracy, consistency and sensitivity towards the amount of remaining biofilm on the surface of the teeth [16]. digital plaque image analysis (dpia) could be a noninvasive alternative for measuring biofilm on the tooth surface and has been used in some studies [10]. however, majority of the studies so far have evaluated the efficacy of dye containing toothpaste in professional care, hence it warranted its evaluation in self-care as well. hence, the main objective of the present study was to compare the effectiveness of a plaque-disclosing toothpaste and a regular fluoridated toothpaste in removing the supragingival biofilm, using dpia as an adjunct to clinical measurement, during routine oral hygiene maintenance. material and methods this was a randomized controlled trial with two parallel arms. the trial was registered at clinicaltrials.gov with the id no nct03287011 prospectively. ethical considerations the study was approved by the institutional ethics committee with ec number segi/ec/2017-18/11. informed and written consent was obtained from all the participants prior to the commencement of the trial. the trial followed the consort guidelines and was designed in compliance with the declarations of helsinki act [17], with principles of good clinical practice. study participants study participants included healthy male and female patients in the age range of 18-25 years visiting segi oral health centre, kota damansara, malaysia, with chronic generalized gingivitis. patients who were pregnant or lactating, who had used anti-inflammatory and antibiotics two weeks prior, undergone any dental treatment within the past 30 days, and persons undergoing orthodontic treatment were excluded from the study. one hundred and seven people were screened for eligibility between january 2018 and march 2018 and forty of them were recruited for the study based on the inclusion and the exclusion criteria (fig.1). sample size the sample size was calculated using g power software. with a 95% confidence interval, 80% power and an effect size of 90%, a sample size of n=40 (20 in each group) was determined. randomization and concealment study participants were randomized into two groups based on the lottery method. allocation concealment was ensured by providing sealed envelopes with participants’ http://dentistry3000.pitt.edu/ effects of a novel dye containing toothpaste versus regular fluoridated toothpaste for supragingival biofilm reduction in patients with chronic gingivitis: a randomized controlled trial vol 9 no 1 (2021) doi 10.5195/d3000.2021.117 http://dentistry3000.pitt.edu assignment to a dental hygienist not involved in the study. study design at baseline, participants were instructed to refrain from routine oral hygiene practices for 24 hours before their visit to our oral health centre. upon their arrival, modified bass technique of brushing [18] was demonstrated to all of them. both the groups were provided with a standardized toothbrush (colgate® 360°® advanced optic white® manual toothbrush 360) and a similar amount of regular fluoridated toothpaste (colgate total® clean mint toothpaste) was dispensed. all follow up analysed (n=20) • excluded from analysis (n=0) • confirm appropriate hygiene cessation • brush teeth with control toothpaste • take intraoral photo • silness & loe plaque index • dismiss participants • review informed consent • confirm appropriate hygiene • brush teeth with control toothpaste • take intraoral photo • silness & loe plaque index • dismiss participants control group a (n=20) analysed (n=20) • excluded from analysis (n=0) assessed for eligibility (n=107) randomized (n=40) • confirm appropriate hygiene cessation • brush teeth with experimental toothpaste • take intraoral photo • silness & loe plaque index • questionnaire • dismiss participants experimental group b (n=20) enrollment excluded (n=67) • not meeting inclusion criteria (n=56) • declined to participate (n=7) • other reasons (n=4) • review informed consent • confirm appropriate hygiene cessation • brush teeth with control toothpaste • take intraoral photo • silness & loe plaque index • dismiss participants • first visit analysis figure 1. experimental design http://dentistry3000.pitt.edu/ effects of a novel dye containing toothpaste versus regular fluoridated toothpaste for supragingival biofilm reduction in patients with chronic gingivitis: a randomized controlled trial vol 9 no 1 (2021) doi 10.5195/d3000.2021.117 http://dentistry3000.pitt.edu participants were asked to brush their teeth in front of a mirror to eliminate any compounding factors. immediately after brushing, a disclosing agent (gc tri plaque id gel tm) was applied to all tooth surfaces of all the participants with a micro brush to disclose the remaining plaque left after toothbrushing in both groups. the area was gently rinsed, and a cheek retractor was placed. intraoral frontal photographs were taken by a dslr camera with a macro lens for the analysis of the dpia (fig.2). photographs were taken following the standard frontal photography requirements. figure 2. intraoral frontal photographs taken for dpi analysis. next, biofilm left after toothbrushing was recorded using the silness & loe plaque index (pi) [19]. each of the four surfaces of the teeth (buccal, lingual, mesial and distal) were given a score from 0-3, where score 0 was absence of plaque, score 1 was a film of plaque adhering to the free gingival margin, score 2 was moderate plaque accumulation in sulcus or pocket and score 3 was abundance of soft matter in sulcus or pocket. the values obtained from this visit were recorded as baseline values for the clinical trial. participants were then allowed to brush off the disclosing agent, and recalled after one month. participants in both the groups were instructed not to use any other oral hygiene aids other than their toothbrush during this period. two trained examiners were calibrated in recording biofilm, prior to the commencement of the study, to ensure inter recorder reliability. the clinical parameters of 10% of the volunteers were evaluated in duplicate to obtain diagnostic confidence using the kappa agreement test, whose inter-rater agreement was k = 0.89. the examiners were blinded to the group allocation. the second visit was planned one month after the first visit. prior to the second visit, all participants were requested to refrain from brushing for 24 hours. on arrival at the oral health centre, the control group brushed with the regular fluoridated toothpaste (colgate total® clean mint toothpaste) and the experimental group brushed with the experimental toothpaste (shoplaq® disclosing toothpaste). both the toothpastes contained fluoride, and were provided by the investigator. the experimental group was instructed to use one drop of dye containing toothpaste on a dry brush, and then to brush all the teeth and rinse, check teeth and gums for pink stains, and gently brush stains off. the brushing, disclosing, photographic procedures and plaque score recording were similar to the baseline visit. additionally, the experimental group was given a self-administered questionnaire at the end of the follow-up visit, to provide feedback towards the toothpaste used and side-effects if any. the primary outcome was the amount of supragingival dental biofilm between the groups at the end of study and the secondary outcome was any side effects in the experimental group. digital image analysis of dental biofilm in addition to the clinical plaque scores, frontal photographs of the anterior teeth of subjects in both groups were taken after brushing and were subjected to dpia analysis. frontal photographs were analyzed using computer software (matlab 2016 version). the photographs underwent a rough standardization of raw image size and were masked by a blinded investigator to define the area of analysis. rgb standard deviation detected the stains on the photographs. a pixel was deemed to be stained if the standard deviation (sd) of the rgb value exceeded a set percentage of the maximum sd observed in the image. http://dentistry3000.pitt.edu/ effects of a novel dye containing toothpaste versus regular fluoridated toothpaste for supragingival biofilm reduction in patients with chronic gingivitis: a randomized controlled trial vol 9 no 1 (2021) doi 10.5195/d3000.2021.117 http://dentistry3000.pitt.edu a pixel at position x, y was deemed stained if the following conditions were met: where 𝑚𝑎𝑥[𝑆𝐷𝑥,𝑦(𝑅𝑥,𝑦, 𝐺𝑥,𝑦, 𝐵𝑥,𝑦)] was the maximal sd value for all pixels in the processed image and 𝜎 (ranging from 0 to 1) was a user-set sensitivity. for image presentation, all stained pixels were then overlaid with a transparent cyan mask for ease of comparison. (fig.3a&3b) a simple relative area calculation was performed to quantify plaque coverage, as below: figure 3a & 3b. stained pixels overlaid with a transparent cyan mask to calculate percentage of plaque coverage after baseline and follow up visits respectively. statistical analysis data was analyzed using spss version 22 (ibm corporation, armonk, ny, usa). independent sample t-test was used to compare the mean plaque scores and mean dpia scores between groups. p<0.05 was considered significant. visual analogue scale (vas) scores were expressed as a percentage. results one hundred and seven patients were initially screened for eligibility. of these, sixty-seven (n=67) patients were excluded due to missing anterior teeth, ongoing orthodontic treatment, refusal to participate or if they did not meet the inclusion criteria. the remaining 40 subjects who were enrolled, completed the study, without any drop outs during the study period. the mean age of the participants was 22.2+ 0.6 years in control group and 21.8+0.3 years in experimental group respectively. about 40% were males and 60% were females in both the groups. no differences were seen in the baseline plaque score of the groups with a mean plaque score of about 1.15 in both the groups. however, the follow up results showed that the mean plaque score was lesser (0.98+0.41) in the group that used the dye containing toothpaste as compared to the other group (1.20+0.57). but this difference was not significant with p=0.059. (table 1) similarly, no significant differences were seen in the mean anterior plaque scores calculated using digital image analysis, either at the baseline or at the follow up visit with p=0.98 and 0.93 respectively. (table 2) the questionnaire responses are shown in table 3 and table 4. about 65% of the participants in experimental group liked the color, freshness and cleansing action of the visit group n mean standard deviation p-value baseline control 20 1.16 0.49 0.882 experimental 20 1.14 0.29 follow up control 20 1.20 0.57 0.059 experimental 20 0.98 0.41 𝑆𝐷𝑥,𝑦 (𝑅𝑥,𝑦 , 𝐺𝑥,𝑦 , 𝐵𝑥,𝑦) max{ 𝑆𝐷𝑥,𝑦 (𝑅𝑥,𝑦, 𝐺𝑥,𝑦, 𝐵𝑥,𝑦)} ≥ 𝜎 %𝑃𝑙𝑎𝑞𝑢𝑒 𝐶𝑜𝑣𝑒𝑟𝑎𝑔𝑒 = 𝑛𝑜 𝑜𝑓 𝑝𝑖𝑥𝑒𝑙𝑠 𝑤𝑖𝑡ℎ 𝑠𝑡𝑎𝑖𝑛 𝑡𝑜𝑡𝑎𝑙 𝑡𝑒𝑒𝑡ℎ 𝑝𝑖𝑥𝑒𝑙𝑠 × 100% table 1. comparison of mean plaque scores between control and experimental groups http://dentistry3000.pitt.edu/ effects of a novel dye containing toothpaste versus regular fluoridated toothpaste for supragingival biofilm reduction in patients with chronic gingivitis: a randomized controlled trial vol 9 no 1 (2021) doi 10.5195/d3000.2021.117 http://dentistry3000.pitt.edu dye containing toothpaste and about 70% felt motivated to brush using the dye containing toothpaste. however, about 40% felt that it took a longer time to brush with the dye containing toothpaste. discussion the pathogenic nature of dental biofilm can be counteracted by maintaining effective oral hygiene, using mechanical and chemical approaches [20-22]. home oral hygiene maintenance has shown to be ineffective in adults [23]. on average, 60% of the biofilm is generally left behind after brushing [24]. a simple means to improve one’s oral hygiene habits is by improving skills involved in removal of dental biofilm [25]. a disclosing agent as the name itself suggests, changes the color of dental biofilm due to the dye present in it, which reveals the locations more clearly. this is due to the ability of dental biofilm to retain a large number of dye substances due to the interaction between the polarity difference of biofilm and dyes. however, the availability of these disclosing dyes in toothpaste is rare outside the dental offices [26]. hence, in our study, the toothpaste shoplaq® was provided to the participants. the dye, ci-45410, present in this toothpaste gave it a pinkish color that adhered to the intraoral biofilm. the other ingredients included fluoride, natural herbs with no sodium lauryl sulphate added and it was a nonabrasive toothpaste. in our study, no significant difference was observed in the overall mean plaque scores between the groups, which was similar to a study which also reported that disclosing agents did not play a critical role in improving plaque control [27]. however, studies by stevens et al [10], and ower et al [28] showed better plaque removal with the dye containing toothpaste. this could be attributed to the differences in the dye containing tooth pastes used, oral hygiene instructions provided prior to brushing and the indices used to record biofilm. we felt that most areas such as the posterior teeth and lingual surfaces of the maxillary teeth which are often missed, cannot be seen while visit group n mean standard deviation p-value baseline control 20 11.40 3.61 0.98 experimental 20 11.29 4.01 follow up control 20 10.53 2.91 0.93 experimental 20 10.25 2.87 items assessed very good good not sure bad very bad cleansing action 4 (20%) 9 (45%) 6 (30%) 1 (5%) 0 colour 1 (5%) 11(55%) 4 (20%) 3 ( 15%) 1 (5%) freshness 1 (5%) 13 (65%) 5 (25%) 1 (5%) 0 time taken to brush 1 (5%) 6 (30%) 5 (25%) 5 (25%) 3 (15%) table 2. comparison of mean dpia scores between control and experimental groups. table 3: experimental group’s feedback on dye containing toothpaste (n=20). http://dentistry3000.pitt.edu/ effects of a novel dye containing toothpaste versus regular fluoridated toothpaste for supragingival biofilm reduction in patients with chronic gingivitis: a randomized controlled trial vol 9 no 1 (2021) doi 10.5195/d3000.2021.117 http://dentistry3000.pitt.edu brushing. and not many people brush their teeth visualizing all areas in the mirror. hence, the pink stains of the disclosed plaque could go unnoticed, even when a dye containing tooth paste was used. hence, in our study, the biofilm on the anterior teeth was assessed using dpia, anticipating that patients may visualize their anterior teeth better while brushing. however, no significant differences were seen in mean plaque scores between both groups using digital images. this indicates that correct technique of tooth brushing by patients is more important than merely using a disclosing dye containing toothpaste, for effective plaque removal. items assessed responses n=20 (%) side effects yes 0 no 20 (100%) motivation to brush yes 14 (70%) no 6 (30%) overall satisfaction satisfied 7 (35%) neutral 12 (60%) unsatisfied 1 (5%) establishing an accurate, visual method of assessing biofilm remaining after brushing is especially advantageous for children, elderly and patients undergoing orthodontic treatment for good oral hygiene. adding a disclosing agent into the toothpaste, makes it more convenient for chairside education and is easily obtainable for the patient to bring home and can cultivate an effective brushing habit [29]. our questionnaire responses showed that 70% of participants who used disclosing tooth paste, felt more motivated to brush their teeth. hence, we can infer that disclosing toothpastes can still be a useful tool for educational and motivational purposes. the limitations of this study were that the brushing technique was demonstrated only once and the time taken for brushing was not standardized. also, the effectiveness of plaque removal was assessed after just one brushing, as our objective was solely to compare the effectiveness of the plaque disclosing toothpaste and not the effectiveness of patient’s brushing technique. hence, long term studies and different patient population pools are needed to verify the effectiveness of such products in future. conclusion a dye containing toothpaste may not significantly improve plaque removal effectiveness as seen in our study; nevertheless, it may serve as a potential educational tool and motivate patients to maintain proper oral hygiene through a conscious effort. acknowledgement the authors would like to thank the subjects for their patience and participation, mr. yeoh chin vern for his help in digital plaque analysis, dr khairiyah abd muttalib, dr bennete fernandes and dr daniel devaprakash for their critical evaluation and support throughout the study period. references 1. oral microbes, biofilms and their role in periodontal and peri-implant diseases. lasserre jf, brecx mc, toma s. materials (basel). 2018 sep 22;11(10):1802. doi: 10.3390/ma11101802. pmid: 30248991; pmcid: pmc6213094. 2. gbd 2016 disease and injury incidence and prevalence collaborators. global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 19902016: a systematic analysis for the global burden of disease study 2016. lancet. 2017 sep 16; 390(10100):1211-1259. doi: 10.1016/s0140-6736(17)321542. 3. the antibacterial and antiplaque effectiveness of mouthwashes containing cetylpyridinium chloride with and without alcohol in improving gingival health. williams mi. j clin dent. table 4: experimental group’s feedback on dye containing tooth paste. http://dentistry3000.pitt.edu/ effects of a novel dye containing toothpaste versus regular fluoridated toothpaste for supragingival biofilm reduction in patients with chronic gingivitis: a randomized controlled trial vol 9 no 1 (2021) doi 10.5195/d3000.2021.117 http://dentistry3000.pitt.edu 2011;22(6):179-82. pmid: 22403972. 4. global oral health inequalities: task group--periodontal disease. jin lj, armitage gc, klinge b, lang np, tonetti m, williams rc. adv dent res. 2011 may;23(2):221-6. doi: 10.1177/0022034511402080. pmid: 21490234. 5. prevalence of periodontal disease, its association with systemic diseases and prevention. nazir ma. int j health sci (qassim). 2017 aprjun;11(2):72-80. pmid: 28539867; pmcid: pmc5426403. 6. a global perspective on changes in the burden of caries and periodontitis: implications for dentistry. baelum v, van palenstein helderman w, hugoson a, yee r, fejerskov o. j oral rehabil. 2007 dec;34(12):872-906; discussion 940. doi: 10.1111/j.13652842.2007.01799.x. pmid: 18034671. 7. a short-term clinical study design to investigate the chemical plaque inhibitory properties of mouthrinses when used as adjuncts to toothpastes: applied to chlorhexidine. owens j, addy m, faulkner j, lockwood c, adair r j clin periodontol. 1997 oct;24(10):732-7. doi: 10.1111/j.1600051x.1997.tb00190.x. pmid: 9350557. 8. pannuti cm, saraiva mc, ferraro a, falsi d, cai s, lotufo rf. efficacy of a 0.5% chlorhexidine gel on the control of gingivitis in brazilian mentally handicapped patients. j clin periodontol. 2003 jun;30(6):573-6. doi: 10.1034/j.1600051x.2003.00299.x. pmid: 12795797. 9. the importance of health education in prevention of oral health in children. igić m, apostolović m, kostadinović l, tricković-janjić o, surdilović d. med pregl. 2008 jan-feb;61(12):65-70. serbian. doi: 10.2298/mpns0802065i. pmid: 18798477. 10. evaluation of plaque removal efficacy of a novel dyecontaining toothpaste: a clinical trial. stevens k, belavsky bz, evans ca, viana g, wu cd.int j dentistry oral sci. 03(1), 185189.doi : dx.doi.org/10.19070/2377-80751600039 11. disclosing wafers in an oral hygiene instruction program. bellini ht, anerud a, moustafa mh. odontol revy. 1974;25(3):247-53. pmid: 4140484. 12. the quantification of dental plaque using an image analysis system: reliability and validation. smith rn, brook ah, elcock c. j clin periodontol. 2001 dec;28(12):1158-62. doi: 10.1034/j.1600051x.2001.281211.x. pmid: 11737514. 13. computerized planimetric method for clinical plaque measurement. söder po, jin lj, söder b. scand j dent res. 1993 feb;101(1):21-5. doi: 10.1111/j.16000722.1993.tb01640.x. pmid: 8441891. 14. a study to assess the efficacy of a new detergent free, whitening dentifrice in vivo using qlf planimetric analysis. pretty ia, edgar wm, higham sm.br dent j. 2004 nov 13;197(9):561-6; discussion 551. doi: 10.1038/sj.bdj.4811809. pmid: 15543118. 15. refinement of the modified navy plaque index to increase plaque scoring efficiency in gumline and interproximal tooth areas. rustogi kn, curtis jp, volpe ar, kemp jh, mccool jj, korn lr. j clin dent. 1992;3(suppl c):c9-12. pmid: 1306676. 16. new portable system for dental plaque measurement using a digital single-lens reflex camera and image analysis: study of reliability and validation. rosa gm, elizondo ml. j indian soc periodontol. 2015 mayjun;19(3):279-84. doi: 10.4103/0972-124x.152415. pmid: 26229267; pmcid: pmc4520111. 17. world medical association declaration of helsinki: ethical principles for medical research involving human subjects. world medical association. jama. 2013 nov 27;310(20):2191-4. doi: 10.1001/jama.2013.281053. pmid: 24141714. 18. the gingival index, the plaque index and the retention index systems. löe h. j periodontol. 1967 nov-dec;38(6):suppl:6106. doi: 10.1902/jop.1967.38.6.610. pmid: 5237684. 19. comparison of modified bass technique with normal toothbrushing practices for efficacy in supragingival plaque removal. poyato-ferrera m, segura-egea jj, bullónfernández p. int j dent hyg. 2003 may;1(2):110-4. doi: 10.1034/j.16015037.2003.00018.x. pmid: 16451532. 20. self-reported changes in using fluoride toothpaste among older adults in sweden: an intervention study. acta odontol scand. jensen o, moberg sköld http://dentistry3000.pitt.edu/ effects of a novel dye containing toothpaste versus regular fluoridated toothpaste for supragingival biofilm reduction in patients with chronic gingivitis: a randomized controlled trial vol 9 no 1 (2021) doi 10.5195/d3000.2021.117 http://dentistry3000.pitt.edu u, birkhed d, gabre p. 2015 jan;73(1):48-56. doi: 10.3109/00016357.2014.949847 . epub 2014 sep 3. pmid: 25183252. 21. efficacy of two mouthwashes on 3-day supragingival plaque regrowth: a randomized crossover clinical trial. marchetti e, casalena f, capestro a, tecco s, mattei a, marzo g int j dent hyg. 2017 feb;15(1):73-80. doi: 10.1111/idh.12185. epub 2015 nov 2. pmid: 26522915. 22. oral hygiene measures and promotion: review and considerations. choo a, delac dm, messer lb. aust dent j. 2001 sep;46(3):166-73. doi: 10.1111/j.18347819.2001.tb00277.x. pmid: 11695154. 23. a systematic review of the effectiveness of self-performed mechanical plaque removal in adults with gingivitis using a manual toothbrush. van der weijden ga, hioe kp. j clin periodontol. 2005;32 suppl 6:214-28. doi: 10.1111/j.1600051x.2005.00795.x. pmid: 16128840. 24. plaque-left-behind after brushing: intra-oral reservoir for antibacterial toothpaste ingredients. otten mp, busscher hj, abbas f, van der mei hc, van hoogmoed cg. clin oral investig. 2012 oct;16(5):143542. doi: 10.1007/s00784-0110648-2. epub 2011 dec 13. pmid: 22160537; pmcid: pmc3443356. 25. the effect of oral care feed-back devices on plaque removal and attitudes towards oral care. schäfer f, nicholson ja, gerritsen n, wright rl, gillam dg, hall c. int dent j. 2003 dec;53(6 suppl 1):404-8. doi: 10.1111/j.1875595x.2003.tb00917.x. pmid: 14725386. 26. mechanism of action of a twotone plaque disclosing agent. gallagher ih, fussell sj, cutress tw. j periodontol. 1977 jul;48(7):395-6. doi: 10.1902/jop.1977.48.7.395. pmid: 69704. 27. the role of visual feedback by a disclosing agent in plaque control. tan ae, wade ab. j clin periodontol. 1980 apr;7(2):1408. doi: 10.1111/j.1600051x.1980.tb01957.x. pmid: 6155386. 28. the role of self-administered plaque control in the management of periodontal diseases: i. a review of the evidence. ower p. dent update. 2003 mar;30(2):60-4, 66, 68. doi: 10.12968/denu.2003.30.2.60. pmid: 12705026. 29. detection of dental plaque with disclosing agents in the context of preventive oral hygiene training programs. fasoulas a, pavlidou e, petridis d, mantzorou m, seroglou k, giaginis c. heliyon. 2019 jul 10;5(7):e02064. doi: 10.1016/j.heliyon.2019.e02064. pmid: 31334380; pmcid: pmc6624240. http://dentistry3000.pitt.edu/ microsoft word 77 2017.docx   vol  5,  no  1  (2017)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2017.77         http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     class  iii  malocclusion:  an  argument  for  early  orthodontic  treatment   eric  moe1      1university  of  pi.sburgh,  school  of  dental  medicine abstract   class   iii  malocclusion   is   a   complex  mul0factorial   condi#on  with  many   gene#c   and   environmental   influences.  most   o!en  the  condi*on  is  inherited  in  a  mendelian  autosomal  dominant  pa3ern.  early  referral  and  treatment  can  lead  to   be#er   outcomes   in   orthodon#c   therapy.   the   subject   presented   for   an   early   orthodon#c   referral   at   age   5.5   and   showed  signs  of   future  class   iii  malocclusion.  a   rapid  maxillary  expander  was  given  as   interven$onal   treatment   for   one  year.  at  age  11.5  the  subject  was  treated  with  braces  for  2  years.  the  outcome  was  a  normal  class  i  occlusion.   the  subject’s  mother  had  class  iii  malocclusion  but  was  not  evaluated  early  and  was  only  able  to  establish  an  edge-­‐to-­‐ edge  class  iii  malocclusion  as  the  best  treatment  outcome  without  orthognathic  surgery.  the  subject’s  grandmother   was  also  class  iii,  establishing  an  autosomal  dominance  pa6ern  of  inheritance  in  the  family.  this  case  demonstrates   the  importance  of  the  general  den1st  educa1ng  families  about  malocclusion  and  making  orthodon-c  referrals  as  early   as  possible  so  the  best  treatment  outcomes  can  be  reached  through  orthodon3c  therapy.     cita%on:  moe,  e.  (2017)  class  iii  malocclusion:   an  argument  for  early  orthodon0c  treatment.   den$stry  3000.  1:a001   doi:10.5195/d3000.2017.77   received:    may  22,  2017   accepted:    may  26,  2017   published:    june  19,  2017   copyright:  ©2017  moe,  e.  this  is  an  open  access   ar!cle   licensed   under   a   crea!ve   commons   a!ribu"on  work  4.0  united  states  license.   email:  edm27@pi).edu   introduction   malocclusion  is  the  wide-­‐ ranging  diagnosis  given  when  the   dentition  from  the  two  arches  oc-­‐ clude,  or  come  together,  abnor-­‐ mally.  the  effects  of  malocclusion   can  include  decreased  masticatory   function,  compromised  esthetics,   and  heightened  risk  of  dental   trauma  [1].  depending  on  the  se-­‐ verity  of  the  malocclusion,  the   overall  effect  is  that  quality  of  life   can  be  compromised  [1].  one  of   the  most  common  classification   systems  of  malocclusion  is  angle’s   classification  which  designates   malocclusion  into  three  catego-­‐ ries:  class  i,  ii,  and  iii.  this  case   report  focuses  on  class  iii  maloc-­‐ clusion,  which  is  defined  as  the   mesiobuccal  cusp  of  the  maxillary   first  permanent  molar  occluding   distally  to  the  buccal  groove  of  the   mandibular  first  molar,  instead  of   aligning  with  it.  class  iii  malocclu-­‐ sion  can  result  in  anterior  cross-­‐ bite  and  negative  overjet  or  an   edge-­‐to-­‐edge  incisal  relationship.   it  is  estimated  that  up  to  4%  of   european-­‐americans  and  23%  of   eastern  asians  are  affected  by   class  iii  malocclusion  [2].  the  con-­‐ dition  can  be  diagnosed  from  a   young  age  and  it  often  becomes   more  evident  with  age  and  is  one   of  the  main  reasons  for  orthodon-­‐ tic  treatment  or  orthognathic  sur-­‐ gery  [2].  a  better  understanding  of   the  genetic  and  environmental   etiology  of  this  condition  could   lead  to  earlier  and  more  effective   treatment  options  for  those  af-­‐ fected.   class  iii  malocclusion  is  a   diverse  and  complex  phenotype   that  can  have  several  etiologies.   the  class  iii  phenotype  is  often   described  as  mandibular  progna-­‐ thism,  maxillary  retrognathism,  or   a  combination  of  both.  one  study   analyzing  lateral  cephalometric   variables  further  described  the   class  iii  phenotype  into  five  clini-­‐ cally  meaningful  phenotypes  [3]:   combination  of  mild  maxillary  ret-­‐ rognathism  and  mandibular  prog-­‐ nathism  with  a  flat  mandibular   plane,  combination  of  mild  maxil-­‐ lary  retrognathism  and  mandibular   prognathism  with  a  normal  man-­‐ dibular  plane,  large  mandible  ex-­‐ pressed  vertically,  severe  mandib-­‐ ular  prognathism,  and  severe  max-­‐ illary  retrognathism  [3].  the  ability   to  classify  the  phenotypes  associ-­‐ ated  with  class  iii  malocclusion  is  a   good  step  towards  discovering  the   genetic  etiology  of  each  pheno-­‐ type  [3].  with  the  complexity  of   development  and  the  numerous   ways  class  iii  malocclusion  can  oc-­‐ cur,  it  is  reasonable  that  class  iii   malocclusion  is  considered  a  mul-­‐ tifactorial  condition  with  both  ge-­‐ netic  and  environmental  influ-­‐ ences.    class  iii  malocclusion:  an  argument  for  early  orthodon6c  treatment   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.77    http://dentistry3000.pitt.edu   the  etiology  of  class  iii   malocclusion  is  multifactorial  and   involves  different  genes  and  envi-­‐ ronmental  factors.  different   chromosomal  locations  and  genes   have  been  associated  with  class  iii   malocclusion  in  different  ethnic   populations  [2].  family  studies  al-­‐ so  support  a  polygenic  multifacto-­‐ rial  inheritance  pattern,  as  13%  of   siblings  of  subjects  with  class  iii   malocclusion  also  exhibited  class   iii  malocclusion  [4].  other  studies   analyzing  twins  have  found  that   the  rate  of  mandibular  progna-­‐ thism  in  monozygotic  twins  is  six   times  higher  than  dizygotic  twins,   also  suggesting  polygenic  inher-­‐ itance  and  etiology  [4].  however,   pedigree  analysis  of  class  iii  mal-­‐ occlusion  in  families  has  followed   an  autosomal  dominant  mendeli-­‐ an  pattern  of  inheritance  with  in-­‐ complete  penetrance  in  a  majority   of  analyses  [2].  the  conclusion  of   many  of  these  studies  is  that  class   iii  malocclusion  within  families  is   explained  by  some  dominant  gene   being  inherited  in  an  autosomal   dominant  pattern,  while  likely  be-­‐ ing  modified  by   other  genes  and   environmental   influences  [2].   unfortu-­‐ nately,  while   class  iii  malocclu-­‐ sion  can  often  be   described  as  a   mendelian  auto-­‐ somal  dominant   inherited  condi-­‐ tion,  studies  have   already  located   many  genes  and   chromosomal  regions  that  are  as-­‐ sociated  with  this  condition,  show-­‐ ing  that  its  genetic  etiology  is  still   very  complex  and  not  easily  de-­‐ fined  to  one  or  two  genes  and  not   every  case  analyzed  is  easily  gen-­‐ eralized  to  all  people  of  all  ethnici-­‐ ties  [2].  for  example,  analyses  of   korean  and  japanese  families   showed  mandibular  prognathism   is  associated  with  chromosome   regions  1p36,  6q25,  and  19q13.2   [5].  colombian  families  have   shown  class  iii  malocclusion  is  as-­‐ sociated  with  regions  1p22.1,   3q26.2,  11q22,  12q13.13,  and   12q23  [6].  analyses  of  han  chi-­‐ nese  people  showed  regions   14q24.3-­‐q31.2  influence  mandible   growth  [7].  an  estonian  family  was   described  as  having  the  mutation   p.ser182phe  in  dusp6  leading  to   class  iii  malocclusion  [2].  all  of   these  cases  show  how  class  iii  oc-­‐ clusion  has  a  complex  genetic  eti-­‐ ology  that  is  multifactorial  and   variable.  furthermore,  there  are   environmental  factors  that  may   contribute  to  the  development  of   class  iii  malocclusion.  the  man-­‐ dibular  bone  is  developed  from   condylar  cartilage,  and  mechanical   loading  may  influence  the  re-­‐ sponse  of  the  condylar  cartilage,   leading  to  growth  of  the  mandible   through  various  growth  factors  or   signaling  molecules.  these  factors   include  indian  hedgehog  homolog,   parathyroid-­‐hormone  like  hor-­‐ mone,  insulin-­‐like  growth  factor-­‐1,   and  vascular  endothelial  growth   factor.  it  is  possible  that  genes   coding  for  these  factors  may  ex-­‐ press  the  factors  at  levels  that   have  interactions  with  certain  en-­‐ vironmental  forces  at  play  that   combine  to  predispose  the  devel-­‐ opment  of  a  class  iii  phenotype   [4].   in  summary,  there  is  much   more  that  needs  to  be  learned   about  the  etiology  of  class  iii  mal-­‐ occlusion,  but  current  knowledge   shows  there  are  many  genes  and   environmental  factors  that  can   lead  to  the  condition.  interesting-­‐ ly,  while  it  has  such  a  complex   multifactorial  genetic  and  envi-­‐ ronmental  etiology,  it  more  often   than  not  follows  an  autosomal   dominant  mendelian  inheritance   with  incomplete  penetrance  that   is  likely  influenced  by  other  genes   and  environmental  factors.   because  of  the  complex   and  poorly-­‐understood  etiology  of   class  iii  malocclusion,  treatment   plans  focus  on  fixing  symptoms   instead  of  preventing  etiology  [1].   the  two  main  concepts  to  consid-­‐ er  for  a  treatment  plan  are  its  tim-­‐ ing  and  type  of  appliance  [8].  of-­‐ ten,  early  treatment  can  help  pre-­‐ vent  the  need  for  future  orthog-­‐ nathic  surgery,  so  a  fixed  ortho-­‐ dontic  treatment,  like  braces,  is    class  iii  malocclusion:  an  argument  for  early  orthodon6c  treatment   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.77    http://dentistry3000.pitt.edu   sufficient  instead.  currently,  there   is  lacking  evidence  to  support  the   long  term  benefits  of  early  treat-­‐ ment,  but  short  term  evidence   shows  both  skeletal  and  dental   beneficial  effects  of  early  treat-­‐ ment  in  class  iii  malocclusion  [8].   case  presentation   the  angle’s  class  iii  maloc-­‐ clusion  subject  is  a  caucasian  male   who  first  presented  for  orthodon-­‐ tic  evaluation  at  5.5  years  of  age.   the  subject  presented  with  a   probable  class  iii  growth  pattern   that  may  become  more  obvious  as   growth  continued.  he  had  a  com-­‐ plete  anterior  crossbite  and  nor-­‐ mal  amount  of  anterior  overbite.   he  had  moderate  maxillary  crowd-­‐ ing  and  adequate  mandibular  arch   length.  the  maxillary  arch  was   constricted.  the  profile  of  the  sub-­‐ ject  was  prognathic.  the  subject   received  early  interventional  or-­‐ thodontic  treatment  with  a  bond-­‐ ed  rapid  maxillary  expander  and   reverse  pull  headgear  at  night  to   protract  the  maxilla  for  one  year.   the  subject  was  reevaluat-­‐ ed  after  interventional  treatment.   he  still  showed  a  possible  class  iii   growth  pattern  that  may  become   more  obvious  as  growth  contin-­‐ ues.  however,  the  interventional   treatment  was  successful  because   the  subject  had  normal  overjet,   indicating  the  complete  anterior   crossbite  was  eliminated.  the  sub-­‐ ject  had  adequate  maxillary  and   mandibular  arch  length,  indicating   the  moderate  maxillary  crowding   was  relieved.  the  evaluation  indi-­‐ cated  that  comprehensive  ortho-­‐ dontic  treatment  would  be  need-­‐ ed  in  the  future  and  that  the  sub-­‐ ject  should  be  periodically  reeval-­‐ uated.   at  11.5  years  of  age  the   subject  was  evaluated  to  have  an   edge-­‐to-­‐edge  incisal  relationship.   his  mandibular  midline  was  also   shifted  1-­‐2mm  to  the  right.  the   maxillary  arch  was  constricted   again.  at  this  time,  the  subject  re-­‐ ceived  full  fixed  braces  treatment   to  correct  the  edge-­‐to-­‐edge  incisal   relationship  and  other  findings.   the  expected  treatment  duration   was  24-­‐30  months  and  the  ex-­‐ pected  outcome  was  a  class  i   normal  occlusion.  after  24  months   of  treatment,  the  subject  finished   the  course  of  treatment  with  a   class  i  normal  occlusion  at  13.5   years  of  age.  figure  1  shows  be-­‐ fore  (age  11.5)  and  after  (age  13.5)   pictures  of  the  subject’s  dentition.   family  history  was  gath-­‐ ered  about  the  subject  in  terms  of   class  iii  malocclusion.  the  pedi-­‐ gree  for  all  known  relatives’  occlu-­‐ sion  before  any  orthodontic   treatments  is  shown  in  figure  2.   the  subject’s  mother  and  his   grandmother  on  his  mother’s  side   also  had  class  iii  malocclusion.  no   one  else  in  the  family,  including   his  sister,  father,  and  all  other  rel-­‐ atives  had  class  iii  malocclusion.   a  limited  dental  history   was  obtained  from  the  subject’s   mother  in  regards  to  her  class  iii   malocclusion.  the  mother  had  a   similar  class  iii  malocclusion  to  the   subject,  but  she  did  not  receive   any  orthodontic  evaluation  or  in-­‐ terventional  orthodontic  treat-­‐ ment  until  she  was  13  years  of   age.  her  outlook  at  that  time  was   either  orthognathic  surgery  or  a   probable  best  case  scenario  of  an   edge-­‐to-­‐edge  incisal  relationship   after  braces  treatment.  she  opted   for  braces  and  the  result  was  the   predicted  edge-­‐to-­‐edge  incisal  re-­‐ lationship,  which  was  better  than   the  more  severe  class  iii  malocclu-­‐ sion  at  the  beginning  of  treat-­‐ ment.  no  detailed  history  about   the  grandmother’s  class  iii  maloc-­‐ clusion  or  treatment  could  be  ob-­‐ tained.    class  iii  malocclusion:  an  argument  for  early  orthodon6c  treatment   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.77    http://dentistry3000.pitt.edu   discussion   this  case  presents  a  pedi-­‐ gree  with  a  mendelian  inheritance   pattern  of  autosomal  dominance   as  the  subject,  his  mother,  and  his   grandmother  all  have  class  iii   malocclusion.  while  the  pedigree   available  is  a  small  sample  size,  in   each  case  of  inheritance,  50%  of   the  children  received  the  class  iii   malocclusion  phenotype,  which  is   to  be  expected  with  an  autosomal   dominant  inheritance  pattern.  this   type  of  inheritance  pattern   matches  what  the  literature  re-­‐ ports  for  class  iii  malocclusion  be-­‐ ing  multifactorial  in  nature,  but   often  inherited  in  an  autosomal   dominant  manner.  it  is  likely  that   if  the  subject  has  offspring,  they   will  each  have  a  50%  chance  of   developing  a  class  iii  malocclu-­‐ sion.     this  case  also  demon-­‐ strates  the  importance  of  general   dentists  educating  families  with   young  children  about  malocclusion   early  on  and  making  early  referrals   to  orthodontists  for  evaluation  so   early  interventional  treatment  can   be  provided,  if  needed.  the  sub-­‐ ject  of  this  case  received  an  early   referral  at  the  age  of  5.5  and  re-­‐ ceived  a  rapid  maxillary  expander   for  one  year  before  fixed  appli-­‐ ance  braces  at  11.5  years  of  age   for  2  years  of  treatment.  it  is  likely   that  a  normal  occlusion  was  possi-­‐ ble  due  to  the  interventional   treatment  received  at  a  young   age.  this  course  of  treatment  can   be  compared  to  the  subject’s   mother  who  did  not  receive  any   interventional  treatment  or  evalu-­‐ ation  at  an  early  age.  her  best   non-­‐surgical  treatment  outcome   was  a  class  iii  edge-­‐to-­‐edge  incisal   relationship.  this  shows  that  den-­‐ tists  have  the  ethical  implication   to  be  knowledgeable  and  thor-­‐ ough  in  recognizing  early  maloc-­‐ clusion  and  making  referrals.  this   includes  the  ethical  necessity  of   educating  the  family  about  why   following  up  with  the  orthodontic   referral  is  important.  the  dentist  is   also  in  an  advantageous  position   to  start  educating  the  family  when   the  children  are  born  about  the   possibilities  of  their  child  requiring   orthodontic  treatment  in  the  fu-­‐ ture  so  they  can  begin  to  plan  for   this  treatment  as  early  as  possible.   the  knowledgeable  dentist  will  be   able  to  educate  families  with  par-­‐ ents  that  have  class  iii  malocclu-­‐ sion  that  it  is  likely  that  their  chil-­‐ dren  may  also  have  class  iii  mal-­‐ occlusion  that  requires  treatment.   it  is  also  thorough  to  inform  par-­‐ ents  that  had  normal  occlusion   that  malocclusion,  and  specifically   class  iii  malocclusion,  is  complex   and  can  be  sporadic  as  well,  so  the   parents  know  it  is  possible  that   orthodontic  therapy  may  be  re-­‐ quired  for  their  children  even  if   they  did  not  require  it  themselves.   overall,  this  case  shows  that  early   patient  education  and  early  or-­‐ thodontic  referral  and  treatment   can  help  the  patient  achieve  the   best  possible  outcome,  in  this  case   a  normal  occlusion,  when  present-­‐ ing  with  class  iii  malocclusion.   conclusion     class  iii  malocclusion  has   several  phenotype  subgroups  that   result  from  a  complex  multifacto-­‐ rial  etiology  with  many  genes  and   environmental  factors  influencing   its  development.  in  many  cases,   class  iii  malocclusion  is  inherited   in  a  mendelian  autosomal  domi-­‐ nant  pattern.  early  treatment  of   class  iii  malocclusion  can  aid  in   reaching  a  normal  occlusion,  and   dentists  can  utilize  early  patient   education  and  orthodontic  refer-­‐ rals  to  facilitate  this  early  treat-­‐ ment.  special  emphasis  should  be   given  to  educate  patients  with   parents  who  have  or  had  class  iii   malocclusion,  as  they  are  most   susceptible  to  the  condition.   references   1.  moreno  uribe  lm,  miller  sf.   genetics  of  the  dentofacial  varia-­‐ tion  in  human  malocclusion.  or-­‐ thod  craniofac  res.  2015  apr;18   suppl  1:91-­‐9.  doi:   10.1111/ocr.12083.  pubmed   pmid:  25865537;  pubmed  central   pmcid:  pmc4418210.   2.  nikopensius  t,  saag  m,   jagomägi  t,  annilo  t,  kals  m,  kivis-­‐ tik  pa,  milani  l,  metspalu  a.  a   missense  mutation  in  dusp6  is   associated  with  class  iii  malocclu-­‐ sion.  j  dent  res.  2013   oct;92(10):893-­‐8.  doi:   10.1177/0022034513502790.   epub  2013  aug  21.  erratum  in:  j   dent  res.  2016  jul;95(8):955.   pubmed  pmid:  23965468.   3.  moreno  uribe  lm,  vela  kc,   kummet  c,  dawson  dv,  southard   te.  phenotypic  diversity  in  white   adults  with  moderate  to  severe   class  iii  malocclusion.  am  j  orthod   dentofacial  orthop.  2013   jul;144(1):32-­‐42.  doi:   10.1016/j.ajodo.2013.02.019.   pubmed  pmid:  23810043;  pub-­‐  class  iii  malocclusion:  an  argument  for  early  orthodon6c  treatment   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.77    http://dentistry3000.pitt.edu   med  central  pmcid:   pmc3964133.   4.  xue  f,  wong  rw,  rabie  ab.   genes,  genetics,  and  class  iii  mal-­‐ occlusion.  orthod  craniofac  res.   2010  may;13(2):69-­‐74.  doi:   10.1111/j.16016343.2010.01485.x .  review.  pubmed  pmid:   20477965.   5.  yamaguchi  t,  park  sb,  narita  a,   maki  k,  inoue  i.  genome-­‐wide   linkage  analysis  of  mandibular   prognathism  in  korean  and  japa-­‐ nese  patients.  j  dent  res.  2005   mar;84(3):255-­‐9.  pubmed  pmid:   15723866.   6.  frazier-­‐bowers  s,  rincon-­‐ rodriguez  r,  zhou  j,  alexander  k,   lange  e.  evidence  of  linkage  in  a   hispanic  cohort  with  a  class  iii   dentofacial  phenotype.  j  dent  res.   2009  jan;88(1):56-­‐60.  doi:   10.1177/0022034508327817.   pubmed  pmid:  19131318;  pub-­‐ med  central  pmcid:   pmc2777651.   7.  li  q,  li  x,  zhang  f,  chen  f.  the   identification  of  a  novel  locus  for   mandibular  prognathism  in  the   han  chinese  population.  j  dent   res.  2011  jan;90(1):53-­‐7.  doi:   10.1177/0022034510382546.   epub  2010  nov  1.  pubmed  pmid:   21041550.   8.  woon  sc,  thiruvenkatachari  b.   early  orthodontic  treatment  for   class  iii  malocclusion:  a  systematic   review  and  meta-­‐analysis.  am  j   orthod  dentofacial  orthop.  2017   jan;151(1):28-­‐52.  doi:   10.1016/j.ajodo.2016.07.017.  re-­‐ view.  pubmed  pmid:  28024779.     is youtubetm an accurate source of patient-information for awareness about periodontal diseases? vol 9 no 1 (2021) doi 10.5195/d3000.2021.134 http://dentistry3000.pitt.edu is youtubetm an accurate source of patient-information for awareness about periodontal diseases? berceste güler, büşra özaltun kutahya health sciences university, faculty of dentistry, department of periodontology, kutahya, turkey. abstract objectives: oral hygiene education and patient awareness are crucial for prevention and the sustainability of its treatment because of high prevalence of periodontal diseases. this study aimed to evaluate youtube videos related to periodontal diseases and assess them comprehensively, reliability, and quality for non-professional internet users. methods: youtube search was performed using the three keywords: ‘periodontal disease’, ‘gingival disease,’ and ‘gum disease’. video lengths, duration, numbers of total views, likes, dislikes, comments values were recorded. the interaction index, viewing rates and video power index (vpi) were calculated. comprehensiveness tailor-made index was assessed for content, global quality scale (gqs), and discern scales were used for reliability and quality of videos. results: a total of 210 videos were evaluated, and 79 videos were included in the study. while 69 of the videos are useful videos, 9 of them have misleading video content. vpi values were found 2.88±0.67 for useful videos and 1.78±0.66 for misleading videos. the mean gqs value of the videos has seen as 2.76±0.7. according to the discern score, 41.8% of the videos show poor quality. the number of videos with comprehensiveness value (2) score is 44 and the number of videos (1) score is 35. conclusions: youtube videos may be used as an education source about periodontal disease for nonprofessional users; however, videos need to be improved in terms of content and quality. these and similar publications may be supported for the optimization of videos to be shared on youtube with periodontal disease and oral hygiene education. keywords: youtube; oral hygiene; periodontitis; awareness; information. citation: güler b, et al. (2021) is youtubetm an accurate source of patient-information for awareness about periodontal diseases? dentistry 3000. 1:a001 doi:10.5195/d3000.2021.134 received: january 9, 2021 accepted: march 7, 2021 published: april 1, 2021 copyright: ©2021 güler b, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: berceste43@gmail.com introduction periodontal diseases (pds) affect about 90% of the worldwide population [1] and defined as the inflammatory process affecting the soft and hard tissues around teeth. [2] gingivitis is a reversible inflammation, and periodontitis is an irreversible disease that results in alveolar marginal bone loss and clinical attachment loss. [2] the most crucial etiological cause of gingival inflammation in periodontal diseases is plaque accumulation and biofilm formation. [3] the bacterial biofilm layer is colonized on the root surfaces, and many undesirable consequences can occur, ranging from tooth mobility, loss of function, aesthetic disorders, and tooth loss. [4] due to the systemic low-grade chronic inflammation of periodontitis, it can cause or induce detrimental effects on the occurrence, course, or prognosis of many different diseases. [4] in cases where good oral hygiene and periodontal condition are healthy, general, and oral healthrelated quality of life has also been well reported. [5] thus, it is reported that dentists play an important role in preventing general health problems. [6] one of the most critical issues for the treatment of periodontal disease is that the patient understands the cause of the disease and is conscious and willing about oral hygiene and treatment. a study has shown that regular dental participants had more http://dentistry3000.pitt.edu/ is youtubetm an accurate source of patient-information for awareness about periodontal diseases? vol 9 no 1 (2021) doi 10.5195/d3000.2021.134 http://dentistry3000.pitt.edu information about periodontitis; however, about one-third of regular dental care users never heard of periodontitis, and the other twothirds had better knowledge about periodontal health. [3] in the study conducted in partially edentulous patients, 68.5% of the patients were previously informed about oral care; however, only 17.3% of patients are reported to have information about the gingival disease. [7] a study has conducted that 72% of internet users were reported to seek health information on the internet. [8] however, according to statistical information, 40% of the health content shared on social media is fake news, and 20% of it comes from the same source. [9] however, in a study evaluating the impact of social media on oral health literacy in adolescents, it was reported that youtube and facebook are the most effective social media. [10] youtube is the most widely used video sharing site in the world since 2005, and a significant number of broadcasts are added to the site dynamically every day. [11] different results have been reached in the studies evaluating the content and quality of the publications presented in the dentistry field on youtube. while the quality and content of videos shared on youtube regarding gingival recessions were weak [12], it was revealed that youtube videos related to diabetes and oral health could be educational. [13] there are many studies related to youtube and medical and dental diseases, but according to our information, no studies have been found in the literature on periodontal diseases. this study investigates whether youtube can be used as a quality, educational, and accurate information source in terms of periodontal disease for nonprofessional. materials and methods study design this study was a cross-sectional trial of periodontal disease evaluation on the youtubetm website. the study did not require the approval of an ethics board. in order to evaluate the videos and eliminate the bias, the entire search history on the search engine to be evaluated has been cleared. an account was opened with a new mail address on youtube. in order to evaluate the information accessed by non-professional youtube users about periodontal disease, three keywords were determined: "periodontitis" "gingivitis" and "gum disease." the present study followed the preferred reporting items for systematic reviews and meta-analysis (prisma) guidelines [14] (http://www.prismastatement.org/), illustrating the outcomes of the video searches and selection process have shown in figure 1. the search process of determining keywords on youtubetm was made on 04.21.2020. eighteen thousand nine hundred videos for "periodontitis," 37700 videos for "gingivitis" and 30900 videos for "gum disease" were shared from the keywords related to the study. the first 60 videos for each keyword are included in the study. [12] all of the videos included in the study were recorded on the youtube account opened with a separate mail address. simultaneously, all videos were downloaded and saved in a separate folder on the hard disk. the evaluation of the videos was completed between 05.15.2020 and 05.22.2020. a single researcher evaluated all videos. for the researcher's intraexaminer reliability calibration, each keyword, ten videos out of 70 selected videos were included and evaluated twice with discern scores at three-week intervals. the overall intraexaminer reliability observer agreement calculated as weighted kappa score was 0.98 (range: 0.9520.997). inclusion and exclusion criteria as a criterion for inclusion in the study, it is based on the videos being published for patient educational information with content related to the clinical presentation, aetiological factors, and periodontal disease treatment options. english and audible videos are also included in the study. the criteria for exclusion are as follows: 1) non-english, 2) conference or school lectures, 3) non-relevant videos are defined as videos not communicating any of the abovementioned aspects of periodontal diseases, 3) duplicate http://dentistry3000.pitt.edu/ is youtubetm an accurate source of patient-information for awareness about periodontal diseases? vol 9 no 1 (2021) doi 10.5195/d3000.2021.134 http://dentistry3000.pitt.edu figure 1: prisma flow chart search strategy videos identified using the key word ‘’gum disease’’ n=30900 videos identified using the keyword ‘’gingivitis’’ n=37700 videos identified using the key word ‘’periodontitis’’ n=18900 the 1st 70 videos for each key word (n =210) videos after duplicates removed (n=28) videos screened n =182 videos assessed for eligibility n =182 103 videos were excluded; non-english =36 irrelevant n=41 non-audible n=1 comments are closed n=5 training video n=5 surgical procedure n=11 rejenerative surgical procedure n=1 phase i treatment for periodontitis n =3 videos included in qualiative and quantitative analysis (n =79) (gum disease= 40; gingivitis= 20; periodontitis =19) http://dentistry3000.pitt.edu/ is youtubetm an accurate source of patient-information for awareness about periodontal diseases? vol 9 no 1 (2021) doi 10.5195/d3000.2021.134 http://dentistry3000.pitt.edu videos, 4) about regenerative/respective surgical technique, 5) videos that give information about periodontology in academic language for professionals, 6) videos that closed to comments, 7) non-audible (table 1). table 1. exclusion reasons demographic measurements each term was searched separately to gather the following quantitative information: category (useful, misleading, personal experience), total view, video length, mean duration, view count, number of likes and dislikes, number of comments, video quality degree, duration of the video (min) were recorded. the video's interaction index, viewing rate, and video power index (vpi) were calculated using the following formulas [15,16]: viewing rate (%) = [number of views / number of days since upload] x 100 interaction index (%) = [number of likes − number of dislikes / total number of views]x100. video power index (vpi) = (number of likes / number of likes – number of dislikes) x100. videos meeting including criteria were evaluated, and the above‐ mentioned information was recorded in a microsoft excel© 2016 spreadsheet. video classification all selected videos were as two classified systems: based on the useful, misleading, or as personal experience-based [17] useful: scientifically correct information about the etiology, treatment or prognosis of the disease; (b) misleading: containing scientifically unproven and false information currently available (herbal therapy, alternative medicine); (c) personal experience: during personal periodontal treatment / after periodontal treatment. categorized according to source into 5 groups: (a) independent users; (b) government/news agencies; (c) university channels/professional organizations; (d) health information websites; (e) medical advertisements/for-profit companies. video quality evaluation indexes global quality scale the quality of the information provided by included videos was evaluated called global quality scale (gqs), based on the quality of the video, the availability of information, and the usefulness for the patients. [18] discern scoring system the reliability was assessed using the discern tool (https:// www.discern.org.uk/discern_instrum ent.php), a questionnaire based on a standardized set of criteria for judging the reliability and quality of written health information on treatment choices. discern instrument focusing on the source of information, including references and dates of publication, description of treatments, short-term and long-term benefits of the treatments including risks, and the effects of treatment of choice on the search term reason gum disease gingivitis periodontitis total irrelevant 14 11 16 41 duplicate 0 12 16 28 non-english 2 22 12 36 non-audible 1 0 0 1 closed comments 1 2 2 5 education for professionals 0 2 3 5 surgical procedure 10 1 0 11 regenerative surgical procedure 0 0 1 1 phase i treatment 2 0 1 3 total 30 50 51 131 http://dentistry3000.pitt.edu/ is youtubetm an accurate source of patient-information for awareness about periodontal diseases? vol 9 no 1 (2021) doi 10.5195/d3000.2021.134 http://dentistry3000.pitt.edu quality of life. [19] both gqs and discern surveys are scored on a 5-point likert scale. (5 = good quality criteria, 2–4 = quality criteria partially good, 1 = bad quality criteria). comprehensiveness tailor-made index a tailor-made comprehensiveness index (ci) was used to evaluate the videos based on their content in terms of clinical presentation, aetiological factors, and management options. [12] the following scores were used: (0) when videos did not mention any clinical presentation, aetiological factors or management options; (1) when videos mentioned one clinical presentation and one aetiological factor and did not describe treatment options; (2) for videos that reported at least two clinical presentations, at least two aetiological factors, and at least one treatment information. statistical analyses statistical analysis was carried out on the ibm spss statistics for windows, version 22.0 statistical software (ibm corporation, new york, ny, usa). the cohen j statistical method was used to calculate intra-examiner reliability. the normality of continuous variables was investigated through the use of the kolmogorov–smirnov test, and categorical variables were analyzed using the χ² test. logistic regression analysis was performed to measure the extent to which the independent variable explained the dependent useful videos mean ± sd (min max) misleading videos mean ± sd (min max) patient views mean ± sd (min max) total number of videos (%) 69 (87.3%) 9 (11.3%) 1 (1.2%) 79 total view 95358.7±03743.8 (39-183512) 78259.6±91243.7 (2313 253645) 13590 92375.5±28 5300.5 video length 3.03±2.61 (0.39 13.42) 4.09±4.18 (1.08 14.35) 10.38 3.24±2.92 duration on youtube( month) 60.71 (3 137) 58.56±47.37 (10 154) 58 60.43±41.7 number of likes 282.25±727.45 (0 4945) 384.33±450.81 (0 1395) 58 291.04±695 .7 number of dislikes 26.22±88.95 (0 646) 19.44±24.64 (0 67) 3 25.15±83.5 interactio n index% 0.74±1.12 (-0.69 7.69) 1.20±1.08 (0 3.05) 0.4 0.78±1.11 viewing rate 48.73±137.03 (0 1018) 45.56±61.40 (2 200) 7 47.84±129. 5 number of comment s 38.88±104.73 (0 586) 31.89±27.87 (0 77) 12 37.75±98.2 6 video power index 104.96±36.16 94.85±36.3 105.45 103.81±35. 85 global quality scale 2.88±0.67 1.78±0.66 3 2.76±0.7 table 2. video characteristics http://dentistry3000.pitt.edu/ is youtubetm an accurate source of patient-information for awareness about periodontal diseases? vol 9 no 1 (2021) doi 10.5195/d3000.2021.134 http://dentistry3000.pitt.edu variable. statistical significance was set at p < 0.05. results video characteristic a total of 210 videos were evaluated; however, 79 videos were included in the study to be evaluated for three keywords in total. after the initial evaluation, 131 videos were excluded, and exclusion reasons were shown in table 1. information about whether the videos included in the study are animated or not was recorded. 19 of the 79 videos included are animated videos. while 69 (87.3%) of the included videos were useful videos, 9 (11.3%) were included in the misleading, and one was included in the patient view classification. the view values for useful videos is 95358.68 ± 303743.79, while it is 78259.56 ± 91243.71 for misleading and 13590 for patients' views. while mean length video was 3.03 ± 2.61 min, it was found 4.09 ± 4.18 min in misleading videos, and 10.38 min in patients' view. the mean duration on youtube was 60.71 months for useful videos, 58.56 ± 47.37 months for misleading videos, 58 months for patients' views. mean likes değerleri 282.25 ±727.45 for useful videos, 384.33 ±450.81 for misleading videos, 58 for patients’ views. mean dislike values were founded 26.22 ±88.95 for useful videos, 19.44±24.64 for misleading videos, 3 for patient’s views. mean comment number values were found 38.88±104.73 for useful videos, 31.89 ±27.87 for misleading videos, 12 for patient views. video characteristics are shown in table 2. mean interaction index for useful videos 0.74±1.12%, misleading videos 1.20±1.08%, patients views 0.4%. mean viewing rate 48.73 ±137.03 for useful videos, misleading videos 45.56 ± 61.40, patient views for 7. video power index values were found 2.88 ± 0.67 for useful videos and 1.78 ± 0.66 for misleading videos. (table 2) the videos included in the study; 25 (31.6%) are independent users, 4 (5.1%) are university channel professional organization, 6 (7.6%) are health information websites; 44 (55.7%) were published by medical advertisements/for-profit companies. (figure 2). global quality scale scores when the quality assessments of all periodontal disease-related videos were made with gqs, it was reported that no videos published to date on youtube show excellent quality. while 9 (11.3%) of the included videos received the definition of good quality and generally good flow, that is (4) score, 45 (56.9%) received the score with the definition of moderate quality (3). 22 (27.8%) of the videos got the (2) score with the definition of generally poor quality and flow. eventually, 3 (3.8%) of the videos received a score with the definition of poor quality, poor flow of the video, most information missing, (1). mean gqs was found to be 2.76±0.7 (table 3). score definition number of videos (%) 1 poor quality, poor flow of the video, most information missing, not at all useful for patient 3 (3.8%) 2 generally poor quality and flow, some information listed but many important topics missing, of very limited use to patients 22 (27.8%) 3 moderate quality, suboptimal flow, some important information adequately discussed but others poorly discussed, somewhat useful for patient 45 (56.9%) 4 good quality and generally good flow. most of the relevant information is listed but some topics are not listed. useful for patient 9 (11.3%) 5 excellent quality and flow, very useful for patient 0 (0%) table 3. global quality scale (gqs) http://dentistry3000.pitt.edu/ is youtubetm an accurate source of patient-information for awareness about periodontal diseases? vol 9 no 1 (2021) doi 10.5195/d3000.2021.134 http://dentistry3000.pitt.edu discern scale the average discern scale score was 2.01±1.06. in the questions used for discern scoring, how many answers were given for each likert scale is shown in table 4 in detail. a significant portion of the uploaded videos clearly stated their purpose and provided information on the subject. however, when 90% of the information sources used to create the video and which information sources are used are not explicitly mentioned in the video. it has been reported that 51% of the videos are partially balanced and unbiased. while 45% of the videos do not give information about additional sources of information, 73% do not refer to the uncertain areas. 65% of the videos talk about the treatment options partially or entirely, but 30% do not give the benefits of the treatments. only 13% of videos about future situations in untreated cases provide full information, while 93.7% do not give any information about the effect of treatment options on quality of life. as a result, only 2.5% of the videos can be used as a reliable source in terms of broadcast quality. one reference is provided in only one video. comprehensive tailor-made index content evaluation of the evaluated videos related to periodontal disease was done with the comprehensive tailor-made index. according to this index, approximately 50% of the clinical presentation videos mention gingival bleeding, gingival erythema, gingival edema, and tooth loss. approximately 25% of the videos mention gingival recession and halitosis. (table 5). as aetiological factors, 74.6% of the videos mentioned plaque and biofilm. poor oral hygiene and calculus were mentioned in the video by 31(39.2%) and 28(35.4%). incompatible prosthetic-orthodontic structures, restorations, and fillings etiological factor is mentioned in 5 (6.3%) videos. the secondary effect of parafunctional movements on periodontal tissues is not mentioned in any of the included videos. information about phase i periodontal treatment and modification of brushing techniques, proper oral hygiene education for the treatment of periodontal diseases was mentioned by 33 (41.7%), and 39 (49.4%) videos, respectively. while the number of videos mentioning phase ii surgery periodontal treatment is 11 (13.9%); the number of videos mentioning the elimination of risk factors is 10 (12.65%). while the number of videos providing information about the use of mouthwash is 18 (22.8%), the use of desensitizing agents after periodontal treatment is not mentioned in any video. thirty-five of the videos scored (1) according to this index, while 44 videos scored (2). 3.5. relationship between dependent and independent variables as a result of the regression analysis between discern and video length, gqs, and video length, no statistically significant relationship was shown. (p> 0.05) again, no statistically significant relationship was found in the regression analysis conducted between discern and viewing ratio and gqs and viewing ratio. while analyzing the relationship between vpi and gqs (p> 0.05), no statistically significant relationship was shown (p = 0.729), but a statistically significant independent users 32% university channelprofessional organization 5% health informaton websites 7% medical advertisement s/for-profit companies 56% figure 2. source of uploaded videos. http://dentistry3000.pitt.edu/ is youtubetm an accurate source of patient-information for awareness about periodontal diseases? vol 9 no 1 (2021) doi 10.5195/d3000.2021.134 http://dentistry3000.pitt.edu relationship was provided between vpi and discern (p = 0.036). table 4: discern questions scores frequencies according to likert scale http://dentistry3000.pitt.edu/ is youtubetm an accurate source of patient-information for awareness about periodontal diseases? vol 9 no 1 (2021) doi 10.5195/d3000.2021.134 http://dentistry3000.pitt.edu discussion there are studies evaluating videos on youtube as periodontal disease, gingival recession model, the relationship between diabetes and oral health, parent education about oral hygiene. however, to our knowledge, this study is the first study to evaluate the quality of video content as a periodontal inflammatory disease. a significant part of the videos evaluated as a result of this study provides comprehensiveness values that provide useful information about periodontal disease. however, when the quality and reliability of the videos were evaluated, low values were obtained. [12,13] most studies utilizing youtube as a search engine have used 60-200 videos. [20] the majority of youtube users scan only the first 30 videos. [21] therefore, 70 videos for each keyword were included in our study. in one study, the researchers cleaned the internet search browser and scanned through the incognito tab to prevent robot learning [22]; however, in another study, no information was found to clear the browser history [23]. in one study, 81.9% of the videos do not provide information on where to find oral cancer or more information. [15] a report on the gingival recession reported that 37% of the videos had reliable reference documents according to discern scores. [12] in our study, it is not clear what information sources are used to compile the broadcast in 91.1% of the videos. in a study that questioned whether youtube could be an information source about oral cancer, the interaction index was found to be 0.3±0.68. [15] in a study presented, 30% of all evaluated videos contain misleading information, while 76% of these videos are reported to be of poor quality. [23] in a study evaluating youtube videos about genioplasty, interaction index, like and dislike number is not a suitable source of information for genioplasty, since it gives higher wrong information in videos that fall under the misleading group. [24] longer videos have been reported to contain more misleading information for genioplasty. [24] in our study, the interaction index was higher in misleading videos than useful videos. while the duration of misleading and usefulness videos on youtube was similar, the number of likes was found higher in misleading videos in a study by koller et al. about hip arthritis, only 2-4% of youtube videos were reported to be of good quality. [25] in the studies about videos on youtube about dental implants, it was table 5: comprehensiveness tailor-made index frequencies of evaluated videos. http://dentistry3000.pitt.edu/ is youtubetm an accurate source of patient-information for awareness about periodontal diseases? vol 9 no 1 (2021) doi 10.5195/d3000.2021.134 http://dentistry3000.pitt.edu also emphasized that they are in poor quality and content in terms of education. [23,26,27] when all reliability scores are evaluated, it can be said that the quality content of the videos related to periodontal disease can be very diverse, and the content quality is moderate in this study. less than 10% of videos about burning mouth syndrome are categorized as good/excellent, and lower quality videos have been reported to show higher views, and it has also been reported that the videos uploaded by professionals take longer, but patient views have more views and likes. [28] mean completeness score related to root canal treatment is only 22% complete when evaluated in terms of all etiology, symptoms, procedure, postoperative approach, and progression. [29] in a presented study, it has been reported that the content of youtube videos evaluated for parent oral hygiene education will be useful but contains incomplete information. it has been reported that healthcare professionals can think of uploading more content to youtube as a reason for having more useful content. [30] as a result of this study, a significant portion of the videos evaluated was included in the classification providing useful information about periodontal disease. when comprehensiveness was evaluated, it was found that at least 55.6 percent, at least two etiological factors, at least two clinical findings, and at least one treatment method were mentioned. the videos that he never mentioned about treatment constitute 44.4%. compared to the studies presented concerning periodontal disease, it can be said that in our study, it provided more information in terms of content related to periodontal inflammation. however, low values were found in terms of reliability and quality of the studies. 42.7% of videos about oral cancer were uploaded by healthcare professionals; in educational videos related to oral cancer, most of the videos talk about etiology, risk factors, and early detection, while the percentage of videos talking about management and prognosis is around 11-17%. [15] it has been reported that useful videos about oral cancer have been seen more recently. [15] in the study presented by menziletoglu et al., it was reported that better quality videos take longer. [26] in a study evaluating youtube videos about disc herniation, the discern reliability index and jama index with vpi were found, and no statistically significant difference was found. [16] however, videos uploaded by physicians have been reported to have higher content and quality. [16] however, in our study, a statistically significant relationship was found between discern scores and vpi. it can be interpreted that higher quality and reliable videos can have more power. in our study, 210 videos were included for three different keywords, but 41 of them were excluded because they were irrelevant, 28 were due to duplication, and 36 were non-english. in this study, only the english videos were evaluated. however, evaluating youtube sources that provide information about periodontal diseases related to each country's language can be considered an information source for patients worldwide. there are some limitations to our study. firstly, although the search history has been cleared and a search is made through a new mail account, the geographic region where the search was done may affect the results, in order not to affect the search results on youtube. in our study, only three keywords related to periodontal disease were selected. however, searches based on symptoms such as "gingival bleeding, gingival edema" may give different results. also, as the third limitation, youtube is a dynamic social media platform, and videos can be deleted continuously, and new videos can be added. therefore, the quality and content of the videos may change according to the search time. there are many filters in the youtube filter section, such as 4k, hd, subtitle, creative commons, 3d, and view count. in articles about youtube and dentistry, filters are not generally used, but different results can be obtained after applying them. only english videos were included in our study; however, videos uploaded in other languages with english subtitles were not evaluated. it may be important to include these videos as http://dentistry3000.pitt.edu/ is youtubetm an accurate source of patient-information for awareness about periodontal diseases? vol 9 no 1 (2021) doi 10.5195/d3000.2021.134 http://dentistry3000.pitt.edu well, to increase the number of data analyzed. it has been reported that patients do not tend to share or change the information they have previously obtained from different sources with their medical doctors. [31] in the study have mentioned that the videos uploaded by medical professionals to youtube are reported to be unprofessional in terms of content, reliability, and quality. [16] health professionals were considering that youtube can be an essential source of dental treatment leading today. conclusion the study has shown that youtube videos about the periodontal disease can be illuminating in content but contain incomplete information. the fact that there were no high-quality informative videos in our study suggests that there is a need to create more professional, evidencebased, and higher-quality videos. youtube videos may be used as an education source about periodontal disease for non-professional users; however, videos need to be improved in terms of content and quality. acknowledgments the authors do not have any conflict of interest in the companies whose any concern are included in this article. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. references 1. prevalence of periodontal disease, its association with systemic diseases and prevention. nazir ma. int j health sci (qassim). 2017 aprjun;11(2):72-80. pmid: 28539867. 2. a new classification scheme for periodontal and peri-implant diseases and conditions introduction and key changes from the 1999 classification. caton jg, armitage g, berglundh t, chapple ilc, jepsen s, kornman ks, mealey bl, papapanou pn, sanz m, tonetti ms. j clin periodontol. 2018 jun;45 suppl 20:s1-s8. pmid: 29926489. 3. regular dental attendance and periodontal health knowledge: a cross-sectional survey. varela-centelles p, diziglesias p, estany-gestal a, blanco-hortas a, bugaríngonzález r, seoane-romero jm. oral dis. 2020 mar;26(2):419428. pmid: 31785179. 4. supportive periodontal therapy (spt) for maintaining the dentition in adults treated for periodontitis. manresa c, sanz-miralles ec, twigg j, bravo m. cochrane database syst rev. 2018 jan 1;1(1):cd009376. pmid: 29291254. 5. the benefit of dental care to an elderly population assessed using a sociodental measure of oral handicap. fiske j, gelbier s, watson rm. br dent j. 1990 feb 24;168(4):153-6. pmid: 2310635. 6. oral hygiene and the need for treatment of the dependent institutionalised elderly. montal s, tramini p, triay ja, valcarcel j. gerodontology. 2006 jun;23(2):67-72. pmid: 16677178. 7. evaluation of partially dentate patients' knowledge about caries and periodontal disease. ribeiro dg, jorge jh, varjão fm, pavarina ac, garcia pp. gerodontology. 2012 jun;29(2):e253-8. pmid: 21235624. 8. evaluating asthma websites using the brief discern instrument. banasiak nc, meadows-oliver m. j asthma allergy. 2017 jun 16;10:191-196. pmid: 28670135. 9. the spread of medical fake news in social media–the pilot quantitative study. waszak pm, kasprzycka-waszak w, kubanek a. health policy and technology, 2018 7(2), 115-118. 10. social media in adolescent health literacy education: a pilot study. tse ck, bridges sm, srinivasan dp, cheng bs. jmir res protoc. 2015 mar 9;4(1):e18. pmid: 25757670. 11. social media use by health care professionals and trainees: a scoping review. hamm mp, chisholm a, shulhan j, milne a, scott sd, klassen tp, hartling l. acad med. 2013 sep;88(9):137683. pmid: 23887004. 12. medical reliability of a video-sharing website: the gingival recession model. hamdan aa, shaqman m, abu karaky a, hassona y, bouchard p. eur j dent educ. 2019 may;23(2):175-183. pmid: 30633844. 13. youtube information about diabetes and oral healthcare. pons-fuster e, ruiz roca j, tvarijonaviciute a, lópez-jornet p. odontology. 2020 http://dentistry3000.pitt.edu/ is youtubetm an accurate source of patient-information for awareness about periodontal diseases? vol 9 no 1 (2021) doi 10.5195/d3000.2021.134 http://dentistry3000.pitt.edu jan;108(1):84-90. pmid: 31396751. 14. the prisma statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration. liberati a, altman dg, tetzlaff j, mulrow c, gøtzsche pc, ioannidis jp, clarke m, devereaux pj, kleijnen j, moher d. plos med. 2009 jul 21;6(7):e1000100. pmid: 19621070. 15. youtube as a source of information on mouth (oral) cancer. hassona y, taimeh d, marahleh a, scully c. oral dis. 2016 apr;22(3):202-8. pmid: 26718020. 16. a quality analysis of disc herniation videos on youtube. gokcen hb, gumussuyu g. world neurosurg. 2019 feb 2:s1878-8750(19)30246-3. pmid: 30721774. 17. youtube as a source of information on cardiopulmonary resuscitation. murugiah k, vallakati a, rajput k, sood a, challa nr. resuscitation. 2011 mar;82(3):332-4. pmid: 21185643. 18. youtube as a source of information on covid-19 and rheumatic disease link. kocyigit bf, akaltun ms, sahin ar. clin rheumatol. 2020 jul;39(7):20492054. pmid: 3244760. 19. discern: an instrument for judging the quality of written consumer health information on treatment choices. charnock d, shepperd s, needham g, gann r. j epidemiol community health. 1999 feb;53(2):105-11. pmid: 10396471. 20. is content really king? an objective analysis of the public's response to medical videos on youtube. desai t, shariff a, dhingra v, minhas d, eure m, kats m. plos one. 2013 dec 18;8(12):e82469. pmid: 24367517. 21. youtube: a gauge of public perception and awareness surrounding epilepsy. lo as, esser mj, gordon ke. epilepsy behav. 2010 apr;17(4):541-5. pmid: 20236867. 22. who is providing dental education content via youtube? dias da silva ma, pereira ac, walmsley ad. br dent j. 2019 mar;226(6):437-440. pmid: 30903071. 23. quality of youtube tm videos on dental implants. abukaraky a, hamdan aa, ameera mn, nasief m, hassona y. med oral patol oral cir bucal. 2018 jul 1;23(4):e463-e468. pmid: 29924766. 24. are youtube™ videos a reliable source of information about genioplasty? ayranci f, buyuk sk, kahveci k. j stomatol oral maxillofac surg. 2021 feb;122(1):39-42. pmid: 32360751. 25. youtube provides irrelevant information for the diagnosis and treatment of hip arthritis. koller u, waldstein w, schatz kd, windhager r. int orthop. 2016 oct;40(10):1995-2002. pmid: 27029480. 26. are youtube videos related to dental implant useful for patient education? menziletoglu d, guler ay, isik bk. j stomatol oral maxillofac surg. 2020 dec;121(6):661-664. pmid: 32045688. 27. social media patient testimonials in implant dentistry: information or misinformation? ho a, mcgrath c, mattheos n. clin oral implants res. 2017 jul;28(7):791-800. pmid: 27279455. 28. the usefulness of youtube™ videos as a source of information on burning mouth syndrome. fortuna g, schiavo jh, aria m, mignogna md, klasser gd. j oral rehabil. 2019 jul;46(7):657-665. pmid: 30919986. 29. youtube as a patientinformation source for root canal treatment. nason k, donnelly a, duncan hf. int endod j. 2016 dec;49(12):11941200. pmid: 26551481. 30. youtube™ quality as a source for parent education about the oral hygiene of children. duman c. int j dent hyg. 2020 aug;18(3):261-267. pmid: 32416034. 31. youtube for information on rheumatoid arthritis--a wakeup call? singh ag, singh s, singh pp. j rheumatol. 2012 may;39(5):899-903. pmid: 22467934. http://dentistry3000.pitt.edu/ a study on patient behaviors towards dental visits and oral health during covid-19 outbreak vol 9 no 1 (2022) doi 10.5195/d3000.2022.183 http://dentistry3000.pitt.edu a study on patient behaviors towards dental visits and oral health during covid-19 outbreak marriam mahmood, bds independent researcher, san jose, ca, usa. abstract objectives: investigating the impact of covid-19 pandemic on oral health and patient attitudes towards a dental visit is essential to improving access to oral health care during the pandemic. this calls for studying the chief patient fears, preferences and future intentions related to dental visits during the covid-19 pandemic. methods: a web-based survey was administered to the public within the us in december 2020. the participants (n=502) answered questions about their oral health, trust level for dental offices, and chief concerns and preferences related to dental visits during the covid-19 outbreak. we used descriptive statistics (e.g., chi-square, shapiro-wilk and kruskall-wallis tests) to find if demographics, oral health history or social behaviors were associated with the relevant patient attitudes. results: when queried about covid-19’s impact on oral health, 62.5% respondents reported a minimal impact, 7.2% a positive impact, and 30.3% a negative impact. there were statistically significant differences in responses based on respondents’ ethnicity (n=502, p=.033) and dental visit history during the pandemic (n=502, p=.008). the public trust rating for covid-19-related precautions was more favorable towards the medical offices than the dental offices (n=502, p<.001). the majority considered contracting covid-19 from the other patients in the dental office waiting area (60.4%), from the dentist/hygienist/dental assistants (54.2%), and from the aerosols (50.8%) as their chief concerns. 20.1% respondents preferred no other patient, 25.1% only one more patient, and 31.1% up to three more patients in the waiting area during a dental visit. conclusion: the reported impact of covid-19 on self-perceived oral health was modest for most respondents. patients’ chief concerns for a dental visit include contracting covid-19 infection from other patients, dentists, and aerosols in the operatory. it may be beneficial if dental practices avoid scheduling multiple patients in the waiting area to improve access to oral health care. keywords: covid-19; dentistry; patient comfort; oral health. citation: mahmood, m (2022). a study on patient behaviors towards dental visits and oral health during covid-19 outbreak dentistry 3000. 1:a001 doi:10.5195/d3000.2022.183 received: may, 7, 2021 accepted: september, 28, 2021 published: june, 7, 2022 copyright: ©2022 mahmood, m. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: marriammahmood12@gmail.com introduction coronavirus disease 2019 (covid19) is caused by the severe acute respiratory syndrome coronavirus 2 virus (sars-cov-2) [1]. it spreads through respiratory droplets and aerosols produced by the infected person’s breathing, coughing, speaking and sneezing [2,3]. a person can get infected if these droplets/aerosols settle on the mucous membranes or are inhaled [2–4]. although sars-cov-2 is usually transmitted via close person-person contact, it can also spread through contaminated surfaces [2–4]. in dental settings, sars-cov-2 can potentially be transmitted through aerosols generating dental procedures (agdp) [5–8]. covid-19 has had a profound impact on all aspects of dentistry [9–11]. the american dental association (ada) health policy institute (hpi) has been regularly polling dentists to track the economic impact of covid-19 on dental practices [12]. in the early days of the pandemic (march 2020), most dental practices were closed except for emergency http://dentistry3000.pitt.edu/ a study on patient behaviors towards dental visits and oral health during covid-19 outbreak vol 9 no 1 (2022) doi 10.5195/d3000.2022.183 http://dentistry3000.pitt.edu services. the patient volume has since gradually risen to 81% of the pre-pandemic levels as per the recent hpi poll (february 15, 2021) [13]. the reduced patient volume during the pandemic can be attributed to the fear of contracting covid-19 at the dental office. however, there is no existing detailed study on the specific concerns of the patients regarding dental visits during the pandemic. moreover, the information available on how covid-19 has impacted the oral health is rather limited [14]: an increased prevalence of stressrelated conditions such as bruxism and clenching during the pandemic has been reported [13]. an association between poor oral health and the severity of covid19 symptoms has also been suggested [15]. covid-19 related restrictions are expected to cause a degradation in the oral health of vulnerable populations such as older adults [16,17]. as the existing work is mostly based on information gathered from dental providers, the patients’ perspectives on oral health during covid-19 remain little-known. we aim to address these gaps by providing further insights on these aspects in this study. in this cross-sectional study, we investigate the impact of covid-19 on oral health and dental visits from a patient’s perspective. it has three main objectives. firstly, assess if the ongoing pandemic has affected the patients’ selfperceived oral health. secondly, quantify how much the patients trust the covid-19 related precautionary measures taken by the dental offices versus those by (non-dental) medical offices. thirdly, identify the chief patient fears related to dental visits during the covid-19 pandemic and learn about their intentions for a future visit under different scenarios. we use statistical analysis of the collected data to see if demographics, oral health history or social behaviors impact the relevant patient attitudes. methods we administered a web-based survey hosted on google forms and surveymonkey during dec 3dec 24, 2020. the survey was circulated using e-mail and messaging apps to the public within the us. the ethical review and approval of the study was provided by hml irb (fwa# 1102). the survey was completely anonymous, and the informed consent of the participant was obtained prior to accessing the survey questions. there was no monetary benefit offered to the respondents. the designed survey included 27 closed-ended questions on the following topics: demographics; oral health and the impact of covid-19 pandemic; future intentions and history for dental visits during covid-19 pandemic; trust level for dental offices and key concerns for dental visits amid the covid-19 pandemic; and lifestyle. the questionnaire contained both mandatory and optional questions. the section on demographics contained questions on gender, age group, ethnicity, race, state of residence, and living status (with or without family). respondents were asked to rate their current oral health (excellent, good, fair/poor), caries risk category based on the number of needed dental interventions (low risk: once a year, moderate risk: 2-3 times a year, high risk: 4 or more times a year), and oral hygiene habits (twice daily, once daily, irregular) for brushing and flossing. we then asked how the covid-19 pandemic had affected their oral health and oral hygiene routines (positive impact, little or no impact, negative impact). this was followed by a set of questions related to whether they had visited the dental office during the pandemic, and if they intend to do so in the next 6 months. we asked the respondents about their trust level for the covid-19 http://dentistry3000.pitt.edu/ a study on patient behaviors towards dental visits and oral health during covid-19 outbreak vol 9 no 1 (2022) doi 10.5195/d3000.2022.183 http://dentistry3000.pitt.edu related preventive measures taken by dentists/dental offices and doctors/medical offices using a 5point likert scale (1 = lowest trust level, 5 = highest trust level). we also asked them to identify their greatest concern(s) for a dental visit amid covid-19. options included contracting covid-19 infection from: the dentist/dental assistant/hygienist; front desk staff; patients in the waiting area; physical contact with dental equipment/office surfaces; and aerosols/air droplets in the operatory. we further asked if the number of patients present in the waiting area of a dental office was an important consideration when planning a dental visit. we also queried about the preferred number of patients in the waiting area (1, 2, 3-4, any number of patients). there was also a question about their attitudes towards a dental visit in the postpandemic era (same as in prepandemic times, same as during the pandemic, somewhere inbetween). finally, they were asked about their general lifestyle: whether working from home, outdoor activity level (regular, sometimes, rare) and shopping style (online, curbside, in-store) during the preceding three-month period. we performed the statistical analysis on the collected survey data using ibm spss software. we used the chi-square test and the exact fisher test to check for statistical independence between categorical variables. we used the shapiro-wilk test to check for normality of ordinal data. we used non-parametric tests (mannwhitney u and kruskall-wallis 1way anova) to check for association between ordinal and categorical variables. we used the wilcoxon signed rank test to compare the results of two ordinal variables. we used a p-value of .05 to determine the statistical significance. results demographics we received n=502 completed responses. the completion rate of individual questions varied as not all questions were mandatory. the demographics, social behaviors and self-reported dental history are provided in table 1. we note that the racial demographics resemble that of an urban metropolitan region [18]. oral health during covid-19 outbreak we asked the respondents about the impact of the covid-19 outbreak on their oral health and oral hygiene routine. for oral health, 62.5% of the respondents reported a minimal impact, 7.2% reported a positive impact while 30.3% reported a negative impact (see figure 1). http://dentistry3000.pitt.edu/ a study on patient behaviors towards dental visits and oral health during covid-19 outbreak vol 9 no 1 (2022) doi 10.5195/d3000.2022.183 http://dentistry3000.pitt.edu table 1 demographics, social behaviors, and dental history of respondents. characteristics % (no.) demographics gender (n=502) male 46.6 (234) female 52.5 (266) prefer not to say .004 (2) age group (n=502) 18-24 15.7 (79) 25-34 36.3 (182) 35-44 19.3 (97) 45-54 14.1 (71) 55-64 9.0 (45) 65+ 5.6 (28) ethnicity (n=502) hispanic or latino 12 (60) not hispanic or latino 88 (442) race (n=502) american indian or alaska native 0.4 (2) asian 31.7 (159) black or african american 5.2 (26) native hawaiian or pacific islander 0.8 (4) white 44.8 (225) other 17.1 (86) us census region (n=497) midwest 14.1 (71) northeast 16.1 (81) south 27.9 (140) west 40.8 (205) living status (n=430) single 20.23 (87) with family 79.77 (343) social behaviors nature of work (n=419) working from home 75.18 (315) going to work 24.82 (104) outdoor exercise (n=428) rarely 21.73 (93) sometimes 41.59 (178) regularly 36.68 (157) shopping style (n=427) online 17.09 (73) curb-side pickup 12.18 (52) in-store 70.73 (302) dental history caries risk category (n=423) low risk 39.24 (166) moderate risk 52.72 (223) high risk 8.04 (34) current oral health (n=432) excellent 20.14 (87) good 64.58 (279) fair/poor 15.28 (66) brushing routine (n=431) twice daily 64.73 (279) once daily 31.55 (136) irregular 3.71 (16) flossing routine (n=418) twice daily 11.00 (46) once daily 32.30 (135) irregular 56.70 (237) dental visit history in the 5 years period before covid-19 (n=432) twice a year or more 49.07 (212) once a year 26.16 (113) once every 2 years 10.19 (44) once or twice in 5 years 14.58 (63) http://dentistry3000.pitt.edu/ a study on patient behaviors towards dental visits and oral health during covid-19 outbreak vol 9 no 1 (2022) doi 10.5195/d3000.2022.183 http://dentistry3000.pitt.edu figure 1 impact of covid-19 outbreak on respondents’ oral health and oral hygiene routine. there were statistically significant differences in responses based on ethnicity (n=502, p=.033) due to a considerably higher negative impact reported by the hispanic respondents (see table 2). there were statistically significant differences in the responses depending on the respondent’s current oral health (n=432, p<.001), brushing habits (n=431, p<.001), flossing habits (n=419, p<.001), and whether they had visited a general dentist during the covid-19 pandemic (n=502, p=.008). there was also an association between the caries risk category that the respondents belonged to and the impact of covid-19 pandemic on respondent’s self-perceived oral health (n=423, p=.013). as shown in table 2, the self-reported negative impact was most prevalent among those with poor oral hygiene habits. conversely, those with good oral hygiene habits reported the highest positive impact on their oral health. similarly, the negative impact was disproportionately high among those with poor oral health and/or belonging to “high risk” caries category. finally, a lower negative impact was observed among those who sought routine dental care during the pandemic. when asked about covid-19 outbreak’s impact on oral hygiene routine, 76.16% of the respondents reported a minimal impact, 10.88% reported a positive impact whereas 12.96% reported a negative impact (see figure 1). there were statistically significant differences in the responses depending on the respondent’s gender (n=432, p=.019), current oral health (n=432, p<.001), and oral hygiene habits ((n=432, p<.001) for brushing; (n=419, p<.001) for flossing). the disruption in oral hygiene routines due to the pandemic was observed to be higher among females than males. similarly, the association between oral health and hygiene follows from the earlier explanation provided for covid-19’s impact on oral health. dental visits during covid-19 outbreak trust level: dentists vs. doctors http://dentistry3000.pitt.edu/ a study on patient behaviors towards dental visits and oral health during covid-19 outbreak vol 9 no 1 (2022) doi 10.5195/d3000.2022.183 http://dentistry3000.pitt.edu our intention was to know how much the public trusts the covid19 safety precautions followed by dental practices. we also wanted to learn if the public trust ratings for doctors was any different than that towards dentists. we found that the doctors/medical offices received a more favorable rating than dentists/dental offices (see figure 2): the average score was 3.74 (std. 1.076) for dentists/dental offices and 3.91 (std. 1.03) for doctors/medical offices (n=502, p<.001). table 2 impact of covid-19 outbreak on oral health and oral hygiene routine cross-tabulated with other variables. variables categories covid-19 impact on oral health covid-19 impact on oral hygiene positive no negative p-value, n positive no negative p-value, n % (no.) % (no.) gender male 8.5 (20) 64.1 (150) 27.4 (64) .277, 500 14.0 (29) 76.8 (159) 9.2 (19) .019, 430 female 6.0 (16) 61.2 162) 32.8 (88) 8.0 (18) 75.6 (168) 16.4 (37) ethnicity hispanic 6.7 (4) 48.3 (29) 45.0 (27) .033, 502 13.7 (7) 68.6 (35) 17.6 (9) .399, 432 not hispanic 7.2 (32) 64.5 (285) 28.3 (125) 10.5 (40) 77.2 (294) 12.3 (47) brushing habits irregular 0 (0) 25.0 (4) 75.0 (12) <.001, 431 0 (0) 31.3 (5) 68.8 (11) <.001, 431 once daily 2.9 (4) 68.4 (93) 28.7 (39) 7.4 (10) 77.2 (105) 15.4 (21) twice daily 7.9 (22) 63.1 (176) 29.0 (81) 12.9 (36) 78.5 (219) 8.6 (24) flossing habits irregular 3.4 (8) 64.6 (153) 32.1 (76) <.001, 418 7.2 (17) 74.7 (177) 18.1 (43) <.001, 418 once a day 3.7 (5) 63.7 (86) 32.6 (44) 7.4 (10) 84.4 (114) 8.1 (11) twice a day 23.9 (11) 56.5 (26) 19.6 (9) 32.6 (15) 65.2 (30) 2.2 (1) current oral health fair/poor 4.5 (3) 31.8 (21) 63.6 (42) <.001, 432 7.6 (5) 60.6 (40) 31.8 (21) .005, 432 good 4.7 (13) 67.0 (187) 28.3 (79) 10.4 (29) 78.5 (219) 11.1 (31) excellent 11.5 (10) 75.9 (66) 12.6 (11) 14.9 (13) 80.5 (70) 4.6 (4) caries risk category low risk 4.8 (8) 70.5 (117) 24.7 (41) .013, 423 7.2 (12) 80.7 (134) 12.0 (20) .206, 423 medium risk 6.3 (14) 60.5 (135) 33.2 (74) 12.6 (28) 74.0 (165) 13.5 (30) high risk 11.8 (4) 41.2 (14) 47.1 (16) 17.6 (6) 64.7 (22) 17.6 (6) dental visit during covid-19 no 5 (15) 60.5 (181) 34.4 (103) .008, 502 9.3 (24) 73.7 (191) 17.0 (44) .005, 432 yes 10.3 (21) 65.5 (133) 24.1 (49) 13.3 (23) 79.8 (138) 6.9 (12) http://dentistry3000.pitt.edu/ a study on patient behaviors towards dental visits and oral health during covid-19 outbreak vol 9 no 1 (2022) doi 10.5195/d3000.2022.183 http://dentistry3000.pitt.edu figure 2 trust ratings based on a 5-point likert scale for dental offices/dentists and medical offices/doctors. 1 is the lowest and 5 is the highest trust rating. figure 3 patients’ chief sources of concern about contracting covid-19 during a dental visit. we found a statistically significant difference in the trust level for dentists based on the respondent’s age (n=502, p=.004). moreover, there were also statistically significant differences depending on whether the patient had visited a dental office (n=502, p<.001) during the covid-19 pandemic, as well as the dental visit history prior to the covid-19 pandemic (n=432, p=.009). as shown in table 3, dentists enjoyed a higher trust level among the respondents who had visited their dentists during the pandemic. similarly, the respondents who regularly visited their dentists prior to the pandemic also gave a higher trust rating compared to infrequent dental patients. patient concerns when asked about their greatest concerns regarding visiting the dental offices during the current pandemic, the majority was concerned about contracting covid-19 from the patients in the waiting area (60.4%), the dentists/hygienists/dental assistants (54.2%), and the agdp (50.8%). a relatively smaller proportion regarded dental equipment/office surfaces (43%) and front desk staff (28.5%) as their major concerns (see figure 3). there were statistically significant differences depending on the patient’s trust level for dentists and dental offices (n=432, p=.002). furthermore, as shown in table 4, we also found a significant association with certain demographics. for instance, a significantly higher proportion of female respondents was concerned about contracting covid-19 from agdp than the male respondents. number of patients in the waiting area we wanted to learn if the public was concerned about the patient traffic at a dental office while planning their dental visits. a majority (84.9%) of the respondents agreed that the number of patients present in the waiting area of the dental office was an important consideration even if covid-19 precautions were followed. moreover, there was a statistically significant association between the response to this question and the patient trust level http://dentistry3000.pitt.edu/ a study on patient behaviors towards dental visits and oral health during covid-19 outbreak vol 9 no 1 (2022) doi 10.5195/d3000.2022.183 http://dentistry3000.pitt.edu table 3 average scores for trust level for dentists/dental offices for different categories. table 4 patients’ concerns about contracting covid-19 during a dental visit for different us census regions, age, and gender. (no.) p-value age group (n=502) 18-24 (79) 25-34 (182) 35-44 (97) 45-54 (71) 55-64 (45) 65+ (28) .004 mean 3.49 3.83 3.47 3.97 3.73 4.25 routine dental visit during covid-19 outbreak (n=502) yes (203) no (299) <.001 mean 4.01 3.46 general dentist visits in last 5 years before covid-19 outbreak (n=432) twice a year or more (212) once a year (113) less than once a year (107) .009 mean 3.89 3.64 3.63 greatest concern % (no.) p-value us census region (n=497) midwest (71) northeast (81) south (140) west (205) patients 63.38 (45) 60.49 (49) 55.71 (78) 62.93 (129) ns (.553) dentists 49.29 (35) 41.98 (34) 55 (77) 60.0 (123) .039 aerosols 47.89 (34) 35.80 (29) 48.57 (68) 60.0 (123) .002 equipment 45.07 (32) 35.80 (29) 40.0 (56) 47.8 (98) ns (.235) front desk 30.99 (22) 19.75 (16) 21.43 (30) 35.61 (73) .008 age group (n=502) 18-24 (79) 25-34 (182) 35-44 (97) 45-54 (71) 55-64 (45) 65+ (28) patients 74.7 (59) 61 (111) 58.8 (57) 49.3 (35) 48.9 (22) 67.9 (19) .018 dentists 49.4 (39) 55.5 (101) 60.8 (59) 49.3 (35) 62.2 (28) 35.7 (10) ns (.141) aerosols 48.1 (38) 56 (102) 53.6 (52) 52.1 (37) 31.1 (14) 42.9 (12) ns (.068) equipment 51.9 (41) 46.7 (85) 45.4 (44) 28.2 (20) 40 (18) 28.6 (8) .026 front desk 34.2 (27) 33.5 (61) 29.9 (29) 19.7 (14) 13.3 (6) 21.4 (6) .034 gender (n=502) male (234) female (268) http://dentistry3000.pitt.edu/ a study on patient behaviors towards dental visits and oral health during covid-19 outbreak vol 9 no 1 (2022) doi 10.5195/d3000.2022.183 http://dentistry3000.pitt.edu for the dental offices (n=502, p=.026). we then asked a more specific question about the number of patients in the waiting area that the respondents feel comfortable with during their dental visits. we found that about 20.1% respondents were comfortable with having no other patient, 25.1% with only 1 other patient, 31.1% with 2-3 other patients, and 23.7% with any of these options as long as covid-19 safety precautions (social distancing and face coverings) were followed. there were no statistically significant differences between the responses based on the demographics. however, there were statistically significant differences depending on how much the respondents trust the measures taken by dental offices to prevent the spread of covid-19 (n=502, p<.001). as illustrated in figure 4, we observe that the higher the trust rating, the more indifferent the respondents were to the number of patients in the waiting area provided covid-19 precautions were followed. we also asked the respondents about their attitudes towards a dental visit in a post-pandemic scenario (n=429). 45.2% (no.=194) responded that it will be the same as before the pandemic, 13.5% (no.=58) believed that it will be same as during the pandemic, whereas 41.3% (no.=177) said that it will be somewhere in between the two extremes. this is plausible given the disruptive nature of the social and psychological changes brought about by the pandemic. future visits we also asked the respondents if they intended to visit a dental office in the 6-month period following the survey (i.e., until june 2021). the majority (55.6%, no.=279) answered yes, 29.5% (no.=147) were undecided while 15.1% (no.=76) said no. there were statistically significant differences in the received responses depending on the patients’ trust rating for dentists/dental offices (n=502, p<.001). patients with a higher trust level were more willing to visit a dentist than those with a lower trust level. social behaviors we also asked the respondents about their activities outside the household in the three-month period preceding the survey. there was a statistically significant association between the preferred number of patients in the waiting area and the respondents’ shopping style (n=427, p=0.012). we observed that online/curb-side shoppers preferred a smaller number of patients than in-store shoppers. this is plausible as instore shoppers are likely to be more comfortable in crowded areas (in the context of covid-19) than others. furthermore, in-store shoppers assigned a higher trust rating to dentists/dental offices than others. this led to a statistically significant association between the trust rating for dentists and shopping style (n=427, p=.008). we did not find any other significant association with the social behaviors. discussion in this exploratory study, we have found the self-perceived impact of covid-19 pandemic on oral health patients 56.83 (133) 63.43 (170) ns (.132) dentists 55.12 (129) 53.36 (143) ns (.691) aerosols 44.01 (103) 56.72 (152) .005 equipment 41.45 (97) 44.40 (119) ns (.505) front desk 26.92 (63) 29.85 (80) ns (.468) http://dentistry3000.pitt.edu/ a study on patient behaviors towards dental visits and oral health during covid-19 outbreak vol 9 no 1 (2022) doi 10.5195/d3000.2022.183 http://dentistry3000.pitt.edu figure 4 respondents’ preferences for the number of patients in the dental office waiting area. a stratification based on the respondents’ trust rating for dental offices is also shown. to be modest. our preliminary findings indicate a more adverse impact on the oral health for the hispanic population. however, more specialized studies are needed for vulnerable populations as the covid-19 pandemic may disproportionately affect the oral health care for marginalized populations. furthermore, we found the adverse impact to be higher among those with a poor oral health or a poor oral hygiene routine. we also found that dental offices have a lower trust rating among the public when it comes to covid19 precautionary measures, as compared to medical offices. we further observed that most patients were mainly concerned about contracting covid-19 infection from the dental staff in the operatory, from the other patients in the waiting area and from agdp. recent studies have shown the prevalence of covid-19 among dentists [19] and dental hygienists [20] to be extremely low. the ongoing mass vaccination drives are further expected to boost the patient confidence for dental visits [21]. we found that a significant proportion of the patients prefers to have no other patients in the waiting area of the dental office. it may be beneficial to have the patients wait in their cars instead, a protocol which many dental practices are already implementing. teledentistry, though still in its infancy, may also prove useful in this regard [22,23]. as for agdp, significant research efforts are needed to establish the risk factors and proper guidelines. the onset of the pandemic has already catalyzed the research on agdp [24,25], which will prove useful in the future. to conclude, we have identified the key patient concerns and offered insights into patient attitudes regarding a dental visit during covid-19 pandemic. dental practices may benefit by incorporating patient preferences in their protocols and proactively communicating relevant information to patients, thus easing access to oral health care during the ongoing pandemic. acknowledgements the authors acknowledge hml irb for conducting the ethical review of this study. they also appreciate the valuable feedback provided by talha khan, ph.d. on the statistical analysis in this study. conflicts of interest the authors declare no competing interest. references 1. naming the coronavirus disease (covid-19) and the virus that causes it. world health organ. braz j implantol health sci. 2020 feb;2(3). http://dentistry3000.pitt.edu/ a study on patient behaviors towards dental visits and oral health during covid-19 outbreak vol 9 no 1 (2022) doi 10.5195/d3000.2022.183 http://dentistry3000.pitt.edu 2. the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak. rothan ha, byrareddy sn. j autoimmun. 2020 may;109:102433. doi: 10.1016/j.jaut.2020.102433. epub 2020 feb 26. pmid: 32113704; pmcid: pmc7127067. 3. pathophysiology, transmission, diagnosis, and treatment of coronavirus disease 2019 (covid-19): a review. wiersinga wj, rhodes a, cheng ac, peacock sj, prescott hc. jama. 2020 aug 25;324(8):782-793. doi: 10.1001/jama.2020.12839. pmid: 32648899. 4. the transmission modes and sources of covid-19: a systematic review. rahman hs, aziz ms, hussein rh, othman hh, salih omer sh, khalid es, abdulrahman na, amin k, abdullah r. int. j. surg. open 2020 sep;26:125-136. doi: 10.1016/j.ijso.2020.08.017. 5. dental procedure aerosols and covid-19. epstein jb, chow k, mathias r. lancet infect dis. 2021 apr;21(4):e73. doi: 10.1016/s1473-3099(20)306368. epub 2020 aug 10. pmid: 32791041; pmcid: pmc7417139. 6. the debate: what are aerosol-generating procedures in dentistry? a rapid review. virdi mk, durman k, deacon s. jdr clin trans res. 2021 apr;6(2):115-127. doi: 10.1177/2380084421989946. epub 2021 jan 29. pmid: 33509030; pmcid: pmc7961632. 7. possible aerosol transmission of covid-19 and special precautions in dentistry. ge zy, yang lm, xia jj, fu xh, zhang yz. j zhejiang univ sci b. 2020 may;21(5):361-368. doi: 10.1631/jzus.b2010010. epub 2020 mar 16. pmid: 32425001; pmcid: pmc7089481. 8. cdc updates guidance on covid-19 transmission in dental settings: concerns about aerosols, splashing. evans g. hosp. infect. control prev. 2020;47. 9. editorial: how will covid19 change dentistry? eckert s. int. j. oral maxillofac. implants. 2020 may;35(3). doi:10.11607/jomi.2020.3.e. 10. impact on dental economics and dental healthcare utilization in covid-19: an exploratory study. patel n. j. adv. oral res. 2020 nov;11(2):128136. doi:10.1177/2320206820941365. 11. impact of covid-19 on dental education in the united states. iyer p, aziz k, ojcius dm. j dent educ. 2020 jun;84(6):718722. doi: 10.1002/jdd.12163. epub 2020 apr 27. pmid: 32342516. 12. covid-19 economic impact on dental practices. health policy institute, a.d.a. available online: https://www.ada.org/en/science -research/health-policyinstitute/covid-19-dentistseconomic-impact (accessed on mar 27, 2021). 13. adanews hpi poll: dentists see increased prevalence of stress-related oral health conditions. available online: https://www.ada.org/en/publica tions/ada-news/2021archive/march/hpi-poll-dentistssee-increased-prevalence-ofstress-related-oral-healthconditions (accessed on mar 27, 2021). 14. the impact of coronavirus infectious disease 19 (covid-19) on oral health. dziedzic a, wojtyczka r. oral dis. 2021 apr;27 suppl 3:703-706. doi: 10.1111/odi.13359. epub 2020 may 6. pmid: 32304276; pmcid: pmc7264805. 15. is there an association between oral health and severity of covid-19 complications? botros n, iyer p, ojcius dm. biomed j. 2020 aug;43(4):325327. doi: 10.1016/j.bj.2020.05.016. epub 2020 may 29. pmid: 32713780; pmcid: pmc7258848. 16. covid-19 pandemics and oral health care for older adults. marchini l, ettinger rl. spec care dentist. 2020 may;40(3):329-331. doi: 10.1111/scd.12465. epub 2020 may 11. pmid: 32391586; pmcid: pmc7272993. http://dentistry3000.pitt.edu/ a study on patient behaviors towards dental visits and oral health during covid-19 outbreak vol 9 no 1 (2022) doi 10.5195/d3000.2022.183 http://dentistry3000.pitt.edu 17. the challenges of dental care provision in patients with learning disabilities and special requirements during covid-19 pandemic. picciani bls, bausen ag, michalski dos santos b, marinho ma, faria mb, bastos lf, dziedzic a. spec care dentist. 2020 sep; 40(5):525-527. doi: 10.1111/scd.12494. epub 2020 jul 2. pmid: 32614970; pmcid: pmc7361526. 18. what unites and divides urban, suburban and rural communities. parker k, horowitz j, brown a, fry r, cohn d, igielnik r. pew res. cent. 2018. 19. estimating covid-19 prevalence and infection control practices among us dentists. estrich cg, mikkelsen m, morrissey r, geisinger ml, ioannidou e, vujicic m, araujo mwb. j am dent assoc. 2020 nov;151(11):815-824. doi: 10.1016/j.adaj.2020.09.005. pmid: 33071007; pmcid: pmc7560385. 20. covid-19 prevalence and related practices among dental hygienists in the united states. estrich cg, gurenlian jr, battrell a, bessner sk, lynch a, mikkelsen m, morrissey r, araujo mwb, vujicic m. j dent hyg. 2021 feb;95(1):6-16. pmid: 33627448. 21. covid-19 vaccines and dentistry. samaranayake l, fakhruddin ks. dent. update 2021 jan;48(1):76:81. doi:10.12968/denu.2021.48.1.76 . 22. teledentistry from a patient perspective during the coronavirus pandemic. rahman n, nathwani s, kandiah t. br dent j. 2020 aug 14:1–4. doi: 10.1038/s41415-020-1919-6. epub ahead of print. pmid: 32801323; pmcid: pmc7427495. 23. can teledentistry improve the monitoring of patients during the covid-19 dissemination? giudice a, barone s, muraca d, averta f, diodati f, antonelli a, fortunato l. a descriptive pilot study. int j environ res public health. 2020 may 13;17(10):3399. doi: 10.3390/ijerph17103399. pmid: 32414126; pmcid: pmc7277372. 24. is there an upside to covid19 for dentistry? klemmedson d. j am dent assoc. 2020 oct;151(10):713-715. doi: 10.1016/j.adaj.2020.08.005. epub 2020 aug 14. pmid: 32979943; pmcid: pmc7427589. 25. can extraoral suction units minimize droplet spatter during a simulated dental procedure? chavis se, hines se, dyalram d, wilken nc, dalby rn. j am dent assoc. 2021 feb;152(2):157-165. doi: 10.1016/j.adaj.2020.10.010. pmid: 33494869; pmcid: pmc7826119. http://dentistry3000.pitt.edu/   vol 1, no 1 (2013)  issn 2167‐8677 (online)  doi 10.5195/d3000.2013.9     http://dentistry3000.pitt.edu this work is licensed under a creative commons attribution 3.0 united states license.  this site is published by the university library system, university of pittsburgh as part of its d‐scribe digital publishing program and is cosponsored  by the university of pittsburgh press.  lack of association between irf6 polymorphisms and nonsyndromic oral clefts in south indian population venkatesh babu gurramkonda1, jyotsna murthy2, altaf hussain syed2, and bhaskar vks lakkakula1   1 department of biomedical sciences, sri ramachandra university, chennai, india.  2 department of plastic surgery, sri ramachandra university, chennai, india.  abstract objec ve:  this present  study  is  aimed  to  inves gate  the  associa on between  interferon  regulatory factor 6 (irf6), single nucleo de polymorphisms (snps), and nonsyndromic cle   lip  without  without  cle   palate  (nsclp)  in  the  south  indian  popula on.   subject and methods: for this study, 190 unrelated nsclp pa ents and 189 controls with‐ out cle s were genotyped with rs2235371 (v2741) and rs642961 snps using pcr‐rflp. the  associa ons between nsclp groups and  irf6 gene polymorphisms, as well as haplotypes,  were analyzed using chi‐squared test and 95% confidence interval (95%ci) of the odds ra ‐ os were calculated with the control groups as reference.    results: for controls,  the minor allele  frequencies of both variants, v2741 and  rs642961,  were 7.1%  and 21.1%,  respec vely. genotype data  for both  variants  in  control  and  cle   groups follow the hardy weinberg equilibrium. between cases with nsclp and controls, the  two snps  showed no differences  in  frequencies of  the genotypes or alleles. the pairwise  linkage  disequilibrium  (ld)  values  (d’=1  and  r2=0.027)  between v2741  and  rs642961  re‐ vealed that these two snps are not  in strong ld. haplotype g‐t showed a significantly re‐ duced  risk  for  oral  cle s  (p<0.001)  and  haplotype  a‐t  increased  the  risk  for  oral  cle s  (p=0.043). gene‐gene  interac on showed that the higher risk group contains more gg‐cc  combina on of cases that the controls, but this model was not significantly associated with  cle  status (p=0.136)  conclusion: in conclusion, while irf6 is strongly associated in other popula ons, this study  demonstrated that variants  in irf6 may play a role  in nsclp  in a south indian popula on,  but other genes are expected to play a role in this popula on as well.   cita on: gurramkonda vb, murthy j, syed ah, and  lakkakula bvks (2013) evidence of associa on between  irf6 polymorphisms and nonsyndromic oral cle s in  south indian popula on. den stry 3000 1:a001  doi:10.5195/d3000.2013.9  received: march 25, 2013  accepted: june 20, 2013  published: august 1, 2013  copyright: ©2013 gurramkonda et al. this is an open‐ access ar cle licensed under a crea ve commons  a ribu on 3.0 united states license.  email: lvksbhaskar@gmail.com introduc on cleft lip with or without cleft palate is an extremely complex orofacial birth defect and is found to be more common in asian and asian‐american populations and less common in africans and african‐americans. roughly 70% of clp cases are nonsyndrom‐ ic, occurring as an isolated condition, while the remaining 30% of clp cases are present in association with syndromes [1]. converg‐ ing lines of evidence suggest that the non‐ syndromic cleft lip with or without cleft palate (nsclp) involves interplay of both genetic and environmental factors. nsclp most often occurs as an isolated de‐ fect in families with no history of clefts. nsclp gene identification is difficult be‐ cause of varying levels of penetrance, sex differences, and environmental overlays that increase etiological heterogeneity [2]. despite having a substantial genet‐ ic component, only a fraction of all predis‐ posing genes have been convincingly con‐ firmed as playing role in nsclp. many genes associated with syndromic cases of clp have been identified to contribute to the incidence of nsclp [3]. this approach led to the identification of several genes that con‐ tribute to the isolated clefting [4‐9]. van der woude syndrome (vws; omim 119300) is the most common autosomal dominant clefting syndrome and is distinguished by the presence of highly characteristic pitting of lower lip mucosa and clp [10]. the vws locus was initially mapped to hu‐ man chromosome 1q32–q41 region that harbors interferon regulatory factor 6 (irf6) gene [11]. numerous mutations in the irf6 were reported to cause vws [11‐ 14]. the irf6 gene encodes a transcription fac‐ tor characterized by a highly conserved dna‐binding domain in addition to a less well‐conserved protein interaction domain [15]. several polymorphisms in the irf6 gene have been studied to check their asso‐ ciation with cleft lip and palate, but the re‐ sults are inconclusive. genome‐wide and candidate gene studies and a subse‐ quent meta‐analysis have identified irf6 as a plausible gene contributing to cleft lip and palate in different ethnicities [16‐18]. the present study is aimed to investigate the association between irf6 single nucleotide polymorphisms and nsclp in a south indi‐ an population. we chose two markers:   lack of associa on between irf6 polymorphisms and nonsyndromic oral cle s in south indian popula on  vol 1, no 1 (2013)    doi 10.5195/d3000.2013.9  http://dentistry3000.pitt.edu 2 rs2235371 (v274i), a non‐synonymous snp, and rs642961, located in the newly identified irf6 enhancer region. in addition to this, hapmap data on these polymor‐ phisms in gih samples (gujarati ‐ a west indian population) showed that these snps are polymorphic and located in two differ‐ ent linkage disequilibrium (ld) blocks. since the present study population is near‐ est to gih, it is likely that these two poly‐ morphisms are the best fit for the study undertaken. materials and methods subjects: this study included a total of 379 individu‐ als of south indian origin. one hundred ninety unrelated nsclp patients who were admitted for palatoplasty and lip repair were ascertained at sri ramachandra cleft and craniofacial centre, sri ramachandra university, chennai, india. to determine their individual phenotype status, all of the cases were examined by two clinicians. oral clefts with other congenital malfor‐ mations or major developmental disorders were excluded from the study. all of the cases are isolated, nonsyndromic oral clefts and were classified into two groups: cleft lip with or without cleft palate (clp) and cleft palate only (cpo). all patients with oral clefts were screened for history, consan‐ guinity, affected members in family and relatives, gestational history, drug intake, smoking, alcohol consumption, etc. we re‐ cruited 189 age‐ and sex‐matched normal children without family history of birth de‐ fects and considered this as the control group. power and sample size calculation software (version 2.1.31) was used to calcu‐ late the sample sizes in the study. we used an uncorrected chi‐squared statistic to evaluate this null hypothesis. based on power analysis, a study with 190 case pa‐ tients and 190 controls was large enough to detect a significant odds ratio (or) of 0.5 with a power of 85.8% and an alpha of 5% and a minor allele frequency difference of 0.15 between cases and controls. the study was approved by the institutional ethics committee of sri ramachandra university, chennai, india. as many of the subjects are under 15 years of age, consent was request‐ ed from their parents. three milliliters of blood sample was collected from all the participants after obtaining the informed consent. genotyping: genomic dna was extracted from the sam‐ ples by phenol chloroform extraction and ethanol precipitation protocol [19]. the v274i and rs642961 snps of the irf6 gene were amplified by polymerase chain‐ reaction (pcr) with primers published elsewhere [20]. genotyping for the two snps was carried out by restriction diges‐ tion of the pcr products with mbol and bstni restriction enzymes, respectively. for the v274i polymorphism, mbol digests the g allele in five fragments [322, 177, 80, 35, and 33 base pairs (bp)], whereas the a allele adds another restriction site, allowing the 322 bp fragment to be digested into two smaller pieces of 235 and 87 bp. digestion of the rs642961 pcr products with bstni results in three fragments (213, 33, and 30 bp) for the c allele and two fragments (246 and 30 bp) for the t allele [20]. statistical analysis: allele frequencies were estimated based on the gene count method. the genotypic fre‐ quencies for v274i and rs642961 were evaluated for hardy‐weinberg equilibrium by using a monte carlo permutation test implemented in the hwsim program [21]. all frequencies were in agreement with hardy‐weinberg equilibrium. the associa‐ tions between nsclp groups and irf6 gene polymorphisms, as well as haplotypes, were analyzed using chi‐squared test, and 95% confidence interval (95% ci) for the odds ratios were calculated with the control group as reference. linkage disequilibrium values of d′ and r2 were estimated using haploview 3.12 [22]. haplotypes were con‐ structed using arlequin program [23]. multifactor dimensionality reduction (mdr) 2.0.beta.8.4 software was used to detect the gene‐gene interactions[24]. results genotyping by rflp and electrophoresis on the dna samples of all cleft and control individuals was performed. hardy‐ weinberg expectations were fulfilled in controls for both v274i (p=0.964) and rs642961 (p=0.260) snps. for v274i, the distribution of mutant allele and genotypes was not significantly different between nsclp groups and controls (table 1). mu‐ tant allele (ile) was less in controls (7.1%) than the cleft groups (clp 9.9% and cpo 11.7%) and the difference is not statistically significant for clp and cpo with ors of 1.41 (ci, 0.80‐2.50; p=0.208) and 1.73 (ci, 0.60‐ 4.33; p=0.219), respectively (table 2). comparison of individual genotype fre‐ quencies between nsclp groups and con‐ trols did not reveal association between the v274i genotype and type of cleft (table 1). although, in the present study the mutant genotypes (ga+aa) increased the risk in both nsclp groups, the increase in risk is not statistically significant (table 1). simi‐ larly, the enhancer polymorphism (rs642961) also failed to demonstrate sig‐ nificant differences in genotype frequencies between controls and nsclp groups (table 1). the pairwise ld values (d′=1 and r2=0.027) between v274i and rs642961 also revealed that these two snps are not in strong ld. the haplotypes, constructed by using two polymorphic snps, are provided in table 3. the g‐t haplotype was the sec‐ ond major haplotype in both cases (10.0%) and controls (20.0%) and showed a signifi‐ cantly reduced risk for oral clefts (p<0.001). whereas, the rare haplotype a‐t, formed of two minor alleles of v274i and rs642961 polymorphisms, increased the risk for oral clefts (p=0.043). using the mdr analysis, the best mdr models for the studied snp snp. id  group  gg (%)  ga (%)  aa (%)  p value  or (95% ci) gg vs. ga  or (95% ci)  gg vs. aa  or (95% ci) gg vs. (ga+aa)  v274i  control  164(86.32)  25(13.16)  1(0.53) clp  129(81.24)  29(8.24)  1(0.63) 0.419 1.47(0.82‐2.64) 1.27(0.07‐20.52)  1.47(0.82‐2.60) cpo  23(76.67)  7(23.33)  0(0.00) 0.318 1.99(0.77‐5.13) ‐  1.73(0.60‐4.33) total  152(80.42)  36(19.5)  1(0.53) 0.295 1.55(0.86‐2.81) 1.08(0.01‐85.2)  1.4(0.86‐3.75)   group  cc (%)  ct (%)  tt (%)  p value  or (95% ci) cc vs. ct  or (95% ci)  cc vs. tt  or (95% ci) cc vs. (ct+tt)  rs642961  control  121(63.68)  58(30.53)  11(5.79) clp  90(56.60)  55(34.59)  14(8.81) 0.323 1.27(0.80‐2.01) 1.7(0.74‐3.94)  1.34(0.87‐2.06) cpo  22(73.33)  8(26.57)  0(0.00) 0.326 0.76(0.31‐1.80) ‐  0.64(0.26‐1.50) total  112(59.26)  63(34.59)  14(8.81) 0.633 1.17(0.74‐1.87) 1.38(0.56‐3.41)  1.21(0.78‐1.86) table 1: results of association tests with irf6 gene polymorphisms in case and control groups.   lack of associa on between irf6 polymorphisms and nonsyndromic oral cle s in south indian popula on  vol 1, no 1 (2013)    doi 10.5195/d3000.2013.9  http://dentistry3000.pitt.edu 3 combination is shown in figure 1. the two‐ snp model containing the irf6 v274i and rs642961 markers had a testing accuracy (ta) of 0.541 and cross‐validation con‐ sistency (cvc) of 10/10. however, this model was not significantly associated with cleft status (p=0.136); the higher risk group contains more gg‐cc combination of cases than the controls (figure 1). discussion interferon regulatory factor 6 (irf6) is a member of the irf family of transcription factors that share a highly conserved helix– turn–helix dna‐binding domain and a less conserved protein‐binding domain. the function of irf6 is still unknown because it is not linked to the regulatory pathways or functions associated with other irf family members. however, amino acid sequence alignment analysis demonstrated that the irf6 is exhibiting 89% similarity with irf5, which plays a major role in interferon acti‐ vation and tumor suppression [25]. although a role for irf6 during embryonic development has been identified, its func‐ tion and regulation remain unknown [26, 27].however, recent studies show that irf6 is regulated primarily through the enhancer where rs642961 resides by p63 and ap2‐ alpha [28]. under normal conditions, the vertical pala‐ tal shelves elevate above the tongue, grow horizontally towards each other, and come in contact at the medial edge epithelium (mee) region along the facial midline. sub‐ sequently, the thin medial edge epithelial lining is eliminated and the surrounding mesenchyme migrates inward and fuses the palatal cleft. during this process, irf6 ex‐ pression in the mee rapidly increases, lead‐ ing to dramatic morphological and cell spec‐ ification changes, allowing palatal fusion [12]. a recent study demonstrated that irf6 knockout mice failed to express tgfa in palatal tissues [29]. furthermore, irf6 ex‐ pression in mme is mediated by tgfβ sig‐ naling [30]. the functional polymorphism rs2235371(820g>a) replaces a valine with an isoleucine at amino acid position 274 (v274i) of the smir‐binding domain of irf6. another variant, rs642961 (g>a), is located 10 kilobases upstream of the tran‐ scription start site of irf6. this disrupts the binding site of the transcription factor ap‐ 2α, which plays role in craniofacial devel‐ opment [31]. analysis of two important irf6 gene poly‐ morphisms in 190 nsclp patients and 189 controls of south indian origin has not sup‐ ported the association at neither genotype nor allele level with nsclp. haplotype anal‐ ysis, however, provided indication that irf6 contributes to nsclp in the studied popula‐ tion. over‐transmission of 274v allele in clp subjects has been identified as a risk factor in asians and south americans, but it is not as strong in european populations [16]. a replication study that was conducted using four irf6 snps that have high hetero zygosity showed evidence for altered transmission of three of the four snps in italians with clp [32]. this positive associa‐ tion between nsclp and irf6 was con‐ firmed in american populations [33], and belgian populations [34]. comparison of clp family members and controls revealed that the gg genotype increased the risk of clp in thai populations [35]. the v274i polymorphism was significantly associated with nsclp in different populations, such as south america [36], chile [37], west china [38], honduras [39], norway [40] and span‐ ish honduras [39]. conversely, positive association was not reported in a german population where the frequency of 274v allele is 99.4% [41]. although tdt haplo‐ type analysis showed significant association between nsclp and irf6 haplotypes, v274i is not associated with nsclp in chileans and chinese, where the v274 allele frequen‐ cy is 74% and 75%, respectively [37, 42]. in an indian population, the v274i alone con‐ tributed to minor risk, but the risk is in‐ creased when the v274 allele is present in homozygous condition in combination with mthfr 677ct [43]. based on the published sources, the v274i polymorphism showed wide variations in the minor allele frequencies (274i allele); africans 0%, europeans 0% to 10% [16], hispanic and non‐hispanic populations 7% and 22%, respectively [44]. however, east asians and southeast asian populations reported the highest frequencies: 34% and 42%, respectively [16]. hapmap data also showed wide variations in the v274i minor allele frequency in world populations with highest frequency in chd (42.1 %), chb (41.1 %), jpt (40.6 %) and mex (16.7 %) populations, whereas lowest frequency was observed in yri (0.7 %), lwk (0.5 %), asw (1.8 %), tsi (0.5 %) and ceu (3.2 %) popu‐ lations. gujarati indian population (gih; 8.6 %) exhibited fairly lesser frequencies than the east asian populations. this high fre‐ quency of the 274v allele in indian and eu‐ ropean populations yielded a nonsignificant trend of positive association with cleft lip and palate [16]. the rs642961 snp showed slight variations in european populations, ranging from 24% to 25% [31], and in hispanic and non‐       v274i        group  g (%)  a (%)  p value  or (95% ci) control  353(92.8)  27(7.10)  reference clp  287(90.25)  31(9.74)  0.208  1.41(0.80‐2.50) cpo  53(88.3)  7(11.66)  0.219  1.73(0.60‐4.33) total  340(89.94)  38(10.05)  0.17  1.46(0.85‐2.52)       rs642961        c (%)  t (%)  p value  or (95% ci) control  300(78.94)  80(21.05)  reference clp  235(73.89)  83(30.81)  0.116  1.32(0.92‐1.91) cpo  52(86.66)  8(0.13)  0.164  1.58(0.24‐1.32) total  287(75.92)  91(25.07)  0.319  1.190(0.83‐1.70) haplotype control (%) oral clefts (%)  or(95%ci) p‐value g‐c 277 (72.9) 296 (78.3)  reference g‐t 76 (20.0) 38 (10.0)  0.47(0.31 ‐ 0.71) <0.001 a‐c 23 (6.1) 31 (8.3)  1.26(0.72 ‐2.22) 0.419 a‐t 4 (1.1) 13 (3.3)  3.04(0.98 ‐ 9.44) 0.043 table 2: results of allelic association tests  for  irf6 gene polymorphisms  in case  and control groups.  figure 1: graphical representation of inter‐ action analysis between v274i and rs642961 in nsclp by mdr. value within each cell is combined genotypes and color‐coding represents degree of risk. dark grey is high‐risk, light grey is low risk, empty cell is not a possible combination. table 3: associa on between irf6 haplotypes and nsclp.    lack of associa on between irf6 polymorphisms and nonsyndromic oral cle s in south indian popula on  vol 1, no 1 (2013)    doi 10.5195/d3000.2013.9  http://dentistry3000.pitt.edu 4 hispanic populations 22% and 25%, respec‐ tively [44]. however, in han chinese popu‐ lations, it was reported as low as 15% [45]. the irf6 enhancer polymorphism (rs642961) exhibited a dose‐dependent effect of a allele with cleft lip alone, but not cleft palate alone [31, 46]. a highly signifi‐ cant association between rs642961 and nsclp was observed in central europe [47, 48], poland [49], and china populations [42, 50, 51]. in contrast to this, negative associa‐ tion was found in brazilian [20, 52], spanish honduras [39], swedish, and finnish nsclp families [53]. in conclusion, while irf6 is strongly associ‐ ated in other populations, this study demonstrated that variants in irf6 may play a role in nsclp in a south indian popu‐ lation, but it is expected that other genes may play a role in this population as well. conflict of interest: there are no conflicts of interests. acknowledgements: the authors acknowledge funding from the indian coun‐ cil of medical research (icmr), government of india (project ref. no. 56/15/2007‐bms). references 1. current  concepts  in  genetics  of  nonsyndromic  clefts; murthy  j, bhaskar l;  indian  j plast surg.  2009;42(1):68‐81.  epub  date:  2009/11/03.  pimd:19881024.  2. face  facts:  genes,  environment,  and  clefts;  murray  jc;  am  j  hum  genet.  1995;57(2):227‐ 32.epub date:1995/08/01. pimd:7668246.  3. genetics  of  cleft  lip  and  palate:  syndromic  genes  contribute  to  the  incidence  of  non‐ syndromic  clefts;  stanier  p,  moore  ge;  hum  mol  genet.  2004;13  spec  no  1:r73‐81.epub  date:2004/01/15.pimd:14722155.  4. splitting p63; van bokhoven h, brunner hg; am  j  hum  genet.  2002;71(1):1‐13.epub  date:2002/05/31.pimd:12037717.  5. craniofacial  expression  of  human  and murine  tbx22 correlates with the cleft palate and anky‐ loglossia  phenotype  observed  in  cpx  patients;  braybrook c, lisgo s, doudney k, henderson d,  marcano ac, strachan t, et al.; hum mol genet.  2002;11(22):2793‐804.epub  date:2002/10/11.pimd:12374769.  6. tbx22 mutations are a  frequent  cause of  cleft  palate; marcano ac, doudney k, braybrook c,  squires  r,  pa�on ma,  lees mm,  et  al.;  j med  genet.  2004;41(1):68‐74.epub  date:2004/01/20.pimd:14729838.  7. mutations in foxc2 (mfh‐1), a forkhead family  transcription factor, are responsible for the he‐ reditary  lymphedema‐distichiasis  syndrome;  fang j, dagenais sl, erickson rp, arlt mf, glynn  mw,  gorski  jl,  et  al.;  am  j  hum  genet.  2000;67(6):1382‐8.epub  date:2000/11/15.pimd:11078474.  8. foxc2  truncating  mutation  in  distichiasis,  lymphedema, and cleft palate; bahuau m, hou‐ dayer  c,  tredano  m,  soupre  v,  couderc  r,  vazquez mp; clin genet. 2002;62(6):470‐3.epub  date:2002/12/18.pimd:12485195.  9. a  novel  loss‐of‐function  mutation  in  ttf‐2  is  associated  with  congenital  hypothyroidism,  thyroid  agenesis  and  cleft palate; castanet m,  park sm, smith a, bost m, leger j, lyonnet s, et  al.; hum mol genet. 2002;11  (17):2051‐9.epub  date:2002/08/08.pimd:12165566.  10. fistula  labii  inferioris congenita and  its associa‐ tion with cleft lip and palate; van der woude a;  am j hum genet. 1954;6(2):244‐56.epub   11. a preliminary gene map for the van der woude  syndrome critical region derived from 900 kb of  genomic  sequence  at  1q32‐q41;  schutte  bc,  bjork  bc,  coppage  kb, malik mi,  gregory  sg,  scott  dj,  et  al.;  genome  res.  2000;10(1):81‐ 94.epub date:2000/01/25.pimd:10645953.  12. mutations  in  irf6  cause  van  der woude  and  popliteal  pterygium  syndromes;  kondo  s,  schutte bc, richardson rj, bjork bc, knight as,  watanabe  y,  et  al.; nat genet.  002;32(2):285‐ 9.epub date:2002/09/10.pimd:12219090.  13. a combined targeted mutation analysis of  irf6  gene would be useful  in  the  first  screening of  oral  facial  clefts; wu‐chou yh,  lo  lj, chen kt,  chang  cs,  chen  yr;  bmc  med  genet.  2013;14:37.  epub  date:2013/03/21.  pimd:23510002.  14. comparative analysis of irf6 variants in families  with  van  der woude  syndrome  and  popliteal  pterygium syndrome using public whole‐exome  databases; leslie ej, standley j, compton j, bale  s,  schutte  bc,  murray  jc;  genet  med.  2012.epub date:2012/11/17.pimd:23154523.  15. irf family of transcription factors as regulators  of host defense; taniguchi t, ogasawara k, ta‐ kaoka  a,  tanaka  n;  annu  rev  immunol.  001;19:623‐55.  epub  date:2001/03/13.  pimd:11244049.  16. interferon  regulatory  factor 6  (irf6) gene vari‐ ants and  the  risk of  isolated cleft  lip or palate;  zucchero  tm,  cooper  me,  maher  bs,  daack‐ hirsch  s,  nepomuceno  b,  ribeiro  l,  et  al.;  n  engl  j  med.  2004;351(8):769‐80.  epub  date:2004/08/20. pimd:15317890.  17. breakthroughs in the genetics of orofacial cleft‐ ing; mangold e, ludwig ku, nothen mm; trends  mol  med.  2011;17(12):725‐33.epub  date:2011/09/03.pimd:21885341.  18. genome‐wide meta‐analyses  of  nonsyndromic  cleft lip with or without cleft palate identify six  new  risk  loci; ludwig ku, mangold e, herms s,  nowak s, reutter h, paul a, et al.; nat genet.  2012;44(9):968‐71.epub  date:2012/08/07.pimd:22863734.  19. molecular cloning: a laboratory manual., cold  spring  harbor  laboratory  press,  cold  spring  harbor, ny.; sambrook j, fritsch ef, maniatis t.  1989. epub   20. lack  of  association  between  irf6  polymor‐ phisms  (rs2235371  and  rs642961)  and  non‐ syndromic  cleft  lip and/or palate  in a brazilian  population; paranaiba lm, bufalino a, martelli‐ junior h, de barros  lm, graner  e,  coletta rd;  oral  dis.  2010;16(2):193‐7.epub  date:2009/09/29. pimd:19780991.  21. population  genetics  of  a  functional  variant  of  the  dopamine  betahydroxylase  gene  (dbh);  cubells  jf,  kobayashi  k,  nagatsu  t,  kidd  kk,  kidd  jr,  calafell  f,  et  al.;  am  j  med  genet.  1997;74(4):374‐9.epub  date:1997/07/25.  pimd:9259372.  22. haploview: analysis and visualization of ld and  haplotype maps; barrett jc, fry b, maller j, daly  mj;  bioinformatics.  2005;21(2):263‐5.epub  date:2004/08/07. pimd:15297300.  23. arlequin suite ver 3.5: a new series of programs  to perform population genetics analyses under  linux and windows; excoffier l, lischer he; mol  ecol  resour.  2010;10(3):564‐7.epub   date:2011/05/14. pimd:21565059.  24. multifactor  dimensionality  reduction  software  for detecting gene‐gene and gene‐environment  interactions; hahn  lw, ritchie md, moore  jh;  bioinformatics.  2003;19(3):376‐82.epub  date:2003/02/14. pimd:12584123.  25. virus‐induced heterodimer  formation between  irf‐5 and irf‐7 modulates assembly of the ifna  enhanceosome in vivo and transcriptional activ‐ ity  of  ifna  genes;  barnes  bj,  field  ae,  pitha‐ rowe  pm;  j  biol  chem.  2003;278  (19):16630‐ 41.epub date:2003/02/26. pimd:12600985.26.   26. abnormal  skin,  limb  and  craniofacial morpho‐ genesis  in mice deficient  for  interferon  regula‐ tory  factor  6  (irf6);  ingraham  cr,  kinoshita  a,  kondo  s, yang b,  sajan  s, trout kj, et al.; nat  genet.  2006;38(11):1335‐40.  epub  date:2006/10/17. pimd:17041601.  27. irf6  is  a  key  determinant  of  the  keratinocyte  proliferation‐differentiation  switch;  richardson  rj, dixon  j, malhotra s, hardman mj, knowles  l,  boot‐handford  rp,  et  al.;  nat  genet.  2006;38(11):1329‐34.  epub  date:2006/10/17.  pimd:17041603.  28. genome‐wide  analysis  of  p63  binding  sites  identifies  ap‐2  factors  as  coregulators  of  epi‐ dermal  differentiation; mcdade  ss,  henry  ae,  pivato gp, kozarewa  i, mitsopoulos c, fenwick  k,  et  al.; nucleic acids  res.  2012;40(15):7190‐ 206. epub date:2012/05/11. pimd:22573176.  29. interaction  between  irf6  and  tgfa  genes  contribute  to  the  risk  of  nonsyndromic  cleft  lip/palate;  letra  a,  fakhouri  w,  fonseca  rf,  menezes  r,  kempa  i,  prasad  jl,  et  al.;  plos  one. 2012;7(9):e45441. epub date:2012/10/03.  pimd:23029012.  30. smad4‐irf6  genetic  interaction  and  tgf  beta‐ mediated  irf6 signaling cascade are crucial  for  palatal fusion in mice; iwata j, suzuki a, pelikan  rc, ho tv, sanchez‐lara pa, urata m, et al.; de‐ velopment.  2013;140(6):1220‐30.  epub  date:2013/02/15. pimd:23406900.  31. disruption  of  an  ap‐2alpha  binding  site  in  an  irf6  enhancer  is  associated  with  cleft  lip;  rahimov  f, marazita ml,  visel  a,  cooper me,  hitchler  mj,  rubini  m,  et  al.;  nat  genet.  2008;40(11):1341‐7.epub  date:2008/10/07.  pimd:18836445.   32. strong  evidence  of  linkage  disequilibrium  be‐ tween  polymorphisms  at  the  irf6  locus  and  nonsyndromic  cleft  lip  with  or  without  cleft  palate, in an italian population; scapoli l, palm‐ ieri a, martinelli m, pezzetti f, carinci p, togn‐ on m, et al.; am j hum genet. 2005;76(1):180‐ 3.epub date:2004/11/24. pimd:15558496.    lack of associa on between irf6 polymorphisms and nonsyndromic oral cle s in south indian popula on  vol 1, no 1 (2013)    doi 10.5195/d3000.2013.9  http://dentistry3000.pitt.edu 5 33. variation  in  irf6  contributes  to  nonsyndromic  cleft lip and palate; blanton sh, cortez a, stal s,  mulliken  jb,  finnell  rh,  hecht  jt;  am  j med  genet  a.  2005;137a(3):259‐62.epub  date:2005/08/13. pimd:16096995.  34. interferon  regulatory  factor‐6:  a  gene  predis‐ posing to isolated cleft lip with or without cleft  palate  in  the  belgian  population; ghassibe m,  bayet b, revencu n, verellen‐dumoulin c, gille‐ rot  y,  vanwijck  r,  et  al.;  eur  j  hum  genet.  2005;13(11):1239‐42.epub  date:2005/09/01.  pimd:16132054.  35. significant  association  between  irf6  820g‐>a  and  non‐syndromic  cleft  lip  with  or  without  cleft  palate  in  the  thai  population;  srichomthong  c,  siriwan  p,  shotelersuk  v;  j  med  genet.  2005;42(7):e46.  epub  date:2005/07/05. pimd:15994871.  36. 844ins68  in  the  cystathionine  beta‐synthase  gene  in  israel  and  review of  its distribution  in  the world; zoossmann‐diskin a, gazit e, peleg l,  shohat  m,  turner  d;  anthropol  anz.  2004;62(2):147‐55.  epub  date:2004/07/02.  pimd:15228193.  37. linkage  disequilibrium  between  irf6  variants  and nonsyndromic cleft  lip/palate  in  the chile‐ an population; suazo j, santos jl, jara l, blanco  r;  am  j  med  genet  a.  2008;146a(20):2706‐ 8.epub date: 2008/09/18. pimd:18798331.  38. association between  irf6  snps and oral  clefts  in west china; huang y, wu j, ma j, beaty th,  sull  jw,  zhu  l,  et  al.;  j  dent  res.  2009;88(8):715‐8.epub  date:2009/09/08.  pimd:19734457.  39. association of common variants, not rare muta‐ tions,  in  irf6  with  nonsyndromic  clefts  in  a  honduran  population;  larrabee  yc,  birkeland  ac,  kent  dt,  flores  c,  su  gh,  lee  jh,  et  al.;  laryngoscope.  2011;121(8):1756‐9.  epub  date:2011/07/28. pimd:21792966.  40. genetic  variants  in  irf6  and  the  risk  of  facial  clefts: single‐marker and haplotype‐based anal‐ yses  in  a  population‐based  case‐control  study  offacial clefts in norway; jugessur a, rahimov f,  lie rt, wilcox aj, gjessing hk, nilsen rm, et al.;  genet  epidemiol.  2008;32(5):413‐24.  epub  date:2008/02/19. pimd:18278815.  41. the  irf6  p.274v  polymorphism  is  not  a  risk  factor  for  isolated  cleft  lip;  hering  r,  grund‐ mann k; genet med. 2005;7(3):209; author  re‐ ply  ‐10.  epub  date:2005/03/19.  pimd:15775759.  42. association  between  interferon  regulatory  factor  6  gene  polymorphisms  and  nonsyn‐ dromic cleft lip with or without cleft palate in  a chinese population; zhou q, li m, zhu w, guo  j, wang y, li y, et al.; cleft palate craniofac  j.  2013. epub date:2013/03/21. pimd:23509905.  43. mthfr 677tt alone and  irf6 820gg  together  with mthfr  677ct,  but  not mthfr  a1298c,  are  risks  for  nonsyndromic  cleft  lip  with  or  without cleft palate in an indian population; ali  a,  singh  sk,  raman  r;  genet  test  mol  bi‐ omarkers.  2009;13(3):355‐60.epub  date:2009/05/08. pimd:19419265.  44. ethnic heterogeneity of irf6 ap‐2a binding site  promoter snp association with nonsyndromic  cleft lip and palate; blanton sh, burt a, garcia  e, mulliken jb, stal s, hecht jt; cleft palate cra‐ niofac  j.  2010;47(6):574‐7.  epub  date:2010/11/03. pimd:21039277.  45. low erythrocyte  folate status and polymorphic  variation  in folate‐related genes are associated  with risk of neural tube defect pregnancy; rel‐ ton  cl, wilding  cs,  laffling aj,  jonas  pa,  bur‐ gess  t,  binks  k,  et  al.;  mol  genet  metab.  2004;81(4):273‐81.epub  date:2004/04/03.  pimd:15059614.  46. genome  scan,  fine‐mapping,  and  candidate  gene analysis of nonsyndromic cleft  lip with or  without cleft palate  reveals phenotype‐specific  differences  in  linkage  and  association  results;  marazita  ml,  lidral  ac,  murray  jc,  field  ll,  maher  bs,  goldstein mchenry  t,  et  al.;  hum  hered.  2009;68(3):151‐70.epub  date:2009/06/13. pimd:19521098.  47. polymorphisms  in  the  folate‐metabolizing  genes mtr, mtrr, and cbs and breast cancer  risk; weiner  as,  boyarskikh  ua,  voronina  en,  selezneva  ia,  sinkina  tv,  lazarev  af,  et  al.;  cancer  epidemiol.  2012;36(2):e95‐e100.  epub  date:2012/01/13. pimd:22236648.  48. irf6 gene variants in central european patients  with  non‐syndromic  cleft  lip  with  or  without  cleft palate; birnbaum s, ludwig ku, reutter h,  herms s, de assis na, diaz‐lacava a, et al.; eur j  oral  sci.  2009;117(6):766‐9.  epu‐ date:2010/02/04. pimd:20121942.  49. association between genetic variants of report‐ ed candidate genes or  regions and  risk of cleft  lip  with  or  without  cleft  palate  in  the  polish  population; mostowska a, hozyasz kk, wojcicki  p,  biedziak  b,  paradowska  p,  jagodzinski  pp;  birth  defects  res  a  clin  mol  teratol.  2010;88(7):538‐45.  epub  date:2010/06/15.  pimd:20544801.  50. single‐nucleotide polymorphisms  (snps) of  the  irf6 and tfap2a in nonsyndromic cleft lip with  or without  cleft  palate  (nsclp)  in  a  northern  chinese population; shi j, song t, jiao x, qin c,  zhou  j;  biochem  biophys  res  commun.  2011;410(4):732‐6.epub  date:2011/06/21.  pimd:21683068.  51. irf6  polymorphisms  are  associated with  non‐ syndromic  orofacial  clefts  in  a  chinese  han  population; pan y, ma j, zhang w, du y, niu y,  wang  m,  et  al.;  am  j  med  genet  a.  2010;152a(10):2505‐11. epubdate:2010/08/28.  pimd:20799332.  52. irf6 is a risk factor for nonsyndromic cleft lip in  the  brazilian  population;  brito  la,  bassi  cf,  masotti c, malcher c, rocha km, schlesinger d,  et al.; am j med genet a. 2012;158a(9):2170‐5.  epub date:2012/08/14. pimd:22887868.  53. association and mutation analyses of  the  irf6  gene  in families with nonsyndromic and syn‐ dromic  cleft  lip  and/or  cleft  palate;  pegelow  m, koillinen h, magnusson m,  fransson  i, un‐ neerg p, kere  j,  et al.; cleftpalate craniofac  j.  2013. epub date:2013/02/12. pimd:23394314.  assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography aslıhan akbulut1, beyza ballı akgöl2, kaan orhan3†, merve bayram4 1 phd. assistant professor, istanbul medipol university, school of dentistry, department of dentomaxillofacial radiology, istanbul, turkey 2 phd. assistant professor, istanbul medipol university, school of dentistry, department of pediatric dentistry, istanbul, turkey 3 prof. dr. ankara university, school of dentistry, department of dentomaxillofacial radiology, ankara, turkey †ankara university medical design application and research center (meditam), ankara, turkey 4 phd. assistant professor, istanbul medipol university, school of dentistry, department of pediatric dentistry, istanbul, turkey abstract objective: to define the prevalence of dehiscence and fenestration and classify them in terms of the localization of fenestrations in a random sampled group of children and adolescent patients using conebeam computed tomography (cbct). methods: cbct performed at the department of oral and maxillofacial radiology of patients referred by the paediatric dentistry clinic were included in this retrospective study. image evaluations were performed by dentomaxillofacial radiologist (aa, asst. prof.), and these images were examined in three dimensions of the axial, coronal, and sagittal planes. intraexaminer agreement for the evaluations were found acceptable. the presence/absence of dehiscence and/or fenestration, fenestration’s classification type, and localization of defects were recorded. moreover, the presence/absence of periapical lesion in related root with dehiscence and fenestration was noted. for statistical analysis, the chi-square test, fisher freeman halton test, and yates' continuity of correction were used. results: 3061 roots in 1801 teeth of 120 cases were analyzed. the mean age was 9.97±2.22 years. dehiscence was detected in 261(8.5%) roots of 161(8.9%) teeth, and fenestration was detected 63(2%) roots of 36(2%) teeth. the most common fenestration type was type i, followed by type ii and iv. dehiscence was observed more frequently in primary teeth than permanent teeth, and the difference was statistically significant (p:0.000). dehiscence and fenestration incidence in maxillary teeth was significantly higher than in the mandibular teeth (pdehiscence:0.000, pfenestration:0.004). apical lesions were observed more in primary teeth than permanent teeth for both defects. conclusion: this study concludes that alveolar dehiscence and fenestrations are more common in primary teeth than permanent teeth. moreover, these defects were detected more for the teeth in the maxilla. concerning endodontic and orthodontic therapies in maxilla, use of cbct is useful in determining the region's anatomical structure accurately in suspected cases of child and adolescent patients. keywords: alveolar bone defect; cone-beam computed tomography; dehiscence; fenestration; primary tooth. citation: akbulut a, et al. (2021) assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography. dentistry 3000. 1:a001 doi:10.5195/d3000.2021.143. dentistry 3000. 1:a001 doi:10.5195/d3000.2021.143 received: february 3, 2020 accepted: april 1, 2021 published: july 28, 2021 copyright: ©2021 akbulut a, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: mbayram@medipol.edu.tr introduction diagnosis of alveolar bone defects is crucial for dental practitioners due to the fact that the teeth sockets are formed and supported by the alveolar bone. shape of the defects as well as the position is not predictable and specific to the corresponding region [1, 2]. the http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu alveolar bone structure should be correctly assessed for accurate diagnosis and appropriate treatment which affects positive prognosis[1, 3]. destruction of the alveolar bone, caused by gingival inflammation spreading to the bone, is the leading cause of periodontal disease unless treated properly [4, 5]. this process is exacerbated by anatomical defects in the cortical bone layer that covers the root surfaces [6, 7]. the terms fenestration and dehiscence according to the glossary of endodontic terms are described as follows [8]: “fenestration a windowlike opening or defect in the alveolar plate of bone frequently exposing a portion of the root; usually located on the facial aspect of the alveolar process dehiscence a narrow, vertical defect in the alveolar plate of bone over a root extending from the crestal area apically; usually located on the facial aspect of the alveolar process.” fenestration and dehiscence have been observed with various reasons such as: i. endodontic and periapical disease ii. trauma iii. bruxism iv. tooth movement v. thin cortical bone vi. the tooth/jaw ratio vii. position of the teeth viii. orthodontic problems such as rapid maxillary expansion (rme), orofacial clefts, occlusal forces, posterior crossbite [2, 3, 5–7, 9– 12]. although some patients experienced a variety of clinical signs and symptoms of fenestration (i.e. persistent pain, discomfort, abscess, and other complex symptoms) [2], others who presented root fenestration did not suffer from any detectable symptoms or discomfort [13]. the outcome of root canal treatment may be affected by preoperative factors such as periapical lesions and periodontal bone defects [14, 15]. when patient suffers from persistent periapical inflammatory symptoms following endodontic treatment, fenestration should be kept in mind as a potentially missing diagnosis [16]. moreover, dental arch expansion and orthodontic movements of teeth which may cause teeth to move out of the alveolar bone socket leading to dehiscence and fenestration, may be observed primarily in child and adolescent patients. it was indicated that rme may cause fenestration and/or dehiscence in the buccal side of the maxillary teeth [17]. therefore, detailed imaging is essential to detect problems related to alveolar morphology as it can vary the course of the orthodontic treatment process [3, 10–12, 17– 21]. considering the fact that alveolar bone dehiscence is covered with soft tissues; it is invisible to the naked eye and is often difficult to observe clinically. radiographic assessment is recommended to aid clinical examination for relatively more dependable findings [1, 4, 9, 22–25]. the dehiscence, which occurs on the buccal or lingual side, cannot be observed in routine radiographic diagnosis due to the overlapping images of the surrounding alveolar bone. therefore, 3d imaging techniques such as cone-beam computed tomography (cbct) are preferable to 2d to acquire a better view of surrounding tissues and overcome the issues of superimposition and magnification [9, 17, 19–24, 26]. however, non-essential cbct use is not recommended for certain patients, in particular children and adolescents, due to the high http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu radiation exposure [27]. on the other hand, intraoral radiographs have a sensitivity of 63–67% in detection and classification of bone defects, whereas cbct has a reported [6, 22] sensitivity of 80– 100%. dehiscence and fenestration were observed to have a close relationship with endodontic and/or orthodontic problems and their treatments, as previously mentioned. therefore, the motivation of this research had been the anticipation of observation of these defects in child and adolescence patients, as well as adults. previous research in dental literature has predominantly focused on factors affecting the occurrence of dehiscence and fenestration in permanent teeth, and to our knowledge no study has been published evaluating the prevalence of dehiscence and fenestration in primary teeth. the aim of this study was to investigate prevalence of dehiscence and fenestration in children and adolescence, and to classify according to the localization employing cbct. the study hypothesis was that dehiscence and fenestration prevalence was different in primary teeth than permanent teeth. methods study sample ethical approval was obtained from the ethics committee of xxxx university, clinical research ethics committee, no: xxxxx. the written informed consent forms signed by the parent of the patient as a routine procedure before dental examinations or interventions. sample size was calculated using the following assumptions; alpha significance level= 0.05, study power= 80%, anticipated dehiscence/fenestration prevalence= 10 % based on a previous study [6] and prevalence of study group= 20 %. the minimum required sample size was calculated to be 85 cases. cbct images of patients who applied for dental care in the paediatric clinic referred to the department of oral and maxillofacial radiology due to cbct image requirement for various reasons were evaluated in this retrospective study. the 3d cbct images were extracted randomly from the archives of the department of oral and maxillofacial radiology. no cbct was requested from patients for this study. the patients' demographic data (age, gender, health (systemic disease) status) were recorded. inclusion criteria in this study setup, three main inclusion criteria were identified to determine the study sample, listed as follows: i. child and adolescent patients with cbct image. ii. complete development of the roots in primary and permanent teeth, observed in cbct images. iii. cbct images with clear visibility of the entire root and surrounding alveolar bone. exclusion criteria four main exclusion criteria were identified while determining the study group, listed as follows: i. children with malocclusions and/or orthodontic treatment history ii. children with systemic disease, syndromes or congenital anomalies iii. presence of maxillofacial fracture or trauma history iv. cbct images with obvious pathology (cyst or tumour). among the overall identified group http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu of 162 patients, the cbct images of children who had orthodontic treatment history (2 patients), systemic disease (5 patients), congenital anomalies (2 patients), obvious pathology (29 patients), trauma history (4 patients) were excluded from the analysis in the study. a total of 120 cbct images fulfilled the above-listed inclusion criteria, and were included in the study. all primary and permanent teeth in mandibula and maxilla couldn’t be evaluated for each case, because of limited field view based on the reason of image request. therefore, 578 primary and 1223 permanent teeth were examined in total. cbct acquisition and evaluation this retrospective study was carried out using the cbcts taken during the period between july 2011july 2020. image evaluations were performed by the same examiner (aa, asst. prof., dentomaxillofacial radiologist, with eighteen years of experience) experienced in cbct scans, and these images were examined in three dimensions of the axial, coronal, and sagittal planes. for determining the intraexaminer reliability associated with cbct evaluations, 15 cbct images were selected randomly, and these corresponding evaluations were repeated two weeks after the first evaluations carried out by the same examiner. kappa statistics were used to determine intraexaminer agreement for the two sets of evaluations and found to be higher than 0.90, indicating that all evaluations' reliability was acceptable. scans were obtained using cbct (icat®, model 17–19, imaging sciences international, hatfield, pa) with a single 360° rotation and a voxel size of 0.3 mm with following settings: i. x-ray emission time was 3.5 seconds ii. exposures were made with 5.0 ma, 120 kv and an exposure time of four seconds iii. the axial slice thickness was 0.3 mm the images were transformed into data using invivo 5 ver. 5.2 anatomage (san jose, california, usa) to diagnose the presence of dehiscence and fenestration. each root of erupted primary and permanent teeth was evaluated in axial and cross-sectional slices at the buccal and lingual surfaces. an alveolar defect was identified when there was no cortical bone around the root in at least three sequential views. when the alveolar bone height was more than 2 mm. from the cementoenamel junction, it was classified as dehiscence. in addition to this, if the defect did not involve the alveolar crest, the case was classified as fenestration [2, 3, 6, 22]. based on the classification by pan et al. [9], fenestrations were classified into the following six types (fig.1): http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu figure 1. fenestrations based on the classification by pan et al. a-type i; b-type ii; c-type iii; d-type iv; e-type v; f-type vi. “type i protrusion of the apical 1/3 of the root regardless of whether the apical foramen or anatomical apex is involved. type ii protrusion of the middle 1/3 of the root. type iii protrusion of the coronal 1/3 of the root but excluding the alveolar margin. type iv protrusion of the apical and middle parts of the root regardless of whether the apical foramen or apical apex is involved but excluding the coronal 1/3 of the root. type vprotrusion of the middle and coronal part of the root without the involvement of the alveolar margin but excluding the apical 1/3 of the root. type vi protrusion of the whole root but excluding the alveolar margin.” retrospective data acquisition patients were identified by a patient protocol number and case number generated from database records, to protect confidentiality. the case number, age (at the time of the http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu cbct was taken), gender, health status, tooth number, number of roots for each tooth, presence/absence of a dehiscence (fig. 2) or fenestration (fig 3.), fenestration’s classification type, and localizations of defects were recorded in a spreadsheet. moreover, the presence/absence of periapical lesion in related root with dehiscence and fenestration was noted. all data was evaluated by one independent examiner (mb). figure 2. presence of a dehiscence. figure 3. presence of fenestration type i. statistical analysis raw patient data extracted from the hospital database was structured using microsoft excel (microsoft corporation, redmond, wa, usa) for further analysis. ibm spss statistics 22 for statistical analysis (ibm corporation, armonk, ny, usa) was used for statistical analysis. the descriptive statistics (mean, standard deviation, frequency) were recorded. the chi-square test, fisher freeman halton test, and yates' continuity of correction were used to compare qualitative data. the significance level was set as < 0.05. results in this study, 3061 roots in 1801 teeth of 120 cases (64 male and 56 female) were evaluated. the ages of the cases ranged between 6 15, and the mean age was 9.97 ± 2.22 years. overall prevalence of dehiscence was 40.8 % whereas fenestration was 2.5 %. dehiscence was detected in 261 (8.5 %) roots of 161 (8.9 %) teeth, and fenestration was detected 63 (2 %) roots of 36 (2 %) teeth. the presence/absence, and side of dehiscence and fenestrations for concerning root are laid out in table 1. the dehiscence and fenestration were most frequently observed in the buccal/labial side of the root. the most common fenestration type was type i (63.9 %) (fig. 3), followed by type ii (11.1 %) and iv (11.1 %) (fig. 4) (table 2). http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu table 1. findings related to dehiscence and fenestration: dehiscence n (%) fenestration n (%) defect (teeth; n=1801) presence 161(8.9) 36 (2) absence 1640 (91.1) 1765 (98) defect side (teeth; ndehiscence=161 nfenestration=36) labial/buccal 146 (90.7) 32 (88.9) palatinal/lingual 8 (5) 4 (11.1) labial/buccal and palatinal/lingual 7 (4.3) na related root (teeth; ndehiscence=161 nfenestration=36) buccal root 4 (2.5) 6 (16.7) distobuccal root 3 (1.9) 5 (13.9) mesial root 3 (1.9) na mesial root, distal root 6 (3.7) 1 (2.8) mesiobuccal root 2 (1.2) 4 (11.1) mesiobuccal root, distobuccal root 27 (16.8) 1 (2.8) mesiobuccal root, distobuccal root, palatinal root 3 (1.9) na mesiobuccal root, palatinal root 4 (2.5) na palatinal root 5 (3.1) na single root 104 (64.6) 19 (52.8) na: non-applicable http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu table 2. types of fenestration: figure 4. presence of fenestration type iv. the distribution of dehiscence and fenestrations according to the primary/permanent tooth, jaw and tooth type is demonstrated in table 3. dehiscence was observed more frequently in primary teeth (21.5 %) than permanent teeth (3 %). the difference of dehiscence presence between primary and permanent teeth was statistically significant. (p:0.000, table 4). incidence of dehiscence in maxillary teeth (10.9 %) was higher than mandibular teeth (4.6 %) (p:0.000, table 4). the highest rate of dehiscence was observed in canine (14.9 %) teeth, whereas the lowest rate of dehiscence was noticed in the second premolar (2.7 %). a significant difference for dehiscence frequency was found between tooth types (p:0.000, table 4). table 3. frequency of dehiscence / fenestration, based on tooth type: primary/permanent jaw tooth type dehiscence fenestration n (%) n (%) permanent tooth maxilla incisor 14 (4.5) 12 (3.8) canine 3 (5.2) 0 (0) first premolar 4 (5) 5 (6.3) second premolar 1 (1.6) 2 (3.2) first molar 2 (1) 0 (0) second molar 3 (7.1) 0 (0) mandible incisor 3 (1.7) 0 (0) canine 2 (3.9) 0 (0) first premolar 2 (3.4) 0 (0) second premolar 2 (4.1) 0 (0) fenestration types n % type i 23 63.9 type ii 4 11.1 type iii 1 2.8 type iv 4 11.1 type v 2 5.6 type vi 2 5.6 http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu first molar 1 (1.1) 0 (0) second molar 0 (0) 0 (0) primary tooth maxilla incisor 36 (50) 2 (2.8) canine 35 (23.5) 2 (1.3) first molar 21 (17.6) 8 (6.7) second molar 16 (11.9) 2 (1.5) mandible incisor 2 (28.6) 0 (0) canine 4 (10.8) 2 (5.4) first molar 5 (19.2) 1 (3.8) second molar 5 (14.7) 0 (0) table 4. tooth and jaw relations with dehiscence and fenestration: dehiscence n (%) p-value fenestration n (%) p-value primary/permanent permanent teeth 37 (3) †0.000 19 (1.6) ‡0.074 primary teeth 124 (21.5) 17 (2.9) jaw maxilla 135 (10.9) †0.000 33 (2.7) ‡0.004 mandible 26 (4.6) 3 (0.5) tooth type incisor 55 (9.6) †0.000 14 (2.4) §0.437 canine 44 (14.9) 4 (1.4) first premolar 6 (4.3) 5 (3.6) second premolar 3 (2.7) 2 (1.8) first molar 29 (6.6) 9 (2.1) second molar 24 (9.8) 2 (0.8) http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu † chi-square test ‡yates' continuity of correction §fisher freeman halton test table 5. distribution of periapical lesion in teeth with dehiscence and fenestration: dehiscence n (%) fenestration n (%) total 161 (8.9) 36 (2) periapical lesion 32 (19.8) 2 (5.5) primary teeth 28 (87.5) 2 (100) permanent teeth 4 (12.5) 0 (0) dehiscence of different tooth types was demonstrated in fig. 5. fenestration was observed in 1.6 % of permanent teeth and 2.9 % of primary teeth, the difference was not statistically significant (p>0.05, table 4). fenestrations were more frequent in maxillary teeth (2.7 %) than in mandibular teeth (0.5 %). fenestration was not observed in any permanent mandibular teeth. there was a significant difference for fenestration frequency between the lower and upper teeth (p:0.004, table 3). the highest incidence of fenestration was observed in the first premolar (3.6 %) teeth, whereas the lowest incidence of teeth with fenestration was in the second molar (0.8 %). there was no statistically significant difference between fenestration incidence according to tooth types (p>0.05, table 4). table 5 lays out the number and frequency of apical lesion presence in teeth with seen dehiscence and fenestration. the periapical lesions were observed more frequently in teeth with dehiscence 19.8 % than with fenestration 5.5 %. periapical lesions were observed more in primary teeth than permanent teeth for both defects. discussion in this retrospective study 1801 teeth were inspected for dehiscence and fenestration defects in child and adolescent figure 5. dehiscence of different tooth types. http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu patients. moreover, fenestration classification types were evaluated. to our knowledge, no previous studies have analyzed the prevalence of dehiscence and fenestration in primary teeth. the limitations of this study are as follows: i. thickness of the cortical bone may be influenced from hormonal and functional changes which is associated with the study group. the age range was between 6 – 15 years, and this study group contained growing patients. ii. all teeth in both jaws couldn’t be evaluated for each case due to the retrospective acquisition of cbct records, which included limited field view based on the reason of image request. among the 1801 teeth evaluated, (8.9 %) had alveolar dehiscence. this result was higher than the previous reported dry human skulls investigation studies (3.2 % 7.1 %), yet was similar to a previous cbct study (8.6 %). the current study showed that dehiscence (8.9 %) was seen more frequently than fenestration (2%), which is in line with previous researches [10, 11, 19, 21]. 90.7% of the dehiscence was on the buccal/labial side while 5% were on the lingual/palatinal side, and these findings are consistent with previous studies [9, 12, 18, 19]. the highest rate of dehiscence was seen in single root teeth (64.6%), followed by mesiobuccal and distobuccal roots (16.8%). on the other hand, dehiscence, only involving the mesiobuccal root was 1.2%. in the past, fenestration and dehiscence in dry human skulls and flap surgery on cadaver heads were used merely to investigate the prevalence. until the introduction of computer tomography (ct), visualization of labial/buccal and lingual bone plates was not possible because of the image superimposition associated with conventional radiographs [10]. 2d radiographic imaging is not sufficient to show the alveolar bone defects, especially in the posterior area. when significant buccolingual tooth movement or tipping is required during orthodontic treatment, it becomes necessary to be aware of existing bone defects in the alveolar area which may make complex the orthodontic treatment process [28]. cbct has provided to visualize these defects three-dimensionally [11]. ct images can now show bone dehiscence and fenestration utilizing high definition and sensitivity.[18] cbct also yields high-definition images of teeth and bone at a far lower dosage of radiation that was formerly used with medical imaging and closer to the range of standard dental film series [12]. as can be seen in various animal experiments, the loss of thin bone plates may be induced by orthodontic tooth movement [17]. therefore, the treatment plan's choice should depend on bone morphology and the position of the teeth [19, 20]. imaging anatomic details of the patients and comprehension of tooth movement collateral effects allow us to safely realize the borders and practice, unlike the traditional method of investigating the prevalence and morphology of fenestration on dry human skulls. visual examination and direct measurement made the traditional methods using dry human skulls are highly accurate and reliable despite its disadvantages as dried skulls studies offer no clinical information. moreover, this method could never be applied to clinical endodontic diagnosis. however, it must be taken into consideration that both in vivo and ex vivo studies have indicated that cbct is likely to be a useful and http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu more practical clinical tool than digital subtraction radiography or ct, for almost all endodontic and periodontal periapical applications [29]. in this respect, the low doses of radiation and superior image quality of cbct compared with conventional ct were worthwhile in diagnosing dehiscence and fenestration defects [30]. following the proper root canal treatment, teeth are generally asymptomatic; the pain symptoms are not associated with bone defect. however, mechanical and chemical irritation caused by contact of the periosteum and mucosa with foreign material after overflowing root canal filling may cause temporary or permanent pain. in the presence of dehiscence, direct contact of the canal filling material with the mucosa may cause spontaneous pain. apical fenestration may be considered an anatomic predisposing factor for persistent pain following root canal treatment which often is misdiagnosed [31]. when planning for a root canal treatment, a careful examination should be carried out not only for the root but also the tissues around the root. dehiscence and fenestration may cause ongoing pain, unsuccessful treatment outcome accompanied by periodontal problems, especially following endodontic treatment. therefore, the possibility of apical fenestration should be precisely considered with cbct image when necessary. in the current study, addition to prevalence of dehiscence and fenestration, periapical lesions were detected in these teeth with dehiscence and fenestrations defects, leading to pathological changes. dehiscence and fenestration were more frequently observed in maxillary teeth than in mandibular teeth in this research which is in line with previous studies [5, 6, 9, 12, 17, 18]. although this phenomenon has not been understood yet, nimigean et al. [2] has hypothesized that potential reason for seeing more fenestrations in the maxillary arch is root tip inclination to the labial/buccal for teeth in maxilla. the current study strengthens the previous findings [6, 7, 9, 12, 18, 19] reporting far more frequent dehiscence and fenestration on the labial/buccal side, than on the palatal/lingual. in a previous study, frequency of permanent tooth types with dehiscence was found highest in maxillary first permanent molars and least in mandibular permanent molars. the present study demonstrated that maxillary permanent canine has the highest dehiscence frequency, where mandibular permanent molars have the lowest. ectopic eruption may be considered a potential reason for the high observation frequency in the upper canine tooth. the study group was mostly in the mixed dentition stage, and ectopic eruption was clearly one of the aetiological factors that affects bone volume housing the teeth [32]. in the maxilla, frequency of permanent tooth types with fenestration was higher in premolar teeth followed by incisors. in addition to this, fenestration was not observed in any permanent tooth type in mandible. previous researchers [2, 33] reported different pattern for relative fenestration frequency in permanent tooth types as follows: maxillary first molar, maxillary/mandibular canines and mandibular lateral incisors. the difference is probably due to the fact that the above-mentioned researches could make an assessment including all permanent teeth. considering our study group which included patients in mixed dentition stage, it was not possible to evaluate all permanent teeth. http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu there is a tendency to perceive fenestration as a rare condition [9] and there exists a few case reports, [13, 16, 31, 34] among which only one related with children. the frequency of root fenestrations in this study was found low (2 %), and type i fenestrations was the highest proportion (63.9 %) among all types. type of fenestration findings in the current study was in line with the chinese subpopulation study [9]. in this study, 5.5 % of teeth with fenestration had periapical lesions, all of which was associated with primary teeth. besides, the presence of periapical lesions was found to be higher in teeth with dehiscence compared to with fenestration. alveolar bone loss caused by periapical lesions may also be related to dehiscence and fenestration. during the pretreatment period, clinicians are generally focused on the clinical status of supporting tissues and implications on treatment. treatment plan gets complicated by the complex modelling of the alveolus in a growing patient by growth rotations of the skeletal structures that affect the direction of compensatory eruptive movements. this study concludes that alveolar dehiscence and fenestration defects are more common in primary teeth than permanent teeth. moreover, the teeth in the maxilla were identified as highly affected from these defects. concerning endodontic and orthodontic therapies in maxilla, use of cbct is useful in determining the region's anatomical structure accurately in suspected cases of child and adolescent patients. persistent pain following root canal treatment in children should be further investigated for possible dehiscence and fenestration presence. this study's clinical relevance is providing the result of high defect prevalence in primary teeth, to assist dental professionals perceive which teeth are most often associated with alveolar defects, which may be considered on the diagnosis and treatment plans. conflict of interest: the authors declare that they have no conflict of interest. funding: the authors declare that this study has received no financial support. ethical approval: ethics committee approval was received for this study from the ethics committee of istanbul medipol university (no: 10840098-604.01.01-e.6363) author contributions: ko conceptualized the manuscript. bba and aa carried out methodology; mb carried out data analysis, drafted and edited the manuscript. all authors subsequently revised the draft. all authors read and approved the final manuscript. references 1. comparison of linear and volumetric measurements obtained from periodontal defects by using cone beam-ct and micro-ct: an in vitro study. tayman ma, kamburoğlu k, küçük ö, et al (2019). clin oral investig 23:2235–2244. pmid: 30284102 2. alveolar bone dehiscences and fenestrations: an anatomical study and review. nimigean vr, nimigean v, bencze ma, et al (2008). rom j morphol embryol 50:391–397. pmid: 19690764 3. accuracy of cone-beam computed tomography in detecting alveolar bone dehiscences and fenestrations. sun l, zhang l, shen g, et al (2015). am j orthod dentofac orthop 147:313–323. pmid: http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu 25726398 4. classification of alveolar bone destruction patterns on maxillary molars by using cone-beam computed tomography. ozcan g, sekerci ae (2017). niger j clin pract 20:1010–1019. pmid: 28891547 5. cone-beam computed tomography evaluation of alveolar bone dehiscences on mesiobuccal root region of upper first molar teeth. nalcı g, alaçam t, salmanov e, et al (2020). turkiye klin j dent sci 26:195–200 6. kktc toplumunda fenestrasyon ve dehisens prevalansının konik ışınlı bilgisayarlı tomografi ile değerlendirilmesi. aksoy u, aksoy s (2019). 317–322 7. alveolar bone fenestrations and dehiscences in dry bedouin jaws. edel a (1981). j clin periodontol 8:491–499. pmid: 6949922 8. glossary of endodontic terms. american association of endodontists (2015). gloss endod terms 9:43 9. use of cone-beam computed tomography to evaluate the prevalence of root fenestration in a chinese subpopulation. pan hy, yang h, zhang r, et al (2014). int endod j 47:10– 19. pmid: 23701176 10. dehiscence and fenestration in skeletal class i, ii, and iii malocclusions assessed with cone-beam computed tomography. yagci a, veli i, uysal t, et al (2012). angle orthod 82:67–74. pmid: 21696298 11. dehiscence and fenestration in patients with different vertical growth patterns assessed with cone-beam computed tomography. enhos s, uysal t, yagci a, et al (2012). angle orthod 82:868–874. pmid: 22356702 12. appraisal of the relationship between tooth inclination, dehiscence, fenestration, and sagittal skeletal pattern with cone beam computed tomography. coskun i, kaya b (2019). angle orthod 89:544–551. pmid: 30741575 13. management of mucosal fenestrations by interdisciplinary approach: a report of three cases. jhaveri hm, amberkar s, galav l, et al (2010). j endod 36:164–168. pmid: 20003959 14. outcome of secondary root canal treatment: a systematic review of the literature. ng y-l, mann v, gulabivala k (2008). int endod j 41:1026–1046. pmid: 19133093 15. a prospective study of the factors affecting outcomes of nonsurgical root canal treatment: part 1: periapical health. ng y-l, mann v, gulabivala k (2011). int endod j 44:583–609. pmid: 21366626 16. persistent pain related to root canal filling and apical fenestration: a case report. boucher y, sobel m, sauveur g (2000). j endod 26:242–244. pmid: 11199728 17. evaluation of alveolar bone loss following rapid maxillary expansion using cone-beam computed tomography. baysal a, uysal t, veli i, et al (2013). korean j orthod 43:83–95. pmid: 23671833 http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu 18. detection of bone defects using cbct exam in an italian population. gambarini g, miccoli g, gaimari g, et al (2017). int j dent 2017: 19. prevalence of posterior alveolar bony dehiscence and fenestration in adults with posterior crossbite: a cbct study. choi jy, chaudhry k, parks e, ahn jh (2020). prog orthod 21:. pmid: 32173764 20. evaluation of dehiscence and fenestration in adolescent patients affected by unilateral cleft lip and palate: a retrospective cone beam computed tomography study. buyuk sk, ercan e, celikoglu m, et al (2016). angle orthod 86:431–436. pmid: 26284755 21. dehiscence and fenestration in patients with class i and class ii division 1 malocclusion assessed with cone-beam computed tomography. evangelista k, vasconcelos kdf, bumann a, et al (2010). am j orthod dentofac orthop 138:133.e1-133.e7. pmid: 20691344 22. comparison of the influence of fov sizes and different voxel resolutions for the assessment of periodontal defects. kolsuz me, bagis n, orhan k, et al (2015). dentomaxillofacial radiol 44:. pmid: 25900235 23. detection of simulated periodontal defects using cone-beam ct and digital intraoral radiography. bayat s, talaeipour ar, sarlati f (2016). dentomaxillofacial radiol 45: 24. diagnostic accuracy of cbct for periodontal lesions. braun x, ritter l, jervøestorm pm, frentzen m (2014). clin oral investig 18:1229–1236. pmid: 24048949 25. comparison of intraoral radiography and cone-beam computed tomography for the detection of periodontal defects: an in vitro study. bagis n, kolsuz me, kursun s, orhan k (2015). bmc oral health 15:1–8. pmid: 26016804 26. diagnostic validity (or acuity) of 2d ccd versus 3d cbct-images for assessing periodontal breakdown. vandenberghe b, jacobs r, yang j (2007). oral surgery, oral med oral pathol oral radiol endodontology 104:395–401. pmid: 17613257 27. indications for cone beam computed tomography in children and young patients in a turkish subpopulation. i̇şman ö, yılmaz hh, aktan am, yilmaz b (2017). int j paediatr dent 27:183–190. pmid: 27452447 28. changes of alveolar bone dehiscence and fenestration after augmented corticotomy-assisted orthodontic treatment: a cbct evaluation. sun l, yuan l, wang b, et al (2019). prog orthod 20:. pmid: 30773604 29. cone-beam ct diagnostic applications: caries, periodontal bone assessment, and endodontic applications. tyndall da, rathore s (2008). dent clin north am 52:825–841. pmid: 18805231 30. digital volume tomography for diagnostics in periodontology. kasaj a, willershausen b (2007). int j comput dent 10:155–168. http://dentistry3000.pitt.edu/ assessment of dehiscence and fenestration in children and adolescents using cone beam computed tomography vol 9 no 1 (2021) doi 10.5195/d3000.2021.143 http://dentistry3000.pitt.edu pmid: 17899891 31. atypical facial pain related to apical fenestration and overfilling. pasqualini d, scotti n, ambrogio p, et al (2012). int endod j 45:670– 677. pmid: 22309707 32. pattern of bone resorption in vertically fractured, endodontically treated teeth. lustig jp, tamse a, fuss z (2000). oral surg oral med oral pathol oral radiol endod 90:224–227. pmid: 10936842 33. prevalence of dehiscences and fenestrations in modern american skulls. rupprecht rd, horning gm, nicoll bk, cohen me (2001). j periodontol 72:722–729. pmid: 11453233 34. the management of mucosal fenestration: a report of two cases. chen g, fang ct, tong c (2009). int endod j 42:156–164. pmid: 19134044 http://dentistry3000.pitt.edu/ microsoft word trevillatto 27 dec 11.docx vol  3,  no  1  (2015)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2015.27   http://dentistry3000.pitt.edu   new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.   this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.   polymorphism  in  the  tumor  necrosis  factor-­‐alpha  gene  (tnfa  -­‐308   g/a)  is  not  associated  with  susceptibility  to  chronic  periodontitis   in  a  brazilian  population   paula  cris*na  trevila/o1,  rui  barbosa  de  brito  jr2,  raquel  mantuaneli  scarel-­‐caminaga3,  ana  paula  de  souza  pardo4,  cleber  machado  de  souza1,   antonio  wilson  sallum4,  sérgio  roberto  peres  line4   1school  of  health  and  biosciences  at  pon23cia  universidade  católica  do  paraná  (pucpr),  curi'ba,  pr,  brazil   2são  leopoldo  mandic  dental  research  ins$tute,  campinas,  sp,  brazil   3department  of  diagnosis  and  surgery,  school  of  den7stry,  unesp  -­‐  são  paulo  state  university,  araraquara,  sp,  brazil   4department  of  morphology,  dental  school  of  piracicaba,  state  university  of  campinas,  piracicaba,  sp,  brazil   abstract   objec&ve:   tumor   necrosis   factor-­‐alpha   (tnf-­‐α)   is   a   major   mediator   of   the   immune-­‐ inflammatory   response  and  may  play  an   important   role   in   the  pathogenesis   and  progres-­‐ sion  of  chronic  periodon--s.  polymorphisms  in  the  promoter  of  the  tnfa  gene  have  been   associated  with   some   types  of   inflammatory  diseases.   the  present   study   inves1gated   the   associa&on   between   a   single-­‐nucleo'de   polymorphism   (snp)   of   the   tnfa   (g-­‐308a)   gene   and  chronic  periodon,,s   in  brazilians.  methods:  one  hundred  and  thirteen  (113)  over  25   years  were  divided  according  to  the  severity   level  of  periodontal  disease:  44  healthy   indi-­‐ viduals  (control  group),  31  subjects  with  moderate  and  38  pa*ents  with  severe  periodon*-­‐ !s.  genomic  dna  was  obtained  from  epithelial  cells.  the  samples  were  analyzed  for  tnfa   (g-­‐308a)  polymorphism  using  polymerase  chain  reac6on-­‐restric'on  fragment   length  poly-­‐ morphism   techniques.   the   significance   of   the   differences   in   the   genotype   frequencies   of   the  polymorphism  was  assessed  by  chi-­‐square  test  (p<0.05).  results:  no  significant  differ-­‐ ences   in   the  genotype  distribu1on  and  allele   frequency  were   found  between  control   and   groups   with   periodon//s.   conclusion:   it   was   concluded   that   tnfa   (-­‐308)   polymorphism   was   not   associated  with   chronic   periodon//s.  other   polymorphisms   in   this   or/and   other   genes  of  the  host  inflammatory  response  might  be  involved  in  determining  suscep1bility  to   periodon((s  in  the  study  popula(on.   cita%on:   trevila(o   p,   de   brito   jr.   r,   scarel-­‐ caminaga  r,  pardo  a,  de  souza  c,  sallum  a,  line   s.   (2015)   polymorphism   in   the   tumor   necrosis   factor-­‐alpha  gene  (tnfa  -­‐308  g/a)  is  not  associ-­‐ ated   with   suscep-bility   to   chronic   periodon--s   in  a  brazilian  popula,on.  den$stry  3000.  1:a001   doi:10.5195/d3000.2015.27   received:  september  24,  2014   accepted:    november  25,  2015   published:    december  18,  2015   copyright:   ©2015   trevilatto p, de brito jr. r, scarel-caminaga r, pardo a, de souza c, sallum a, line s. this   is   an   open   access   ar!cle   licensed   under   a  crea%ve   commons   a"ribu%on   wor k   4.0   united   states  license.   email:  pctrev@yahoo.com.br introduction   bacterial  lipopolysaccaha-­‐ rides  (lps)  activate  monocytes  of   inflamed  periodontal  tissues  to   produce  cytokines.  tumor  necrosis   factor-­‐alpha  (tnf-­‐α)  is  an  im-­‐ portant  immune  mediator  and  a   pluripotent  proinflammatory  cyto-­‐ kine  that  is  able  to  induce  bone  re-­‐ sorption  [1].     tnfa  gene  is  encoded  on   chromosome  6  inside  the  hla  re-­‐ gion  [2]  and  its  biological  activities   have  raised  the  possibility  that  pol-­‐ ymorphisms  within  this  gene  might   contribute  to  genetic  association  to   diverse  immune-­‐inflammatory  dis-­‐ eases.  in  fact,  polymorphisms  in   tnfa  gene  have  been  implicated  in   the  pathogenesis  of  a  large  number   of  human  diseases.  within  the  pro-­‐ moter  region  of  tnfa,  a  biallelic   polymorphic  site  at  position  -­‐308   has  been  reported  to  influence  the   production  of  tnf-­‐α  protein  [3].   allele  a  of  tnfa  (-­‐308)  polymor-­‐ phism  has  been  positively  associat-­‐ ed  with  asthma  [4]  and  ulcerative   colitis  [5].   polymorphism  in  the  tumor  necrosis  factor-­‐alpha  gene  (tnfa  -­‐308  g/a)  is  not     associated  with  suscep.bility  to  chronic  periodon..s  in  a  brazilian  popula.on   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.27    http://dentistry3000.pitt.edu   2     epidemiological  studies  in-­‐ dicate  that  chronic  periodontitis   (cp)  is  widespread  among  the  bra-­‐ zilian  population  [6,  7].  tnfa   mrna  was  significantly  more  fre-­‐ quent  in  diseased  than  in  healthy   gingival  biopsies  [8],  suggesting  a   role  of  this  cytokine  in  the  devel-­‐ opment  of  cp.  increased  levels  of   tnf-­‐α  in  periodontal  tissues  were   associated  with  genotypes  carrying   allele  a  [9].  because  of  the  increas-­‐ ing  evidence  that  genetic  variations   within  the  tnfa  locus  are  im-­‐ portant  in  determining  suscepti-­‐ bility  to  periodontitis,  this  study   aimed  to  investigate  the  possible   link  between  the  polymorphism  at   position  -­‐308  (g→a)  of  tnfa  gene   promoter  and  chronic  periodontitis   in  a  brazilian  population.     materials  and  methods     subject  selection     a  convenience  sample  of   113  unrelated,  non-­‐smoking  sub-­‐ jects  >25  years  of  age,  were  recruit-­‐ ed  for  study  from  the  patient  pool  at   the  dental  clinics  of  the  faculty  of   dentistry  of  piracicaba  -­‐  unicamp.   the  patients  are  from  the  south-­‐ eastern  region  of  brazil.  the  base-­‐ line  clinical  parameters  for  the  sub-­‐ ject  population  are  presented  in  ta-­‐ ble  1.       all  subjects  were  in  good   general  health  and  had  at  least  20   teeth  in  the  mouth.  subjects  were   not  included  if  presented:  diseases   of  the  oral  hard  or  soft  tissues  ex-­‐ cept  caries  and  periodontal  disease;   use  of  orthodontic  appliances;  need   for  pre-­‐medication  for  dental   treatment;  chronic  usage  of  anti-­‐ inflammatory  drugs;  a  history  of   diabetes,  hepatitis  or  hiv  infection;   immunosuppressive  chemotherapy;   history  of  any  disease  known  to  se-­‐ verely  compromise  immune  func-­‐ tion;  present  acute  necrotizing  ul-­‐ cerative  gingivitis  or  current  preg-­‐ nancy  or  lactation.  subjects  com-­‐ pleted  personal  medical  and  dental   history  questionnaires,  and  within  a   protocol  approved  by  an  institu-­‐ tional  review  board,  signed  a  con-­‐ sent  form  after  being  advised  of  the   nature  of  the  study  (approved  by   the  ethical  committee  in  research   at  fop/unicamp,  protocol  63/99).     diagnosis  and  classification   of  disease  severity  were  made  on   the  basis  of  clinical  parameters  and   consisted  of  physical  examination,   medical  and  dental  history,  probing   depth,  assessment  of  clinical  at-­‐ tachment  loss  (cal),  tooth  mobility,   gingival  recession  and  observation   of  bleeding  on  probing,  according  to   lindhe  et  al.  [10].  measurements  of   probing  depth  and  attachment  level   were  recorded  at  6  points  around   each  tooth.  subjects  were  included   in  clinical  categories  according  to:       cp  severity:     1)    healthy  group:  subjects  found  to   exhibit  no  signs  of  periodontal  dis-­‐ ease  as  determined  by  the  absence   of  clinical  attachment  loss  and  no   sites  with  probing  depth  >3  mm  (n   =  44)  in  at  least  3  teeth  in  at  least  2   different  quadrants;   2)    moderate  periodontitis:  patients   with  tooth  sites  exhibiting  ≥3  mm   and  <7  mm  cal  (n  =  31)  in  at  least   3  teeth  in  at  least  2  different  quad-­‐ rants;   3)    severe  periodontitis:  patients   with  teeth  exhibiting  ≥7  mm  cal  (n   =  38)  in  at  least  3  teeth  in  at  least  2   different  quadrants.     sampling     the  sampling  of  epithelial   buccal  cells  was  performed  as  de-­‐ scribed  by  trevilatto  and  line  [11].   briefly,  113  individuals  undertook  a   mouthwash  after  1  min,  containing   5  ml  3%  glucose.  following  mouth-­‐ wash,  a  sterile  wood  spatula  was   used  to  scrape  oral  mucosa.  the  tip   of  the  spatula  was  then  shaken  into   the  retained  mouthwash  solution.   buccal  epithelial  cells  were  pelleted   by  centrifugation  at  2000  rpm  for   10  min.  the  supernatant  was  dis-­‐ carded  and  the  cell  pellet  resus-­‐ pended  in  500  μl  of  extraction  buff-­‐ er  [10  mm  tris-­‐hcl  (ph  7.8),  5  mm   edta,  0.5%  sds].  the  samples  were   then  frozen  at  -­‐20oc  until  used  for   dna  extraction.       dna  extraction   table 1. baseline clinical parameters of the subject population (n=113). healthy (n=44) moderate (n=31) severe (n=38) age (years) mean (± sd) 43.2 (±14.0) 36.9 (±11.2) 43.6 (±14.4) gender % female male 68.2 80.6 84.2 31.8 19.4 15.8 ethnic group % caucasian 84.1 77.4 68.4 afro-american 6.8 16.1 13.2 mulatto 6.8 6.5 18.4 japanese 2.3 0.0 0.0 polymorphism  in  the  tumor  necrosis  factor-­‐alpha  gene  (tnfa  -­‐308  g/a)  is  not     associated  with  suscep.bility  to  chronic  periodon..s  in  a  brazilian  popula.on   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.27    http://dentistry3000.pitt.edu   3     after  defrosting,   samples  were  incubated   overnight  (on)  with  100   ng/ml  proteinase  k  (sig-­‐ ma  chemical  co.,  st.  lou-­‐ is,  mo,  usa)  at  37°c  with   agitation.  dna  was  then   purified  by  sequential   phenol/chloroform  ex-­‐ traction  and  salt/ethanol   precipitation.  dna  was  dissolved  in   70  μl  te  buffer  [10  mm  tris  (ph   7.8),  1  mm  edta].  the  concentra-­‐ tion  was  estimated  by  measure-­‐ ments  of  od  260.     polymerase  chain  reaction  (pcr)   and  restriction  fragment  poly-­‐ morphism  (rflp)     polymorphism  in  the  tnf-­‐α   gene  at  position  -­‐308  (rs1800629)   the  oligonucleotides  5’-­‐ aggcaataggttttgagggccat-­‐3’   and  5’-­‐tcctccctgctccgattccg-­‐ 3’.  amplification  reactions  were   performed  with  500  ng  genomic   dna  in  a  total  volume  of  50  μl,  con-­‐ taining  10  mm  tris-­‐hcl  (ph  8.3),  50   mm  kcl,  2  μm  of  each  primer,  200   μm  each  datp,  dctp,  dgtp  and   dttp,  and  2.5  units  taq  dna  poly-­‐ merase  (amersham  pharmacia  bio-­‐ tech,  uppsala,  sweden).  cycling  was   2  cycles  of  3  min  at  94°c,  1  min  at   60°c  and  1  min  at  72°c,  followed  by   35  cycles  of  1  min  at  94°c,  1  min  at   60°c  and  1  min  at  72°c,  with  a  final   extension  of  7  min  at  72°c.  the   products  were  digested  with  3  u   per  25  μl  reaction  of  ncoi  at  37°c   on  to  detect  allele  1  [g]  (20  bp  +  87   bp)  and  allele  2  [a]  (107  bp).       electrophoresis  gel     restriction  products  were   visualized  by  electrophoresis  on   vertical  10%  non-­‐denaturing  poly-­‐ acrylamide  gels  in  1x  tbe  (89  mm   tris-­‐borate,  89  mm  boric  acid,  2   mm  edta),  followed  by  silver  stain-­‐ ing  (bio-­‐rad  silver  stain  kit).         statistical  analysis   the  allele  ratio  and  genotype  distri-­‐ bution  of  periodontitis  patients  and   healthy  control  subjects  were  ana-­‐ lyzed  with  chi-­‐square  test.  a  p-­‐ value  <0.05  was  considered  signifi-­‐ cant.         results       there  was  no  significant   difference  in  the  genotype  distribu-­‐ tion  (p=0.289)  between  healthy   control  and  periodontitis  patients  in   the  polymorphism  tnfa  (-­‐308),   neither  was  observed  statistical   variation  in  the  frequency  of  the   alleles  (p=0.674).  the  distribution   of  the  genotype  and  the  frequencies   of  the  alleles  for  the  polymorphism   studied  are  shown  in  tables  2  and  3,   respectively.  allele  2  was  carried  by   19.5%  (22/113)  of  the  subjects,   with  1.8%  (2/113)  of  homozygous.     the  allele  distribution  in  the   study  population  was  consistent   with  hardy-­‐weinberg  equilibrium.   the  moderate  and  severe  groups   (table  1)  were  combined  and  com-­‐ pared  to  the  healthy  group  in  order   to  seek  a  better  biological  explana-­‐ tion  of  the  results.  the  results  of   analyzes  of  the  additive  model  (p  =   0.503),  a  dominant  model  for  the  l   (p  =  0.559)  and  for  the  recessive   allele  g  (p  =  0.255)  allele  were  not   significant.       discussion       because  of  their  frequent   presence  in  diseased  sites,  it  has   been  postulated  that  cytokines  such   as  interleukin-­‐1  (il-­‐1)  and  tnf-­‐α   may  be  indicators  of  the  active   phase  of  periodontal  disease  [12].   tumor  necrosis  factor-­‐alpha,  a  pro-­‐ inflammatory  cytokine,  has  been   detected  in  gingival  crevicular  fluid   and  gingival  tissues  from  individu-­‐ als  with  periodontitis  [13].  this  cy-­‐ tokine  is  a  potent  immunologic  me-­‐ diator  that,  in  addition  to  its  in-­‐ flammatory  effects,  increases  bone   resorption  and  regulates  fibroblast   proliferation  [14].       a  number  of  single  nucleo-­‐ tide  polymorphisms  (snps)  has   been  identified  in  the  tnfa  pro-­‐ moter  and  associated  with  certain   chronic  inflammatory  diseases,  in-­‐ cluding  cerebral  malaria  [15],  mul-­‐ tiple  sclerosis  [16],  ulcerative  colitis   [17],  alzheimer's  disease  [18],   chronic  bronchitis  [19]  and  rheu-­‐ matoid  arthritis  [20].  in  cerebral   malaria  tnf-­‐α  is  of  major  im-­‐ portance  for  the  pathology  [15]  and   individuals  homozygous  for  the   tnfa  (-­‐308)  a  allele  carry  a  7-­‐fold   higher  risk  of  death  or  severe  neu-­‐ rological  sequelae  due  to  cerebral   malaria.     in  this  study,  no  association   of  tnfa  (-­‐308)  polymorphism  with   cp  was  found.  however,  in  another   brazilian  population,  trombone  et   al.  [9]  found  a  frequency  (17.7%)  of   the  tnfa  (-­‐308)  a  allele  in  the  cp   group.  kornman  et  al.  [21]  found  an   allele  a  carriage  rate  of  28.5%  at   table 2. distribution of the tnf-α genotypes in the healthy group and in groups with moderate and severe chronic periodontitis. group gg ga aa p value healthy 37 (84.0) 7 (16.0) 0 (0.0) 0.289 moderate 24 (77.4) 7 (22.5) 0 (0.0) severe 31 (81.6) 0 (0.0) 2 (5.3) polymorphism  in  the  tumor  necrosis  factor-­‐alpha  gene  (tnfa  -­‐308  g/a)  is  not     associated  with  suscep.bility  to  chronic  periodon..s  in  a  brazilian  popula.on   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.27    http://dentistry3000.pitt.edu   4   tnfa  (-­‐308)  locus  in  north-­‐ american  caucasians,  but  no  asso-­‐ ciation  with  cp.  we  found  an  allele   a  carriage  rate  of  20.7%  (18/87)  for   our  caucasians.       although  the  study  sample   was  mostly  composed  by  cauca-­‐ sians,  the  brazilian  white  popula-­‐ tion  is  heterogeneous.  recent  arti-­‐ cle  has  not  suggested  grouping  bra-­‐ zilians  into  ethnic  groups  based  on   color,  race  and  geographical  origin   because  brazilian  individuals  classi-­‐ fied  as  white  or  black  have  signifi-­‐ cantly  overlapping  genotypes,   probably  due  to  miscegenation  [22].   reporting  the  caucasoid  popula-­‐ tion,  there  is  a  predominance  of  ital-­‐ ian,  spanish,  and  portuguese  herit-­‐ age.  in  a  meta-­‐analysis  by  ni-­‐ kolopoulos  et  al.  [23]  the  great  ma-­‐ jority  of  studies  were  carried  out  in   caucasian  populations  and  no  asso-­‐ ciation  of  tnfa  (-­‐308)  was  found   neither  with  aggressive  nor  chronic   periodontal  diseases.  a  lack  of  asso-­‐ ciation  of  this  polymorphism  in  pa-­‐ tients  with  early-­‐onset  periodontitis   was  also  found  in  a  japanese  popu-­‐ lation  [24].  however,  lin  et  al.  [25]   observed  that  allele  a  may  increase   the  odds  of  having  moderate-­‐to-­‐ advanced  aggressive  periodontitis.   galbraith  et  al.  [26]  reported  the   frequency  of  tnfa  (-­‐308)  allele  g   significantly  greater  in  patients  with   advanced  periodontitis.  recently,  a   new  meta-­‐analysis  study  was  per-­‐ formed  [27]  and  stratification  by   ethnicity  showed  that  the  a  allele   was  associated  with  periodontitis  in   brazilians,  in  spite  of  the  lack  of  as-­‐ sociation  showed  in  the  studies  in-­‐ cluded  in  the  meta-­‐analysis  for  cp   [28-­‐30].     there  has  been  an  evidence   that  allele  a  is  over-­‐represented  in   diseases  where  tnf-­‐α  levels  are   associated  with  poor  prognosis   [31].  allele  a  of  tnfa  (-­‐308)  poly-­‐ morphism  seems  to  be  associated  to   increase  in  the  production  of  tnf-­‐α   cytokine  [9].  although  the  role  tnf-­‐ α  plays  in  leading  to  tissue  destruc-­‐ tion,  it  is  worth  mentioning  the   need  for  evaluation  of  other  poten-­‐ tial  candidate  genes  as  contributors   to  periodontitis,  since  chronic  peri-­‐ odontitis  may  possibly  represent  a   polygenic  disease.       analysis  in  some  of  other   study  cytokine  genes  (il4,  mmp1,   vdr,  il1)  revealed  positive  associa-­‐ tions  with  chronic  periodontitis  in   the  same  brazilian  patients  [32-­‐35].   those  studies  may  provide  pieces  of   information  with  regard  to  which   factors  could  indeed  be  implicated   in  the  process  of  disease  in  this   population.  in  spite  of  the  reduced   number  of  individuals  in  each   group,  it  seems  that  this  sample  was   composed  of  well  characterized   phenotypes,  once  positive  associa-­‐ tions  could  be  identified  for  some   gene  polymorphisms  in  previous   studies  [33,  35,  36].  besides,  only  a   small  functional  part  of  the  gene   was  investigated,  which  does  not   mean  that  this  gene  could  not  be   involved  in  the  susceptibility  con-­‐ trol  of  chronic  periodontitis.  thus,   an  investigation  of  tag  snps,  which   physically  represent  the  gene  as  a   whole,  should  be  recommended.     it  was  concluded  that  tnfa   (-­‐308)  polymorphism  was  not  asso-­‐ ciated  with  chronic  periodontitis  in   the  population  studied.       acknowledgements       this  study  was  supported   by  fapesp  grants  99/05610-­‐2  and   99/06094-­‐8.  there  are  no  conflicts   of  interest  to  report.     references   1.  proinflammatory  cytokines  and   osteoporosis.  mclean  rr.  curr  os-­‐ teoporos  rep.  2009  dec;7:134-­‐9.   pmid:  19968917.           2.  an  allelic  polymorphism  within   the  human  tumor  necrosis  factor   alpha  promoter  region  is  strongly   associated  with  hla  a1,  b8,  and   dr3  alleles.  wilson  ag,  de  vries  n,   pociot  f,  di  giovine  fs,  van  der   putte  lb,  duff  gw.  j  exp  med.  1993   feb;177:557-­‐60.  pmid:  8426126.       3.  effects  of  a  polymorphism  in  the   human  tumor  necrosis  factor  alpha   promoter  on  transcriptional  activa-­‐ tion.  wilson  ag,  symons  ja,   mcdowell  tl,  mcdevitt  ho,  duff   gw.  proc  natl  acad  sci  usa.  1997   apr;94:3195-­‐9.  pmid:  9096369.       4.  the  specific  features  of  tnf-­‐ alpha  gene  polymorphism  in  asth-­‐ matic  patients  and  their  relatives.   cherkashina  ii,  nikulina  s,  log-­‐ vinenko  ni,  voevoda  mi,  maksimov   vn,  liberdovskaia  ed.  probl  tuberk   bolezn  legk.  2009;8:51-­‐6.  pmid:   19803352.       5.  polymorphisms  in  the  promoter   region  of  tumor  necrosis  factor  al-­‐ pha  (tnf-­‐alpha)  and  the  hla-­‐drb1   locus  in  mexican  mestizo  patients   table 3. distribution of the tnf-α alleles in healthy, moderate and severe groups with chronic periodontitis. allele healthy n (%) moderate n (%) severe n (%) p value g 81 (92.0) 55 (88.7) 67 (88.0) 0.674 a 7 (8.0) 7 (11.3) 9 (12.0) polymorphism  in  the  tumor  necrosis  factor-­‐alpha  gene  (tnfa  -­‐308  g/a)  is  not     associated  with  suscep.bility  to  chronic  periodon..s  in  a  brazilian  popula.on   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.27    http://dentistry3000.pitt.edu   5   with  ulcerative  colitis.  yamamoto-­‐ furusho  jk,  uscanga  lf,  vargas-­‐ alarcón  g,  rodríguez-­‐pérez  jm,  zu-­‐ ñiga  j,  granados  j.  immunol  lett.   2004;95:31-­‐5.  pmid:  15325795.       6.  prevalence  of  bone  loss  in  a   group  of  brazilian  teenagers  as-­‐ sessed  on  bite-­‐wing  radiographs.   gjermo,  p.  bellini,  h.t.  pereira  san-­‐ tos  v,  martins  jg,  ferracyoli  jr.  j   clin  periodontol.  1984;11:104-­‐13.   pmid:  6583209.       7.  periodontal  conditions  and   treatment  needs  (cpitn)  in  a  work-­‐ er  population  in  araraquara,  sp,   brazil.  dini  el,  guimaraes  lo.  int   dent  j.  1994;44:309-­‐11.  pmid:   7822055.       8.  quantitative  assessment  of  in-­‐ flammatory  cytokine  gene  expres-­‐ sion  in  chronic  adult  periodontitis.   roberts  fa,  hockett  rd,  bucy  rp,   michalek  sm.  oral  microbiol  immu-­‐ nol.  1997;12:336-­‐44.  pmid:   9573807.       9.  tumor  necrosis  factor-­‐alpha  -­‐ 308g/a  single  nucleotide  polymor-­‐ phism  and  red-­‐complex  periodon-­‐ topathogens  are  independently  as-­‐ sociated  with  increased  levels  of   tumor  necrosis  factor-­‐alpha  in  dis-­‐ eased  periodontal  tissues.  trom-­‐ bone  ap,  cardoso  cr,  repeke  ce,   ferreira  sb,  martins  w,  campanelli   ap,  avila-­‐campos  mj,  trevilatto  pc,   silva  js,  garlet  gp.  j  periodontal  res.   2009;44:598-­‐608.  pmid:  19076989.       10.  consensus  report:  chronic  per-­‐ iodontitis.  lindhe  j,  lamster  ri,   charles  a,  chung  cp,  flemmig  t,   kinane  d.  ann  periodontol.   1999;4:38.   11.  use  of  buccal  epithelial  cells  for   pcr  amplification  of  large  dna   fragments.  trevilattopc,  line  srp.  j   forensic  odontostomatol.   2000;18:6-­‐9.  pmid:  11324090.       12.  gene  polymorphisms  in  pro-­‐ inflammatory  cytokines  are  associ-­‐ ated  with  systemic  inflammation  in   patients  with  severe  periodontal   infections.  d'aiuto  f,  parkar  m,   brett  pm,  ready  d,  tonetti  ms.  cy-­‐ tokine.  2004;28:29-­‐34.  pmid:   15341923.       13.  role  of  cytokines  and  inflamma-­‐ tory  mediators  in  tissue  destruc-­‐ tion.  birkedal-­‐hansen  h.  j  periodon-­‐ tal  res.  1993;28:500-­‐10.  pmid:   8263720.       14.  role  of  cytokines  in  bone  re-­‐ sorption.  manolagas  sc.  bone.  1995;   17:63s-­‐67s.  pmid:  8579900.       15.  cytokines  and  adhesion  mole-­‐ cules  expression  in  the  brain  in  hu-­‐ man  cerebral  malaria.  armah  h,   wired  ek,  dodoo  ak,  adjei  aa,   tettey  y,  gyasi  r.  int  j  environ  res   public  health.  2005;2:123-­‐31.   pmid:  16705810.       16.  il-­‐1,  il-­‐1r  and  tnfalpha  gene   polymorphisms  in  iranian  patients   with  multiple  sclerosis.  sarial  s,   shokrgozar  ma,  amirzargar  a,   shokri  f,  radfar  j,  zohrevand  p,  ar-­‐ jang  z,  sahraian  ma,  lotfi  j.  iran  j   allergy  asthma  immunol.   2008;7:37-­‐40.  pmid:  18322311.       17.  effect  of  the  polymorphism  of   tumor  necrosis  factor-­‐alpha-­‐308   g/a  gene  promoter  on  the  suscepti-­‐ bility  to  ulcerative  colitis:  a  meta-­‐ analysis.  lu  z,  chen  l,  li  h,  zhao  y,   lin  l.  digestion.  2008;78:44-­‐51.   pmid:  18827481.       18.  systematic  review  by  meta-­‐ analyses  on  the  possible  role  of   tnf-­‐alpha  polymorphisms  in  asso-­‐ ciation  with  alzheimer's  disease.  di   bona  d,  candore  g,  franceschi  c,   licastro  f,  colonna-­‐romano  g,   cammà  c,  lio  d,  caruso  c.  brain  res   rev.  2009;61:60-­‐8.  pmid:   19445962.       19.  nuclear  factor  kappab-­‐mediated   down-­‐regulation  of  adhesion  mole-­‐ cules:  possible  mechanism  for  in-­‐ hibitory  activity  of  bigelovin  against   inflammatory  monocytes  adhesion   to  endothelial  cells.  nam  kw,  oh  gt,   seo  ek,  kim  kh,  koo  u,  lee  sj,  mar   w.  j  ethnopharmacol.   2009;123:250-­‐6.  pmid:  19429369.       20.  associations  between  tumor   necrosis  factor-­‐{alpha}  (tnf-­‐ {alpha})  -­‐308  and  -­‐238  g/a  poly-­‐ morphisms  and  shared  epitope  sta-­‐ tus  and  responsiveness  to  tnf-­‐ {alpha}  blockers  in  rheumatoid  ar-­‐ thritis:  a  metaanalysis  update.  lee   yh,  ji  jd,  bae  sc,  song  gg.  j  rheu-­‐ matol.  2010;37:740-­‐6.  pmid:   20194454.       21.  the  interleukin-­‐1  genotype  as  a   severity  factor  in  adult  periodontal   disease.  kornman  ks,  crane  a,   wang  hy,  di  giovine  fs,  newman   mg,  pirk  fw,  wilson  jr  tg,  hig-­‐ ginbottom  fl,  duff  gw.  j  clin  perio-­‐ dontol.  1997;24:72-­‐77.  pmid:   9049801.       22.  color  and  genomic  ancestry  in   brazilians.  parra,  f.c.  amado,  r.c.   lambertucci  jr,  rocha  j,  antunes   cm,  pena  sd.  proceedings  of  the  na-­‐ polymorphism  in  the  tumor  necrosis  factor-­‐alpha  gene  (tnfa  -­‐308  g/a)  is  not     associated  with  suscep.bility  to  chronic  periodon..s  in  a  brazilian  popula.on   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.27    http://dentistry3000.pitt.edu   6   tional  academy  of  sciences  of  the   united  states  of  america.   2003;100:177-­‐82.  pmid:  12509516.       23.  cytokine  gene  polymorphisms   in  periodontal  disease:  a  meta-­‐ analysis  of  53  studies  including   4178  cases  and  4590  controls.  ni-­‐ kolopoulos  gk,  dimou  nl,  hamo-­‐ drakas  sj,  bagos  pgj.  clin  periodon-­‐ tol.  2008;35:754-­‐67.  pmid:   18673406.       24.  analysis  of  single  nucleotide   polymorphisms  in  the  5'-­‐flanking   region  of  tumor  necrosis  factor-­‐ alpha  gene  in  japanese  patients   with  early-­‐onset  periodontitis.  endo   m,  tai  h,  tabeta  k,  kobayashi  t,   yamazaki  k,  yoshie  h.  j  periodontol.   2011;72:1554-­‐9.  pmid:  11759867.       25.  study  on  the  correlation  of  cyto-­‐ kine  gene  polymorphism  with   chronic  periodontitis.  lin  l,  pan  yp,   yin  ly.  shanghai  kou  qiang  yi  xue.   2003;12:456-­‐9.  pmid:  14966591.       26.  polymorphic  cytokine  geno-­‐ types  as  markers  of  disease  severity   in  adult  periodontitis.  galbraith   gmp,  hendley  tm,  sanders  jj,   palesch  y,  pandey  jp.  j  clin  perio-­‐ dontol.  1999;  26:705-­‐9.  pmid:   10589805.       27.  association  between  tumor  ne-­‐ crosis  factor-­‐α  promoter  -­‐308  a/g,  -­‐ 238  a/g,  interleukin-­‐6  -­‐174  g/c   and  -­‐572  g/c  polymorphisms  and   periodontal  disease:  a  meta-­‐ analysis.  song  gg,  choi  sj,  ji  jd,  lee   yh.  mol  biol  rep.  2013;40:5191-­‐ 203.  pmid:  23657600.       28.  interleukin-­‐6  (g-­‐174c)  and  tu-­‐ mour  necrosis  factor-­‐alpha  (g-­‐ 308a)  gene  polymorphisms  in  geri-­‐ atric  patients  with  chronic  perio-­‐ dontitis.  costa  am,  guimarães  mc,   de  souza  er,  nóbrega  ot,  bezerra   ac.  gerodontology.  2010;27(1):70-­‐ 5.  pmid:  19555357.       29.  lack  of  association  between  the   tnf-­‐alpha  -­‐308  (g/a)  genetic  pol-­‐ ymorphism  and  periodontal  disease   in  brazilians.  menezes  ng,  colombo   ap.  braz  oral  res.  2008;22(4):322-­‐ 7.  pmid:  19148387.       30.  analysis  of  il-­‐1a(-­‐889)  and   tnfa(-­‐308)  gene  polymorphism  in   brazilian  patients  with  generalized   aggressive  periodontitis.  maria  de   freitas  n,  imbronito  av,  neves  ac,   nunes  fd,  pustiglioni  fe,  lotufo  rf.   eur  cytokine  netw.   2007;18(3):142-­‐7.  pmid:   17823082.       31.  tnf  concentration  in  fatal  cere-­‐ bral,  non-­‐fatal  cerebral,  and  un-­‐ complicated  plasmodium  falcipa-­‐ rum  malaria.  kwiatkowski  d,  hill   av,  sambou  i,  twumasi  p,  castra-­‐ cane  j,  manogue  kr,  cerami  a,   brewster  dr,  greenwood  bm.  lan-­‐ cet.  1990;336:1201-­‐4.  pmid:   1978068.       32.  frequencies  of  the  -­‐330  (t  -­‐-­‐>  g)   il-­‐2  and  -­‐590  (t  -­‐-­‐>  c)  il-­‐4  gene   polymorphisms  in  a  population   from  south-­‐eastern  brazil.  scarel-­‐ caminaga  rm,  trevilatto  pc,  souza   ap,  brito  rb,  line  sr.  eur  j  immu-­‐ nogenet.  2002;29:293-­‐6.  pmid:   12121273.       33.  mmp-­‐1  promoter  polymor-­‐ phism:  association  with  chronic   periodontitis  severity  in  a  brazilian   population.  de  souza  ap,  trevilatto   pc,  scarel-­‐caminaga  rm,  de  brito   rb,  line  sr.  j  clin  periodontol.   2003;30:154-­‐8.  pmid:  12622858.       34.  polymorphisms  in  the  vitamin  d   receptor  gene  are  associated  with   periodontal  disease.  brito  júnior   rb,  scarel-­‐caminaga  rm,  trevilatto   pc,  de  souza  ap,  barros  sp.  j  perio-­‐ dontol.  2004;75:1090-­‐95.  pmid:   15455736.       35.  association  of  il1  gene  poly-­‐ morphisms  with  chronic  periodon-­‐ titis  in  brazilians.  trevilatto  pc,  de   souza  pardo  ap,  scarel-­‐caminaga   rm,  de  brito  jr  rb,  alvim-­‐pereira  f,   alvim-­‐pereira  cc,  probst  cm,  garlet   gp,  sallum  aw,  line  sr.  arch  oral   biol.  2011;56:54-­‐2.  pmid:   20934174.       36.  polymorphism  at  position  -­‐174   of  il-­‐6  gene  is  associated  with  sus-­‐ ceptibility  to  chronic  periodontitis   in  a  caucasian  brazilian  population.   trevilatto  pc,  scarel-­‐caminaga  rm,   de  brito  rb  jr,  de  souza  ap,  line  sr.   j  clin  periodontol.  2003;30(5):438-­‐ 42.  pmid:  12716337.         microsoft word 73 2017.docx   vol  5,  no  1  (2017)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2017.73           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     behavioral  genetics  and  thumb  sucking  in  adolescents     jessica  hatala1      1university  of  pi.sburgh,  school  of  dental  medicine abstract   thumb  sucking  is  a  common  habit  developed  by  children  and  begins  as  early  as  in  utero.  however,  it  becomes  prob-­‐ lema%c  when  a  child  con%nues  to  suck  their  thumb  past  the  age  of  4  years,  when  their  secondary  den,,on  is  develop-­‐ ing   and  preparing   to   erupt.   prolonged   thumb   sucking   into   adolescence   can  have  deleterious   effects  on  dental   and   skeletal   structures   based   on   the   dura0on   and   how   frequent   the   child   engages   in   this   nonnutri0ve   sucking   habit.     thumb  sucking  can  lead  to  various  types  of  effects  such  as  increased  overjet,  anterior  open  bite,  posterior  cross  bite,   maxillary  arch  constric-on,  high  palatal  vault,  and  class   ii  malocclusion.  this  paper  presents  a  case  study,  which  fo-­‐ cuses  on  chronic   thumb  sucking   in  a   family,   the  dental  and  skeletal  changes   that   the   family  members  experienced,   and  whether  or  not  this  prolonged  behavior  can  be  gene2cally  influenced.  based  on  this  case  study  and  the  field  of   behavioral  gene-cs  it  is  possible  that  there  can  be  a  gene)c  component  to  the  dura)on  of  thumb  sucking  into  adoles-­‐ cence.   cita%on:   hatala,   j.   (2017)   behavioral   gene-cs   and   thumb   sucking   in   adolescents.   den$stry   3000.  1:a001  doi:10.5195/d3000.2017.73   received:    may  5,  2017   accepted:    may  26,  2017   published:    june  19,  2017   copyright:  ©2017  hatala,   j.  this   is   an  open  ac-­‐ cess  ar!cle   licensed  under  a  crea!ve  commons   a"ribu%on  work  4.0  united  states  license.   email:  jeh193@pi*.edu   introduction   nonnutritive  sucking  is  a   very  common  habit  that  occurs  in   children  and  can  persist  through   adolescence  and  sometimes  even   adulthood.    studies  have  suggest-­‐ ed  that  70-­‐90%  of  children  take   part  in  some  form  of  nonnutritive   sucking  in  their  lifetime  [1].     nonnutritive  sucking  can  include   thumb  and  finger  sucking  and  pac-­‐ ifier  use,  with  thumb  sucking  being   the  most  prevalent.  evidence  has   shown  that  thumb  sucking  can   start  as  early  as  29  weeks  of  gesta-­‐ tion.  at  birth,  sucking  is  a  natural   reflex  for  newborns  as  it  enables   the  child  to  breastfeed.  however,   it  can  be  engrained  in  a  habit  that   is  very  hard  to  break  for  some   children.  most  children  stop  suck-­‐ ing  their  thumbs  by  the  age  of  4,   however  some  children  continue   to  suck  their  thumbs  into  adoles-­‐ cence  and  sometimes  even  into   adulthood.     even  though  thumb  suck-­‐ ing  in  infants  and  toddlers  is  nor-­‐ mal,  prolonged  thumb  sucking  has   been  shown  to  have  deleterious   effects  on  the  occlusion  of  chil-­‐ dren  and  adolescents.  primary,   mixed,  and  secondary  dentition   can  all  be  affected.    the  effects  on   the  dentition  and  maxillofacial   structures  varies  between  individ-­‐ uals  due  to  the  frequency  and  du-­‐ ration  of  the  thumb  sucking  in  that   individual.     thumb  sucking  can  be  a  re-­‐ sult  of  environmental  factors,  but   there  has  been  some  speculation   that  prolonged  thumb  sucking  into   adolescence  may  have  some  ge-­‐ netic  influence.  behavioral  genet-­‐ ics  is  a  branch  of  genetics  that  fo-­‐ cuses  on  how  behavior  and  per-­‐ sonality  can  be  influenced  by   genes.  this  paper  focuses  on   thumb  sucking,  the  deleterious   effects  it  can  cause  to  the  oral  and   maxillofacial  structures,  and  how   it  may  be  associated  with  behav-­‐ ioral  genetics.     malocclusion   chronic  nonnutritive  suck-­‐ ing,  particularly  thumb  sucking  can   have  some  deleterious  effects  on   the  primary,  mixed,  and  perma-­‐ nent  dentitions.  studies  have   shown  that  in  the  primary  denti-­‐ tion,  nonnutritive  sucking  habits   were  associated  with  anterior   open  bite,  increased  overjet,  and   class  ii  canine  and  molar  relation-­‐ ships  [2].  several  studies  have  also   shown  that  prolonged  nonnutri-­‐ tive  sucking  can  cause  decreased   maxillary  arch  width  and  an  in-­‐ creased  palatal  depth.  a  cohort   study  conducted  by  warren  et  al   [2]  consisted  of  sending  question-­‐ naires  to  mothers  regarding   whether  or  not  their  child  took   part  in  any  nonnutritive  sucking   habit.  questionnaires  were  sent   out  at  3,  6,  9,  12,  and  20  months   behavioral  gene-cs  and  thumb  sucking  in  adolescents     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.73    http://dentistry3000.pitt.edu   to  assess  nutritive  and  nonnutri-­‐ tive  behaviors.  questionnaires   were  subsequently  sent  out  at  24   months  and  then  yearly  thereaf-­‐ ter.  study  models  of  children  4-­‐5   years  of  age  were  then  studied  to   looks  for  any  type  of  malocclusion.   children  who  continued  their  digit   habits  until  48  months  of  age  or   older  presented  with  more  con-­‐ stricted  maxillary  arch  widths,  in-­‐ creased  maxillary  arch  depth,  in-­‐ creased  overjet,  and  anterior  open   bite  compared  to  children  with  a   shorter  habit  duration  or  those   who  had  no  habit  at  all.     studies  focusing  on  the   mixed  dentition  are  less  common   than  the  primary  dentition  be-­‐ cause  children  should  normally   stop  any  form  of  nonnutritive   sucking  before  the  age  of  4.  how-­‐ ever,  the  few  studies  that  were   conducted  show  that  there  is  an   increase  in  malocclusion  in  those   who  continue  to  suck  their  thumb   into  their  mixed  dentition  stage.   common  forms  of  malocclusion   that  are  often  seen  in  the  mixed   dentition  stage  include  anterior   open  bite,  posterior  cross  bite,  bi-­‐ lateral  class  ii  molar  relationship,   and  overjet  >4  mm  [3].  in  a  longi-­‐ tudinal  study  conducted  by  war-­‐ ren  et  al.  [3],  dental  examinations   were  conducted  on  630  children,   and  524  sufficient  study  models   were  obtained  in  order  to  evaluate   the  effects  of  chronic  nonnutritive   sucking  habits  on  the  mixed  denti-­‐ tion.  the  children  in  the  study   were  7-­‐11  years  of  age,  with  al-­‐ most  90%  of  those  children  being   8  or  9  years  old.  fifty-­‐five  percent   of  the  children  had  some  sort  of   malocclusion.  the  study  showed   that  for  anterior  open  bite  and   posterior  cross  bite,  the  preva-­‐ lence  increased  significantly  with  a   longer  duration  of  digit  sucking.  in   addition,  there  was  a  significant   increase  in  the  prevalence  of  a   class  ii  type  malocclusion.  the   same  dental  and  skeletal  manifes-­‐ tations  can  then  persist  into  the   permanent  dentition  phase.   behavioral  genetics     it  has  been  speculated  that   human  behavior  and  personality   are  influenced  by  our  genetic   makeup.    the  study  of  behavioral   genetics  has  been  a  topic  of  inter-­‐ est  among  many  researchers.  in   the  case  of  thumb  sucking,  genes   can  be  influencing  the  behavior  of   children  and  can  cause  some  chil-­‐ dren  to  suck  their  thumbs  for  a   prolonged  period  of  time.  children   usually  suck  their  thumbs  when   they  are  bored,  stressed,  anxious,   or  nervous.  according  to  lubitz   [4],  children  suck  their  thumbs  due   to  developmental  problems  and   environmental  disturbances  and   that  thumb  sucking  is  a  way  of  re-­‐ laxation  for  many  children.  our   genetic  composition  can  make   some  people  more  susceptible  to   these  emotions  than  others  and   can  lead  to  differences  in  behav-­‐ ior.     children  usually  stop  suck-­‐ ing  their  thumbs  by  the  age  of  4   years.  however,  a  child  can  con-­‐ tinue  to  suck  their  thumbs  into   adolescence.  adolescence  is  a  de-­‐ velopmental  period  that  marks   many  changes  in  physical  growth,   maturation,  and  psychological  de-­‐ velopment  [5].  the  children  who   do  suck  their  thumbs  past  the  age   of  4  years  usually  stop  sucking   their  thumbs  during  adolescence.   rarely,  do  people  suck  their   thumbs  into  adulthood.  it  is  possi-­‐ ble  that  people  stop  sucking  their   thumbs  during  adolescence  be-­‐ cause  they  are  transitioning  into   being  their  own  person.  it  is  possi-­‐ ble  that  behavioral  genetics  is   causing  a  switch  in  the  decision-­‐ making  process  in  those  individu-­‐ als.  a  study  by  dick  et  al  [5]  focus-­‐ es  on  adolescents  and  how  their   genes  can  affect  their  behavior   and  decision  making  processes.   they  looked  at  literature  related   to  alcohol  use  in  adolescents  and   then  concluded  that  there  is  a  ge-­‐ netic  association  with  their  deci-­‐ sion-­‐making  process.  they  believe   during  that  period  in  their  lives,   adolescents  are  becoming  their   own  person  and  are  more  likely  to   experiment  with  different  things   like  alcohol  use  and  cigarette  use.   this  study  isn’t  related  to  thumb   sucking,  but  it  can  be  used  to   show  that  adolescence  is  a  very   dynamic  period  where  individuals   are  trying  to  become  their  own   person  and  change  their  behav-­‐ iors.  that  could  be  a  possible  ex-­‐ planation  why  people  who  suck   behavioral  gene-cs  and  thumb  sucking  in  adolescents     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.73    http://dentistry3000.pitt.edu   their  thumbs  for  a  prolonged  peri-­‐ od  of  time  discontinue  sucking   their  thumb  during  adolescence.     most  of  the  studies  that   looked  at  behavior  and  genetics   consisted  of  twin  study  models.   these  models  allowed  researchers   to  determine  whether  genetics  is   responsible  for  the  behavior  or  if  it   is  mainly  the  environment  that  is   influencing  the  behavior.  in  these   studies,  if  dizygotic  twin  behavior   is  very  similar  to  monozygotic  twin   behavior,  their  environment  could   play  a  larger  role  in  the  develop-­‐ ment  of  that  particular  behavior   [5].  on  the  other  hand,  if  a  behav-­‐ ior  is  seen  more  in  monozygotic   twins  than  dizygotic  twins,  genet-­‐ ics  is  the  most  likely  explanation   since  they  have  the  same  dna.  a   japanese  twin  study  by  ooki  et  al   [6]  evaluated  the  association  of   genetics  with  thumb  sucking  and   nail  biting  in  monozygotic  and   dizygotic  twins.    the  results  of  this   study  have  shown  a  greater  preva-­‐ lence  of  thumb  sucking  in   monozygotic  twins  than  dizygotic   twins,  suggesting  that  there  is   most  likely  a  genetic  component   to  thumb  sucking.   case  study   a  family  where  chronic   thumb  sucking  was  common  was   studied  to  show  if  there  is  a  genet-­‐ ic  component  for  thumb  sucking  in   families.  chronic  thumb  sucking  in   this  study  was  defined  by  an  indi-­‐ vidual  sucking  their  past  the  age  of   4  years  old.  the  study  was  per-­‐ formed  by  interviewing  the  family   members  that  were  chronic   thumb  suckers  and  observing  their   dentition  and  skeletal  orientation.   the  family  members  were  asked   what  age  did  they  discontinue   sucking  their  thumb,  whether  or   not  they  had  undergone  ortho-­‐ dontic  therapy,  and  what  made   them  discontinue  this  habit.  in  ad-­‐ dition,  the  dental  and  skeletal  re-­‐ lationship  was  observed  to  deter-­‐ mine  whether  they  had  any  sort  of   malocclusion.     the  family  members  who  chroni-­‐ cally  sucked  their  thumb  included   the  proband  in  the  third  genera-­‐ tion,  a  paternal  aunt  in  the  second   generation,  and  a  paternal  first   cousin  in  the  third  generation.  on-­‐ ly  females  were  shown  to  suck   their  thumb  for  a  prolonged  peri-­‐ od  of  time.  two  of  the  three  rela-­‐ tives  presented  with  signs  of  mal-­‐ occlusion  or  changes  to  their  skel-­‐ etal  structures.         the  proband  is  a  23  year-­‐ old  caucasian  female.  she  discon-­‐ tinued  sucking  her  thumb  at  the   age  of  12  years.  the  patient  cur-­‐ rently  presents  with  a  class  i  oc-­‐ clusion  and  straight  profile.  the   proband’s  occlusion  is  normal  and   ideal.  however,  the  proband  had   undergone  orthodontic  treatment   at  the  age  of  13  years  to  correct   her  crowding.  before  orthodontic   treatment,  the  proband  had  in-­‐ creased  maxillary  overjet.  the   proband  currently  has  a  very  high   palatal  vault,  which  is  a  common   finding  in  individuals  with  a  chron-­‐ ic  history  of  thumb  sucking  (figure   1).  force  of  the  thumb  onto  the   palate  during  growth  and  devel-­‐ opment  causes  the  maxillary  vault   to  increase  in  depth.  the  proband   stopped  sucking  her  thumb  at  the   age  of  12  years  due  to  peer  ac-­‐ ceptance.  she  was  going  into  mid-­‐ dle  school  and  did  not  want  any-­‐ one  to  see  her  and  make  fun  of   her.  also,  she  was  planned  to  start   orthodontic  treatment  in  the  next   year.       the  proband’s  paternal   aunt  is  a  57  year-­‐old  caucasian   female.  she  stopped  sucking  her   thumb  at  the  age  of  15  years.  the   behavioral  gene-cs  and  thumb  sucking  in  adolescents     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.73    http://dentistry3000.pitt.edu   subject  presents  with  a  unilateral   posterior  cross  bite  on  her  left  side   with  her  mandibular  premolars   and  molars  positioned  in  front  of   her  maxillary  premolars  and  mo-­‐ lars  (figure  2).  the  subject  also   presents  with  a  very  constricted   and  high  maxillary  palatal  vault.  in   addition,  the  subject  presents  with   an  angle’s  class  ii  malocclusion,   with  her  mandible  being  slightly   retrognathic  (figure  3).  the  class  ii   malocclusion  is  apparent  when   you  observe  the  position  of  her   canines  as  seen  in  the  pictures  be-­‐ low.  her  canines  line  up  instead  of   her  mandibular  canine  being   slightly  ahead  of  the  maxillary  ca-­‐ nine  as  seen  in  an  ideal  class  i  oc-­‐ clusion.  the  subject  never  re-­‐ ceived  any  orthodontic  treatment   to  correct  her  malocclusion.    she   discontinued  sucking  her  thumb  at   the  age  of  15  because  she  didn’t   want  people  to  make  fun  of  her.   she  was  able  to  break  this  long-­‐ term  habit  with  the  use  of  a  pala-­‐ tal  appliance.   the  next  subject  in  the   study  is  the  proband’s  first  cousin,   a  17  year-­‐old  caucasian  female.   she  stopped  sucking  her  thumb  at   the  age  of  10  years.  she  presents   with  no  forms  of  dental  and  skele-­‐ tal  malocclusion.  she  had  never   had  an  orthodontic  treatment  per-­‐ formed  on  her.  this  is  most  likely   because  she  did  not  suck  her   thumb  as  long  as  the  other  sub-­‐ jects  in  the  study.  the  subject   stated  that  she  stopped  sucking   her  thumb  for  social  acceptance   and  so  people  would  not  make  fun   of  her.       by  looking  at  the  pedigree   on  the  next  page  and  seeing  that   three  people  on  one  side  of  the   family  all  sucked  their  thumbs  into   adolescence,  there  can  be  possible   genetic  component  to  this  distri-­‐ bution  of  traits  in  a  family  (figure   4).    the  field  of  genetics  that  is   most  probable  for  influencing  this   behavior  is  behavioral  genetics.   there  is  very  little  research  on   chronic  thumb  sucking  and  behav-­‐ ioral  genetics.  however,  it  can  be  a   possible  explanation  for  the  preva-­‐ lence  of  this  habit  in  families.      the   most  likely  mode  of  inheritance   for  this  trait  is  a  multifactorial   mode  of  inheritance  where  both   genes  and  environment  play  a   role.  the  genes  involved  are  most   likely  genes  that  affect  our  per-­‐ sonality  and  decision  making.   children  often  suck  their  thumbs   in  the  presence  of  stress,  anxiety,   and  nervousness.  it  is  possible  that   the  subjects  in  the  study  had  an   increased  level  of  stress  which   could  be  influenced  by  genetics.   the  family  members  who  had  this   habit  all  stopped  sucking  their   thumbs  during  adolescence.  ado-­‐ lescence  is  a  transitional  period   where  children  are  starting  to   make  their  own  decisions  and  try-­‐ ing  to  become  more  independent.   in  all  three  cases,  the  subjects   wanted  to  stop  sucking  their   thumbs  for  peer  acceptance.  per-­‐ haps,  behavioral  genes  are  affect-­‐ ing  the  decision  making  process,   and  that  is  why  the  family  mem-­‐ bers  discontinued  sucking  their   thumbs  during  adolescence.   conclusion   nonnutritive  sucking  is  a   common  phenomenon  that  occurs   in  young  children,  but  when  the   habit  becomes  engrained,  the  in-­‐ dividual  can  continue  to  suck  their   thumb  into  adolescence  and   sometimes  even  into  adulthood.   there  are  many  deleterious  ef-­‐ fects  that  can  result  from  the  forc-­‐ es  that  are  applied  by  the  thumb   onto  the  oral  structures.  the  dura-­‐ tion  and  frequency  of  the  sucking   influences  how  severe  these  ef-­‐ fects  can  be  on  the  dental  and   skeletal  structures.  thumb  sucking   is  a  very  difficult  topic  to  study.     very  little  research  has  been  per-­‐ formed  on  whether  or  not  there  is   genetic  component  to  chronic   thumb  sucking.  however,  there  is   some  speculation  that  there  can   behavioral  gene-cs  and  thumb  sucking  in  adolescents     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.73    http://dentistry3000.pitt.edu   be  a  behavioral  genetic  compo-­‐ nent  related  to  prolonged  nonnu-­‐ tritive  sucking.  often  children  who   suck  their  thumbs  are  nervous,   anxious,  or  bored.  studies  have   shown  that  there  are  some  behav-­‐ ioral  genes  that  could  be  influenc-­‐ ing  those  emotions  and  subse-­‐ quent  behaviors.  the  case  study   presented  in  this  paper,  shows   that  thumb  sucking  can  possibly   have  a  genetic  component.  three   individuals  on  one  side  of  a  family   all  sucked  their  thumb  into  ado-­‐ lescence.  adolescence  is  a  transi-­‐ tional  period  with  many  develop-­‐ mental  changes  that  often  affect   the  individual’s  personality,  emo-­‐ tions,  and  decision  making.  it  is   believed  that  genes  can  affect  all   of  these  changes  throughout  ado-­‐ lescence.  it  can  be  hypothesized   that  the  behavioral  genes  play  a   role  in  the  prolonged  habit  of   thumb  sucking  and  then  the  sub-­‐ sequent  discontinuation  of  the   habit  in  adolescence.  however,  it   is  still  uncertain  whether  this  is   true  and  more  research  needs  to   be  conducted  to  obtain  more  in-­‐ formation  on  this  possible  associa-­‐ tion.     references   1. thumb  and  finger  sucking.  da-­‐ vidson  l.  pediatr  rev.  2008   jun;  29(6):  207   208.    doi:  10.1542/pir.29-­‐6-­‐ 207;  pmid:  18515338.     2. duration  of  nutritive  and   nonnutritive  sucking  behaviors   and  their  effects  on  the  dental   arches  in  the  primary  denti-­‐ tion.  warren  jj,  bishara  se.  am   j  orthod  dentofacial  or-­‐ thop.  2002  apr;  121(4):  347– 356.  pmid:  11997758.     3. effects  of  nonnutritive  sucking   habits  on  occlusal  characteris-­‐ tics  in  the  mixed  dentition.   warren  jj,  slayton  rl,  bishara   se,  levy  sm,  yonezu  t,  kanellis   mj.  pediatr  dent.  2005  nov-­‐ dec;  27(6):  445–450.  pmid:   16532883.   4. nail  biting,  thumb  sucking,   and  other  irritating  behaviours   in  childhood.  lubitz  l.  aust   fam  physician.  1992   aug;21(8):1090-­‐4.  pmid:   1530488.   5. genetic  influences  on  adoles-­‐ cent  behavior.  dick  dm,  adkins   ae,  i-­‐chun  kuo  si.  neurosci  bi-­‐ obehav  rev.  2016  nov;  70:   198–205.    doi:   10.1016/j.neubiorev.2016.07.0 07.  epub  2016  jul  12.  pmid:   27422449.   6. gene$c  and  environmental   influences  on  finger-­‐sucking   and  nail-­‐bi#ng  in  japanese   twin  children.  ooki  s.  twin  res   hum  genet.  2005  aug;  8(4):   320-­‐ 327.    doi:  10.1375/183242705 4936637.  pmid:  16176716.     effect of chlorhexidine, honey and propolis on streptococcus mutans counts: in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.166 http://dentistry3000.pitt.edu effect of chlorhexidine, honey and propolis on streptococcus mutans counts: in vitro study abdulrhman alkhaled1, laith alsabek2, maher al-assaf3, faek badr1 ¹ department of pediatric dentistry, faculty of dentistry, tishreen university, syria. 2 faculty of dentistry, damascus university, damascus, syria. 3 department of oral histology and pathology, faculty of dentistry, damascus university, damascus, syria. abstract objective: streptococcus mutans is considered one of the bacteria that causes necrosis, as it metabolism sugar to obtain energy, producing an acidic environment and causing depression in the enamel surface, which causes the dissolution of the calcium molecule, a problem of cavited in the tooth; so the aim of the research is to evaluate the effect of mouthwashes chlorhexidine 0.12% honey 50% propolis 5% on streptococcus mutans in children. study design: a laboratory study to assess the efficiency of chlorhexidine, honey and propolis on streptococcus mutans counts. the sample included 60 children divided into: g1 (chlorhexidine 0.12% = 20 children), g2 (propolis 5% = 20 children), g3 (honey 100% = 20 children). the data was analyzed using the statistical analysis program spss, version 13.00, at a confidence level of 95% (p <0.05(. paired sample t test was used to compare the variable means of the values of the studied variables. results: there is significant difference between three groups. the reduction ratio was 89.97% in g1, 85.49% in g2 and 76.29% in g3. conclusion: this study demonstrated the effectiveness of mouthwashes of chlorhexidine, propolis and honey on the bacteria of streptococcus mutans. keywords: streptococcus mutans; chlorhexidine; propolis; honey citation: alkhaled a, et al. (2021) effect of chlorhexidine, honey and propolis on streptococcus mutans counts: in vitro study. dentistry 3000. 1:a001 doi:10.5195/d3000.2021.166 received: april 03, 2021 accepted: april 21, 2021 published: november 19, 2021 copyright: ©2021 alkhaled a, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: dr.maher.assaf1990@gmail.com introduction streptococcus mutans are grampositive bacteria of oval or aciduric shapes that are lined up in the form of acidogenic and aciduric nodules or swabs. they survive at low phs and also metabolize carbohydrates in an acid medium [1]. streptococcus mutans grows at temperatures ranging between 18-40 degrees celsius, and it is considered one of the bacteria that causes necrosis, as it metabolism sugar to obtain energy, producing an acidic environment and causing depression in the enamel surface, which causes the dissolution of the calcium molecule, a problem of cavited in the tooth [2]. it found that streptococcus mutans is more prevalent in pit and groove and there are a few of them on the buccal surfaces, where the mutant streptococcus forms 39% of the total streptococci within the oral cavity [3]. a study by köhler et al.1948 indicated a relationship between early colonization of the streptococcus mutans in the mouths of children and a high rate of caries [4], whereas aas et al. 2008 study showed that streptococcus mutans is mainly responsible for the initiation of dental caries despite the involvement of other oral bacteria in the development of dental caries such as lactobacillus and veillonella [5]. there are two types of oral washes (chemical and http://dentistry3000.pitt.edu/ effect of chlorhexidine, honey and propolis on streptococcus mutans counts: in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.166 http://dentistry3000.pitt.edu herbal). chlorhexidine is chemical. while many plant extracts have antibacterial properties, they are used effectively in oral hygiene. natural herbs such as miswak, honey, coconut oil, ginger, clove oil, and many other types [6]. for a long time, chlorhexidine has been the most widely used oral washes for reducing plaque and gingivitis, and there is no difference between an alcoholbased or a water-based lotion, and it is considered the gold standard among oral washes [7] , chlorhexidine is considered a broad-spectrum antibacterial against gram-positive and gramnegative bacteria, fungi, and some viruses, and it has an active effect against streptococcus mutans [8]. propolis or what is known as bee gum or glue is a natural resinous substance that bees collect from the buds and parts of the plant, mixed with bee enzymes, pollen and wax. it has a complex formula with a wide range of antibacterial, antiviral, antifungal, and antioxidant effects [9]. propolis has important uses in dentistry and preventive oral medicine, as studies in this field have shown the protective ability of propolis extracts when used to reduce the amount of bacterial plaque, thus reducing dental caries [10], barrientos et al 2013 confirmed the promising efficacy of propolis in the prevention of dental caries and some diseases of the oral cavity, when they demonstrated the bio-efficacy of the ethanoli extract and metanoli of the chilean propolis samples against the bacteria that cause caries , streptococcus sobrinus and streptococcus mutans [11] , honey is a natural, viscous, sweettasting food compound, ranging in color from light brown to dark, resulting from the collection of nectar flowers or sugar secretions from some trees by bees and the addition of compounds to it that these bees secrete [12]. honey has antibacterial activity which is proven as honey is a broad-spectrum inhibitor of many types of bacteria , including aerobic and anaerobic bacteria , the pros and cons of bacteria , as one laboratory study showed that methanol, ethanol, and ethyl acetate extracted from honey showed antibacterial activity against gram-positive (staphylococcus aureus, bacillus subtilis, bacillus cereus, enterococcus faecalis) and against gram-negative bacteria (escherichia coli, pseudomonas aeruginosa, salmonella typhi) [13]. the aim of this study is to evaluate the effect of mouthwashes chlorhexidine 0.12% honey 50% propolis 5% on streptococcus mutans in children. material and methods the sample included 60 children (30 males 30 females) aged between 6-9. the tishreen university research and ethics committee at the faculty of medicine and dentistry approved the study protocol, and subjects gave their informed consent to participate. the study was conducted in accordance with the consort statement. inclusion criteria: cooperative children aged from 6 to 9 years old. healthy patients with no history of previous systematic diseases that can affect their oral health. exclusion criteria: patients with gingivitis, having fixed or movable braces, and children who is taking any antibiotic before enrollment. inform consent was taken from the volunteers before starting the study according to a special form designed for the research that includes details of the research and the materials used in the research. we used local products that are widespread in pharmacies. the children were divided into 3 groups, each group containing 20 http://dentistry3000.pitt.edu/ effect of chlorhexidine, honey and propolis on streptococcus mutans counts: in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.166 http://dentistry3000.pitt.edu children (20 chlorhexidine 20 honey 20 propolis). saliva collection mechanism: [14] the child was brought to the clinic and the guardian requested the following: avoiding food with a high content of acids and sugars 60 minutes before work in order to cause it to reduce the ph of saliva, which leads to an increase in bacterial growth. and, avoiding foods and drinks containing caffeine for 12 hours before work. that the sample be collected between 9 12 p.m. emphasis on not brushing the teeth on the same day in order to preserve the oral flora and not to have bleeding that affects the accuracy of reading the results. patients did not use any oral rinse or paste containing chlorhexidine 7 days before the start of the clinical procedures. a first saliva sample was taken before using rinsing by using a sterile salivary swab for this procedure. the swab included passing the swab head over the vestibular surfaces of the teeth, the palate, the floor of the mouth, and the vestibule of the cheek. then, the child was asked to rinse his mouth with solution using 10 ml of solution for a period of time (30) seconds, after that, we took a second swab for the child, similar to its procedures for the first one, and the child's data were recorded on it (name age gender swab before / after) on each cotton swab and sent to the bacterial culture laboratory at hama national hospital hama city syria, to start the laboratory work procedures. chlorhexidine sample (20 children) the product used is a 0.12% chlorhexidine solution ready for use, produced by a national company. propolis sample (20 children) the product used is a 5% propolis solution ready for use, produced by a national company. honey sample (20 children) the honey was 100% concentrated from the packing of a national company. we drew 5 ml of honey and added it to 5 ml of distilled water in a sterile sample collection package, and we mixed it and then gave it to the child to rinse with it. procedures for bacterial culture in the laboratory: sampling extension: we dilate the saliva samples in two stages to reduce the bacterial load for ease of counting, provided that the real concentration of germs is calculated later as follows: stage one: use of 10000 microliters (10 ml) of salin by disposable one use syringe and put it in the glass tube. then, we remove 100 microns. thus, we get 9900 microliters in the tube. then we add 100 microns of the saliva sample by micropipette. finally, we get dilate of saliva sample in 1/100 radio; 10-2. then we mixed the homogenous dilate saliva sample on viberator for 30 seconds. stage two: repeating the previous stages but by adding 100 microliters of dilate solution to 9900 microliters from the saline in the other glass tube and the ratio became 1/ 10000 and then repeated this homogenous process by vibrator. bacterial culture: culture medium: preparation of the culture medium for streptococcus mutans. we add 90 g of mitis salivarius agar (msa) powder to 1 liter (1000 ml) of distilled water, then sterilize it in autoclave at 121 c for 20 minutes under 15 lbs pressure, and when cooled to a temperature of 45-50 ° c, 1 ml of 1% potassium tellurite solution is added to it, mixing the components well and pouring the mixture into sterile petri dishes in the airflow chamber, according to the instructions of the http://dentistry3000.pitt.edu/ effect of chlorhexidine, honey and propolis on streptococcus mutans counts: in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.166 http://dentistry3000.pitt.edu manufacturer of the culture medium. culture method: we take 10 microliters of dilate solution by micropipette. and then we spread it on the surface of culture media in petri dish by sterilize platin loop tool in way that we get a distinct bacterial colony. and placed upside down within the incubator at 37 ° c for 48 hours in the presence of 5-10% of carbon dioxide. bacterial appearance: the msa culture medium performs selective isolation of streptococcus mitis, streptococcus salivirus, streptococcus mutans, streptococcus sanguis, enterococci. streptococcus mutans appear convex, crested, blue, pale, with a grainy appearance, as shown in figure 1 and figure 2. colonies counting: we count the colonies by using an electron microscope, after the colonies counting is complete. this number presents the number of colonies in 10 dilate microliter. thus, we multiply this number with 10000 which is the dilation ratio then we divide the number by 1000 to get the final number of bacterial colonies in ml (cfu/ml). statistical analysis: the data were analyzed using the statistical analysis program spss, version 13.00, at a confidence level of 95% (p <0.05(. we used paired sample t test to compare the variable means of the values of the studied variables. results table no. 1 shows descriptive statistical measures of the number of streptococcus mutans among the 60 patients (20 patients in each group), while fig. no. (3) shows the arithmetic averages of the number of streptococcus mutans in the research patients. figure 1 indicates the number of streptococcus mutans before applying honey. figure 2 indicates the number of streptococcus mutans after applying honey. http://dentistry3000.pitt.edu/ effect of chlorhexidine, honey and propolis on streptococcus mutans counts: in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.166 http://dentistry3000.pitt.edu table no. 2 shows the results of using paired samples t test when comparing the arithmetic means of the number of streptococcus mutans in each patient group before and after the experiment. table no. 3 shows the percentages of the amount of decrease in number of streptococcus mutans between the two times (before using the substance and after using the substance) between the three experimental groups. as for figure no. 4, it shows the percentage values of the amount of decrease in number of streptococcus mutans between the two times (before using the substance and after using the substance) between the three experimental groups. max value minimal value sd mean studied variables sample material 222 96 43.11 161.30 number of streptococcus mutans before using honey 20 honey 53 23 10.23 38.25 number of streptococcus mutans after using honey 222 101 40.24 158.45 number of streptococcus mutans before using propolis 20 propolis 25 9 4.48 15.90 number of streptococcus mutans after using propolis 250 117 33.87 173.70 number of streptococcus mutans before using chlorhexidine 20 chlorhexidine 37 17 4.94 25.20 number of streptococcus mutans after using chlorhexidine table no 1. statistical measures of the number of streptococcus mutans in the research patients figure 3 mean of the number of streptococcus mutans in the research patients. http://dentistry3000.pitt.edu/ effect of chlorhexidine, honey and propolis on streptococcus mutans counts: in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.166 http://dentistry3000.pitt.edu sig: significance. *: statistically significant. sig p-value df t test value difference between arithmetic means comparisons materials * 0.000 19 16.731 123.05 number of streptococcus mutans before using honey number of streptococcus mutans after using honey pair 1 honey * 0.000 19 22.876 148.50 number of streptococcus mutans before using propolis number of streptococcus mutans after using propolis pair 1 propolis * 0.000 19 17.428 142.55 number of streptococcus mutans before using chlorhexidine number of streptococcus mutans after using chlorhexidine pair 1 chlorhexidine table no. 2. results of using paired samples t test when comparing the arithmetic means of the number of streptococcus mutans before and after the experiment. http://dentistry3000.pitt.edu/ effect of chlorhexidine, honey and propolis on streptococcus mutans counts: in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.166 http://dentistry3000.pitt.edu discussion the saliva count is a reasonable indicator of a complete bacterial load in the oral cavity, and the count of streptococcus mutans or lactobacillus in total dental plaque samples does not explain the variation in the incidence of caries as well as it does when counted in induced saliva [15]. in this study we studied the count of streptococcus mutans in saliva because there is an association between the count of streptococcus mutans in saliva and the occurrence of dental caries in children, and often there is a correlation between the levels of the streptococcus mutans and caries activity. identification persons with a high risk of caries [16]. bacteria have developed antibiotic-resistant strains, and reduction ratio % the amount of decrease streptococcus mutans variable value after using the material streptococcus mutans variable value before using the material number of patients materials 76.29% 123 38.25 161.30 20 honey 85.49% 149 25.20 173.70 20 propolis 89.97% 143 15.90 158.45 20 chlorhexidine 76.19% 125 39.167 164.48 60 total table no. 3. the percentages of the amount of decrease in number of streptococcus mutans between the two times (before using the substance and after using the substance) between the three experimental groups. figure 4 the percentages of the amount of decrease in number of streptococcus mutans between the two times (before using the substance and after using the substance) between the three experimental groups. http://dentistry3000.pitt.edu/ effect of chlorhexidine, honey and propolis on streptococcus mutans counts: in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.166 http://dentistry3000.pitt.edu pharmaceutical factories found it difficult to develop new antibacterial agents to meet this new challenge due to the high costs of drug research, which prompted researchers to search for an alternative to low-cost natural materials [17]. reports have shown the efficacy of honey against a wide spectrum of clinically resistant multibacteria, so its use has emerged as a promising alternative to industrial pharmaceutical products [17]. the rate of decrease in the count of streptococcus mutans was statistically significant after one minute of using chlorhexidine, as it reached 89.97%, and in propolis it was 85.49%, and in honey it was 76.29%. the results of this study showed that the three oral rinse had an immediate and direct effect on the bacteria of the streptococcus mutans, as chlorhexidine had a higher direct efficacy on the count of streptococcus mutans , then propolis and then honey. the results of this study regarding the antibacterial effect of honey oral washes are in agreement with the results of rupesh et al. 2014, which confirmed the antibacterial properties of new zealand honey oral washes [18]. the results of our study also agreed with the study of nassar and colleagues 2012, where they compared natural honey with synthetic honey in its effect on the streptococcus mutans found in plaque, and found the effect of natural honey much higher than synthetic [19], this negates the effect of the high sugar content in honey on the oral bacteria. we agreed with the results of hegde and colleagues' 2013 study in which they indicated that propolis has anti-streptococcus mutans activity in the oral cavity and can be used as a measure to prevent dental caries [20]. we also agreed with the results of the study by elbaz and colleagues 2012, where the study compared new zealand honey and egyptian propolis extract on streptococcus mutans and lactobacillu, and the study draw a conclusion that egyptian propolis was superior to new zealand honey in affecting the count of streptococcus mutans and lactobacillus [21]. the results of our study on the effect of chlorhexidine on streptococcus mutans are in agreement with the results of the fredri lundström and krasse 1987 study where the use of chlorhexidine reduced the count of streptococcus mutans in fixed orthodontic patients [22]. conclusion this study demonstrated the effectiveness of mouthwashes of chlorhexidine, propolis and honey on the bacteria of streptococcus mutans. the results of the present study showed the effectiveness of chlorhexidine mouthwashes 0.12% honey 50% propolis 5% in reducing oral bacterial count rates. chlorhexidine showed the maximum effectiveness, followed by propolis, then honey. acknowledgment none. references 1. dental caries is a preventable infectious disease. balakrishnan m, simmonds rs, tagg jr. aust dent j. 2000 dec;45(4):235-245. doi: 10.1111/j.1834-7819.2000.tb00257.x. pmid: 11225524. 2. streptococcus mutans, caries and simulation models. forssten sd, björklund m, ouwehand ac. nutrients. 2010 mar;2(3):290-298. doi: 10.3390/nu2030290. pmid: 22254021. 3. mode of delivery and other maternal factors influence the acquisition of streptococcus mutans in infants. li y, caufield pw, dasanayake ap, wiener hw, vermund sh. j dent res. 2005 sep;84(9):806-811. doi: 10.1177/154405910508400905. pmid: 16109988. http://dentistry3000.pitt.edu/ effect of chlorhexidine, honey and propolis on streptococcus mutans counts: in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.166 http://dentistry3000.pitt.edu 4. the effect of cariespreventive measures in mothers on dental caries and the oral presence of the bacteria streptococcus mutans and lactobacilli in their children. köhler b, andréen i, jonsson b. arch oral biol. 1984 29(11):879-883. doi: 10.1016/0003-9969(84)90086-4. pmid: 6596034. 5. bacteria of dental caries in primary and permanent teeth in children and young adults. aas ja, griffen al, dardis sr, lee am, olsen i, dewhirst fe, leys ej, paster bj. j clin microbiol. 2008 apr;46(4):1407-1417. doi: 10.1128/jcm.01410-07. pmid: 18216213. 6. in vitro amoebicidal effect of aloe vera ethanol extract and honey against acanthamoeba spp. cysts. kadry gm, ismail mam, el-sayed nm, el-kholy hs, el-akkad dmh. j parasit dis. 2021 mar;45(1):159-168. doi: 10.1007/s12639-020-01292-8. pmid: 33746401. 7. evaluation of new seawaterbased mouth rinse versus chlorhexidine 0.2% reducing plaque and gingivitis indexes. a randomized controlled pilot study. calvo-guirado jl, fernandez dominguez m, aragoneses jm, martinez gonzalez jm, fernández-boderau e, garcésvillalá ma, et al. appl. sci. 2020 feb;10(3):982. doi: 10.3390/app10030982. 8. adjunctive use of chlorhexidine in oral candidoses: a review. ellepola an, samaranayake lp. oral dis. 2001 jan;7(1):11-17. pmid: 11354914. 9. management strategies of honey bee diseases. arbia a, babbay b. journal of entomology. 2011 ;8(1):1-15. doi: 10.3923/je.2011.1.15. 10. does propolis help to maintain oral health?. więckiewicz w, miernik m, więckiewicz m, morawiec t. evid based complement alternat med. 2013 jan;2013:351062. doi: 10.1155/2013/351062. pmid: 23365605. 11. chemical and botanical characterization of chilean propolis and biological activity on cariogenic bacteria streptococcus mutans and streptococcus sobrinus. barrientos l, herrera cl, montenegro g, ortega x, veloz j, alvear m, cuevas a, saavedra n, salazar la. braz j microbiol. 2013 oct 30;44(2):577-585. doi: 10.1590/s1517-83822013000200038. pmid: 24294257. 12. clinical usage of honey as a wound dressing: an update. molan pc, betts ja. j wound care. 2004 oct;13(9):353-356. doi: 10.12968/jowc.2004.13.9.26708. pmid: 15517742. 13. antibacterial efficacy of raw and processed honey. mohapatra dp, thakur v, brar sk. biotechnol res int. 2011 dec;2011:917505. doi: 10.4061/2011/917505. pmid: 21350671. 14. individual differences in salivary cortisol: associations with common over-the-counter and prescription medication status in infants and their mothers. hibel lc, granger da, kivlighan kt, blair c. horm behav. 2006 aug;50(2):293300. doi: 10.1016/j.yhbeh.2006.03.014. pmid: 16682032. 15. number of mutans streptococci or lactobacilli in a total dental plaque sample does not explain the variation in caries better than the numbers in stimulated whole saliva. sullivan a, borgström mk, granath l, nilsson g. community dent oral epidemiol. 1996 jun;24(3):159-163. doi: 10.1111/j.1600-0528.1996.tb00834.x. pmid: 8871012. 16. caries risk assessment from dental plaque and salivary streptococcus mutans counts on two culture media. sánchez-pérez l, acosta-gío ae. arch oral biol. 2001 jan;46(1):49-55. doi: 10.1016/s00039969(00)00095-9. pmid: 11163595. 17. antibacterial activity of selected malaysian honey. zainol mi, mohd yusoff k, mohd yusof my. bmc complement altern med. 2013 jun;13:129. doi: 10.1186/1472-688213-129. pmid: 23758747. 18. evaluation of the effects of manuka honey on salivary levels of mutans streptococci in children: a pilot study. rupesh s, winnier jj, nayak ua, rao ap, reddy nv, peter j. j indian soc pedod prev dent. 2014 jul-sep;32(3):212-219. doi: http://dentistry3000.pitt.edu/ effect of chlorhexidine, honey and propolis on streptococcus mutans counts: in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.166 http://dentistry3000.pitt.edu 10.4103/0970-4388.135827. pmid: 25001440. 19. effect of honey on streptococcus mutans growth and biofilm formation. nassar hm, li m, gregory rl. appl environ microbiol. 2012 jan;78(2):536-540. doi: 10.1128/aem.05538-11. pmid: 22038612. 20. effect of propolis on streptococcus mutans counts: an in vivo study. hegde ks, bhat ss, rao a, sain s. int j clin pediatr dent. 2013 jan;6(1):22-25. doi: 10.5005/jpjournals-10005-1180. pmid: 25206182. 21. comparison of the antimicrobial effect of egyptian propolis vs new zealand propolis on streptococcus mutans and lactobacilli in saliva. elbaz ga, elsayad ii. oral health prev dent. 2012;10(2):155160. doi: 10.3290/j.ohpd.a28003. pmid: 22763595. 22. streptococcus mutans and lactobacilli frequency in orthodontic patients; the effect of chlorhexidine treatments. lundström f, krasse b. eur j orthod. 1987 may;9(2):109-116. doi: 10.1093/ejo/9.2.109. pmid: 3472888. http://dentistry3000.pitt.edu/ vol 1, no 1 (2013) issn 2167-8677 (online) doi 10.5195/d3000.2013.10 http://dentistry3000.pitt.edu this work is licensed under a creative commons attribution 3.0 united states license. this site is published by the university library system, university of pittsburgh as part of its d-scribe digital publishing program and is cosponsored by the university of pittsburgh press. sexual dimorphism involved in the mesiodistal and buccolingual dimensions of permanent teeth ticiana medeiros sabóia 1 , patricia nivoloni tannure 2 , ronir raggio luiz 5 , marcelo de castro costa 1 , jose mauro granjeiro 3 , erika calvano küchler 3 , leonardo santos antunes 4 1department of pediatric dentistry and orthodontics, school of dentistry, federal university of rio de janeiro, rj, brazil 2discipline of pediatric dentistry, school of dentistry, veiga de almeida university, rj, brazil 3unit of clinical research, fluminense federal university, niterói, rj, brazil 4department of specific formation, school of dentistry, fluminense federal university, nova friburgo, rj, brazil 5 institute of public health studies, federal university of rio de janeiro, rj, brazil abstract studies indicate that tooth crown diameters are clinical markers for sex differentiation. therefore, the aim of this study was to assess the degree of sexual dimorphism in different teeth. maximum mesiodistal (md) and buccolingual (bl) dimensions of 2400 permanent teeth from 100 pretreatment orthodontic dental study casts and clinical records (50 males and 50 females) from the department of pediatric dentistry and orthodontics, federal university of rio de janeiro, brazil, were examined. comparison of the md and bl dimensions between males and females was performed using the student’s t test with alpha 0.05, effect size, and discriminant function analysis. comparisons in md and bl widths between sexes demonstrated that the combined mean in the female group presented reduction when compared with the male group, except for the bl dimension of tooth 26. in regard to the md dimensions, statistically significant differences were observed in various dental groups. the greatest sexual dimorphism was observed in the left mandibular canine (p<0.001) with effect size over 0.8 (0.94), which characterizes large effect. in bl dimension, numerous teeth demonstrated statistical differences between the sexes. our findings reinforced the magnitude of sexual dimorphism in tooth size, and, in addition, highlighted the differences in specific dental groups. citation: sabóia tm, tannure pn, luiz rr, costa mc, granjeiro jm, küchler ec, antunes ls. (2013) sexual dimorphism involved on the mesiodistal and buccolingual dimensions of permanent teeth. dentistry 3000 1:a003 doi: 10.5195/d3000.2013.10 received: may 06, 2013 accepted: november 01, 2013 published: november 19, 2013 copyright: ©2013 sabóia et al. this is an open-access article licensed under a creative commons attribution 3.0 united states license. license. email: leonardoantunes@id.uff.br introduction the sexual dimorphism is explained by different effects of the human x and y chromosome genes on various somatic features, such as the frequency of some dental anomalies and the tooth crown size [1-3]. various explanations for tooth-size dimorphism between males and females have been proposed: the differences in hormonal balance [4], the effect of the y chromosome in increasing mitotic activity within the developing dental lamina, and the fact that the chromosome x is known to be involved in the enamel formation [2]. some authors suggested that this difference is due to the amount of enamel [5], while others found significant differences in the amounts of dentine [6]. the tooth crown size is a valuable tool and provides significant information on human evolution [7] and biological alterations [8], a in forensic evaluation [9,10] and clinical odontology [11]. tooth crown diameters are reasonably accurate predictors of sex and are good adjuncts for sex differentiation [12]. although the degree of dimorphism varies within different populations, generally, males have larger tooth crowns than females [13-16]. therefore, the present study was conducted to assess the degree of sexual dimorphism in permanent teeth of patients recruited at the federal university of rio de janeiro, brazil. materials and methods a total of 2400 permanent teeth from 100 pretreatment orthodontic dental study casts and clinical records from the department of pediatric dentistry and orthodontics, federal university of rio de janeiro, brazil, were examined. patients with underlying syndromes and oral clefts were not included in this study. patients who had their pretreatment orthodontic records performed between 2000 to 2010 were available for this study. fifty males and 50 females were selected at random. ethical approval was obtained from the human ethics committee of the health department of the city of rio de janeiro, rio de janeiro, brazil (113/09). written informed consent was obtained from all participating individuals or parents/legal guardians. the population included in this study resides in the metropolitan area of rio de janeiro, brazil, and is comprised of a mixture of caucasians (mainly european desce http://dentistry3000.pitt.edu/ http://creativecommons.org/licenses/by-nc-nd/3.0/us/ http://creativecommons.org/licenses/by-nc-nd/3.0/us/ http://www.library.pitt.edu/ http://www.pitt.edu/ http://www.library.pitt.edu/articles/digpubtype/index.html http://upress.pitt.edu/ sexual dimorphism involved in the mesiodistal and buccolingual dimensions of permanent teeth vol 1, no 1 (2013) doi 10.5195/d3000.2013.10 http://dentistry3000.pitt.edu 2 ndants (53.6%) and african descendants (33.6% obviously mixed portuguese, 12.3% not obviously mixed africans). the remaining 0.5% of the population is amerindian or asian descendants. the ethnicity was based on the self-reported description. assessment of tooth dimensions dental casts were used to obtain data regarding tooth dimensions. a dental diagram with the tooth nomenclature used in this work is presented in figure 1. dental casts were excluded from assessment if they fell into one of the following criteria: teeth with restorations extending both for the mesiodistal and for the buccolingual surfaces, teeth displaced or crowded, and teeth not fully erupted. second and third molars were excluded due to the young age of many of the subjects. maximum mesiodistal (md) and buccolingual (bl) dimensions of fully erupted permanent teeth were measured to the nearest 0.1 millimeters using the digital mitutoyo caliper. md is defined as the maximum distance between the most mesial and the most distal point of the crown, whereas bl is defined as the maximum distance between the most lingual/palatal and the most buccal/labial point of the crown. the dimensions were recorded for each tooth using the method proposed by moorrees and reed [17]. all measurements were undertaken by one operator (t.m.s.), with a strict criterion in order to reduce variation. five models were analyzed per day during two months to prevent depletion of the operator. each tooth was measured three times and, if the measurements differed by more than 0.2 millimeters, was measured three times again. statistical analysis an intra-class correlation coefficient was calculated to assess random error of intraobserver variability. the dental casts of ten subjects were selected and all tooth measurements were assessed on two occasions at least two weeks apart. the level of agreement was equal to 0.99, indicating an excellent level of reproducibility of the tooth dimension measurements. the descriptive values of md and bl dimensions (means and standard deviations) were recorded. comparison between md and bl dimensions of the male and female permanent teeth was performed using the student’s t test with an alpha of 0.05. effect sizes were calculated as the difference between the means (measurements for each tooth in relation to sex) divided by standard deviation of either group. the widely used benchmarks, suggested by cohen [18], indicated the magnitude of the change observed. that is, effect sizes of 0.2 were taken to be small, 0.5 to be moderate, and 0.8 or above to be large. comparison between md table1.characteristics of the individuals studied. male female p-value mean age (standard deviation) 17.2(±4.6) 19.8(±6.3) 0.02* ethnicity (%) caucasian 37 (74%) 30 (60%) 0.1** african descendant 13 (26%) 20 (40%) note: * t-test; ** chi-square test; bold emphasis indicates statistical significance (p≤0.05). figure 1. dental diagram. this diagram represents the permanent dentition. second molars are presented in gray color since they were not evaluated in this work. third molars are not presented in this diagram. this diagram is labeled according to the federation dentaire internationale numbering system. figure 2. box plot showing quartiles and mean of the mesiodistal dimensions of upper and lower teeth. statistical difference between genders is represented in table 2. tooth numbers correlate to the numbers in figure 1. http://dentistry3000.pitt.edu/ sexual dimorphism involved in the mesiodistal and buccolingual dimensions of permanent teeth vol 1, no 1 (2013) doi 10.5195/d3000.2013.10 http://dentistry3000.pitt.edu 3 and bl dimensions of the caucasian and african descendants’ permanent teeth was also performed using the student’s t test with an alpha of 0.05. for the dimensions that were different between ethnicities, logistic regression analyses were performed including ethnicity as covariates. results were reported according to the strobe guidelines for cohort. results the univariate analysis showed that there are no significant differences in ethnicity between male and female (p=0.1). the demographic characteristics of the studied individuals are summarized in table 1. in both comparisons, md and bl widths between sex demonstrated that the combined mean of each tooth in the female group presented reduction when compared with the male group. out of the 48 variables measured, male teeth exceeded female teeth significantly in 26 variables (p<0.05). of these, 13 belonged to the maxillary and 13 were bl dimensions. in regard to the md dimensions, statistically significant differences were observed in various teeth, the greatest of which were observed in the mandibular left canine (33) (p<0.001), with effect size over 0.8 (0.94), and in the mandibular right canine (43) (p<0.001), with effect size over 0.8 (0.88). in bl dimension, many teeth demonstrated statistical differences between the sexes. these results were observed in table 2. a graphical representation of md and bl dimensions are also provided in figure 2 and figure 3. in regard to the differences between the ethnicities, caucasians presented statistically significant reduced dimensions in specifics cases. in males, differences were observed in the md dimension of upper right second premolar (15) and in the bl dimension of upper right (12) and left (22) lateral incisor. in females, differences were observed in the md dimension of the first molars (16, 26, 36 and 46) and the lower left lateral incisor (32). differences were also observed in the bl dimensions of the lower second right premolar (45). the results of the logistic regression analyses for these dimensions, using the ethnicity as covariant, are presented in table 3. discussion the general structure and morphology of the teeth are similar in both men and women, however, there are subtle differences, such as variation in dental size, that can give a clue about differences between the sexes. following this pattern, teeth can be considered an important step for sex determination as they are resistant to postmortem destruction and fragmentation. the accessibility for measuring the dimensions of the teeth using morphometric devices would be a reliable method for solving medicolegal investigations and to identify victims of crime, natural disasters, and severe accidents. in this study, we analyzed the degree of the sexual dimorphism in different teeth by measuring the maximum diameters, mesiodistal and buccolingual, of fully erupted permanent teeth from study casts. our results raise an interesting possibility that could be used to clarify this difference. measurement of the md width of the mandibular and maxillary canines provides good evidence of sex identification due to dimorphism, since this measurement showed higher effect sizes than other teeth. these results are consistent with previous studies that found the canine to be one of the most dimorphic teeth [19-21]. it was previously proposed that the magnitude of the difference of canine size is not isolated and that there is a “field” of sexual dimorphism that includes the teeth adjacent to the canines (incisors and premolars) [22, 23]. interestingly, this study provided evidence of strong difference between the first premolars (bl dimensions of 14, 24, 34, 44) and lateral incisors (md dimension of 12 and bl dimension of 22) of males and females. regarding the difference in tooth size between men and women, md and bl dimensions were smaller in females than males in almost all teeth, corroborating with previous studies [20, 24]. several studies have investigated the possible reasons for the morphological and developmental difference in teeth between men and women. animal model studies suggested that specific genetic factors might be involved with specific types of tooth development [25]. according schwartz and figure 3. box plot showing quartiles and mean of the buccolingual dimensions of upper and lower teeth. statistical difference between genders is represented in table 2. tooth numbers correlate to the numbers in figure 1. http://dentistry3000.pitt.edu/ sexual dimorphism involved in the mesiodistal and buccolingual dimensions of permanent teeth vol 1, no 1 (2013) doi 10.5195/d3000.2013.10 http://dentistry3000.pitt.edu 4 dean [6], sex hormone concentrations during development could relate to dental tis sue proportions in teeth forming at different moments. smith et al. [26] obtained histological sections from molars and observed that males showed significantly greater dentine area, enamel—dentine junction length, and bi-cervical diameters in certain tooth types, while women presented significantly thicker average enamel. these results are consistent with the study of saunders et al. [23], who noted that male canines and premolars have significantly more dentine than their female counterparts, as well as relatively more dentine with respect to overall crown size. the female canines and premolars have significantly more enamel relative to overall crown area than those of the males. following this pattern, we presented some evidence that in humans, different dental groups might respond differently to the influence of sex chromosomes/hormones on crown development. another important aspect that should be taken into considerations is the ethnicity differences. caucasian subjects presented some reduced dimensions when compared with african descendants. this should be taken into consideration in mixed populations, like brazilians. in summary, our findings reinforced the magnitude of sexual dimorphism in tooth size, and, in addition, highlighted the differences in specific dental groups. the approach taken in this manuscript (oversimplified with only tooth size variant analyzed) is a limitation to properly assess the explanations for tooth-size dimorphism between males and females. thus, further investigations, based on genetic, evolutionary, and metabolic/hormonal reasons for sexual dimorphism, will hopefully further clarify the etiology of sexual dimorphism in tissue proportions and dental development. in conclusion, our results established the degree of sexual dimorphism in permanent teeth of brazilian individuals. conflict of interest: there are no conflicts of interest to report. table2. effect size of the mesiodistal (md) and buccolingual (bl) dimensions of the permanent teeth between male and female. tooth type male female difference effect size p-value* mean standard deviation mean standard deviation md 33 7.24 0.41 6.85 0.42 0.39 0.94 <0.001 md 43 7.29 0.45 6.90 0.43 0.39 0.88 <0.001 md 13 8.32 0.50 7.94 0.50 0.38 0.76 <0.001 md 23 8.32 0.48 7.96 0.52 0.36 0.72 0.001 bl 25 9.99 0.69 9.55 0.58 0.44 0.69 0.001 bl 15 10.00 0.69 9.58 0.53 0.42 0.68 0.001 bl 21 7.53 0.54 7.21 0.47 0.32 0.63 0.002 bl 24 9.86 0.70 9.51 0.43 0.35 0.61 0.003 bl 33 7.58 0.77 7.18 0.63 0.40 0.57 0.005 md 25 7.16 0.39 6.88 0.59 0.28 0.56 0.006 bl 34 8.25 0.65 7.93 0.47 0.32 0.56 0.006 bl 14 9.86 0.66 9.54 0.44 0.32 0.56 0.006 bl 16 11.63 0.69 11.30 0.57 0.33 0.52 0.010 md 15 7.21 0.40 6.97 0.53 0.24 0.52 0.011 bl 22 6.76 0.69 6.46 0.53 0.30 0.50 0.014 bl 11 7.47 0.60 7.22 0.43 0.25 0.49 0.017 md 21 8.90 0.53 8.65 0.55 0.25 0.47 0.021 bl 44 8.18 0.69 7.90 0.54 0.28 0.45 0.027 md 36 11.47 0.62 11.16 0.75 0.31 0.45 0.027 md 26 10.69 0.63 10.40 0.66 0.29 0.45 0.028 md 12 7.19 0.57 6.95 0.55 0.24 0.43 0.033 md 16 10.79 0.67 10.52 0.62 0.27 0.42 0.038 md 11 8.91 0.55 8.68 0.57 0.23 0.41 0.044 md 46 11.45 0.64 11.18 0.71 0.27 0.40 0.047 bl 23 8.34 0.89 8.04 0.67 0.30 0.38 0.057 bl 45 8.83 0.70 8.60 0.55 0.23 0.38 0.061 bl 12 6.77 0.69 6.53 0.63 0.24 0.38 0.063 bl 36 10.71 0.64 10.49 0.54 0.22 0.36 0.072 bl 13 8.33 0.78 8.08 0.62 0.25 0.36 <0.001 bl 43 7.37 0.77 7.13 0.56 0.24 0.35 <0.001 bl 26 10.71 0.09 10.49 0.07 0.22 0.35 0.071 md 22 7.19 0.63 6.99 0.54 0.20 0.34 0.094 bl 41 6.19 0.56 6.02 0.49 0.17 0.33 0.104 md 24 7.47 0.46 7.32 0.57 0.15 0.30 0.137 bl 46 10.72 0.63 10.55 0.60 0.17 0.27 0.177 bl 35 8.80 0.79 8.62 0.52 0.18 0.27 0.182 bl 31 6.11 0.57 5.97 0.59 0.14 0.26 0.205 md 34 7.49 0.44 7.37 0.54 0.12 0.25 0.210 md 44 7.48 0.42 7.38 0.52 0.10 0.23 0.250 md 32 6.23 0.41 6.13 0.44 0.10 0.23 0.254 bl 42 6.40 0.52 6.29 0.52 0.11 0.22 0.266 md 45 7.60 0.51 7.48 0.52 0.12 0.22 0.270 md 35 7.62 0.53 7.50 0.56 0.12 0.21 0.285 md 14 7.42 0.43 7.33 0.50 0.09 0.21 0.305 bl 32 6.45 0.56 6.36 0.43 0.09 0.19 0.353 md 31 5.60 0.39 5.53 0.36 0.07 0.17 0.399 md 42 6.20 0.42 6.15 0.38 0.05 0.14 0.490 md 41 5.58 0.36 5.54 0.36 0.04 0.11 0.600 note: effect size of the md and bl dimensions of the permanent teeth between male and female. tooth numbers coorelate to the numbers in figure 1. *student’s t test was used to compare the means between males and females. table 3. logistic regression analysis using ethnicity as covariates. tooth type p-value bl 12 0.029 md 15 0.005 bl 22 0.010 md 16 0.021 md 26 0.017 md 32 0.150 md 36 0.012 bl 45 0.029 md 46 0.024 note: using maximum measurements of the mesiodistal (md) and buccolingual (bl) dimensions. tooth numbers coorelate to the numbers in figure 1. http://dentistry3000.pitt.edu/ sexual dimorphism involved in the mesiodistal and buccolingual dimensions of permanent teeth vol 1, no 1 (2013) doi 10.5195/d3000.2013.10 http://dentistry3000.pitt.edu 5 references 1. studies of dental anomalies in a large group of school children. küchler ec, risso pa, costa mde c, modesto a, vieira ar. arch oral biol. 2008 oct;53(10):941-6. doi: 10.1016/j.archoralbio.2008.04.003. pmid: 18490001 2. human sex chromosomes in oral and craniofacial growth. alvesalo l. arch oral biol. 2009 dec;54 suppl 1:s18-24. doi: 10.1016/j.archoralbio.2008.06.004. pmid: 18657798 3. side of dental anomalies and taurodontism as potential clinical markers for cleft subphenotypes. küchler ec, da motta lg, vieira ar, granjeiro jm. cleft palate craniofac j. 2011 jan;48(1):103-8. doi: 10.1597/09-159. pmid: 20507240 4. sex chromosomes and human growth. a dental approach. alvesalo l. hum genet. 1997 nov;101(1):1-5. pmid: 9385359 5. 47,xxx females, sex chromosomes, and tooth crown structure. alvesalo l, tammisalo e, therman e. hum genet. 1987 dec;77(4):345-8. pmid: 3692479 6. sexual dimorphism in modern human permanent teeth. schwartz gt, dean mc. am j phys anthropol. 2005 oct;128(2):312-7. pmid: 15861426 7. sexual dimorphism in primate evolution. plavcan jm. am j phys anthropol. 2001;suppl 33:25-53. pmid: 11786990 8. the effect of prenatal factors on crown dimensions. garn sm, osborne rh, mccabe kd. am j phys anthropol. 1979 nov;51(4):665-78. pmid: 574721 9. use of diagonal teeth measurements in predicting gender in a turkish population. karaman f. j forensic sci. 2006 may;51(3):630-5. pmid: 16696712 10. gender determination by odontometrics in a swedish population. lund h, mörnstad h. j forensic odontostomatol. 1999 dec;17(2):30-4. pmid: 10709560 11. mesiodistal crown diameters of permanent teeth in jordanians. hattab fn, al-khateeb s, sultan i. arch oral biol. 1996 jul;41(7):641-5. pmid: 9015564 12. sex discrimination potential of buccolingual and mesiodistal tooth dimensions. acharya ab, mainali s. j forensic sci. 2008 jul;53(4):790-2. doi: 10.1111/j.1556-4029.2008.00778.x. pmid: 18557797 13. standards for mesiodistal and buccolingual crown size and height of primary molars in a sample of spanish children. barbería e, suárez mc, villalón g, maroto m, garcía-godoy f. eur j paediatr dent. 2009 dec;10(4):169-75. pmid: 20073541 14. the correlation of sexual dimorphism in tooth size and arch form. haralabakis nb, sifakakis i, papagrigorakis m, papadakis g. world j orthod. 2006 fall;7(3):254-60. pmid: 17009475 15. comparison of tooth size and dental arch widths in contemporary japanese and american preschool children. yonezu t, warren jj, bishara se, steinbock kl. world journal of orthodontics 2:356-360, 2001. 16. mesiodistal crown diameters of the primary and permanent teeth in southern chinese--a longitudinal study. yuen kk, so ll, tang el. eur j orthod. 1997 dec;19(6):721-31. pmid: 9458605 17. correlations among crown diameters of human teeth. moorrees cf, reed rb. arch oral biol. 1964 nov-dec;9:685-97. pmid: 14219519 18. cohen j (1988) statistical power analysis for the behavioral sciences (2nd ed.). hillsdale, nj: lawrence earlbaum associates. 19. determining dental sex dimorphism in south indians using discriminant function analysis. anuthama k, shankar s, ilayaraja v, kumar gs, rajmohan m, vignesh. forensic sci int. 2011 oct 10;212(1-3):86-9. doi: 10.1016/j.forsciint.2011.05.018. pmid: 21664775 20. odontometric sex assessment in indians. prabhu s, acharya ab. forensic sci int. 2009 nov 20;192(1-3):129.e1-5. doi: 10.1016/j.forsciint.2009.08.008. pmid: 19744808 21. sexual dimorphism in permanent teeth of modern greeks. zorba e, moraitis k, manolis sk. forensic sci int. 2011 jul 15;210(1-3):74-81. doi: 10.1016/j.forsciint.2011.02.001. pmid: 21371836 22. genetic control of sexual dimorphism in tooth size. garn sm, lewis ab, swindler dr, kerewsky rs. j dent res. 1967 sep-oct;46(5):963-72. pmid: 5234039 23. sexual dimorphism of the dental tissues in human permanent mandibular canines and third premolars. saunders sr, chan ah, kahlon b, kluge hf, fitzgerald cm. am j phys anthropol. 2007 may;133(1):735-40. pmid: 17295299 24. sexual dimorphism in the buccolingual tooth diameter. garn sm, lewis ab, kerewsky rs. j dent res. 1966 nov-dec;45(6):1819. pmid: 5226551 25. oral clefts and syndromic forms of tooth agenesis as models for genetics of isolated tooth agenesis. vieira ar. j dent res. 2003 mar;82(3):162-5. pmid: 12598542 26. modern human molar enamel thickness and enamel-dentine junction shape. smith tm, olejniczak aj, reid dj, ferrell rj, hublin jj. arch oral biol. 2006 nov;51(11):974-95. pmid: 16814245 http://dentistry3000.pitt.edu/ mode of cell death of co-cultured dental mesenchymal stem cells following exposure to endodontic irrigating solutions vol 10 no 1 (2022) doi 10.5195/d3000.2022.146 http://dentistry3000.pitt.edu mode of cell death of co-cultured dental mesenchymal stem cells following exposure to endodontic irrigating solutions eshagh ali saberi, narges farhad mollashahi, mahboobe ahmadi, arezoo pirhaji department of endodontics, faculty of dentistry, oral and dental diseases research center, zahedan university of medical sciences, zahedan, iran. abstract aims: this in vitro study aimed to determine the mode of cell death of co-cultured dental mesenchymal stem cells following exposure to different irrigating solutions. methods and material: mesenchymal stem cells of the apical papilla (scaps) and periodontal ligament stem cells (pdlscs) were isolated from the periapical region of a third molar of an 18-year-old patient and cultured. adequate number of cells in each well, 6-well plate inserts were placed over the wells. the cells were then randomly exposed to 17% ethylenediaminetetraacetic acid (edta), 1.5% sodium hypochlorite (naocl), 2% chlorhexidine (chx) and saline. after 1, 5, and 10 minutes cells were trypsinized and underwent flow cytometry. apoptotic and necrotic cells were quantified. data were analyzed using the one-way anova and tukey’s post-hoc test. results: with increasing exposure time, cell viability is decrease and the mode of cells death was necrosis in chx and naocl groups and necrosis plus late apoptosis in 1 and 10 minutes in edta group. conclusion: maximum cell death occurred following exposure to edta while minimum cell death occurred following exposure to chx. necrosis was the dominant mode of cell death in all groups. keywords: co-culture; apoptosis; necrosis; irrigants; cells; cultured. citation: ali saberi e, et al. (2022) mode of cell death of co-cultured dental mesenchymal stem cells following exposure to endodontic irrigating solutions dentistry 3000. 1:a001 doi:10.5195/d3000.2022.146 received: february 5, 2021 accepted: august 10, 2021 published: march 21, 2022 copyright: ©2022 ali saberi e, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: saberiendo@yahoo.com introduction the mode of cell death is often divided into two groups of apoptosis (programmed cell death) and necrosis (accidental cell death) for the purpose of simplification. however, a more advanced classification defines 11 types of cell death such as apoptosis, necrosis, autophagy, oncosis, pyroptosis, etc. numerous biochemical and morphological differences exist between apoptosis and necrosis. apoptosis is a coordinated biochemical process that leads to cell death and is classified into three types of apoptosis, apoptosis-like and necrosis-like cell death [1]. in this process, the cell, itself, is responsible for its death; thus, it is also referred to as cell suicide. the characteristics of an apoptotic cell include condensed chromatin in cell nucleus, dna fragmentation, transfer of phosphatidylserine from the inner half to the outer half of the cell membrane, membrane ballooning and production of apoptotic bodies. in necrosis, all internal cellular organelles, especially the mitochondria, are swollen, and disintegration of cell membrane and cell lysis occur. cell death by necrosis is associated with extensive tissue destruction. apoptosis can be evaluated by a number of techniques such as evaluation of cytotoxicity and morphological changes, dna laddering, flow cytometry and the tunel test [2]. the basic principles of flow-cytometry are based on the fact that in initial phases of apoptosis, phosphatidylserine that is naturally located in the inner half of the membrane is transferred to the outer half of membrane due to the impaired activity of atp-dependent http://dentistry3000.pitt.edu/ mode of cell death of co-cultured dental mesenchymal stem cells following exposure to endodontic irrigating solutions vol 10 no 1 (2022) doi 10.5195/d3000.2022.146 http://dentistry3000.pitt.edu translocase enzyme or activation of other enzymatic systems such as scramblase. the transfer of phosphatidylserine to the outer half of the cell membrane is a natural signal for detection of apoptotic cells by the macrophages and adjacent cells [1,2]. evidence shows that annexin v, which is an anticoagulation protein with 35 kd molecular weight, has high affinity for binding to phosphatidylserine in presence of calcium. fluorescein isothiocyanate (fitc)-conjugated annexin v is used for quantification of apoptotic cells by flow cytometry [3]. the membrane integrity is lost and the cell membrane becomes highly permeable in necrotic cells. thus, the propidium iodide fluorescent intercalating agent can pass through the membrane and bind to dna. in apoptotic cells, the membrane integrity is preserved. thus, the propidium iodide cannot enter the cells. on the other hand, phosphatidylserine has been transferred to the outer half of the cell membrane in these cells and annexin binds to it. thus, by simultaneous staining of cells with these two compounds, apoptotic and necrotic cells can be differentiated [4]. regeneration is a biological process to replace structures such as dentin, root and dentin-pulp complex. disinfection of the root canal system is an important step in the process of regeneration. ideal disinfecting solutions should have excellent antimicrobial properties and minimal cytotoxicity [5]. evidence shows that irrigating solutions have cytotoxic effects on stem cells and gingival fibroblasts. mollashahi et al. evaluated the cytotoxic effects of commonly used endodontic irrigating solutions on stem cells of the apical papilla (scaps). they found that chlorhexidine (chx) had the highest cytotoxicity followed by naocl, qmix, edta, mtad and saline [6]. barnhart et al. demonstrated the cytotoxic effects of irrigating solutions on gingival fibroblasts [7]. most studies on cytotoxicity have used single culture of cells while stem cells are not independent of other cells in vivo. cell-to-cell interactions in the culture media in vitro are highly complex and play an important role in the scaffold in tissue regeneration [8]. to the best of the authors’ knowledge, no previous study has evaluated the mode of cell death following exposure to different endodontic irrigating solutions. thus, this study aimed to assess the mode of cell death of co-cultured stem cells of the apical papilla(scaps) and periodontal ligament stem cells (pdlscs) following exposure to commonly used endodontic irrigating solutions. materials and methods this study was approved by the ethics committee of zahedan university of medical sciences (ir.zaums.rec.1397.280). the stem cells of the apical papilla and periodontal ligament stem cells were isolated from the periapical region of a third molar tooth of an 18-year-old donor. two-thirds of the root had been formed and the cells were isolated using enzymatic digestion. written informed consent was obtained from the donor. immediately after tooth extraction, the tooth was rinsed with phosphate buffered saline (pbs; gibco brl, grand island, ny, usa) and stored in a sterile solution. the tooth was then decoronated at the cementoenamel junction and its pdl and apical papilla were removed. the tissues were diced into small pieces and immersed in 5 mg/ml of dispase ii (invitrogen, karlsruhe, germany) at 37°c for one hour. single-cell culture was obtained by filtering the solutions using a 70 nm cell filter (bd biosciences, heidelberg, germany). the cells were transferred to cell culture flask with dulbecco’s modified eagle’s medium containing 15% fetal bovine serum and 1% penicillin/streptomycin and incubated in presence of 95% o2/5% co2. the solution was refreshed every 2 days and after reaching 80% cell confluence at the bottom of the flask, the cells were passaged and the flask contents were distributed among three flasks. third passage cells were used for the next experiments (figure 1). characterization of isolated cells in order to characterization of isolated cells, expression of cd105, cd45, cd146, cd73, stro-1, and cd90 surface markers measured by http://dentistry3000.pitt.edu/ mode of cell death of co-cultured dental mesenchymal stem cells following exposure to endodontic irrigating solutions vol 10 no 1 (2022) doi 10.5195/d3000.2022.146 http://dentistry3000.pitt.edu flow-cytometry. for this purpose, after counting the cells, they were fixed in 4% paraformaldehyde at 4°c for 30 min and permeabilized using 0.2% triton-x100 for 30 min. the fixed cells were then rinsed with pbs, and incubated in primary antibodies (table 1) diluted in 10 mg/ml bsa for overnight. finally, following incubation with secondary antibodies (table 1) at 37°c for 2 h, the expression analysis were performed by a facs calibur cytometer (becton dickinson, san jose, ca, usa) and analyzed by winmdi 2.9 software. figure 1. inverted microscopic photographs of the morphology of mesenchymal stem cells assessment of cell death the insert plates (0.4 µm; spl life science, gyeonggi-do, south korea) were used for treatment of the cells. after culturing the cells to reach adequate cell count in each well of a 6-well plate, the inserts were placed over the wells. stem cells of the apical papilla were placed at the top layer while periodontal ligament stem cells were placed at the bottom of the plates (80,000 cells per well) [9]. the wells were then randomly divided into 4 groups for treatment of cells with 2% chx (clorhexidina s, dentscare ltda, joinville, sc, brazil), 17% edta (md-cleanser, meta biomed, chungju, korea), 1.5 % naocl (bojneh, tehran, iran) and saline. the cells were subjected to flow cytometry (becton dickinson) after 1, 5 and 10 minutes of exposure to the irrigating solutions. after treatment of the cells, the overlaying medium was removed and after trypsinization and removal of trypsinized cells, the cell suspension was transferred into a tube for 15 minutes and was then centrifuged at 1200 rpm for 5 minutes. the supernatant was discarded and the cells were washed with pbs. next, 500 µl of the bonding buffer present in the kit and 100 µl of annexin v + fitc were added and the mixture was incubated at room temperature for 15 minutes. the additional dye was washed with pbs, and 5 µl of propidium iodide fluorescent intercalating agent was also added to the cell suspension and around 10,000 cells underwent flow cytometry. annexin v + fitc creates green fluorescence and is used for quantification of apoptotic cells. the propidium iodide with red fluorescence is used for quantification of necrotic cells and their differentiation from apoptotic cells. the fl1 filter was used for annexin and the fl3 filter was used for propidium iodide. data were analyzed using graphpad prism (graphpad software, san diego, ca, usa) software (latest version). the mean and standard deviation of the number of apoptotic and necrotic cells in the groups at different time points were calculated and reported. pairwise comparisons at different time points were carried out using the one-way anova and tukey’s post-hoc test. type one error (alpha) was considered to be 0.05 and each test was repeated in triplicate. results the expression of specific mesenchymal stem cell markers, including cd146, stro-1, cd105, cd90, and cd73 as well as a hematopoietic stem cell marker (cd45) at the passage 4 revealed a substantial expression of all mscrelated markers. figure 2 exhibited a representative histogram for each protein approved that over 95% of the cells expressed mesenchymal cell markers (cd73, cd90, cd105) and around 90% of the cells expressed dental stem cell markers (cd146, stro-1). however, the cells did not express cd45 as a prominent hematopoietic stem cell marker (0.45%). the present results revealed that over 95% of scaps and pdlscs in the control and saline groups were viable in the co-culture, and no significant difference was noted in their viability. the passage of time had no significant effect on their viability either. http://dentistry3000.pitt.edu/ mode of cell death of co-cultured dental mesenchymal stem cells following exposure to endodontic irrigating solutions vol 10 no 1 (2022) doi 10.5195/d3000.2022.146 http://dentistry3000.pitt.edu table 1. antibodies used for identification of mesenchymal cells cat-no company host name mabt117 millipore mouse anti hcd105 05-1413 millipore mouse anti h –cd45 mab16985 chemichon mouse anti h –cd146 mabd122 chemichon(millipore) mouse anti h– cd73 mab4315 millipore mouse antih –stro-1 cbl415 millipore mouse anti h – cd90 672311 mp biomedical goat anti h-mouse igm-fitc p9670 sigma goat antih-mouse igg-pe figure 2. expression of mesenchymal stem cell markers. representative histogram of the expression of cd105, cd90, cd73, cd146, stro-1 and cd45. green histogram indicates cell fluorescence with control antibody (isotype control) and purple histogram indicates cell fluorescence with the respective antibody. table 2 and 3 and figure 3 and 4 shows the viability and mode of cell death of stem cells treated with different irrigating solutions over time. the results showed that increasing the exposure time significantly decreased the viability of stem cells treated with chx, sodium hypochlorite and edta. the majority of the cells underwent necrosis, except in the chx group in which, pdlscs underwent early apoptosis at 1 and 10 min. http://dentistry3000.pitt.edu/ mode of cell death of co-cultured dental mesenchymal stem cells following exposure to endodontic irrigating solutions vol 10 no 1 (2022) doi 10.5195/d3000.2022.146 http://dentistry3000.pitt.edu by an increase in exposure time, the necrotizing effect of sodium hypochlorite and edta increased on stem cells; although no significant difference was noted between sodium hypochlorite and edta at 10 min. in general, the cytotoxicity of the irrigating solutions was as follows: control group=saline<2% chx<1.5% naocl<17% edta table 2. cell viability,early apoptosis, late apoptosis and necrosis in co-cultured pdlscs at different times of treatment with endodontic irrigating solutions. exposure time (minute) group total number of cells viable cells early apoptosis late apoptosis necrosis mean ± sd mean ± sd mean ± sd mean ± sd 1 control 80000 78632a±1.00 432b±59.2 80b±56 848c±256 saline 80000 79504a±176 144b±120 32b±24 320c±120 chx 80000 76360a±106 1864a±1296 960b±280 816c±512 naocl 80000 53248b±2128 2280a±200 3584a±320 20992b±2448 edta 80000 45112b±1632 184b±96 168b±8 34488a±1728 p value 0.00 0.00 0.00 0.00 5 control 80000 78632a±1008 432b±592 80b±56 848b±1056 saline 80000 78888a±568 288b±216 48b±8 784b±728 chx 80000 70448b±3944 3784a±4896 824a±696 4704b±3488 naocl 80000 45848c±1832 48b±16 1568a±48 31888a±1736 edta 80000 45928c±2408 1736a±40 64b±16 32192a±2360 p value 0.00 0.00 0.00 0.00 10 control 80000 78632a±1008 432b±592 80b±56 848b±1056 saline 80000 77896a±72 56b±16 48b±0.00 2000b±48 chx 80000 63984b±3360 9800a±7936 1992b±2280 4216b±5176 naocl 80000 38704c±1512 536b±472 2192b±104 38480a±1184 edta 80000 18288c±2080 216b±24 14288a±520 47512a±1208 p value 0.00 0.043 0.00 0.00 sd: standard deviation; p value: one-way anova.for the comparison of irrigating solutions; a,b,c: comparison of irrigating solutions regarding mode of cell death by tukey’s post-hoc test. similar letters indicate lack of a significant difference. table 3. cell viability,early apoptosis, late apoptosis and necrosis in co-cultured scaps at different times of treatment with endodontic irrigating solutions. http://dentistry3000.pitt.edu/ mode of cell death of co-cultured dental mesenchymal stem cells following exposure to endodontic irrigating solutions vol 10 no 1 (2022) doi 10.5195/d3000.2022.146 http://dentistry3000.pitt.edu exposure time(minute) group total number of cells viable cells early apoptosis late apoptosis necrosis mean ± sd mean ± sd mean ± sd mean ± sd 1 control 80000 79392a±208 240b±72 48c±40 152c±184 saline 80000 78800a±840 232b±64 80c±64 816c±864 chx 80000 65832b±1640 760b±64 1376c±416 11640b±1440 naocl 80000 57976b±1080 6008a±432 6616b±200 9096b±696 edta 80000 31832c±1592 1032b±328 22528a±464 24400a±1208 p value 0.00 0.00 0.00 0.00 5 control 80000 79392a±208 240b±72 48c±40 152d±184 saline 80000 77016a±728 344b±336 192c±272 2520d±768 chx 80000 60080b±600 544b±40 4024b±40 15112c±416 naocl 80000 48992b±680 3456a±464 1368c±64 25528b±496 edta 80000 28472c±872 3608a±40 6880a±288 40664a±608 p value 0.00 0.00 0.00 0.00 10 control 80000 79392a±208 240b±72 48b±40 152c±184 saline 80000 76216a±920 192b±56 72b±56 3240c±760 chx 80000 53512b±3176 5808a±4728 4360b±360 16104b±1600 naocl 80000 36720c±1944 136b±8 800b±8 42760a±2136 edta 80000 16008d±1208 904b±56 28888a±464 33904a±792 p value 0.00 0.00 0.00 0.00 sd: standard deviation; p value: one-way anova.for the comparison of irrigating solutions; a,b,c: comparison of irrigating solutions regarding mode of cell death by tukey’s post-hoc test. similar letters indicate lack of a significant difference. figure 3. flow cytometry graph of annexin/pi for edta treated pdlscs: (a) control group, (b) after 1 minute, (c) after 5 minutes, (d) after 10 minutes. in this graph, the upper left (annexin -/ pi +) indicates the necrotic cells, the lower left (annexin -/ pi -) indicates the viable cells, the upper right (annexin +/ pi +) indicates the late apoptotic cells, and the lower right (annexin +/ pi -) indicates the early apoptotic cells. http://dentistry3000.pitt.edu/ mode of cell death of co-cultured dental mesenchymal stem cells following exposure to endodontic irrigating solutions vol 10 no 1 (2022) doi 10.5195/d3000.2022.146 http://dentistry3000.pitt.edu figure 4. flow cytometry graph of annexin/pi for edta treated scaps: (a) control group, (b) after 1 minute, (c) after 5 minutes, (d) after 10 minutes. in this graph, the upper left (annexin -/ pi +) indicates the necrotic cells, the lower left (annexin -/ pi -) indicates the viable cells, the upper right (annexin +/ pi +) indicates the late apoptotic cells, and the lower right (annexin +/ pi -) indicates the early apoptotic cells. a 0.78% 1.06% 0.1% 98.29% b 43.1% 0.2% 56.39% 0.22% c 40.23% 0.08% 57.41% 2.17% d 59.39% 22.85% 0.26% 17.86% a 0.06 % 99. 24% 0.34% 0.19 % b 39.78% 1.28% 28.15% 30.5% http://dentistry3000.pitt.edu/ mode of cell death of co-cultured dental mesenchymal stem cells following exposure to endodontic irrigating solutions vol 10 no 1 (2022) doi 10.5195/d3000.2022.146 http://dentistry3000.pitt.edu discussion this study compared the mode of cell death of co-cultured cells following exposure to four different endodontic irrigating solutions namely 2% chx, 17% edta, 1.5% naocl and saline after different exposure times. the number of viable cells decreased in all groups following an increase in exposure time. necrosis was the dominant mode of cell death in groups treated with chx, naocl and edta. many studies have evaluated the cytotoxicity of different endodontic irrigating solutions against single culture of cells [3,6,1012]. in this study, we evaluated the mode of cell death of co-cultured cells following their exposure to different irrigating solutions. evaluation of co-culture of cells was the main difference between our methodology and that of previous studies. use of a co-culture of cells helps in assessment of the role of cell interactions and their mutual effects on each other. also, it is a strong tool in tissue engineering and plays a role in regeneration of tissues and organs. it is also effective in guiding and protecting the process of tissue regeneration due to interactions of different cells. the interaction of cells plays an important role in regeneration of tissues and organs, and affects cell mitosis, differentiation and physiology [13,14]. the cytotoxic effects of naocl on l929 fibroblasts [15], human dental pulp stem cells [16] and scaps [17] have been previously confirmed. our results revealed that necrosis was the dominant mode of cell death following exposure to 1.5% naocl such that after 10 minutes of exposure, almost half of the cells died of necrosis. alkahtani et al. showed that 5% naocl induced necrosis of mesenchymal stem cells of the bone marrow. light microscopic assessment revealed that in addition to a reduction in number of viable cells after 2 hours of treatment with naocl, the remaining cells were smaller and rounder. the cells had lost their inter-cellular attachments and their cell wall had been disintegrated. these are typical characteristics of necrotic cells. on the other hand, flow cytometric assessment of cells exposed to naocl indicated positive response to both propidium iodide and annexin dyes [11]. their results were in agreement with our findings. longo et al. indicated that 2% naocl caused apoptosis of human kb cells, which was in contrast to our findings. apoptosis is attributed to generation of free radicals. the chloroamination reactions cause generation of free radicals such as hydroxyl ions, that can cause an alkaline environment and accelerate biological stresses in different parts of the cells. thus, the difference in cell response depends on the concentration of hypochlorite and can be related to higher production of free radicals by cells exposed to 2% naocl. free radicals damage the cell dna and can cause chromosomal injury [18]. the current results revealed that necrosis was the dominant mode of cell death following exposure to different endodontic irrigating solutions. necrosis is the passive form of cell death that occurs as the result of cell membrane rupture and release of cell components into the extracellular matrix [19]. however, apoptosis is the active form of cell death characterized by cell shrinkage, condensation of nuclear chromatin and fragmentation of the nucleus [20]. apoptotic cells, in contrast to necrotic cells, are quickly phagocytosed in vivo without causing inflammatory reactions. for this reason, cell death by apoptosis and necrosis are biologically different. after cell necrosis, severe inflammatory reactions occur, causing severe tissue damage [21]. the present results revealed minimum cell death in scaps treated c 35.58% 4.5% 8.6% 50.83% d 1.12% 20.01% 36.11% 42.37% http://dentistry3000.pitt.edu/ mode of cell death of co-cultured dental mesenchymal stem cells following exposure to endodontic irrigating solutions vol 10 no 1 (2022) doi 10.5195/d3000.2022.146 http://dentistry3000.pitt.edu with chx in the co-culture at 10 min, compared with sodium hypochlorite and edta. the mode of cell death was mainly necrosis in them, followed by early apoptosis; whereas, pdlscs treated with chx mainly underwent early apoptosis at 1 and 10 min. hernandez et al. demonstrated that combination of 0.12% chx and proroot mta induced apoptosis in macrophages and fibroblasts while combination of proroot mta and distilled water could not induce apoptosis. they assessed the cell cycle and concluded that chx increased the ratio of cells in g1 phase and decreased the ratio of cells in s phase. s phase is the dna transcription phase and cell mitosis occurs in this phase. in fact, combination of chx and proroot mta had anti-proliferative effects on the cells [22]. newton et al. showed that chx ruptures the external membrane of the mitochondria and releases apoptosis-inducing proteins such as cytochrome c [23]. faria et al. evaluated apoptosis and necrosis following the use of chx on l929 fibroblasts using flow cytometry after 24 hours. they used 0.000125% to 0.016% concentrations of chx and the results showed that concentrations higher than 0.004% of chx caused cell death by necrosis while lower concentrations induced apoptosis. they suggested increased expression of heat-shock protein as an indicator of cellular stress [15]. their results were in agreement with our findings since in our study, the dominant mode of cell death following exposure to 2% chx was necrosis. li et al, demonstrated that chx had cytotoxic effects on raw264.7 macrophages in a dosedependent and time-dependent manner. type of cell death shifted from apoptosis to necrosis by an increase in concentration of chx, and dose-dependent dna damage occurred [24]. giannelli et al. reported that chx induced apoptosis and necrosis of osteoblasts, endothelial cells and fibroblasts. the viable cell count depends on the dosage and duration of exposure to chx. apoptosis and necrosis are both characterized by impaired mitochondrial function, increased intracellular calcium level and oxidative stress [25]. our results indicated death of around 80% of the cells 10 minutes after exposure to edta; necrosis occurred in 2/3 and late apoptosis occurred in 1/3 of the cells. following an increase in concentration of intracellular calcium due to the activity of ca atpase pump, or increased entry of calcium into the nucleus and mitochondrial organelles, caspases are activated, inducing apoptosis [26]. many studies have suggested the correlation of apoptosis with cationic ions. sakabe et al. showed that edta induced apoptosis of neuro-2a cells and fragmentation of dna [27]. ostby demonstrated that exposure of periapical tissues to 17% edta did not cause any cell or tissue damage. moreover, they did not report any cell necrosis following exposure of dental pulp cells to edta [28], which was in contrast to our findings. amaral et al. showed that 17% edta had direct effects on macrophages and caused some changes in their cell membrane via chelator ions such as magnesium and calcium, which can enhance apoptosis [29]. in brief, the cytotoxicity of chx, naocl and edta significantly increased by increased exposure time. in co-culture, the mode of stem cells death treated with sodium hypochlorite and edta is necrosis, while chlorhexidine at 1 and 10 minutes often causes the pdlscs death through early apoptosis. despite the limitations of this in vitro study, it appears that co-culture better simulates the clinical setting than single culture. references 1. imaging of apoptosis. tait jf. j nucl med. 2008;49:1573-6. 2. cytotoxicity effect of cladribine on the mcf-7 human breast cancer cell line. hashemi m, karami-tehrani f, ghavami s. iran biomed j. 2004;8(1):7-12. 3. evaluation of chlorhexidine toxicity injected in the paw of mice and added to cultured l929 fibroblasts. faria g, celes mr, de rossi a, silva lab, silva js, rossi ma. j endod. 2007;33:715-22. 4. adenosine and deoxyadenosine induces apoptosis in oestrogen receptor-positive andnegative human breast cancer cells via the intrinsic pathway. cell prolif. 2005;38:269-85. 5. evaluation of cytotoxicity of mtad using the mtt-tetrazolium method. zhang w, torabinejad m, li y. j endod. 2003;29:654-7. 6. evaluation of cytotoxic effects of various endodontic irrigation solutions on the survival of stem cell of human apical papilla. mollashahi nf, saberi e, karkehabadi h. iran endod j. 2016;11:293. 7. an in vitro evaluation of the cytotoxicity of various endodontic irrigants on human gingival fibroblasts. barnhart bd, chuang a, dalle lucca jj, roberts s, liewehr f, joyce ap. j endod. 200531:613-5.. 8. cell culture models of higher complexity in tissue engineering and regenerative medicine. kirkpatrick cj, http://dentistry3000.pitt.edu/ mode of cell death of co-cultured dental mesenchymal stem cells following exposure to endodontic irrigating solutions vol 10 no 1 (2022) doi 10.5195/d3000.2022.146 http://dentistry3000.pitt.edu fuchs s, hermanns mi, peters k, unger re.biomaterials. 2007;28:51938. 9. a review of the threedimensional cell culture technique: approaches, advantages and applications. zhang w, zhuang a, gu p, zhou h, fan x. curr stem cell res ther. 2016;11:370-80. 10. cytotoxicity of endodontic irrigants on human periodontal ligament cells. karkehabadi h, yousefifakhr h, zadsirjan s. iran endod j. 2018;13:390. 11. cytotoxicity of qmix™ endodontic irrigating solution on human bone marrow mesenchymal stem cells. alkahtani a, alkahtany sm, mahmood a, elsafadi ma, aldahmash am, anil s. bmc oral health. 2014;14:27 12. effects of chlorhexidine on stem cells from exfoliated deciduous teeth. tu y-y, yang c-y, chen r-s, chen m-h. taiwan yi xue hui za zhi. 2015;114:17-22. 13. controlling cell interactions by micropatterning in co-cultures: hepatocytes and 3t3 fibroblasts. bhatia sn, yarmush ml, toner m. j biomed mater res a. 1997;34:189-99. 14. osteogenic differentiation is selectively promoted by morphogenetic signals from chondrocytes and synergized by a nutrient rich growth environment. gerstenfeld l, barnes g, shea c, einhorn t. connect tissue res. 2003;44:85-91. 15. chlorhexidine-induced apoptosis or necrosis in l929 fibroblasts: a role for endoplasmic reticulum stress. faria g, cardoso cr, larson re, silva js, rossi ma. toxicol appl pharmacol. 2009;234:256-65. 16. effect of sodium hypochlorite on human pulp cells: an in vitro study. essner md, javed a, eleazer pd. oral surg oral med oral pathol oral radiol endod. 2011;112:662-6. 17. effect of irrigants on the survival of human stem cells of the apical papilla in a platelet-rich plasma scaffold in human root tips. trevino eg, patwardhan an, henry ma, perry g, dybdal-hargreaves n, hargreaves km, et al. j endod. 2011;37:1109-15. 18. cytotoxicity and genotoxicity of endodontic irrigants on human cells. longo jpf, valois ca, tapajَs ةcc, santos mdfma, de azevedo rb. arch oral res. 2010;6.135-40 19. adhesive resin induces apoptosis and cell-cycle arrest of pulp cells. mantellini m, botero t, yaman p, dennison j, hanks c, nِr j. j dent res. 2003;82:592-6. 20. the pathways of cell death: oncosis, apoptosis, and necrosis. trump be, berezesky ik, chang sh, phelps pc. toxicol pathol. 1997;25:82-8. 21. the coinitiator dmabee induces death by apoptosis and necrosis in human monoblastoid cells. cimpan mr, matre r, skaug n, lie sa, lygre h. clin oral investig. 2005;9:168-72. 22. effect of proroot® mta mixed with chlorhexidine on apoptosis and cell cycle of fibroblasts and macrophages in vitro. hernandez e, botero t, mantellini m, mcdonald n, nِr j. int endod j. 2005;38:137-43. 23. new data on biological effects of chlorhexidine: fe2+ induced lipid peroxidation and mitochondrial permeability transition. newton apn, cadena sms, rocha mem, carnieri egs, de oliveira mbm. toxicol lett. 2004;151:407-16. 24. cytotoxicity and genotoxicity of chlorhexidine on macrophages in vitro. li yc, kuan yh, lee ss, huang fm, chang yc. environ toxicol. 2014;29:452-8. 25. effect of chlorhexidine digluconate on different cell types: a molecular and ultrastructural investigation. giannelli m, chellini f, margheri m, tonelli p, tani a. toxicol in vitro. 2008;22:308-17. 26. apoptosis in development. meier p, finch a, evan g. nature. 2000;407:796. 27. induction of apoptosis in neuro-2a cells by zn2+ chelating. sakabe i, paul s, dansithong w, shinozawa t. cell struct funct. 1998;23:95-9. 28. chelating in root canal therapy. ethylene-diamine tetraacetic acid for cleansing and widening of root canals. ostby n. odontol tidskr. 1957;65:3-11. 29. cytotoxicity analysis of edta and citric acid applied on murine resident macrophages culture. amaral k, rogero m, fock r, borelli p, gavini g. int endod j. 2007;40:33843. http://dentistry3000.pitt.edu/ microsoft word 96 2020_manuscript.docx vol 8, no 1 (2020) issn 2167-8677 (online) doi 10.5195/d3000.2020.96 http://dentistry3000.pitt.edu new articles in this journal are licensed under a creative commons attribution 4.0 united states license. dental anomalies in orthodontic patients with and without skeletal discrepancies clarissa christina avelar fernandez1, mônica gentil mattos1, christiane vasconcellos cruz alves pereira1, marcelo de castro costa1 1 department of pediatric dentistry and orthodontics, federal university of rio de janeiro, rio de janeiro, brazil abstract objec&ve: to determine whether individuals with skeletal discrepancies of class ii or iii display a higher frequency of dental anomalies in comparison with individuals with class i malocclusion. design: a systematic search of the main electronic medical scienti2ic literature databases was conducted. observational studies were selected if mentioning dental anomalies in the different skeletal malocclusion patterns. results: a total of 4,768 studies were found and the duplicated studies (1,279) were removed, resulting in 3,489 papers to be analyzed. after screening by title, 138 were 2it for screening by abstract. after that, a total of 13 papers were carefully read in full. five studies included dental anomaly frequencies in orthodontic patients and included 7,679 participants. the frequency of dental anomalies ranged from 11.2% to 40.3%. it was observed that individuals with skeletal discrepancies of class ii and iii had more dental anomalies when compared to individuals with class i. conclusion: individuals with skeletal malocclusion patterns have more dental anomalies and there is an association between dental anomalies and skeletal class ii or class iii malocclusion patterns. cita%on: fernandez cca, et al. (2020) dental anomalies in orthodon&c pa&ents with and without skeletal discrepancies. den5stry 3000. 1:a001 doi:10.5195/d3000.2020.96 received: march 30, 2020 accepted: april 5, 2020 published: april 15, 2020 copyright: ©2020 fernandez cca, et al. this is an open access ar5cle licensed under a crea5ve commons ajribu5on work 4.0 united states license. email: pjpo2009@yahoo.com.br introduction dental anomalies are clinical alterations resulting from disturbances during the tooth formation process.1 they represent disturbances of number, size, shape, position and structure of the teeth.1,2 the prevalence of dental anomalies can range from 5.46 to 74.7%,1,3 due to different ethnicities and diagnostic criteria.1,4,5 the etiology of dental anomalies includes genetic and environmental factors.2,5,6 skeletal malocclusions are usually categorized and described by disturbances in the craniofacial and occlusal relationships7 and often appear together with the dental anomalies, asserting their relation and complicating therapy.8 it appears that dental anomalies may be more likely to occur if individuals have class ii or class iii relationships in comparison to class i.9 this sophisticated clinical definition is a base for phenotype-genotype correlation, that may contribute to more accurate treatment predictions and to genetic studies.9 this systematic review aimed to confirm that evidence exists that individuals with class i skeletal malocclusion are less likely to have dental anomalies. material and methods the present systematic review was registered at prospective register of systematic reviews (prospero; centre for reviews and dissemination, university of york; and the national institute for health research) under the registry number dental anomalies in orthodon/c pa/ents with and without skeletal discrepancies vol 8 no 1 (2020) doi 10.5195/d3000.2020.96 http://dentistry3000.pitt.edu crd42016038916 and followed the preferred reporting items for systematic review and meta analyses (prisma) checklist.10 search strategy the following focused question was formulated: “is there a difference in the frequency of dental anomalies in orthodontic patients with class ii or iii versus class i malocclusion?” to develop the focused question, a set of criteria for study eligibility was adopted, based on pecos strategy (population: orthodontic patients; exposure: skeletal discrepancies; comparator: standard of normality; and outcome: dental anomalies), using mesh, entry terms and key words (table 1). the main search was conducted in november 2016 and was updated on january 2019. publications of potential relevance to this study were identified after searching the main electronic medical scientific literature databases, including pubmed, scopus, lilacs, web of science, the cochrane library, and google scholar. additional articles of potential relevance were identified by manual searches. textbooks, dissertations, case reports, case series, review articles, and abstracts were excluded. studies in populations presenting specific systemic diseases, conditions (history of trauma, cleft lip and/or palate, syndromes), endocrine imbalances and/or metabolic disorders (being these sporadic or hereditary), and no descriptions of the three skeletal malocclusions were excluded. two examiners (c.c.a.f. and m.g.m.) independently screened each paper by examining the title, abstract, and keywords. no restriction was applied regarding language or date of publication. the selected studies were described in relation to the type of study, total sample, sex distribution, how the diagnosis of skeletal malocclusion and dental anomalies were performed, and the frequency of dental anomalies in each malocclusion. dental anomalies were also grouped in anomalies of number, shape and position (table 2). results and discussion observational studies were included because they mentioned dental anomalies in the different skeletal malocclusion patterns, enabling comparison between groups (classe i, ii and iii) and different populations. figure 1 shows the flow diagram of the search results from the databases. after search, a total of 4,768 records were found: 750 from cochrane library, 1,616 from pubmed, 911 from scopus, 323 from scopus-med, 1,158 from web of science, four from lilacs and bbo, and six from google scholar. table 1. pecos strategy (popula7on; exposure; comparator; and outcome), using mesh, entry terms and key words dental anomalies in orthodon/c pa/ents with and without skeletal discrepancies vol 8 no 1 (2020) doi 10.5195/d3000.2020.96 http://dentistry3000.pitt.edu table 2. data extracted from the five selected studies. dental anomalies in orthodon/c pa/ents with and without skeletal discrepancies vol 8 no 1 (2020) doi 10.5195/d3000.2020.96 http://dentistry3000.pitt.edu the duplicated studies (1,279) were removed, resulting in 3,489 records to be analyzed. after screening by title, 138 were fit for screening by abstract. after that, a total of 13 records were carefully read in full and five studies containing 7,679 participants were selected. the eight studies were excluded because they did not have the classification of skeletal malocclusions patterns. table 2 shows the extraction of the data from the selected studies. the frequency of dental anomalies ranged from 11.2% to 40.3%.1,9,11,12,13 it was observed that individuals with skeletal discrepancies (class ii and iii) had more dental anomalies when compared to individuals with no skeletal discrepancies (class i).1,9,12,13 the same was observed for dental anomalies of number,1,12,13 shape, and position.10 three studies only made the diagnosis of tooth agenesis11,13 and/or third molar agenesis.11,12 the studies included in this systematic review had a frequency of dental anomalies raging from 11.2% to 40.3%. celicoglu and kamak (2012)12 reported a frequency of 22.7% of dental anomalies whereas fernandez et al. (2018)9 reported a frequency of 15.7% of dental anomalies of number, shape and position. uslu et al. (2009)1 had the highest frequency of dental anomalies among orthodontic patients of the five studies selected (40.3%), having tooth agenesis, evagination, and invagination as the most common dental anomalies. the studies that found the lowest frequency of dental anomalies in orthodontic patients were of young (2010)5 and chung et al. (2008),11 with a frequency of 11.3% and 11.2%, respectively. it is likely that these differences are due to the different populations studied (turkish, brazilian, and korean) and different dental anomaly definitions. conclusion in summary, individuals with skeletal class ii or class iii malocclusion patterns have more dental anomalies than individuals who are class i. in addition, there is an association between dental anomalies and skeletal class ii or class iii malocclusion patterns. acknowledgments this work was developed as part of a course on systematic reviews and meta-analysis taken by c.c.a.f. and m.g.m. we thank lucianne cople maia, marcela baraúna magno, and danielle masterson for this learning experience. references 1. prevalence of dental anomalies in various malocclusions. uslu o, akcam mo, evirgen s, cebeci i. am j orthod dentofacial orthop. 2009 mar;135(3):328-35. doi: 10.1016/j.ajodo.2007.03.030. 2. the class ii division 2 craniofacial type is associated with numerous congenital tooth anomalies. basdra ek, kiokpasoglou m, stellzig a. eur j orthod. 2000 oct;22(5):529-35. 3. [agenesis in permanent dentition]. diaz-perez r, echaverry-navarrete ra. figure 1. flow diagram of the literature search and selec7on process implemented. dental anomalies in orthodon/c pa/ents with and without skeletal discrepancies vol 8 no 1 (2020) doi 10.5195/d3000.2020.96 http://dentistry3000.pitt.edu rev salud publica (bogota). 2009 dec;11(6):961-9. 4. prevalence and distribution of dental anomalies in orthodontic patients. altug-atac at, erdem d. am j orthod dentofacial orthop. 2007 apr;131(4):510-4. 5. investigation of hypodontia as clinically relates dental anomaly: prevalence and characteristics. kim yh. isrn dentistry 2011;article id 246135, 6 pages. 6. studies of dental anomalies in a large group of school children. küchler ec, risso pa, costa mde c, modesto a, vieira ar. arch oral biol. 2008 oct;53(10):941-6. doi: 10.1016/j.archoralbio.2008.04.00. 7. congenital tooth anomalies and malocclusions: a genetic link? basdra ek, kiokpasoglou mn, komposch g. eur j orthod. 2001 apr;23(2):145-51. 8. prevalence of dental anomalies among school going children in india. kathariya md, nikam ap, chopra k, patil nn, raheja h, kathariya r. j int oral health. 2013 oct;5(5):10-4. 9. dental anomalies in different growth and skeletal malocclusion patterns. fernandez cca, pereira cvca, luiz rr, vieira ar, de castro costa m. angle orthod. 2018 mar;88(2):195-201. doi: 10.2319/071917-482.1. 10. preferred reporting items for systematic reviews and metaanalyses: the prisma statement. moher d, liberati a, tetzlaff j, altman dg; prisma group. int j surg. 2010;8(5):336-41. doi: 10.1016/j.ijsu.2010.02.007. 11. the pattern and prevalence of hypodontia in koreans. chung cj, han jh, kim kh. oral dis. 2008 oct;14(7):620-5. doi: 10.1111/j.16010825.2007.01434.x. 12. patterns of third-molar agenesis in an orthodontic patient population with different skeletal malocclusions. celikoglu m, kamak h. angle orthod. 2012 jan;82(1):1659. doi: 10.2319/041911-274.1. long term radiographic evaluation of a potentially impacted and dilacerated tooth into complete transposition vol 9 no 1 (2021) doi 10.5195/d3000.2021.138 http://dentistry3000.pitt.edu long term radiographic evaluation of a potentially impacted and dilacerated tooth into complete transposition hassan ali shafiee 1 , golnaz nahvi 1 *, farzad aghdashi 2 , shahryar karami 3 1department of orthodontics, school of dentistry, shahid beheshti university of medical sciences, tehran, iran 2 department of oral and maxillofacial surgery, school of dentistry, shahid beheshti university of medical sciences, tehran, iran 3 department of orthodontics, school of dentistry, tehran medical sciences, islamic azad university, tehran, iran abstract background: anomalies such as ectopic tooth eruption and transposition are challenging situations that may complicate the orthodontic treatment. some of these anomalies can only be diagnosed and managed by accurate long-term radiographic examination. delayed diagnosis could impede simple preventive orthodontic measurements and cause more costly and consuming future complicated orthodontic treatment. case description: we described in detail the long term follow-up of a young female patient referring with a unilateral ectopic eruption. the patient was under close observation and regular orthodontic treatment to guide a unilateral ectopic eruption into a complete transposition of an upper left canine and first premolar. the long term follow-up revealed stables results. practical implications: early diagnosis and preventive measurements in patients suffering from anomalies such as ectopic eruption is crucial in order to avoid further complications such as tooth impaction and dilaceration formation and adopting the best choice of treatment. keywords: tooth transposition; ectopic eruption; maxillary canine; orthodontic treatment citation: ali shafiee h, et al. (2021) long term radiographic evaluation of a potentially impacted and dilacerated tooth into complete transposition dentistry 3000. 1:a001 doi:10.5195/d3000.2021.138 received: january 14, 2021 accepted: april 22, 2021 published: july 30,2021 copyright: ©2021 ali shafiee h, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: golnaznahvi@gmail.com introduction eruption patterns and timing of individual teeth is affected by various items like ethnicity, geographical area, race, hormonal factors, gene pool, gender, social status, growth and nutrition [1]. the canine is the tooth with the highest percentage of problems of space in the dental arch and its ectopic eruption and eruption in a high zone is common [2-3]. transposition is a kind of ectopic eruption, defined as the positional interchange of two adjacent teeth in the same quadrant of the dental arch [4]. the most common type of transposition is between the canine and first maxillary premolar. unilateral transposition has been reported more often than bilateral transposition, with the left side more frequently involved than the right [5-7]. tooth transposition is a rare anomaly of dentition that interchanges position of two teeth that are adjacent to each other [8]. the period of transition from primary dentitions to permanent one between the age of 6 and 12 years is often a time of worry for parents [9]. in the population the prevalence of dental transposition that is considered as subdivision of the ectopic eruption is about 0.4% [10]. transposition may be complete or incomplete and each of those demands a different management protocol and considerations [11-14]. long term radiographic evaluation of a potentially impacted and dilacerated tooth into complete transposition vol 9 no 1 (2021) doi 10.5195/d3000.2021.138 http://dentistry3000.pitt.edu transposition is often accompanied by other dental anomalies such as dilacerations in the involved teeth [6]. dilacerations of a tooth is when there is a typical bend in the crown or root [14-17]. this complication make endodontic and surgical procedures difficult to carry out [18]. this malformation of the root is not detected in routine clinical examination and as a result it may be underestimated in many cases [19-22]. current research shows a high occurrence of dilaceration concurrent with other dental developmental anomalies such as tooth transposition [23]. the nontreatment approach for an ectopically erupting tooth may lead to root resorption, dilaceration of the neighboring teeth, development of cysts, or ankylosis and the need for more complicated and costly orthodontic treatments. early diagnosis and careful follow-up of developing dentition is crucial for early intervention and correction of eruptive anomalies. sometimes guiding a tooth into a transposed location can be beneficial in order to prevent abnormalities such as dilacerations or resorption and remove the need for tooth extraction and future complex orthodontic measurement. therefore, in the current study we present a case with ectopic canine eruption followed-up regularly for a long-term period and treated into a complete transposition to prevent canine impaction and dilaceration forming in the adjacent first premolar. materials and methods case presentation a healthy seven-year-old patient in the early mixed dentition stage was referred for orthodontic consultation. her extra-oral examination showed symmetrical craniofacial features with average facial proportions. nothing abnormal was detected during a functional evaluation of the stomatognathic system and the temporomandibular joint (tmj). intra-oral examination revealed a mesial step malocclusion in primary dentition and an edge-toedge relationship of left upper and lower lateral incisors (figure 1). radiographic evaluations the upper left canine showed an ectopic position compared to the contralateral upper right canine (figure 2). the patient was treated with removable palatal expander with a tong-guard. she was under regular orthodontic observations until the eruption of figure 1. intra-oral and extra-oral views of the upper and lower dental arch. long term radiographic evaluation of a potentially impacted and dilacerated tooth into complete transposition vol 9 no 1 (2021) doi 10.5195/d3000.2021.138 http://dentistry3000.pitt.edu the lower incisors. at this stage stripping of lower deciduous canines was performed to create enough space for lower incisors eruption. at the two-year follow-up, the ectopic position of upper left canine was still obvious with a close proximity to upper left premolar root (figure 3). at the three-year and four-year follow-ups, the upper right primary canine had exfoliated, leaving its space vacant. the upper right canine showed a downward movement adjacent to right lateral incisor toward the dental arch but the upper left canine did not follow the regular eruptive path (figures 4 and 5). the first and second deciduous molars were extracted with three months interval. at the five-year follow-up at the age of 12, panoramic and cbct views of upper dental arch revealed impaction of upper left canine with the canine crown proximity to upper left first premolar palatal root. left canine long axis was parallel to the premolars, therefore determining a complete transposition anomaly of the canine. in addition, dilaceration formation in buccal root of left first premolar is clearly evident figure 3. the panoramic radiograph of the patient after two years follow-up at the age of 9. figure 4. panoramic radiograph of the patient after three years follow-up at the age of 10. figure 2. the initial panoramic and cephalogram radiographs of the patient at the age of 7. long term radiographic evaluation of a potentially impacted and dilacerated tooth into complete transposition vol 9 no 1 (2021) doi 10.5195/d3000.2021.138 http://dentistry3000.pitt.edu from different views. the follicular space of upper left canine is 2.97 mm and is seen in cbct crosssectional view number 10. all the cbct sections were prepared in real size with the slice thickness and interval of 1.00 mm (figures 6 and 7). the patient was informed that no intervention at this dental developmental stage might complicate future orthodontic mechanics by increasing the risk of canine impaction and first premolar buccal root dilaceration and resorption. therefore two different treatment alternatives were discussed for the patient. treatment alternatives the first treatment option for this case included the extraction of left first premolar to prevent canine impaction, first premolar buccal root dilaceration, resorption and retaining the remaining site for future implant‑supported prosthesis in place of extracted first premolar. the second alternative was to guide the upper left premolar into canine position in order to avoid any extraction and root dilaceration formation and preserving the integrity of upper dental arch. the patient was not willing to extract any permanent figure 5. the panoramic radiograph of the patient after four years follow-up at the age of 11. figure 6. the cbct views of the patient after five years follow-up at the age of 12. figure 7. the panoramic view of the patient after five years follow-up at the age of 12. long term radiographic evaluation of a potentially impacted and dilacerated tooth into complete transposition vol 9 no 1 (2021) doi 10.5195/d3000.2021.138 http://dentistry3000.pitt.edu tooth so the second treatment option was selected by the patient after thorough discussion of the two options. treatment procedure orthodontic treatment was commenced in the upper arch immediately in order to prevent further contact of the canine with premolar buccal root. the primary treatment goal was to reposition the transposed canine into the first premolar site and align the labial segment of both arches. a non-extraction orthodontic treatment was chosen and patient was under rapid maxillary expansion for three months. then fixed appliances were used on all upper teeth. the 0.022× 0.025’’ mbt prescription (3m unitek, usa) was placed with 0.014” nickel titanium [24] wire for initial leveling and alignment of the teeth. as treatment progressed, 0.018” stainless steel wire was placed in the maxillary arch with niti open‑coil spring between maxillary left canine and first premolar for mesialization of maxillary first premolar and creating space for alignment of the permanent maxillary left canine (figure 8). when enough space for the canines was obtained, they were bonded and included in the arch wire. treatment sessions included regular recalls for appliance adjustment and reinforcement of oral hygiene measures. following the transposition of left canine and first premolar, reshaping was also carried out for the maxillary right first premolar to look like canine. the treatment continued to bond the lower arch and establish the final ideal occlusion into class i canine and molar relationships establishment. results clinical results the patient records showed that all treatment objectives were achieved. the eleven year followup of the patient showed that the case's preventive orthodontic treatment was proven successful. at first glance, the possibility of orthognathic surgery in the future for treatment of skeletal class iii was quite possible, but this treatment option was abandoned with preventive treatments such as an expansion removable plate containing a tang guard at the appropriate age, long-term followup, as well as high patient satisfaction with orthodontic treatment. also, the patient's intraoral record shows the complete success of preventive treatment with transposition of first premolar instead of canine. as shown in figure 9, the occlusal view indicates the true transposition of the canine and the first premolar in the left side at the age of 18 years. ideal overjet and overbite were achieved and also transverse correction was done so that no crossbite was seen after treatment. figure 8. the periapical view of upper left canine during orthodontic alignment at the age of 13 (a) and 14 (b). long term radiographic evaluation of a potentially impacted and dilacerated tooth into complete transposition vol 9 no 1 (2021) doi 10.5195/d3000.2021.138 http://dentistry3000.pitt.edu radiographic results the panoramic and periapical radiographs showed good parallelism of roots and normal structure of the periodontium, roots, and surrounding tissues. no sign of root resorption or other damage to the teeth were seen in the eleven-year follow-up period. also, no evidence of caries was found during the long follow-up period, and the final panoramic radiograph was free of any dental pathology, which showed good hygiene and continuous hygiene training during the treatment period (figures 9 and 10). discussion regular patient follow-up from the early stages of tooth development offers opportunities to intercept and correct some types of developmental abnormalities at the proper time, which might be more burdening in future. in this case of ectopic canine eruption timely intervention led to satisfactory results by replacing the canine with adjacent premolar to avoid dilaceration formation in buccal root of the left premolar. correct intercuspation between the upper and lower teeth was achieved, without any aesthetic or functional alterations. orthodontic treatment was conducted without much difficulty or complexity. side effects that may arise in these situations in case of nointervention protocol, such as: root resorption, dilacerations or impactions were prevented by regular long-term observations. previous studies have debated the etiology for variation of root morphology in maxillary first premolar. even though certain genes such as msx1 and wnt have been reported to play a role [2527] most studies indicated that the variation of first premolar roots was related to environmental conditions, particularly the impaction of adjacent canines [28]. the concluded that if canine impaction is not treated it can cause serious harm to adjacent teeth such as root resorption in a large percentage of first premolars [28]. in a recent case report unilateral premolar extraction is conducted in a case with complete transposition of upper canine and first premolar. they recommended that in case of unilateral extraction in transposition cases care must be taken in order to prevent midline shift and development of arch asymmetry during orthodontic intervention [29]. as unilateral extractions in these cases may complicate orthodontic treatment figure 9. intra-oral and extra oral views of the upper and lower dental arch showing successful canine-premolar transposition. long term radiographic evaluation of a potentially impacted and dilacerated tooth into complete transposition vol 9 no 1 (2021) doi 10.5195/d3000.2021.138 http://dentistry3000.pitt.edu choosing a non-extraction protocol to establish a complete transposition could facilitate orthodontic treatment with more predictable mechanics and reasonable results. although canine substitution with premolar may be a suitable alternative treatment in some ectopically displaced canines, it needs some certain considerations in order to achieve optimal aesthetic and functional results such as: controlling the canine torque to move the root palatally to hide the root prominence and the premolar torque to move the root buccally to mimic the canine bulge and prevent occlusal interference of the palatal cusp [30]. in early research results it was speculated that root dilacerations of first maxillary premolars were a probable cause for canine impaction [31]. then, future studies reported that the developing first premolar root may be more susceptible to dilacerations in response to an impacted canine [32-34]. so prevention of canine impaction by transposing it with first premolar is a reasonable treatment approach to prevent dilaceration formation in a first premolar in the stage of root development. thus proper case selection at the suitable age when the premolar root adjacent to ectopic canine is forming is critical because after complete dilaceration occurs any bodily movement to transpose canine with first premolar can lead to serious side effects such as root resorption. this method was conducted in the presented patient and led to successful aesthetic and functional results. the key factor for successful management of this case is long term follow-up, excellent patient cooperation and regular attendance of orthodontic treatment sessions. root morphology is a fundamental issue in planning orthodontic tooth movements as some root variations such as root dilacerations may cause detrimental side effects such as root resorption during orthodontic tooth movements. therefore, a thorough knowledge of root anatomy could be helpful in prevention of some iatrogenic side effects. a recent study on iranian population revealed that the prevalence of root dilacerations is estimated equal between males and females. in addition, the prevalence of dilacerations was estimated 1.65% over ally different teeth types [35]. maxillary canine-first premolar transposition is not a common phenomenon. researches revealed that maxillary canine-first premolar transposition was found to have a lower frequency than the maxillary canine-lateral central transposition [36]. in this case we aimed to intentionally guide a maxillary canine and premolar into a complete transposition so that the potentially first premolar root dilaceration was prevented successfully. figure 10. the panoramic radiograph of the patient after eleven years follow-up at the age of 18 long term radiographic evaluation of a potentially impacted and dilacerated tooth into complete transposition vol 9 no 1 (2021) doi 10.5195/d3000.2021.138 http://dentistry3000.pitt.edu in a study by grisar et al. the relationship between threedimensional position of the impacted canine and associated surrounding tissues abnormality was measured. the authors concluded that the most common associated pathologies was dilaceration of the root and resorption of a neighboring tooth [37]. these findings were in line with the current study results which showed evident dilacerations formation in the first premolar adjacent to the impacted canine in the current case. conclusion based on the results of this study, we can suggest that: careful follow-up of the patient at an early age allows the detection of veiled developmental anomalies. accurate diagnosis and timely intervention in patients with ectopic eruption and other dental eruptive abnormalities could diminish, prevent or eliminate serious complications such as root dilacerations, resorption or canine impaction. prompt intervention can facilitate orthodontic treatment and prevent any complex and timely future treatments. funding there is no financial support. acknowledgments the authors would like to thank the department of orthodontics, school of dentistry, shahid beheshti and islamic azad university of medical sciences, tehran, iran. conflicts of interest the authors declare no conflict of interest. references 1. changes in the sequence of eruption of permanent teeth; correlation between chronological and dental age and effects of body mass index of 515-year-old schoolchildren. khan as, nagar p, singh p, bharti m. international journal of clinical pediatric dentistry. 2020; 13(4):368-380. 2. management of impacted canines. bishara se, kommer dd, mcneil mh, montagano ln, oesterle lj, youngquist hw. american journal of orthodontics. 1976; 69(4):371-387. 3. rohani k, eslamian l, nahvi g. correlation between maxillary cuspid impaction with available space and anomalies of maxillary lateral incisors. dental research journal. 2021 jan 1;18(1):27. 4. tooth transpositions—a review of the literature and treatment considerations. shapira y, kuftinec mm. the angle orthodontist. 1989; 59(4):271-276. 5. canine transposition as autosomal recessive trait in an inbred kindred. feichtinger c, rossiwall b, wunderer h. journal of dental research. 1977; 56(12):1449-1452. 6. maxillary tooth transposition: correct or accept? ciarlantini r, melsen b. american journal of orthodontics and dentofacial orthopedics. 2007; 132(3):385394. 7. the presentations and management of transposed teeth. weeks e, power s. british dental journal. 1996; 181(11):421. 8. canine transposition prevalence, distribution and treatment considerations among orthodontic patients. finkelstein t, shapira y, pavlidi am, davidovitch m, blumer s, schonberger s, shpack n. the journal of clinical pediatric dentistry. 2020; 44(4):268-273. 9. sockalingam snmp, zakaria asi, khan kam, azmi fm, noor nm simple orthodontic correction of rotated malpositioned teeth using sectional wire and 2 × 4 orthodontic appliances in mixeddentition: a report of two cases. case reports in dentistry, 2020. long term radiographic evaluation of a potentially impacted and dilacerated tooth into complete transposition vol 9 no 1 (2021) doi 10.5195/d3000.2021.138 http://dentistry3000.pitt.edu 2020, 6972196 doi: https://doi.org/10.1155/2020/697 2196. 10. orthodontic treatment of unilateral cleft lip and palate associated with maxillary canine/premolar transposition: case report. souza rmd, oliveira htd, farret mm. dental press journal of orthodontics. 2020; 25(3):54-64. 11. orthodontic management of a complete and an incomplete maxillary canine-first premolar transposition. lorente c, lorente p, perez-vela m, esquinas c, lorente t. the angle orthodontist. 0(0):null. 12. isolation and screening of extracellular anticancer enzymes from halophilic and halotolerant bacteria from different saline environments in iran. zolfaghar m, amoozegar ma, khajeh k, babavalian h, tebyanian h. mol. biol. rep. 2019; 46(3):3275-3286. 13. histologic analyses of different concentrations of tritonx-100 and sodium dodecyl sulfate detergent in lung decellularization. tebyanian h, karami a, motavallian e, aslani j, samadikuchaksaraei a, arjmand b, nourani mr. cell mol biol (noisyle-grand). 2017; 63(7):46-51. 14. current and advanced nanomaterials in dentistry as regeneration agents: an update. yazdanian m, rahmani a, tahmasebi e, tebyanian h, yazdanian a, mosaddad sa. minirev. med. chem. 2020; 20(1-21. 15. current biocompatible materials in oral regeneration: a comprehensive overview of composite materials. tahmasebi e, alam m, yazdanian m, tebyanian h, yazdanian a, seifalian a, mosaddad sa. j mater res technol. 2020; 9(5):11731-11755. 16. fabrication and properties of developed collagen/strontiumdoped bioglass scaffolds for bone tissue engineering. mosaddad sa, yazdanian m, tebyanian h, tahmasebi e, yazdanian a, seifalian a, tavakolizadeh m. j. mater. res. technol. 2020; 9(6):14799-14817. 17. current herbal medicine as an alternative treatment in dentistry: in vitro, in vivo and clinical studies. moghadam et, yazdanian m, tahmasebi e, tebyanian h, ranjbar r, yazdanian a, seifalian a, tafazoli a. eur. j. pharmacol. 2020; 889(173665. 18. prevalence of shape-related developmental dental anomalies in india: a retrospective study. goswami m, bhardwaj s, grewal n. international journal of clinical pediatric dentistry. 2020; 13(4):407-411. 19. pawlaczyk-kamieńska t, winiarska h, kulczyk t, cofta s dental anomalies in rare, genetic ciliopathic disorder-a case report and review of literature. international journal of environmental research and public health, 2020. 17, doi: https://doi.org/10.3390/ijerph171 24337. 20. cell toxicity and inhibitory effects of cyperus rotundus extract on streptococcus mutans, aggregatibacter actinomycetemcomitans and candida albicans. khojaste m, yazdanian m, tahmasebi e, shokri m, houshmand b, shahbazi r. eur j transl myol. 2018; 28(4):7917. 21. fabrication and properties of βtcp/zeolite/gelatin scaffold as developed scaffold in bone regeneration: in vitro and in vivo studies. yazdanian m, tabesh h, houshmand b, tebyanian h, soufdoost rs, tahmasebi e, karami a, ghullame s. biocybern biomed eng. 2020; 40(4):16261637. 22. in vitro and in vivo evaluation of novel tadalafil/β-tcp/collagen scaffold for bone regeneration: a rabbit critical-size calvarial defect long term radiographic evaluation of a potentially impacted and dilacerated tooth into complete transposition vol 9 no 1 (2021) doi 10.5195/d3000.2021.138 http://dentistry3000.pitt.edu study. soufdoost rs, yazdanian m, tahmasebi e, yazdanian a, tebyanian h, karami a, nourani mr, panahi y. biocybern biomed eng. 2019; 39(3):789-796. 23. dental developmental alterations in patients with dilacerated teeth. ledesmamontes c, jiménez-farfán md, hernández-guerrero jc. medicina oral, patologia oral y cirugia bucal. 2018; 24(1):e8-e11. 24. a pyk2 inhibitor incorporated into a pegda-gelatin hydrogel promotes osteoblast activity and mineral deposition. posritong s, chavez rf, chu t-mg, bruzzaniti a. biomedical materials. 2019; 14(2):025015. 25. sibling similarities in dental development. garn sm, lewis ab, polacheck d. journal of dental research. 1960; 39(1):170-175. 26. the current markers of cancer stem cell in oral cancers. tahmasebi e, alikhani m, yazdanian a, yazdanian m, tebyanian h, seifalian a. life sci. 2020; 249(117483. 27. surgical suture assembled with tadalafil/polycaprolactone drug-delivery for vascular stimulation around wound: validated in a preclinical model. soufdoost rs, mosaddad sa, salari y, yazdanian m, tebyanian h, tahmasebi e, yazdanian a, karami a, barkhordari a. biointerface res appl chem. 2020; 10(5):63176327. 28. prevalence and risk factors of root resorption of adjacent teeth in maxillary canine impaction, among untreated children and adolescents. rafflenbeul f, gros ci, lefebvre f, bahi-gross s, maizeray r, bolender y. european journal of orthodontics. 2018. 29. orthodontic management of maxillary canine-first premolar transposition by unilateral extraction. adiwirya msk, purwanegara mk. journal of dentistry indonesia. 2019; 26(1):44-50. 30. maxillary canine–first premolar bilateral transposition in a class iii patient: a case report. potrubacz mi, tepedino m, chimenti c. the angle orthodontist. 2016; 86(3):509519. 31. displacement of maxillary canines: a twist in the root. kerrigan j, sandy jr. british journal of orthodontics. 1995; 22(3):275-278. 32. maxillary canine displacement; further twists in the tale. chate r. the european journal of orthodontics. 2003; 25(1):43-47. 33. maxillary canine impaction; a final twist in the tale? chate r. journal of orthodontics. 2004; 31(1):13-14. 34. assessing the prevalence of musculoskeletal disorders and posture conditions during work time of dentists in a dentistry clinic affiliated to a military college in tehran in 2017. yazdanian m, moradi k, sobhani v, akbari h, kazemipour m, shamsoddini a, tahmasbi e. journal-mil-med. 2018; 20(2):222230. 35. prevalence and distribution of dilacerations in the permanent dentition of an iranian population. kuzekanani m, sadeghi mt. eur. j. anat. 2019; 23(4):273-277. 36. transmigration of an impacted mandibular canine. zvolanek j. illinois dental journal. 1986; 55(2):86-87. 37. three‐dimensional position of impacted maxillary canines: prevalence, associated pathology and introduction to a new classification system. grisar k, piccart f, al‐rimawi as, basso i, politis c, jacobs r. clinical and experimental dental research. 2019; 5(1):19-25. vol 7, no 1 (2019) issn 2167-8677 (online) doi 10.5195/d3000.2019.84 http://dentistry3000.pitt.edu new articles in this journal are licensed under a creative commons attribution 4.0 united states license. this journal is published by the university library system, university of pittsburgh as part of its d-scribe digital publishing program and is cosponored by the university of pittsburgh press. the effect of estrogen on craniofacial dimensions: a systematic review marjorie ayumi omori1, mirian aiko nakane matsumoto1, raquel assed bezerra segato,1 léa assed bezerra da silva1, paulo nelson filho1, erika calvano kuchler1 1department of pediatric dentistry, school of dentistry of ribeirão preto (university of são paulo), ribeirão preto/sp brazil abstract sex hormones have an effect on bone metabolism. however, it remains unclear how estrogen hormone affects mandible and maxilla growth and development. the aim of this study was to perform a systematic review to evaluate if estrogen is associated with developmental alterations in the maxilla/mandible phenotype. material and methods: a computer search of the literature was performed using: pubmed medline (1966 – august 2018), google scholar and manual searching. a combination of the terms ‘estrogens', ‘mandible’, ‘dental arch’, ‘maxilla’, ‘craniofacial’, ‘growth’ and ‘development’ was used. studies that used animal models to evaluate the role of estrogen during growth and development on the dimensions of the maxilla and/or mandible were included. results: 5 studies were selected to compose this systematic review. one study used zebrafish as a model. two studies used female mice and 2 studies used female rats as a model. two studies treated the animals with estrogen. four studies demonstrated that estrogen has an effect on mandible dimension and 2 studies demonstrated that estrogen has an effect on maxilla. one study did find an association between estrogen deficiency and mandible/maxilla dimensions conclusion: the current evidence suggested that both, increased and decreased levels of estrogen, have an effect on the maxilla and mandible dimensions. citation: omori m, et al. (2019) the effect of estrogen on craniofacial dimensions: a systematic review. dentistry 3000. 1:a001 doi:10.5195/d3000.2019.84 received: august 7, 2018 accepted: september 18, 2018 published: june 26, 2019 copyright: ©2019 omori m, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email:erikacalvano@gmail.com introduction the understanding of craniofacial growth has been investigated in orthodontic field (1-3). studies have demonstrated that the properly growth and development of the maxilla and mandible requires the coordination of a variety of molecular mechanisms (4-6). in fact, bone growth and development is mediated by different factors, such as hormones and sex hormones. it is well known that sex hormones are important in the regulation of reproductive functions and play an important in bone growth and maintenance (7). estrogen is a sex hormone, mainly produced by ovaries, which has a multifunctional role in growth, development, differentiation and function of various tissues. the role of estrogen in the bone development begins in the embrionary period (8) until the puberty (9). different studies with animal models have suggested that estrogen has an important role on the maxillary arches (9-12). therefore, the aim of this study is to perform a systematic review to evaluate the effect of estrogen and/or estrogen receptors on the maxilla and mandible growth. material and methods focused questions, protocol and data search the present systematic review was conducted in order to answer two focused questions: i) does estrogen levels affect maxilla and mandible growth? ii) does estrogen receptors (ers) affect maxilla and mandible growth? to conduct this review, the prisma statement (figure 1) checklist was used. initially a broad survey was conducted on computer of the literature by using the pubmed medline (1966aug 2018), google scholar (1960aug 2018) and manual search databases. review process http://dentistry3000.pitt.edu/ http://www.library.pitt.edu/ http://www.pitt.edu/ http://www.library.pitt.edu/articles/digpubtype/index.html http://upress.pitt.edu/ http://creativecommons.org/licenses/by/4.0/ the effect of estrogen on craniofacial dimensions: a systematic review vol 7 no 1 (2019) doi 10.5195/d3000.2019.84 http://dentistry3000.pitt.edu the initial selection of the studies was done through the title and abstract. the full text analyses of the potentially relevant studies were made. two examiners selected the studies. when there was some sort of disagreement, a third examiner was requested. terms the search strategy was based on the following medical subject heading terms (mesh) or text word [tw] in different combination strategy. the following terms were used: “estrogens” and “mandible or maxilla or dental arch or craniofacial” and “growth or development”. data was extracted and pooled in a table, to facilitate the visualization of information. inclusion and exclusion criteria studies that used animal models to assess the effect of estrogen during maxilla and mandible growth and have maxilla and mandible dimensions as an outcome were included. there was no language or date restriction. in vitro studies, clinical studies, case reports, case series and review have been excluded, as well as patents and conferences were excluded. data extraction two authors extracted the relevant data from the included papers. from each included study, the description of the animal models used, experimental design, dimensions results and additional information were summarized in tables 1 and 2. results the electronic search of pubmed and google scholar yielded 1.120 citations. a total of 31 titles and abstracts were screened from the selected databases. there were 15 in pubmed, and 16 in google scholar. the duplicates were considered only once, totalizing just 1 paper. additionally, 1 more paper was selected by manual searching. initially, the titles and abstracts not connected with the topic were excluded. afterwards, the papers were selected according to the inclusion and exclusion criteria, then 13 papers were selected for full text analysis. after reading, 8 papers were excluded. thus, 5 full texts were included for this systematic review. http://dentistry3000.pitt.edu/ the effect of estrogen on craniofacial dimensions: a systematic review vol 7 no 1 (2019) doi 10.5195/d3000.2019.84 http://dentistry3000.pitt.edu the summary of the characteristics of the 5 included papers is described in the table 1. the authors followed different protocols to evaluate the effect of estrogen levels or ers in the maxilla and mandible growth. only one study used zebrafish as animal model (13). the others 4 studies used mammals, 2 female rats (10, 11) and 2 female mice (9, 12). two studies ovarectomized the experimental group in order to induce estrogen deficiency (9, 11). others two studies treated with estradiol (e2) to induce an increase of estrogen (10, 13), and only one study treated the animals with estrogen receptors (alfa and beta) antagonists (12). the author who treated the animals with e2 (10) found an increase in mandibular measurements: condylionpogonion (co-pog) and condyliongonion (co-go). fujita et al. (9) induced estrogen deficiency and found some smaller linear distances in the maxilla: mupr (mu: point on intersection between maxillary bone and mesial surface of upper first molar; pr: most inferior and anterior point on alveolar process of premaxilla), a-pr (a: most anterior point on nasal bone; pr: most inferior and anterior point on alveolar process of premaxilla), iu-bu (iu: most prominent point between incisal edges of upper incisors; bu: point on premaxilla between jawbone and lingual surface of upper incisors). in the mandible, the distances pg-gn (pg: point on most inferior contour of lower border of mandible, adjacent to incisors; gn: point on most inferior contour of angular process of mandible), ml-bl (ml: point on intersection between the mandibular alveolar bone and mesial surface of first molar; bl: point on intersection between lingual surface of lower incisor and anteriormost part of lingual alveolar bone), co-co’ (co: most posterosuperior point of condylar process; co’: crossing point on pggn perpendicular to pg-gn from http://dentistry3000.pitt.edu/ the effect of estrogen on craniofacial dimensions: a systematic review vol 7 no 1 (2019) doi 10.5195/d3000.2019.84 http://dentistry3000.pitt.edu co) and il-id (il: most prominent point between incisal edges of lower incisor; id: most inferior and anterior point on alveolar process of mandible) were significantly smaller. besides, no significant differences in all angular measurements were found between the ovarectomized and sham-operated groups. the study that treated zebrafish with different concentrations of e2 detect a deformation of the upper jaw plate, leading to a smaller maxilla and hypoplasia of the lower jaw, leading to a smaller mandible. márquez hernández et al. (12) evaluate the effect of erβ antagonist in the mandible and found that condylar height and mandibular length were significantly smaller; the mandibular growth was significantly affected with the injection. just one author (11) from this systematic review found no significant differences in its study. discussion previous studies have already demonstrated that sex hormones can stimulate osteoclast differentiation directly (14) or indirectly (15) and that estrogen has an effect on bone metabolism (16-18). in cases of low levels of estrogen, there is an increase of the osteoclast count and surface; whereas in cases of high levels of estrogen, the opposite occurs: bone formation, bone healing and regeneration (19, 20). this hormone express its skeletal effects by its binding to estrogen receptors (ers) (21). in the present systematic review, we included studies that used mammals as well as zebrafish as animal models. these studies demonstrated the effect of estrogen on the maxilla (9, 12, 13) and mandible dimensions (9, 10, 12, 13). the zebrafish is an important animal model in health and genetic science nowadays due the fact that its genome has been fully sequenced and evidences revealed that 71.4% of human genes are related to zebrafish genes (22). only one study observed that an increase of estrogen causes a deformation of the upper and lower jaw leading to a smaller maxilla and mandible, interestingly this study used zebrafish as animal model (13). this fact could be explained by the physiological differences that exist between mammals and zebrafish. otherwise, studies that used mice found that a reduction of the craniofacial dimensions occurs when estrogen decreases or when there is a binding in the specific estrogen receptors, blocked through ers antagonists (9, 12). following the same line, singh & gunberg (10) demonstrated an increase in the linear measurements of the mandible when rats receive estrogen daily. these results clearly suggested that estrogen play an important role in mammals maxilla and mandible development. studies that use mammals as an animal model allow more reproducibility for human development understanding once over 90% of human and mouse genomes can be divided into parts with corresponding regions (23) and http://dentistry3000.pitt.edu/ the effect of estrogen on craniofacial dimensions: a systematic review vol 7 no 1 (2019) doi 10.5195/d3000.2019.84 http://dentistry3000.pitt.edu the comparative genomics allow a link between researches using animal model (preclinical researches) and humans (23-25). it is important to highlight that none of the studies evaluated the role of the estrogen during puberty period. in the orthodontic treatment it is crucial to predict the effective period of treatment to improve the skeletal discrepancy between maxillary and mandibular arches, so it is necessary a development of an indicator of bone maturity (for example a hormonal indicator), that would help to establish the beginning and end of growth (12). therefore, more studies should focus in the understanding of the role of the estrogen and estrogen receptor during different periods of the craniofacial development. briefly, these translational investigations are indispensable to clearly demonstrate the influence of sex hormones, specifically estrogen, on craniofacial bone growth. conclusion the current evidence suggested that both, increased and decreased levels of estrogen have an effect on the maxilla and mandible dimensions. acknowledgments funding sources: fundação de amparo à pesquisa do estado de são paulo (fapesp) 2016/13982-4; 2015/ 06866-5. references 1. choi sh, fan d, hwang ms, lee hk, hwang cj. effect of growth hormone treatment on craniofacial growth in children: idiopathic short stature versus growth hormone deficiency. j formos med assoc. 2016. 2. da fontoura cs, miller sf, wehby gl, amendt ba, holton ne, southard te, et al. candidate gene analyses of skeletal variation in malocclusion. j dent res. 2015;94(7):913-20. 3. yoon ss, chung ch. comparison of craniofacial growth of untreated class i and class ii girls from ages 9 to 18 years: a longitudinal study. am j orthod dentofacial orthop. 2015;147(2):190-6. 4. sidlauskas m, salomskiene l, andriuskeviciute i, sidlauskiene m, labanauskas z, vasiliauskas a, et al. heritability of mandibular cephalometric variables in twins with completed craniofacial growth. eur j orthod. 2016;38(5):493-502. 5. pallares lf, carbonetto p, gopalakrishnan s, parker cc, ackert-bicknell cl, palmer aa, et al. mapping of craniofacial traits in outbred mice identifies major developmental genes involved in shape determination. plos genet. 2015;11(11):e1005607. 6. parada c, chai y. mandible and tongue development. curr top dev biol. 2015;115:31-58. 7. lorenzo j. a new hypothesis for how sex steroid hormones regulate bone mass. j clin invest. 2003;111(11):1641-3. 8. ben-hur h, mor g, blickstein i, likhman i, kohen f, dgani r, et al. localization of estrogen receptors in long bones and vertebrae of human fetuses. calcif tissue int. 1993;53(2):91-6. 9. fujita t, ohtani j, shigekawa m, kawata t, kaku m, kohno s, et al. effects of sex hormone disturbances on craniofacial growth in newborn mice. j dent res. 2004;83(3):2504. 10. singh ij, gunberg dl. effect of estrogen treatment on bone cortex of the young rat. anat rec. 1971;171(2):273-82. 11. seifi m, ashiri m, hedayati m. effect of sexual hormone elimination on the changes of craniofacial dimensions in rats. journal of dental school shahid beheshti university of medical sciences. 2008;25(4):365-72. 12. marquez hernandez ra, ohtani j, fujita t, sunagawa h, kawata t, kaku m, et al. sex hormones receptors play a crucial role in the control of femoral and mandibular growth in newborn mice. eur j orthod. 2011;33(5):564-9. 13. fushimi s, wada n, nohno t, tomita m, saijoh k, sunami s, et al. 17beta-estradiol inhibits chondrogenesis in the skull development of zebrafish embryos. aquat toxicol. 2009;95(4):292-8. http://dentistry3000.pitt.edu/ the effect of estrogen on craniofacial dimensions: a systematic review vol 7 no 1 (2019) doi 10.5195/d3000.2019.84 http://dentistry3000.pitt.edu 14. mizuno y, hosoi t, inoue s, ikegami a, kaneki m, akedo y, et al. immunocytochemical identification of androgen receptor in mouse osteoclast-like multinucleated cells. calcif tissue int. 1994;54(4):325-6. 15. bellido t, jilka rl, boyce bf, girasole g, broxmeyer h, dalrymple sa, et al. regulation of interleukin-6, osteoclastogenesis, and bone mass by androgens. the role of the androgen receptor. j clin invest. 1995;95(6):2886-95. 16. riggs bl, melton lj, 3rd. involutional osteoporosis. n engl j med. 1986;314(26):1676-86. 17. turner as, alvis m, myers w, stevens ml, lundy mw. changes in bone mineral density and bone-specific alkaline phosphatase in ovariectomized ewes. bone. 1995;17(4 suppl):395s-402s. 18. harris b, lovett l, smith j, read g, walker r, newcombe r. cardiff puerperal mood and hormone study. iii. postnatal depression at 5 to 6 weeks postpartum, and its hormonal correlates across the peripartum period. br j psychiatry. 1996;168(6):739-44. 19. yamashiro t, takanoyamamoto t. influences of ovariectomy on experimental tooth movement in the rat. j dent res. 2001;80(9):1858-61. 20. haruyama n, igarashi k, saeki s, otsuka-isoya m, shinoda h, mitani h. estrous-cycledependent variation in orthodontic tooth movement. j dent res. 2002;81(6):406-10. 21. gennari l, merlotti d, de paola v, calabro a, becherini l, martini g, et al. estrogen receptor gene polymorphisms and the genetics of osteoporosis: a huge review. am j epidemiol. 2005;161(4):307-20. 22. howe k, clark md, torroja cf, torrance j, berthelot c, muffato m, et al. the zebrafish reference genome sequence and its relationship to the human genome. nature. 2013;496(7446):498-503. 23. waterston rh, lindbladtoh k, birney e, rogers j, abril jf, agarwal p, et al. initial sequencing and comparative analysis of the mouse genome. nature. 2002;420(6915):520-62. 24. lander es, linton lm, birren b, nusbaum c, zody mc, baldwin j, et al. initial sequencing and analysis of the human genome. nature. 2001;409(6822):860-921. 25. gibbs ra, weinstock gm, metzker ml, muzny dm, sodergren ej, scherer s, et al. genome sequence of the brown norway rat yields insights into mammalian evolution. nature. 2004;428(6982):493-521. http://dentistry3000.pitt.edu/ oral health status, attitudes and gender variations among governmental schoolchildren at al-mafraq governate – jordan vol 9 no 1 (2021) doi 10.5195/d3000.2021.141 http://dentistry3000.pitt.edu oral health status, attitudes and gender variations among governmental school children at al-mafraq governate – jordan leena smadi1, lara kharma2, asma’a abu abed3, eman bny mfarej4, asma abdalmohdi 5 1 professor; faculty of dentistry; jordan university; jordan 2 msc foundations in clinical psychology; school of psychology; department of medical sciences; newcastle university; jordan 3 master’s degree in maternal and neonatal health; registered nurse/midwife; head of national woman’s health care center; jordan 4 department of statistics; statistical analysis division; jordan 5 rn/ head of planning; research and information department; national woman’s health care center; jordan abstract objectives: calculate the prevalence and severity of dental caries and periodontal health among school students aged 12-18 years old and their attitudes toward dental health and oral hygiene at al-mafraq governatejordan. materials and methods: demographic and oral behavioral information were collected; students aged 12-18 years old from six public schools distributed over three districts in al-mafraq governate over a sixmonth period had a full oral and dental examination. dmft index (decay, missing, filling, teeth) was used to measure caries experience. plaque index (pi) and gingival index (gi) of löe and silness were used to evaluate the oral health status. t test was used for statistical evaluation. a p-value < 0.05 was considered statistically significant. results: 1165 students were examined, 51% never had been to a dentist or had any previous dental treatment, 39.6% of the students examined never brushed their teeth and 61.85 % had dental caries. the (sic) was 4.72 with a decay representing 93.53%. females brush their teeth more frequent than males with a statistically significant difference; p< 0.001. females showed lower dmft index (1.81) and compared to males (2.25) with a statistically significant difference. females showed lower sic (4.164) compared to males (5.186) with a statistically significant difference. there was no statistically significant difference between males and females in pi p= 0.590, and gi; p= 0.852. conclusions: the prevalence of dental caries among students in al-mafraq governate and the significant caries index were substantially higher than the target goals of who/ fda of year 2015 with more than 90 % of unmet treatment needs. girls showed better oral hygiene habits compared to boys and exhibited less dental caries experience. however, periodontal status did not differ significantly between both genders. keywords: dental health; oral health; adolescents; dental caries. citation: smadi, l, et al. (2021) oral health status, attitudes and gender variations among governmental schoolchildren at al-mafraq governate – jordan. dentistry 3000. 1:a001 doi:10.5195/d3000.2021.141 received: january, 24, 2021 accepted: march, 13, 2021 published: september, 13, 2021 copyright: ©2021 smadi, l, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: alsumadi@yahoo.co.uk introduction oral health is part of the general wellbeing of an individual. [1] caries and periodontal disease are the two most important dental diseases, although common yet, preventable.[2] it is estimated that oral diseases affect nearly 3.5 billion people. more than 530 million children suffer from dental caries of primary teeth. severe http://dentistry3000.pitt.edu/ oral health status, attitudes and gender variations among governmental schoolchildren at al-mafraq governate – jordan vol 9 no 1 (2021) doi 10.5195/d3000.2021.141 http://dentistry3000.pitt.edu periodontal disease, which may result in tooth loss, is also very common, with almost 10% of the global population affected. [3] oral health affects people physically and psychologically and influences how they grow, enjoy life, look, speak, chew, taste food and socialize, as well as their feelings of social well-being. [1] microorganisms found in dental plaque can cause dental caries and periodontal disease.[4] strategies that focus on increasing the awareness and motivation of the population to improve their oral hygiene, implementing prevention methods, regular checkups and making dental care more accessible to those people in underprivileged societies can significantly decrease the prevalence of dental caries and periodontal disease.[5] several dental surveys have been conducted in hashemite kingdom of jordan but unfortunately most of them were focused in amman capital of jordan and examined students in private schools. [6,7] the purpose of this survey and a previous survey [8-11] executed by jordanian national women’s health care center (nwhcc) and a team of researchers from the university of jordan, ministry of health and royal medical services is to identify people who are at a higher risk of developing dental caries and periodontal disease in the most needed societies in governmental schools and peripheral areas. al-mafraq is one of these areas located northeast of jordan with also increasing number of syrian refugees after syrian political crisis in the last few years. our aim from this cross-sectional study was to calculate the prevalence and severity of dental caries and periodontal health among school students aged 12-18 years old and their attitudes toward dental health and oral hygiene at al-mafraq governate jordan. material and methods over 6 month's period from october 2018 until march 2019, the nwhcc pre-trained teams conducted an oral health survey for students aged 12-18 years old, students were recruited from six large public schools distributed over the three main districts of almafraq governorate. ethical approval for the study was granted from the ethical committee of the jordanian ministry of health (code: moh rec 160019). all students were examined after signing an informed consent by them and their parents. demographic and oral behavioral data including age, gender, school, district, oral hygiene methods and previous dental treatment were recorded by using a questionnaire followed by a full oral and dental examination. the data sheet was pre-validated by experts committee of dentists and dental care nurses. a mobile dental clinic was used to examine all students at the schools; disposable examination sets (prepacked mirror and explorer) and disposable latex gloves were used during the examination. we adopted the same methods used in our previous surveys, [8-11] to standardize the examination procedures and to decrease differences in examination outcome, three general practitioner dentists who conducted the clinical examination attended a training course before starting the survey. pre-survey assessment of agreement was performed between examiners, with kappa inter-examiner of 0.93 and intraexaminer of 0.98. tooth decay was defined according to who criteria as ‘cavities with a softened dentine floor’, we had http://dentistry3000.pitt.edu/ oral health status, attitudes and gender variations among governmental schoolchildren at al-mafraq governate – jordan vol 9 no 1 (2021) doi 10.5195/d3000.2021.141 http://dentistry3000.pitt.edu previously used this definition in a previous study. [8] the number of teeth with untreated decay (cavity) was defined as dt (decayed teeth), the number of teeth with decay in the past that had been repaired by operative procedures were defined as ft (filled teeth), and mt (missing teeth) was used to describe teeth removed or extracted because of decay. the sum of dt, mt and ft which represented the total number of teeth affected by tooth decay was referred to as the dmft. the level of tooth decay in a population can be represented by the mean values of dt, mt, ft and dmft as well as by the proportion of the population affected by each type of decay. the dmft ratio represents the total number of teeth affected in the past and present, this value is a cohort that is more representative of the oral health status than the dt, which indicates those teeth requiring attention because of untreated decay; therefore we chose dmft for evaluation and reporting in this study, although this index can be criticized for not counting either enamel caries, or caries activity, or even teeth or surfaces at special risk, in addition to not confirming the reason for decay extraction during examination, especially for deciduous teeth. however, it is good in providing descriptive information for trends monitoring and policy makers’ awareness of dental health. significant caries index (sic) was used in this survey to overcome the limitation in the distribution of dmft. the (sic) refers to the one-third of the population that are more severely affected by dental caries.[12] oral plaque was evaluated by running the side of a ball-tip probe along the inner and outer aspects of the six index teeth, using the criteria of the plaque index of silness and löe. [13] the six indexed teeth are the upper right first molar, upper right lateral incisor, upper left first premolar, lower right first premolar, lower left lateral incisor and first molar. missing teeth are not substituted. the gingival condition was determined for the same teeth using the criteria of the gingival index of löe and silness. [13] objective research was performed to evaluate the state of oral hygiene during the examination as follows: good (plaque index 0.0 i.e., absence of plaque), fair (plaque index 0.1-1.9 i.e., presence of plaque) and bad (plaque index 2.0 3.0 i.e., plaque seen by the naked eye). the gingival index of löe and silness was used to record the severity of gingivitis. the gingival index by löe and silness measures the severity of gingivitis on a scale ranging from 0.1–3.0 (0.1-1.0: mild gingivitis, 1.1-2.0: moderate gingivitis, and 2.1-3.0: severe gingivitis). statistical analysis was performed using spss for windows (version 20, ibm corporation, armonk, ny). the t test was used for statistical evaluation. a p-value < 0.05 was considered statistically significant. results 1165 students were examined 48.4% were males 51.6% were females, age range from 11 to 18 approximately half were in age group from 13 to 15 years old. 94.1 % were jordanian and the rest were syrian refugees. nearly half of the students examined (51%) never had been to a dentist or had any previous dental treatment. oral hygiene habits: more than third of the students examined never brushed their teeth (39.6%) with approximately the same percentage brushed their teeth once daily. teeth brushing was the only oral hygiene method in 87.7 % of the students examined, only 0.4 % claimed to use dental floss, 0.7 % used toothpicks whereas 11.4 % used mouthwash. http://dentistry3000.pitt.edu/ oral health status, attitudes and gender variations among governmental schoolchildren at al-mafraq governate – jordan vol 9 no 1 (2021) doi 10.5195/d3000.2021.141 http://dentistry3000.pitt.edu caries prevalence and caries index: out of 1165 students examined only 38.15 % were caries free (61.85 % have caries). dmft index was found to be 2.037 with a decay representing 92.8 %, missing 2.22% and fillings 5.58%. one third of the students examined were found to have dmft index 4.723 (significant caries index sic) with a decay representing 93.53%, missing 1.62% and fillings 4.83%. (table 1) table 1: dmft index, significant caries index and percentile of d, m, f. dmft index=2.037 sic =4.723 %d 92.18 %d 93.53 %m 2.22 %m 1.62 %f 5.58 %f 4.83 plaque index and gingival index: plaque index (pi) was found to be 0.422 sd 0.5991 whereas gingival index was found to be 0.361  sd 0.585. according to pi approximately two thirds (63.2 %) were classified as having good oral hygiene (pi= 0.0), approximately one third (31.8%) were classified as having fair oral hygiene (pi= 0.11.9) and only 5% were classified as having poor oral hygiene (pi= 2.03.0). according to gi 69.1% were classified as having healthy gingiva (gi= 0.0), approximately a quarter (26%) were classified as having mild gingivitis (gi= 0.1-1.0) less than 5 % (4.5%) were classified as having moderate gingivitis (gi= 1.12.0) and less than 1% (0.30%) were classified as having severe gingivitis. (table 2) gender variations table 3 shows gender variations according to the following 1. frequency of teeth brushing and gender there was a statistically significant difference between males (m= 0.68, sd=0.76) and females (m= 1.03, sd= 0.89) in frequency of teeth brushing; t (1166) = -7.233, p= 0.000 2. caries index and gender: dmft index was 2.254 for males whereas it was 1.814 for females, independent samples t-test showed that there was a statistically significant difference between males (m= 2.25, sd=2.51) and females (m= 1.83, sd= 2.17) in dmf scores; t (1168) = 3.068 p= 0.002 significant caries index was 5.186 for males whereas it was 4.164 for females independent samples ttest showed that there was a statistically significant difference between males (m= 3.46, sd=2.34) and females (m= 2.32, sd= 2.20) in significant caries index scores; t (845) = 7.278 p= 0.000. 3. pi, gi and gender there was no statistically significant difference between males (m= 0.432, sd=0.57) and females (m=0. 423, sd= 0.63) in plaque index scores; t (1166) = 0.539 p= 0.590 there was no significant difference between males (m= 0.37, sd=0.53) and females (m= 0.36, sd= 0.63) in gingival index scores; t (1166) = 0.187 p= 0.852 http://dentistry3000.pitt.edu/ oral health status, attitudes and gender variations among governmental schoolchildren at al-mafraq governate – jordan vol 9 no 1 (2021) doi 10.5195/d3000.2021.141 http://dentistry3000.pitt.edu table 2: percentile of school students according to gingival status: gi gingival status percentage % 0.0 healthy gingiva 69.1% 0.1-1.0 mild gingivitis 26% 1.1-2.0 moderate gingivitis 4.5% 2.1-3.0 severe gingivitis 0.30% table 3: gender variations (independent samples t-test): males females p value number 565 603 d+m+f 1274 1094 dmft index 2.2542.51 1.8142.17 p=002 sic 5.1862.34 4.1642.2 p<001 pi 0.4320.57 0.4320.63 p=0.590 gi 0.370.53 0.360.63 p=0.852 frequency of teeth brushing 0.680.76 1.030.89 p<001  p value shows statistically significant difference discussion the results of this study showed that more than half of the students examined lack any dental care, more than one third of those students never brushed their teeth and almost all of them never used dental floss, this may be explained by poor income, unavailability of dental clinics and unavailability of dental health insurance on one side and lack of motivation, knowledge and dental education on the other side.[14] the prevalence of caries in those students was very high (61.85%), the significant caries index was also high (4.723) with 93.53 % have been left untreated and only 4.83% received dental fillings or have been extracted (1.62%). the high prevalence and severity of dental caries with the unmet treatment needs were consistent with other previous studies. [6-11] the observed prevalence of dental caries among student in the present study was higher than the target established for the year 2000 (50%) by who/fdi [15], also the significant caries index was higher than a global oral health goal for significant caries index of less than 3 dmft in the 12-year-olds in a given population by the year 2015.[12] the results of this survey and previous surveys [8-11] indicate the need for dental education, and motivation for proper oral hygiene methods and to implement dental preventive and treatment methods which may include water fluoridation of drinking water for school children [16]and to provide governmental dental insurance for school children, needless to mention that poor oral status may put future extra burden on the government because of the need for more complicated and expensive dental treatment like for example root canal treatment , fixed or removable prostheses or implants for replacement of missing teeth in addition to the psychological impact of losing teeth on an individual .[17] although 63.2% were classified as having good oral hygiene according to pi, the remains of the students were classified as having poor to fair oral hygiene, similarly approximately one third of those students http://dentistry3000.pitt.edu/ oral health status, attitudes and gender variations among governmental schoolchildren at al-mafraq governate – jordan vol 9 no 1 (2021) doi 10.5195/d3000.2021.141 http://dentistry3000.pitt.edu were classified as having mild, moderate to severe gingivitis which emphasizes the need for dental education for both parents, teachers and students and may be supervised teeth brushing and flossing session [18] obviously, this study shows that females had more positive behavior than males concerning teeth brushing their teeth than males, this is consistent with a previous meta-analysis of 1981, 2000, and 2010 data of greek students by gender which showed that females brushed their teeth significantly more often than males [19] the difference could be attributed to the fact that the society puts more pressure on girls to improve their image or girls tend to be more protective on themselves in a society which is still male driven particularly in areas of poor socioeconomic status. this positive oral hygiene behavior reflected on less caries index in girls than boys with a statistically significant difference, nevertheless it did not reflect on gingival status in this study where there was no statistically significant difference between boys and girls, this may be explained by be hormonal differences between boys and girls at this age and its effect on periodontal status. [20] although the present study is not based on a representative sample of public-school children in almafraq governate because it uses convenience sampling, it does provide insight into the prevalence and severity of caries and gingivitis and lack of oral hygiene practice and dental care among public school children. the results of this survey indicate the need to start a local campaign to raise the public awareness of oral hygiene and the need to modify policies for early access to dental services among school children. conclusions the prevalence of dental caries among students in al-mafraq governate and the significant caries index were substantially higher than the target goals of who/ fda of year 2015 with more than 90 % of unmet treatment needs. girls showed better oral hygiene habits compared to boys and exhibited less dental caries experience. however, periodontal status did not differ significantly between both genders. acknowledgements the authors would like to thank the national woman’s health care centerjordan who organized and executed this survey in collaboration with jordanian ministry of health, jordanian ministry of education and jordanian royal medical services who used their resources and staff to conduct the survey and for their effort to make the data of this survey available for research and publication. competing interests the authors declare that they have no competing interests. references 1. peck c.c. (2017) putting the mouth into health: the importance of oral health for general health. in: sasaki k., suzuki o., takahashi n. (eds) interface oral health science 2016. springer, singapore. https://doi.org/10.1007/97 8-981-10-1560-1_7 2. oral health: the silent epidemic benjamin rm.. public health rep. 2010;125(2):158-159. doi:10.1177/00333549101 2500202 http://dentistry3000.pitt.edu/ oral health status, attitudes and gender variations among governmental schoolchildren at al-mafraq governate – jordan vol 9 no 1 (2021) doi 10.5195/d3000.2021.141 http://dentistry3000.pitt.edu 3. global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990–2017: a systematic analysis for the global burden of disease study 2017. lancet 2018; 392: 1789–8583 4. caries and periodontitis: contesting the conventional wisdom on their aetiology. manji f, dahlen g, fejerskov o: caries res 2018; 52:548564. doi: 10.1159/000488948 5. strategies and approaches in oral disease prevention and health promotion.watt, richard. (2005). bulletin of the world health organisation. 83. 711-718. 6. caries in preschool children in amman, jordan and the relationship to sociodemographic factors. sayegh, a & dini, e.l. & holt, r.d. & bedi, raman. (2002). international dental journal. 52. 87-93. 10.1111/j.1875595x.2002.tb00606. x. 7. early childhood caries and risk factors in jordan. community dent health.rajab ld, hamdan ma. 2002 dec;19(4):224-9. pmid: 12489836. 8. prevalence and severity of dental caries in school students aged 6 11 years in tafelah governorate – south jordan: results of national woman’s health care center survey. smadi l, azab r, rodan r, khlaifat f, abdalmohdi a. ohdm. 2015; 14: 17-22. 9. prevalence and severity of gingivitis in school students aged 6-11 years in tafelah governorate, south jordan: results of the survey executed by national woman's health care center. rodan r, khlaifat f, smadi l, azab r, abdalmohdi a. bmc res notes. 2015 nov 9; 8:662. 10. prevalence and severity of dental caries in school students aged 6-12 years in mafraq governorate: northeast of jordan.smadi l, azab r, khlaifat f, rodan r, abdalmohdi a, maata r, et al. j oral health oral epidemiol 2017; 6(1): 40-7. 11. oral health status and behaviors in jordanian adolescents aged 12-18 years. smadi l. amj 2017;10 (7):587–594. 12. introducing the significant caries index together with a proposal for a new global oral health goal for 12-yearolds. bratthall d. int dent j 2000; 50(6): 378-84. 13. socio-economic inequality in oral healthcare coverage: results from the world health survey. hosseinpoor ar, itani l, petersen pe. j dent res. 2012;91(3):275-281. 14. the gingival index, the plaque index and the retention index systems. j periodontol löe h.. 1967 nov-dec;38(6): suppl:6106. doi: 10.1902/jop.1967.38.6.610 . pmid: 5237684. 15. challenges to improvement of oral health in the 21st century--the approach of the who global oral health programme petersen pe.. int dent j. 2004 dec;54(6 suppl 1):329-43. doi: 10.1111/j.1875595x.2004.tb00009. x. pmid: 15631094. 16. effectiveness of school water fluoridation and dietary fluoride supplements in schoolhttp://dentistry3000.pitt.edu/ https://www.ncbi.nlm.nih.gov/pubmed/?term=rodan%20r%5bauthor%5d&cauthor=true&cauthor_uid=26552995 https://www.ncbi.nlm.nih.gov/pubmed/?term=khlaifat%20f%5bauthor%5d&cauthor=true&cauthor_uid=26552995 https://www.ncbi.nlm.nih.gov/pubmed/?term=khlaifat%20f%5bauthor%5d&cauthor=true&cauthor_uid=26552995 https://www.ncbi.nlm.nih.gov/pubmed/?term=smadi%20l%5bauthor%5d&cauthor=true&cauthor_uid=26552995 https://www.ncbi.nlm.nih.gov/pubmed/?term=azab%20r%5bauthor%5d&cauthor=true&cauthor_uid=26552995 https://www.ncbi.nlm.nih.gov/pubmed/?term=azab%20r%5bauthor%5d&cauthor=true&cauthor_uid=26552995 https://www.ncbi.nlm.nih.gov/pubmed/?term=abdalmohdi%20a%5bauthor%5d&cauthor=true&cauthor_uid=26552995 https://www.ncbi.nlm.nih.gov/pubmed/26552995 https://www.ncbi.nlm.nih.gov/pubmed/26552995 oral health status, attitudes and gender variations among governmental schoolchildren at al-mafraq governate – jordan vol 9 no 1 (2021) doi 10.5195/d3000.2021.141 http://dentistry3000.pitt.edu aged children.horowitz hs. j public health dent. 1989;49(5 spec no):290-6. doi: 10.1111/j.1752-7325. 1989.tb02087. x. pmid: 2681731. 17. oral diseases: a global public health challenge. peres ma macpherson lmd weyant rj et al. lancet. 2019; 394: 249260. 18. effectiveness of supervised toothbrushing and oral health education in improving oral hygiene status and practices of urban and rural school children: a comparative study. damle sg, patil a, jain s, damle d, chopal n. j int soc prev community dent. 2014 sep;4(3):17581. doi: 10.4103/22310762.142021. pmid: 25374836; pmcid: pmc4209617. 19. gender differences in oral health status and behavior of greek dental students: a meta-analysis of 1981, 2000, and 2010 data. mamai-homata e, koletsikounari h, margaritis v. j int soc prev community dent. 2016 jan-feb;6(1):608. doi: 10.4103/22310762.175411. pmid: 27011935; pmcid: pmc4784066. 20. effects of endogenous sex hormones on the periodontium--review of literature. güncü gn, tözüm tf, cağlayan f. aust dent j. 2005 sep;50(3):13845. doi: 10.1111/j.18347819. 2005.tb00352. x. pmid: 16238210. http://dentistry3000.pitt.edu/ vol 3, no 1 (2015) issn 2167-8677 (online) doi 10.5195/d3000.2015.36 http://dentistry3000.pitt.edu pilot study of pre-maxilla replacement and bone graft in a patient with lip and palate clefts: transforaminal relationship between upper canines and bone graft joão luis carlini1, danielle del santo2, kesia keiko asami2 1oral and maxillofacial surgery, chief of stomatology departament, professor at federal university of paraná, staff of caif – center for integral assistance of cleft lip and palate patients, curitiba, paraná, brazil 2dental surgeon, school of dental medicine at federal university of paraná, curitiba, paraná, brazil abstract objective: the aim of this study was to assess patients subjected to replacement surgery of the pre maxilla with bone graft, having as donor area the iliac crest and chin, and the relation with upper canines erupted in grafted areas. materials and methods: 54 records were evaluated from cleft patients that were under treatment at caif center of attention to clefts in curitiba, pr, brazil, both sexes, with unilateral and bilateral lip and palate clefts. patients younger than fourteen years old, both sexes, that lost the surgical segment or did not continue the treatment at same welcoming institution, were excluded. the surgical segment average was 5.72 years. results: using both descriptive and chi-square statistics, we observed a statistically significant distinction regarding the eruption of upper canines in the region of secondary bone grafts when using iliac crest and chin grafts, which 62.8% of the sample that was treated with chin grafts happened to have an eruption of upper canines, and 37.2% were successful with iliac crest grafts (value-p 0.028). conclusions: it is necessary to carry out a study with a larger sample size and where the samples are paired in order to confirm the superiority the iliac crest donor area as the gold standard for this surgical procedure. citation: carlini j, del santo d, asami k. (2015) pilot study of pre maxilla replacement and bone graft in a patient with lip and palate clefts transforaminal– relationship between upper canines and bone graft. dentistry 3000. 1:a001 doi:10.5195/d3000.2015.36 received: july 24, 2015 accepted: september 1, 2015 published: september 29, 2015 copyright: ©2015 carlini j, severo d, asami k. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: kka12@pitt.edu introduction among congenital anomalies of the mouth, oral clefts stand out, ranging from those that may occur as an isolated or associated disorder to anomalies of varying degrees. during january, 1975 until december, 1994, we recorded 16,853 new cases of oral clefts in brazil, having the southeast region contributed with 61% these news cases [1]. the clefted individuals have several problems associated with facial deformity including malocclusion, nasal deformity, problems with feeding, listening and speaking, and the association between clefts with other anomalies is evident. these abnormalities affect the midface, occurring because the upper jaw bones do not fuse during the sixth and tenth week of intrauterine life [2]. currently the classification of spina is the most used to typify the lip and palate clefts [3]. after the primary reconstructive surgeries, lip repair and palatoplasty, becomes necessary http://dentistry3000.pitt.edu/ familial aggregation of anxiety associated with bruxism vol 3, no 1 (2015) doi 10.5195/d3000.2015.36 http://dentistry3000.pitt.edu 2 surgery for correction the cleft alveolar ridge through bone grafts [4]. for the unilateral cleft patient, the secondary bone grafting surgery has been well described in the literature [5,6]. clefts transforaminal patients are the most severely affected due to the absence of alveolar bone support in pre maxilla region, and absence of bone and soft tissue in hard palate region, leading to the projection of the pre maxilla, absence of eruption of the upper permanent canines, and absence of sealing lip [7,8]. having had a bone graft, or with the assistance of bone graft, changes in upper jaw growth are minimized because the majority of bone growth in the time of repositioning of pre maxilla, is already complete [9]. treatment of pre maxilla in patients with bilateral clefts there are surgical procedures, such as complete refurbishment of the pre maxilla, promoting a better sealing lip [10], and the repositioning of pre upper jaw with autogenous bone graft [11]. surgical treatment in bilateral clefts patients can have serious implication, because manipulating the pre maxilla can result in changes of bone growth in the region. there are controversies if the surgical intervention in pre maxilla should be performed before or after the complete facial growth [11]. hedbuchel, kypers-jagtman, and freihofer (1993) studies, demonstrated success with bone graft associated to upper jaw osteotomy and repositioning of pre maxilla, resulting in a well aligned and stabilized dental arch with a significant improvement in incisor inclination. subjects and methods this study was experimental unit with 54 patients, unilateral or bilateral cleft lip and palate according to the classification of spina [12], who were in treatment at center of attention to clefts lip and palate-caif, curitiba-prbrazil. clinical data were collected through medical and dental records, panoramic radiographs, in profile, upper occlusal and periapical the region of cleft. selected patients were older than 14 years old, an age that already set the functional position of the maxillary canines, and both sexes. patients younger than 14 years age, both sexes, that lost the surgical segment or did not continue the treatment at same welcoming institution, were excluded. the patients were submitted to treatment protocol of caif establishing assessment throughout the areas of dentistry, such as speech therapy, psychology, nursing, plastic surgery and pediatrics. in orthodontic intervention breakers appliances were placed in the upper jaw to promote correction of atresia of upper jaw, and to improve the position of the pre maxilla. with this first step performed, the patient was referred to a team of oral and maxillofacial surgery, where the molds of lower and upper arch were made. from the plasters models, surgery models with correct position of the pre maxilla were performed and the confection of surgical guides in autopolymerizing acrylic resin, for this new position. the surgeries were performed under general anesthesia and tracheal intubation. after induction of anesthesia, lidocaine 2% with epinephrine 1:100.000 was infiltrated in the region to be surgically manipulated. 15 scalpel blades was used to vertically incise the margins of the cleft, extending laterally with intrasulcular incision one or two teeth apart from the area of the cleft, where alleviating incisions were made toward the back of the vestibule. the same procedure was performed on the opposite side, followed by the detachment of the mucoperiosteal flap. vertical incisions were also made in the pre maxilla, followed by the detachment of the periosteum on the lateral and palatine portions on both sides. with the assistance of a reciprocating saw the bone situated on the posterior portion of the pre maxilla was segmented. the pre maxilla was anteriorly dislocated; the nasal mucus membrane was separated from the oral mucus membrane and the nasal floor was closed bilaterally using absorbable thread (polyglactin 4.0). once the nasal floor was closed, the palatine mucus membrane was sutured with the remaining palatine mucus membrane existing in the pre maxilla, using absorbable thread (polyglactin 4.0). next, the surgical guide was placed with steel thread nº 0 on the screws that were placed on the pre maxilla and bilaterally on the area of the molars to reposition the pre maxilla. in the donor area, the graft was removed from the iliac crest by the plastic surgeon. the chosen area is the internal (medial) of the crest. the removed block was monocortical, and with the use of a bone scoop, a large quantity of medullary bone was collected. after controlled the bleeding, a suture was performed as planned. the graft block was then prepared. the whole medullary part of the block was removed. the area corresponding to the bilateral cleft was totally filled in with medullary bone, which was collected through curettage and removed from the bone block. the remaining cortical bone plate of the iliac crest was http://dentistry3000.pitt.edu/ familial aggregation of anxiety associated with bruxism vol 3, no 1 (2015) doi 10.5195/d3000.2015.36 http://dentistry3000.pitt.edu 3 then used for the placement of the pre maxilla on the stumps of the remaining upper jaw and supplying the containment of the particulate medullary bone in the area of the clefts. the edges of the cortical block were placed on the distal extremity, corresponding to the alveolar process, with a 1.5 x 7.0mm screw and on the medial extremity, corresponding to the pre maxilla, with a 1.5 x 7.0mm screw. all the cutting edges that could traumatize the mucus membrane were removed with a spherical diamond drill bit in low revolution. the whole grafted area was then covered with the mucoperiosteal flap with the assistance of alleviating incisions in the periosteum. the suture was performed without tension over the flap using a 4.0 nylon thread. in the symphysis donor area, the incision was initiated 1.5cm below the red line of the lower lip, cutting the mucus membrane and muscles, immediately directing the scalpel to the jaw bone. after detaching the periosteum, the osteotomy was performed with an appropriate design to adapt to the jaw defect with a number 701 conical tronca drill in low revolution. the suture of the donor area was performed in anatomic plans with polygalatine 4-0, keeping the external compressive dressings in the chin area to avoid labial ptosis and to offer the patient greater comfort. the placement of the graft was made through miniplates and 1.5mm titanium screws promoting the stability of the graft. a compressive dressing was placed over the area to reduce edema and control possible active bleeding points, kept for a minimum of three days. a regimen of intravenous antibiotics was performed with cefazolin until the release of the patient (that occurs on the first day after the operation) and continued orally until the tenth day after the operation. analgesia was done with dipyrone and supplemented with cetoprofeno. post-operation care included rigorous hygiene of the surgical area with brushing and use of mouth wash with chlorhexidine 0.12%. the follow up was done clinically and the sutures were removed after two weeks. two months after the operation the surgical guide was removed and the stability of the pre maxilla was checked. the screws used in the placement of the graft were removed six months after the operation and, only after this procedure the patients were once again referred for orthodontic treatment. the parameters used to evaluate the success of the treatment were based on periapical x-rays, done six months after surgery, the period during which the filling of the cleft with bone and the immobility of the pre maxilla are observed with a clinical exam. after six months the removal of the placement screws was scheduled, under local anesthetics, and the patient was referred to resume orthodontic treatment. results fifty-four patients with an average age of 19.54 years old were selected based on inclusion criteria presenting a standard deviation of 6.790. the time elapsed from the surgery until the analysis of the medical-dental records had an average of 5.72 years, a standard deviation of 1.867. for the evaluation of the results, the non-parameter chi-squared and the fisher test were used. 33.3% of the patients were female, and 67.4% were male. in 33 table 1. descriptive analysis of the number of patients and region of the donor area frequency percentage iliac crest 24 44.40% chin 30 55.60% total 54 100.00% table 2. comparative analysis between time of surgery and bone graft donor area surgical follow-up donor area frequency average standard deviation p-value iliac crest 24 5.1 1.4 0.033 chin 30 6.2 2.1 total 24 5.7 1.9 http://dentistry3000.pitt.edu/ familial aggregation of anxiety associated with bruxism vol 3, no 1 (2015) doi 10.5195/d3000.2015.36 http://dentistry3000.pitt.edu 4 patients it was verified that the type of cleft was the labiopalatal bilateral type, and 21 possessed unilateral lip and palatal cleft. through the evaluation of the panoramic and profile x-rays, six of the 54 patients evaluated presented with at least one of the permanent upper canines before the surgical procedure, corresponding to 88.9% of the presence of the same teeth included after the evaluation of the pre-surgery xrays. besides the 6 aforementioned patients, 3 of the 54 patients, presented with the absence of both permanent canines, being that 94.4% of the sample presented with two included permanent upper canines in the area of the cleft subsequently operated. in relation to the area originating from the bone graft, 44.4% iliac crest was used, and in 55.6% of the patients chin graft was used (table 2). after the operation, patient check-up and the evaluation of the post-procedure panoramic and profile x-rays, 11.1% of the patients maintained one of the canines included, 11.1% of the sample remained with both of the upper canines enclosed. in relation to the percentage of erupted upper canines in the area of the cleft, after the procedure of the placement of the bone graft and the replacement of the pre maxilla, 14.8% of the patients had at least one erupted canine, and 64.8% of the patients had both upper canines erupted in the area of the cleft. in this way, a succession was observed of approximately 80% of the patients of the sample. beginning with the statistical analysis carried out through the test of averages, 24 patients received an autogenous graft originating from the iliac crest donor area, obtaining a standard deviation of 1.4, and 30 patients had the chin as the donor area, obtaining a standard deviation of 2.1 (table 3). the p value for this case was 0.033, concluding that there was significant statistical evidence of differences between the donor areas. the group of patients that presented with eruption of the upper canines, the proportional test qui-squared was applied, making it possible to verify statistical evidence between the percentages obtained. among the patients that were subjected to the reposition of the pre maxilla, and the filling in of the clefted area with iliac crest bone graft, 34.3% of the patients had upper canines, right and left, erupted. for the patients in whom autogenous bone graft was used, having the chin as the donor area, 65.7% of the patients presented with, after the abovementioned surgical segment, the two upper canines erupted (p≤0.028) (table 4). discussion the rupture of the alveolar bone, exactly at the height of the upper lateral incisors persists after the initial repair surgeries, maintaining the discontinuity of the upper arch [13]. this study of retrospective character evaluated the eruption of the upper canines in areas with an alveolar cleft, in carrier patients with unilateral and bilateral alveolar clefts, treated with chin and iliac crest bone grafts. many studies presented diverse techniques for the reconstruction of alveolar bone defects for these patients, autogenous bone or allogeneous sources being able to be used to provide a bone outline for the reparation of the defect. among the autogenous donor areas most used are the iliac crest, chin, shin, costal arch and skullcap. the benefits of the bone graft in the treatment of cleft patients were well described, mainly when it concerned unilateral clefts. in the bilateral cleft patients, several particularities are noted, such as significant bone and soft tissue deficiency, exaggerated protrusion of the pre maxilla, frequent deviation of the upper dental midline, present malocclusion due to atresia of the upper jaw segments, mobility of the pre maxilla, pronounced buconasal fistulas. these factors damage the aesthetics and social inclusion, phonation and alimentation of these patients, limiting the orthodontic and prosthetic rehabilitations [5 ,14 -16] . from an x-ray point of view, the periapical and occlusal x-rays allow the evaluation of the level of neoformation of the bone in the area of the bone graft [13], as they also allow the visualization of the upper canine eruption process. starting the surgery with bone graft in the area of the cleft, as much for patients with unilateral or bilateral clefts, it is expected that the upper canine erupts in the grafted bone, inducing the deposition of bone in the alveolar crest and increasing the vertical height of the upper jaw [16,17]. in our study we did not contemplate vertical height gain of the upper jaw; we limited ourselves verifying the viability of the eruption of the upper canines in the clefted area. according to cohen et al, the success or failure of the final result of the bone graft in the area of the cleft does not depend specifically on the donor area. other factors influence the final result of the treatment, such as: morbidity, the viability of the autogenous bone, the type of bone, if it is cortical or spongy, and the biological behavior of the graft [5]. the iliac crest is the gold standard for grafts in cleft patients http://dentistry3000.pitt.edu/ familial aggregation of anxiety associated with bruxism vol 3, no 1 (2015) doi 10.5195/d3000.2015.36 http://dentistry3000.pitt.edu 5 since it is a spongy bone which provides advantages, such as: the constant tissue growth of that bone, being remodeled more quickly than the cortical bone, rendering more viable the eruption of the teeth in the area of the cleft. the chin graft also possesses advantages well described in literature, for example: easy access, less morbidity, similar embryonic origin, and quicker revascularization, with a tendency to keep greater post-operation bone volume when compared with the iliac crest graft [18]. mish (2000) affirmed that autogenous bone grafts are osteogenic and capable of forming bone in the absence of undifferentiated mesenchymal cells. the grafted bone forms new bone in three different phases: osteogenesis, osteoinduction, and osteoconduction. he also observed that for the fact of the symphysis area having the same origin intramembranous as the upper jaw, there exists a quicker revascularization than the one of endochondral origin, as in the case of the iliac crest, having the advantage of minimal reabsorption and mobility of the graft, more premature vascularization, ambulation closer to the patient, no visible scar, abundant material, no secondary deformity and reduction of post-operation pain. in our study, we did not evaluate the post-operation conditions of the patients in agreement with the grafted area. regarding the type of graft used and the eruption of the upper canines, we obtained better results with a patient on whom an intramembranous bone graft was used, the chin, possibly due to all the features of that type of bone origin and the receptor location. hedbuchel, kuijpers-jagtman and freihofer performed a study in 1993 with 22 patients who presented with bilateral lip and palate cleft. in all their patients upper jaw osteotomy was performed with the stabilizing of the pre maxilla being that they used various types of grafts, such as: rib, iliac crest, chin, bone bank, and jaw bone. the average age of the patients of the study was 13.3 years old. the results obtained were satisfactory only having suffered the loss of one of the grafts. in our study, the patients were subjected to replacement of the pre maxilla with subsequent bone graft and we only used two types of bone, and the study group was composed of patients with both bilateral and unilateral transformed clefts. we also obtained satisfactory results in both groups, but with a greater statistical value for the group with chin bone graft. silva filho and colleagues (2000), presented a study involving 50 patients (32 men and eighteen women) all with palatine gap and aged from eight to fifteen years old. all the patients were subjected to iliac crest bone graft for the filling of the palatine gap, and they were observed through x-rays for a period of one to five years. in 72% of the cases there was spontaneous eruption of the canines, in 6% of the cases there was a need for orthodontic traction, in the other 22% of cases there was movement of the teeth in question, but they had not erupted yet. our study included 54 patients, a sample similar to the previously cited study, being that 33.3% of the sample composed of male patients and 66.7% of female patients. our patients were monitored through panoramic, profile, upper occlusal and periapical x rays of the upper canine area, in a period of seven days, three months, six months and twelve months following the operation. we obtained both of the erupted upper canines in 34.3% of the cases treated with iliac crest, and 65.7% of the cases treated with a chin graft. cohen and colleagues (1991) performed a study based on the experience of two plastic surgeons of the university of illinois hospital, chicago. the patients were between nine and eleven table 3. comparative analysis between the number of erupted canines regarding the type of donor area erupted canine 1 2 total donor area frequency column % frequency column% frequency column% pvalue iliac crest 4 50% 12 34.30% 16 37.20% 0.028 chin 4 50% 23 65.70% 27 62.80% total 8 100% 35 100% 43 100% http://dentistry3000.pitt.edu/ familial aggregation of anxiety associated with bruxism vol 3, no 1 (2015) doi 10.5195/d3000.2015.36 http://dentistry3000.pitt.edu 6 years old at the time of the surgery, this age being similar to the age in our study, which was from eight to twelve years of age. the surgical technique used was similar in all the patients, only the type of graft being different: iliac crest or skullcap. the patients were monitored between 18 and 60 months and the results were satisfactory and without a significant difference between the two types of grafts [18]. in our study we compared two types of grafts, iliac crest and chin, with an average of 5.72 years of surgical follow-ups, being that we obtained satisfactory results in both groups, but not in all the cases, which affirms the objective of this research: eruption of the upper canines in the area of the bone graft bilaterally or unilaterally. for our research, we obtained a 37.2% success rate for the patients grafted with iliac crest, and a 62.8% success rate for the patients grafted with chin bone. http://dentistry3000.pitt.edu/ familial aggregation of anxiety associated with bruxism vol 3, no 1 (2015) doi 10.5195/d3000.2015.36 http://dentistry3000.pitt.edu 7 references 1. prevale ncia de fissuras orais de 1975 a 1994. prevalence of oral clefts from 1975 to 1994, brazil. loffredo lcm, freitas jasf, grigolli aag. rev sau de pu blica vol.35 no.6 sa o paulo dec. 2001. 2. prevalence of kinds of cleft lip and palate at a pediatric hospital in northeast of brazil. cymrot m; sales fcd; teixeira faa; teixeira jr. faa; teixeira gsb; filho jfc; oliveira nh. rev. bras. cir. pla st. (impr.) vol.25 no.4 sa o paulo oct./dec. 2010. 3. a proposed modification for the classification of cleft lip and cleft palate. spina v. cleft palate j. 1973 jul;10:251-2. no abstract available. pmid: 4513915 4. enxertia o ssea alveolar: uma conduta para todas as fissuras alveolares. roça gb, freitas rs, rodrigues lk, busato ls, canan l. rev. bras. cir. pla st. 2010; 25(supl): 1-102. 5. autogenous cancellous bone and marrow transplants. boyne pj. clin orthop relat res. 1970 nov-dec;73:199209. no abstract available. pmid: 4920990 6. secondary bone grafting of alveolar clefts. a surgical/orthodontic treatment enabling a non-prosthodontic rehabilitation in cleft lip and palate patients. abyholm fe, bergland o, semb g. scand j plast reconstr surg. 1981;15(2):127-40. no abstract available. pmid: 7041248 7. alternative donor site for alveolar bone grafting in adults with cleft lip and palate. so ll, lui ww. angle orthod. 1996;66(1):9-16. review. pmid: 8678350 8. implications and treatment of cleft lip and palate in patients with an autognenous bone graft. rodrigues apgm; castro hcbc; reher p. rev bras de cir bucomaxilofac v. 10 n. 1 p. 91-6, 2010. 9. surgical repositioning of the premaxilla with bone graft in 50 bilateral cleft lip and palate patients. carlini jl, biron c, gomes ku, da silva rm. j oral maxillofac surg. 2009 apr;67(4):760-6. doi: 10.1016/j.joms.2008.07.013 . pmid: 19304031 10. management of the prominent premaxilla in bilateral cleft lip and palate. aburezq h, daskalogiannakis j, forrest c. cleft palate craniofac j. 2006 jan;43(1):92 pmid: 16405381 11. o impacto na qualidade de vida apo s o reposicionamento ciru rgico da pre -maxila em portadores de fissura la bio palatal bilateral estudo de 50 casos. the life quality impact after surgical pre maxilla repositioning in patients with bilateral lip palatal cleft study of 50 cases. gomes ku; rapoport a; lehn cn; denardin ovp; carlini jl.. rev. col. bras. cir. vol.35 no.6 rio de janeiro nov./dec. 2008. 12. an orthodontic and cephalometric study on the results of the combined surgical-orthodontic approach of the protruded premaxilla in bilateral clefts. heidbuchel kl, kuijpers-jagtman am, freihofer hp. j craniomaxillofac surg. 1993 mar;21(2):60-6. pmid: 8450075 13. secondary bone graft and eruption of the permanent canine in patients with alveolar clefts: literature review and case report. da silva filho og, teles sg, ozawa to, filho lc. angle orthod. 2000 apr;70(2):174-8. review. pmid: 10833007 14. secondary bone grafting of residual alveolar and palatal clefts. boyne pj, sands nr. j oral surg. 1972 feb;30(2):87-92. pmid: 4550446 15. bone grafting of alveolar clefts. bertz je. j oral surg. 1981 nov;39(11):874-7. no abstract available. pmid: 7026745 16. o reposicionamento ciru rgico da pre -maxila como determinante da inclusa o e integraça o social, em portadores de fissura la bio palatal bilateral: estudo de 50 casos. gomes, k.u. dissertaça o (mestrado em cie ncias da sau de) hospital helio polis – hosphel sa o paulo. 63 fls, 2008 1. 17. elimination of the residual alveolar cleft by secondary bone grafting and subsequent orthodontic treatment. bergland o, semb g, abyholm fe. cleft palate j. 1986 jul;23(3):175-205. pmid: 3524905 18. iliac versus cranial bone for secondary grafting of residual alveolar clefts. cohen m, figueroa aa, haviv y, schafer me, aduss h. plast reconstr surg. 1991 mar;87(3):423-7; discussion 428. pmid: 1998013 19. late secondary autogenous bone grafting in cleft patients comparing mandibular (ectomesenchymal) and http://dentistry3000.pitt.edu/ http://www.ncbi.nlm.nih.gov/pubmed/4513915 http://www.ncbi.nlm.nih.gov/pubmed/4513915 http://www.ncbi.nlm.nih.gov/pubmed/4513915 http://www.ncbi.nlm.nih.gov/pubmed/4920990 http://www.ncbi.nlm.nih.gov/pubmed/4920990 http://www.ncbi.nlm.nih.gov/pubmed/7041248 http://www.ncbi.nlm.nih.gov/pubmed/7041248 http://www.ncbi.nlm.nih.gov/pubmed/7041248 http://www.ncbi.nlm.nih.gov/pubmed/7041248 http://www.ncbi.nlm.nih.gov/pubmed/7041248 http://www.ncbi.nlm.nih.gov/pubmed/7041248 http://www.ncbi.nlm.nih.gov/pubmed/8678350 http://www.ncbi.nlm.nih.gov/pubmed/8678350 http://www.ncbi.nlm.nih.gov/pubmed/8678350 http://www.ncbi.nlm.nih.gov/pubmed/8678350 http://www.ncbi.nlm.nih.gov/pubmed/19304031 http://www.ncbi.nlm.nih.gov/pubmed/19304031 http://www.ncbi.nlm.nih.gov/pubmed/19304031 http://www.ncbi.nlm.nih.gov/pubmed/19304031 http://www.ncbi.nlm.nih.gov/pubmed/16405381 http://www.ncbi.nlm.nih.gov/pubmed/16405381 http://www.ncbi.nlm.nih.gov/pubmed/16405381 http://www.ncbi.nlm.nih.gov/pubmed/8450075 http://www.ncbi.nlm.nih.gov/pubmed/8450075 http://www.ncbi.nlm.nih.gov/pubmed/8450075 http://www.ncbi.nlm.nih.gov/pubmed/8450075 http://www.ncbi.nlm.nih.gov/pubmed/8450075 http://www.ncbi.nlm.nih.gov/pubmed/8450075 http://www.ncbi.nlm.nih.gov/pubmed/10833007 http://www.ncbi.nlm.nih.gov/pubmed/10833007 http://www.ncbi.nlm.nih.gov/pubmed/10833007 http://www.ncbi.nlm.nih.gov/pubmed/10833007 http://www.ncbi.nlm.nih.gov/pubmed/10833007 http://www.ncbi.nlm.nih.gov/pubmed/4550446 http://www.ncbi.nlm.nih.gov/pubmed/4550446 http://www.ncbi.nlm.nih.gov/pubmed/4550446 http://www.ncbi.nlm.nih.gov/pubmed/7026745 http://www.ncbi.nlm.nih.gov/pubmed/7026745 http://www.ncbi.nlm.nih.gov/pubmed/3524905 http://www.ncbi.nlm.nih.gov/pubmed/3524905 http://www.ncbi.nlm.nih.gov/pubmed/3524905 http://www.ncbi.nlm.nih.gov/pubmed/3524905 http://www.ncbi.nlm.nih.gov/pubmed/3524905 http://www.ncbi.nlm.nih.gov/pubmed/1998013 http://www.ncbi.nlm.nih.gov/pubmed/1998013 http://www.ncbi.nlm.nih.gov/pubmed/1998013 http://www.ncbi.nlm.nih.gov/pubmed/2606985 http://www.ncbi.nlm.nih.gov/pubmed/2606985 http://www.ncbi.nlm.nih.gov/pubmed/2606985 http://www.ncbi.nlm.nih.gov/pubmed/2606985 familial aggregation of anxiety associated with bruxism vol 3, no 1 (2015) doi 10.5195/d3000.2015.36 http://dentistry3000.pitt.edu 8 iliac crest (mesenchymal) grafts. koole r, bosker h, van der dussen fn. j craniomaxillofac surg. 1989 dec;17 suppl 1:28-30. pmid: 2606985 20. implantes denta rios contempora neos. misch, c. e. segunda ediça o. sa o paulo: santos, 2000. 21. secondary bone grafting and orthodontic treatment in patients with bilateral complete clefts of the lip and palate. bergland o, semb g, abyholm f, borchgrevink h, eskeland g. ann plast surg. 1986 dec;17(6):460-74. pmid: 3548551 22. reconstruction of alveolar cleft with allogenous bone graft: clinical considerations. filho ogs; ozawa to; bachega c; ma bachega.. dental press j. orthod. vol.18 no.6 maringa n ov./dec. 2013. http://dentistry3000.pitt.edu/ http://www.ncbi.nlm.nih.gov/pubmed/2606985 http://www.ncbi.nlm.nih.gov/pubmed/2606985 http://www.ncbi.nlm.nih.gov/pubmed/3548551 http://www.ncbi.nlm.nih.gov/pubmed/3548551 http://www.ncbi.nlm.nih.gov/pubmed/3548551 http://www.ncbi.nlm.nih.gov/pubmed/3548551 http://www.ncbi.nlm.nih.gov/pubmed/3548551 vol 7, no 1 (2019) issn 2167-8677 (online) doi 10.5195/d3000.2019.91 http://dentistry3000.pitt.edu new articles in this journal are licensed under a creative commons attribution 4.0 united states license. this journal is published by the university library system, university of pittsburgh as part of its d-scribe digital publishing program and is cosponored by the university of pittsburgh press. evaluation of salivary and oral cell collection methods for genomic dna extraction clarissa christina avelar fernandez1, fernanda farage da costa felipe ferreira1, christiane vasconcellos cruz1, marcelo de castro costa1 1department of pediatric dentistry and orthodontics, federal university of rio de janeiro, brazil abstract the use of saliva and oral cells as sources of biological material has gained attention, due to advantages such as facility, non-invasiveness, and great patient acceptance. the objective of the study was to compare four different types of saliva and oral buccal cell collecting methods for genomic dna extraction: (1)expectoration of saliva, (2)expectoration of saliva with lingual stimulation, (3)scraping with cytological brush, and (4)scraping with cytological brush and expectoration of saliva. the sample was composed of students and employees from the dental school of the federal university of rio de janeiro (n = 20, 10 men and 10 women with mean age of 47.60 ± 15.70 and 20.50 ± 2.1, respectively). the collections were performed with an interval of at least one day between them and the participants were instructed to stay for less than 30 minutes without eating food and brushing teeth. samples were stored at -20°c until dna extraction was performed using a commercially available kit (qiagen®). differences in dna yield between methods were test for statistical significance with an alpha of 0.05. no sexual dimorphism was observed in relation to the concentration of dna (p=0.76), age (p=0.91), and ethnicities (p=0.72). there was no significant difference between the collection methods in relation to the quantity and purity of the extracted dna (p≥0.05). all methods gave lower dna yields than the ones obtained from blood or saliva collected through comercial kits and may be carefully use for clinical diagnostic purposes or for research experiements requiring higher dna concentrations. citation: fernandez cca, et al. (2019) evaluation of salivary and oral cell collection methods for genomic dna extraction. dentistry 3000. 1:a001 doi:10.5195/d3000.2019.91 received: may 17, 2019 accepted: may 23, 2019 published: july 25, 2019 copyright: ©2019 fernandez cca, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: pttpo2009@yahoo.com.br introduction dna for diagnostic or research purposes can be obtained from various biological materials. in the last decades, the dna used in molecular studies was preferentially obtained from blood samples since they provide a large number of cells and, consequently, a good yield [1,2]. however, collection of blood samples has some drawbacks such as the potential non-viability in large populationbased studies. because it is an invasive method, some patients may be resistant to collection, need a specialized professional to perform the procedure, and it is more difficult to perform in the elderly and children [1,3,4]. another factor to be emphasized is that the ferrous ions (fe2 +) present in the blood compete with mg2 + ions, which are inhibitors of pcr (c-reactive protein) [1,2]. saliva has been increasingly used as a source of genomic dna suitable for large studies because it is non-invasive and has diagnostic potential. in addition, when compared to the traditional method of collecting dna from blood, it presents a reduced cost and is a noninvasive method that can be performed more easily in children, the elderly, and patients with special needs [5]. collecting saliva also brings no risk of infections and has the potential to become a first line of clinical diagnosis [6]. a number of methods for collecting saliva and buccal cells have been reported: swabs, brushes, mouthwashes [4], drainage method, spit method, suction method, and stimulation by paraffin mastication and/or by applying citric acid to the tongue [6]. dna quality is essential for the success rates in analyzes and the amount of dna collected is extremely important and we must be attentive to these factors http://dentistry3000.pitt.edu/ http://www.library.pitt.edu/ http://www.pitt.edu/ http://www.library.pitt.edu/articles/digpubtype/index.html http://upress.pitt.edu/ http://creativecommons.org/licenses/by/4.0/ evaluation of salivary and oral cell collection methods for genomic dna extraction vol 7 no 1 (2019) doi 10.5195/d3000.2019.91 http://dentistry3000.pitt.edu during the choice of dna collection method [7]. despite the obvious practicality and advantages of saliva and oral cells in obtaining dna, there is a difference in the amount of dna obtained depending on the method of extraction of genomic dna from saliva used. the objective of the present study was to compare four different methods of collecting saliva and buccal cells for dna extraction evaluation of its concentration and purity. methods 1. sample selection twenty participants [10 males and 10 females, mean age of 36.55 (±15.74) years, 13 white and 7 black] who were students and employees at the school of dentistry of the federal university of rio de janeiro were selected (convenience sample). the inclusion of these individuals occurred after they read and signed a term of free consent. this project was approved by the research ethics committee of the university hospital clementino fraga filho (hucff/ufrj) number 619.096. 2. data collection during the interview, data such as age, sex, and ethnicity were recorded. then, four different protocols randomized for the order that they were performed were used for the collection of saliva and buccal cells by a single researcher (c.c.a.f.), with an interval of at least one day between each other. participants were instructed to not smoke, eat and brush their teeth for at least 30 minutes before each saliva collection. 1. expectoration of saliva; 2. expectoration of saliva with lingual stimulus; 3. scraping with a cytological brush; and, 4. scraping with cytological brush + expectoration of saliva. 2.1 expectoration of saliva: saliva collected was stored in a falcon tube. the researcher instructed each individual to spit until the marking of 2 ml. after the protocol collection was performed, information such as the date of the collection, the method and the study identifier the participant were recorded on the tube and it was protected by a plastic bag and stored at -20°c. this step was performed routinely in all the different protocols carried out in this research. 2.2 expectoration of saliva with lingual stimulus: in this salivary collection method, the individuals were instructed to perform stimulation through the friction of their tongue against intraoral structures and after, they were asked to spit. step by step, this method consisted of rubbing the tongue in the teeth, jugal mucosa, and roof of the mouth 10 times in each hemiarch in order to detach buccal cells from the oral mucosa. 2.3 scraping with a cytological brush: scraping of buccal cells from the oral mucosa was performed with a cytological brush that was then stored in a falcon tube. the procedure was performed by a single researcher (c.c.a.f.) who positioned the brush comfortably in the individual's mouth and rubbed the lower gum 10 times with rotational movements and back and forth. 2.4 scraping with a cytological brush + expectoration of saliva: this method consisted of the combination of the methods scraping with a cytological brush and expectoration of saliva. 3. dna extraction to extract the dna from the samples, the steps of the qiagen® kit proposed by the manufacturer were followed: https://www.qiagen.com/br/qdm/alp/all prep-selectionguide?intcmp=ecom_web_selectiongui des_2610_teaser_tools_allprepguide_ 4. dna quantification the concentration and purity of the dna were determined by optical density spectrophotometer (nanodrop® 2000c) using 2μl of the extracted material. the dna concentration was evaluated at a wavelength of 260nm. the ratio between the values obtained at wavelengths 260nm and 280nm was used to estimate the purity of http://dentistry3000.pitt.edu/ https://www.qiagen.com/br/qdm/alp/allprep-selection-guide?intcmp=ecom_web_selectionguides_2610_teaser_tools_allprepguide_ https://www.qiagen.com/br/qdm/alp/allprep-selection-guide?intcmp=ecom_web_selectionguides_2610_teaser_tools_allprepguide_ https://www.qiagen.com/br/qdm/alp/allprep-selection-guide?intcmp=ecom_web_selectionguides_2610_teaser_tools_allprepguide_ https://www.qiagen.com/br/qdm/alp/allprep-selection-guide?intcmp=ecom_web_selectionguides_2610_teaser_tools_allprepguide_ evaluation of salivary and oral cell collection methods for genomic dna extraction vol 7 no 1 (2019) doi 10.5195/d3000.2019.91 http://dentistry3000.pitt.edu the genomic dna. only dna samples with a 260/280 ratio above 1.7 were considered satisfactory for dna purity. 5. statistical analysis descriptive statistics were applied evaluating the frequency of sex, ethnicity and the mean age of all the studied individuals. one-way anova and t-tests were used determine differences between groups. in addition, chi-square and/or fisher's exact test (p≥0.05) were used to verify statistical differences in frequencies between the dichotomous variables. the data were tabulated and analyzed in the statistical software spss version 20.0 (statistical package for social sciences, spss inc, chicago, iii) and all tests were carried out with a 95% significance level. results the mean concentration of genomic dna was not different between males [6.74 (±1.88) ng/μl] and females [6.88 (±2.31)ng/μl] (p=0.76). this shows that there is no sexual dimorphism regarding the amount of genomic dna recovered from saliva samples of females and males (table 1). the average amount of genomic dna obtained from each method can be observed in table 1. there were no statistical differences between the methods. discussion the four methods of saliva and buccal cell collection did not show dna yield differences but the values obtained are remarkably lower than the ones obtained from kit-based saliva collection methods [8]. most of the studies reported in the literature collected buccal cells and saliva in three ways: rubbing or scraping the oral mucosa with cytological brushes, mouthwash with oral solution and saliva expectoration [1,3,9,10]. the collections are performed through commercial kits and the details of each methodology depend on to the manufacturers' instructions. the most commonly used commercial kit is oragene® (dna genotek) where assessed individuals need only expectorate in a small bottle. when the bottle is closed, a solution containing bactericidal substances mix with the saliva and preserve the dna, allowing the sample to be held for several months at room temperature. this kit has presented favorable amounts of genomic dna and is easy to apply, allowing even self-collection [5,7,11,12]. on the other hand, oragene® kit is expensive, which represents a disadvantage in table 1. characterization of the sample in relation to age, ethnicity and its relationship with dna concentration according to sex. (* p value <0,001. p-value obtained according to ttesta, chi-square testb and anova one-way testc) male female total p value age (±sd) 47.60±15.70 25.50±2.18 36.55±15.74 *a ethnicity: n(%) white 4(40) 9(90) 13(65) black 6(60) 1(10) 7(35) *b dna concentration ±sd 6.74±1.89 6.89±2.31 6.81±2.10 0.57a protocol of saliva collection expectoration of saliva 5.73±1.36 5.06±1.28 5.39±1.33 expectoration of saliva with lingual stimulus 5.66±1.66 7.40±3.70 6.53±2.93 scraping with a cytological brush 8.10±1.56 7.89±1.02 8±1.29 scraping with cytological brush + expectoration of saliva 7.47±1.80 7.19±1.26 7.33±1.52 0.68c http://dentistry3000.pitt.edu/ evaluation of salivary and oral cell collection methods for genomic dna extraction vol 7 no 1 (2019) doi 10.5195/d3000.2019.91 http://dentistry3000.pitt.edu studies with a large sample. in our study, we did not use a method that included a commercial saliva collection kit, however dna concentrations obtained were much lower in comparison to these commercial saliva collection kits8 and this suggests these protocols potentially will not allow for analyses that require higher dna concentrations. all methods gave lower dna yields than the ones obtained from blood or saliva collected through comercial kits and may be carefully use for clinical diagnostic purposes or for research experiements requiring higher dna concentrations. acknowledgements the authors are indebted to the participants of the study. references 1. avaliação do rendimento, pureza e integridade do dna genômico em diferentes protocolos de coleta de células bucais. waltrick-zambuzzi mr, vieira, tcs, romanos hf, macedo ee, granjeiro jm, kuchler ec. int j dent, recife. 2012 jan/mar; 11(1):12-18. 2. comparison of different collection procedures and two methods for dna isolation from saliva. durdiaková j, kamodyivá n, ostatníková d, vlková b, celec p. clin chem lab med. 2012; 50(4):643– 647. 3. determinants of dna yield and purity collected with buccal cell samples. van wieren-de wijer dbma, maitland-van der zee ah, de boer a, belitser sv, kroon aa, de leeuw pw, schiffers p, janssen rgjh, van duijn cm, stricker bhch, klungel oh. eur j epidemiol. 2009; 24: 677-682. 4. comparison of methods for collection of dna samples by mail in the black women´s helth study. cozier yc, palmer jr, rosenberg l. ann epidemiol. 2004; v. 14, p.117122. 5. dna yeld and quality of saliva samples and suitability for large-scale epidemiological studies in children. koni ac, scott ra, wang g, bailey mes, peplies j, bammann k, pirsiladis yp. internt journ of obes. 2011; v. 35. p 113-118. 6. diagnostic potential of saliva: current state and future applications. pfaffe t, cooperwhite j, beyerlein p, kosner k, punyadeera c. clinc chem. 2011; v. 57. p 675-687. 7. quality and quantity of saliva dna obtained from the selfadministrated oragene method a pilot study on the cohort of swedish men. rylander-rudqvist t, hankansson n, tybring g, wolk, alicja. cancer epidemiol biomarkers prev. 2006;15(9). 8. comparative study of five commercially avaiable saliva collection kits for dna extraction. deeley k, noel j, vieira arclin lab. 2016; 1;62(9):1809-1813. 9. collection of buccal cell dna in seventh-grade children using water and a toothbrush. london sj, xia j, lehman ta, yang j, granada e, chunhong l, dubeau l, li t, david-beabes gl, li y. canc epid biom prev, 2001; v.10, p.1227-1230. 10. quality of dna extracted from saliva samples collected with the oragene dna self collection kit. nuner ap, oliveira io, santos br, millech c, silva lp, gonzález da, hallal pc, menezes ambb, araújo cl, barros f. med reserch method. 2012. 11. collection of blood, saliva and buccal cell samples in a pilot study on the danish nurse cohort: comparison of the response rate and quality of genomic dna. hansen to, simonsen mk, nielsen fc, hundrup ya. cancer epidemiol biomarkers prev. 2007; v. 16. p.2072-2076. 12. buccal dna collection: comparison of buccal swabs with fta cards. milne e, bockxmeer fmv, robertson l, brisbane jm, ashton lj, scott rj, armstrong bk. canc epid biom prev. 2006; v.15. p. 816819. http://dentistry3000.pitt.edu/ 1. sample selection 2. data collection 2.1 expectoration of saliva: 2.2 expectoration of saliva with lingual stimulus: 2.3 scraping with a cytological brush: 2.4 scraping with a cytological brush + expectoration of saliva: 3. dna extraction 4. dna quantification 5. statistical analysis prevalence of malocclusion in brazilian quilombola adolescents http://dentistry3000.pitt.edu vol 10 no 1 (2022) doi 10.5195/d3000.2022.155 prevalence of malocclusion in brazilian quilombola adolescents gabrielli oliveira de brito 1 , sarah dos santos martins 1 , ítallo lins paes de araújo 2 , isla camilla carvalho laureano 2 , alessandro leite cavalcanti 2 1 department of dentistry, state university of paraíba, campina grande, paraíba, brazil. 2 post-graduate program in dentistry, department of dentistry, state university of paraíba, campina grande, paraíba, brazil. abstract purpose: to evaluate the prevalence of malocclusion in quilombola adolescents living in rural area in northeastern brazil. materials and methods: this is a cross-sectional study in which 36 quilombola adolescents aged 12-19 years were evaluated. data collection was performed at home between january and april 2020 by a trained researcher using the dental aesthetic index (dai). data were analyzed using the ibm spss software and presented using descriptive statistics. results: more than two thirds of adolescents had already visited the dentist at least once in life (72.2%), with the last dental visit having occurred less than a year ago (57.7%), with predominance of public dental service (84.6%). there was higher frequency of lower anterior misalignment (86.1%) and dental crowding (83.4%) in two segments (55.6%). almost all adolescents presented dental malocclusion (97.2%), with very severe or disabling severity (44.4%). conclusions: the prevalence of very severe malocclusion was high in quilombola adolescents, expressing the need for treatment of this population. keywords: adolescent; epidemiology; ethnic groups; malocclusion. citation: de brito. g, et al. (2022) prevalence of malocclusion in brazilian quilombola adolescents dentistry 3000. 1:a001 doi:10.5195/d3000.2022.155 received: february 26, 2021 accepted: april 05, 2021 published: june 13, 2022 copyright: ©2022 de brito, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: carvalhoisla@gmail.com introduction malocclusion is a condition referred to abnormal occlusion and irregular bone and muscle growth and development, which interferes both in functional aspects of the oral cavity and in the psychosocial aspect of individuals [1-3]. it is considered a public health problem, affecting mainly children [4, 5] and adolescents [6, 7], impacting their quality of life [6, 8, 9]. hereditary, environmental factors or their association are attributed to the etiology of malocclusion and the presence of other dental problems is shown to complicate the maintenance of satisfactory occlusion [1]. malocclusion in adolescents is associated with disadvantageous social position, deleterious habits, poor housing conditions and low family income and schooling levels, in addition to higher occurrence in non-white (those who self-reported as black, pardo, yellow or indigenous) individuals [2, 7, 8, 10, 11]. problems involving chewing, speech and premature tooth loss can be a consequence of malocclusion [12]. quilombolas are ethnic-racial groups with a historical trajectory, with own territorial relations and proud of their black ancestry, who resisted oppression suffered during history [13]. these individuals are on the margins of society, since they make up the prevalence of malocclusion in brazilian quilombola adolescents vol 10 no 1 (2022) doi 10.5195/d3000.2022.155 group of brazilians composed of blacks or browns with lack of access to sanitation services, inadequate housing conditions and few goods [14]. in this context, the remaining quilombola populations are vulnerable due to their precarious living conditions, as they belong to rural communities with low quality of life and unaware of essential health-related information [15] and little knowledge about oral health [16]. low access to water supply and dental services is also reported [17, 18]. regarding malocclusion, a study carried out at kalunka quilombola community in goiás, revealed high prevalence of malocclusions in children, with those in the mixed dentition stage having fewer malocclusions when compared to children with primary dentition [19]. studies enabling early diagnosis of malocclusion, in the early stages of the transition from deciduous to permanent dentition, have been shown to be essential in order to prevent the need for orthodontic treatment during adolescence and adulthood, and consequent negative impacts on quality of life [20]. in addition, identifying vulnerable groups and the influence of sociodemographic aspects on this condition will enable better planning of http://dentistry3000.pitt.edu necessary preventive and therapeutic measures. considering the small number of studies in this minority population group and the importance of carrying out epidemiological studies, this study aimed to assess the prevalence of malocclusion in quilombola adolescents. methods study design and location it was a cross-sectional study carried out at the caiana dos crioulos community, recognized by the palmares cultural foundation as a remaining quilombola area, located in the municipality of alagoa grande, paraíba, brazil. the community has population of 1,500 inhabitants, distributed in 405 families [21]. sample the sample was non-probabilistic and comprised adolescents aged 12 and 15-19 years, age groups based on the brazilian national oral health survey [22]. adolescents of both sexes, selfdeclared black or afrodescendants, and registered with e-sus ab – software to operationalize the information system for health for primary care (sisab) [23] – were included. individuals with inadequate cognitive ability were excluded. data collection data collection was carried out between january and april 2020, at home, being performed by a calibrated postgraduate examiner (kappa = 0.90). theoretical and practical training was performed by gold standard researcher with previous experiences in epidemiological investigations. information regarding demographics factors (sex and age group), oral health habits (visit to the dentist, visit frequency and type of health service used) was collected. for the diagnosis of malocclusion, the dental aesthetic index (dai) [24] was used. dai score was calculated using the regression equation of ten measured components of occlusal morphology, multiplied by the regression coefficients (weights) and added the products plus a constant: (absent teeth × 6) + (crowding) + (spacing) + (midline diastema × 3) + (upper anterior misalignment) + (lower anterior misalignment) + (maxillary overjet × 2) + (mandibular overjet × 4) + (anterior open bite × 4) + (molar relationship × 3) + 13 (constant) [24]. those with dai score less than or equal to 25 were prevalence of malocclusion in brazilian quilombola adolescents vol 10 no 1 (2022) doi 10.5195/d3000.2022.155 considered to have normal occlusion [8]. malocclusion severity was classified as absence of abnormality or mild malocclusion, whose orthodontic treatment is unnecessary (dai ≤ 25); defined malocclusion, whose treatment is elective (dai = 26 to 30); severe malocclusion, whose treatment is highly desirable (dai = 31 to 35) and very severe or disabling malocclusion, whose orthodontic treatment is essential (dai ≥ 36) [24]. statistical analysis data were analyzed using the ibm spss software, version 22.0 for windows (ibm corp., armonk, ny, usa) and presented using descriptive statistics (absolute and percentage distributions). for statistical analysis, variables upper anterior misalignment <2 mm and ≥ 2 mm, lower anterior misalignment <2 mm and ≥ 2 mm, maxillary overjet <4 mm and ≥ 4 mm, mandibular overjet <4 mm and ≥ 4 mm and anterior open bite <2 mm and ≥ 2 mm [25] were dichotomized. ethical aspects this study followed guidelines and a rule established in resolution http://dentistry3000.pitt.edu nº 466/12 of the national health council and the declaration of helsinki (1964) and its subsequent amendments and was approved by the ethics committee on research with human beings of the state of paraíba university, under protocol number 3.272.910. informed consent was obtained from all guardians and individuals included in the study. results regarding the sociodemographic profile, the majority were female (66.7%) aged 1519 years (66.7%), with mean age of 15.92 ± 3.0 years and median of 17 years. a percentage of 72.2% had visited the dentist at some time in their lives, with the last consultation having occurred less than 1 year ago (57.7%) with predominance of public dental services (84.6%) (table 1). lower anterior misalignment was the most prevalent type of malocclusion in the sample (86.1%), followed by dental crowding (83.4%), with frequent table 1. distribution of sociodemographic variables of quilombola adolescents prevalence of malocclusion in brazilian quilombola adolescents vol 10 no 1 (2022) doi 10.5195/d3000.2022.155 presence of two crowded segments (55.6%), and upper anterior misalignment (83.3%). anterior spacing in two segments was found in 27.8% of quilombola adolescents, while midline diastema was present in 50.0% of the sample and half cusp molar ratio in 58.3%. low prevalence of adolescents with absent lower teeth was observed (8.3%), all of them were premolars, in individuals aged 17 and 19 years. the majority had malocclusion cases (97.2%), with very severe or disabling severity (44.4%) (table 2). discussion most of the sample visited the dentist at least once and more than half had the last visit less than a year in public dental services. this finding may be related to the good coverage performance of the public system for this population. however, it is observed that quilombola adolescents have low frequency of visits to the dentist when compared to other adolescents living in rural areas and with the aggravation of being a community with great social vulnerability [26]. http://dentistry3000.pitt.edu table 2. prevalence of malocclusions and components assessed by the dental aesthetic index (dai) among quilombola adolescents prevalence of malocclusion in brazilian quilombola adolescents vol 10 no 1 (2022) doi 10.5195/d3000.2022.155 tooth loss was very low, affecting only lower dental elements. a study by silva et al. [17] associated this condition to the oral hygiene habits of this ethnic group, since most individuals brush their teeth three or more times a day and use dental floss, making it possible to establish a direct relationship between good oral hygiene habits and low occurrence of tooth loss. however, access to oral health services is limited for the quilombolas [17], as already evidenced, as they belong to rural communities with low quality of life and unaware of essential health-related information [15] and little knowledge about oral health [16]. there was predominance of quilombola adolescents with very severe or disabling malocclusion. regarding dai components, occurrence of dental crowding was high, involving at least one segment, with higher occurrence in two segments, and half of the sample presented spacing in one or two anterior segments and midline diastema. these results are in agreement with these obtained by neto et al. [11], who found high occurrence of crowding in non-white brazilian adolescents and data revealed association of types of spacing with skin color. in addition, http://dentistry3000.pitt.edu previous research observed higher likelihood of ethnic groups formed by blacks/browns of having severe and very severe malocclusion [2] which may be due to the racial factor [27]. other factors such as upper and lower anterior misalignment also showed high frequency. however, in a previous study with brazilian adolescents, this condition showed low occurrence [10]. considering overjet, similar results were found [10], in which jaw and mandible, with spaces less than 4 mm were predominant. overjet is considered one of the main characteristics that interfere in social relationships and in the search for orthodontic treatment and is highly prevalent in black adolescents [28]. it was also found that large part of the sample had anterior open bite. this condition was associated with malocclusion severity due to changes in lip tonus and contraction of the periorbicular muscle [29]. considering the stage of life of the analyzed individuals, the presence of anterior open bite can be associated with the occurrence of bullying, generating negative impact on the quality of life and mental health of adolescents [30]. these occlusal characteristics were related with socially vulnerable adolescents, and as already mentioned, to difficult social interaction, which can affect psychological well-being, whereas aesthetic appearance within social standards represents great impact on the interaction among individuals, mainly in this age group [8]. some types of malocclusion promote greater influence on the perception of individuals about their dental aesthetics, as reported in a study that found association between dissatisfaction of adolescents with tooth positioning, with midline diastema being one of the main dai components that increased dissatisfaction [31]. this can probably be explained by the fact that, as it is located in a region of greater disclosure, which is the anterior region, midline diastema draws more attention, and should cause greater aesthetic dissatisfaction for affected individuals. abnormal molar relationship was found in a significant portion of quilombola adolescents in the present study. this dental condition together with jaw irregularity has been associated with increase in the probability of up to 30% of dental caries in adolescents and it is assumed that prevalence of malocclusion in brazilian quilombola adolescents vol 10 no 1 (2022) doi 10.5195/d3000.2022.155 malocclusion, or specific types of it, are predisposing factors for the development of dental caries [32], due to the difficulty for patients to maintain good oral hygiene which results in the increases of biofilm accumulation on the teeth surfaces [33]. although, other studies report that dental caries and its complications leads to alterations in mastication [34], tendency to dental migrate and loss of arch length, may lead to compromised facial growth and development, resulting in malocclusion [1]. almost all quilombola adolescents presented malocclusion in need of treatment. thus, the high prevalence of malocclusion is considered a public health problem and further studies aimed at investigating minority populations and rural areas should be carried out to better understand their characteristics, evaluate possible deleterious habits, since previous researchers have found an association between sleep bruxism [35, 36], tongue thrusting, nail-biting, thumb sucking and mouth breathing [36] and malocclusion and, consequently, to understand the oral health needs of these individuals. cross-sectional studies aimed at analyzing malocclusion conditions http://dentistry3000.pitt.edu and its variables in certain populations help establishing relationships between data found and need for treatment. however, this type of study also has limitations, such as the impossibility of establishing causality. in addition to these factors, the sample size is another limitation, which despite containing expressive data, findings cannot be extrapolated to the entire caiana dos crioulos quilombola population. the scarcity of studies with quilombola adolescent populations was also a limiting factor to confront the findings of this study; however, it is necessary to highlight that despite these aspects, this research is one of the few studies developed in the country with this minority group regarding dental malocclusion. conclusion the prevalence of very severe malocclusion was high in quilombola adolescents, expressing the need for treatment in this population. future studies should be carried out in this community and in other similar groups to establish a diagnosis of the main oral diseases that affect these populations. minority groups, including quilombolas, are subject to greater social vulnerability and their investigation is essential to understand their particularities, needs and the results arising from their socio-cultural process. thus, specialized policies and actions could adapt to the demands of these groups. references 1. zou j, meng m, law cs, rao y, zhou x. common dental diseases in children and malocclusion. int j oral sci. 2018; 10(1):7. pmid: 29540669. 2. rebouças ag, zanin l, ambrosano gmb, flório fm. individual factors associated to malocclusion in adolescents. cien saude colet. 2017; 22(11):37233732. pmid: 29211177. 3. sari cn, jazaldi f, ismah n. association between psychosocial status and orthodontic treatment needs in indonesian high school students. pesqui bras odontopediatria clín integr. 2020; 20:e5649. 4. giffoni tcr, brandt gz, rocha is, ramos al, provenzano mga, fracasso mlc. relation of dental anomalies with occlusal alterations in the pediatric patients. pesquis bras prevalence of malocclusion in brazilian quilombola adolescents http://dentistry3000.pitt.edu vol 10 no 1 (2022) doi 10.5195/d3000.2022.155 odontopediatria clín integr. 2019; 19(1):e4026. 5. assis wc, pereira js, silva ys, brito fr, nunes la, ribeiro ijs, et al. factors associated with malocclusion in preschool children in a brazilian small town. pesquis bras odontopediatria clín integr. 2020; 20:e5351. 6. miotto mhmb, benevides jj, postiglione lrf, dessaune dss, calmon mv, zandonade e. impact produced by oral disorders on the quality of life of brazilian adolescents. pesquis bras odontopediatria clín integr. 2019; 19:e4764. 7. freitas cv, souza jgs, mendes dc, pordeus ia, jones km, martins ameb. need for orthodontic treatment among brazilian adolescents: evaluation based on public health. rev paul pediatr. 2015; 33(2):204-10. pmid: 25769190. 8. martins lp, bittencourt jm, bendo cb, vale mp, paiva sm. malocclusion and social vulnerability: a representative study with adolescents from belo horizonte, brazil. cien saude colet. 2019; 24(2):393-400. pmid: 30726372. 9. mail lr, donassollo sh, donassollo ta. malocclusion diagnosis: normative criteria and self-perception of adolescents. pesquis bras odontopediatria clín integr. 2015; 15(1):197-203. 10. silveira mf, freire rs, nepomuceno mo, martins amebl, marcopito lf. severity of malocclusion in adolescents: populational-based study in the north of minas gerais, brazil. rev saúde pública. 2016; 50:11. pmid: 27143611. 11. neto tan, thomas ebaf, ferreira mc, santos am, queiroz rcs. problemas de espaço dentário em adolescentes brasileiros e fatores associados. ciênc saúde coletiva. 2014; 19(11):4555-4568. [in portuguese]. 12. samohyl m, nadazdyova a, hirjak m, hirosova k, vondrova d, argalasova l, et al. the analysis of selected malocclusion risk factors: a pilot study. pesqui bras odontopediatria clin integr. 2017; 17(1):e3790. 13. brasil. decreto n.º 4.887, de 20 de novembro de 2003. regulamenta o procedimento para identificação, reconhecimento, delimitação, demarcação e titulação das terras ocupadas por remanescentes das comunidades dos quilombos de que trata o art, 68 do ato das disposições constitucionais transitórias. diário oficial da união 21 de nov 2003. [in portuguese]. 14. ibge. diretoria de pesquisas, coordenação de população e indicadores sociais. desigualdades sociais por cor ou raça no brasil. estudos e pesquisas 2019. 41:1-2. [in portuguese]. 15. batista ec, rocha kb. mental health in brazil’s quilombola communities: a systematic review of the literature. interações 2020; 21(1):35-50. 16. rodrigues sa, lucas mg, cerqueira st da s, braga ap da s, vaz lg. educação em saúde em comunidades quilombolas. rev gaucha odontol 2011; 59(3):44551 [in portuguese]. 17. silva ekp, santos pr, chequer tpr, melo cma, santana kc, amorim mm, et al. oral health of quilombola and non-quilombola rural adolescents: a study of hygiene habits and associated factors. ciênc saúde coletiva. 2018; 23(9):2963-2978. pmid: 30281734. 18. dias jg, pereira bl, ribeiro pc, monteiro lrl. vulnerabilidade em saúde bucal: ausência de flúor na água de abastecimento público em uma comunidade remanescente quilombola. j business techn. 2020;13(1):57-69. [in portuguese]. prevalence of malocclusion in brazilian quilombola adolescents vol 10 no 1 (2022) doi 10.5195/d3000.2022.155 http://dentistry3000.pitt.edu 19. rank rcic, collier kf, vilela jer, rank ms, takahashi cm, molina oft. prevalência de oclusopatias em crianças da comunidade kalunga, goiás. cereus 2013; 5(3):86-100. [in portuguese]. 20. guimarães spa, jorge ko, fontes mjf, ramos-jorge ml, araújo ctp, ferreira ef, et al. impact of malocclusion on oral health-related quality of life among schoolchildren. braz oral res. 2018; 32:e95. 21. ibge. instituto brasileiro de geografia e estatística. censo brasileiro de 2010. alagoa grande: ibge, 2010. available from: https://cidades.ibge.gov.br/brasil/ pb/alagoa-grande/panorama&gt;. accessed on october 20, 2018. [in portuguese]. 22. brasil. ministério da saúde (ms). pesquisa nacional de saúde bucal 2010 brasília: ms; 2012. [in portuguese]. 23. brasil, ministério da saúde. esus atenção básica: manual sistema com coleta de dados simplificada: cds. brasília, df, 2013. [in portuguese]. 24. cons nc, jenny j, kohout fj. dai: the dental aesthetic index. iowa city: college of dentistry, university of iowa; 1986. 25. bauman jm, souza jgs, bauman cd, flório fm. sociodemographic aspects related to severity of malocclusion among 12-year-old brazilian children. cien saude colet. 2018; 23(3):723-732. pmid: 29538553. [in portuguese]. 26. silva ekp, medeiros ds. impact of oral health conditions on the quality of life of quilombola and non-quilombola rural adolescents in the countryside of bahia, brazil: a cross-sectional study. health qual life outcomes. 2020; 18(1):318. pmid: 32993764. 27. anthony sn, zimba k, subramanian b. impact of malocclusions on the oral healthrelated quality of life of early adolescents in ndola, zambia. int j dent. 2018; 2018:7920973. 28. farias acr, cangussu mct, ferreira rfa, castellucci m. occlusal characteristics and orthodontic treatment need in black adolescents in salvador/ba (brazil): an epidemiologic study using the dental aesthetics index. dental press j orthod. 2013; 18(1):34.e1-8. 29. yamaguchi h, sueishi k. malocclusion associated with abnormal posture. bull tokyo dent coll. 2003; 44(2):43-54. pmid: 12956088. 30. gatto rcj, garbin aji, corrente je, garbin cas. the relationship between oral health-related quality of life, the need for orthodontic treatment and bullying, among brazilian teenagers. dental press j orthod. 2019; 24(2):73-80. 31. kaieda ak, bulgareli jv, cunha ip, vedovello sas, guerra lm, ambrosano gmb, et al. malocclusion and dental appearance in underprivileged brazilian adolescents. braz oral res. 2019; 33:e014. 32. feldens ca, dullius ais, kramer pf, scapini a, busato als, vargasferreira f. impact of malocclusion and dentofacial anomalies on the prevalence and severity of dental caries among adolescents. angle orthod. 2015; 85(6):1027-34. pmid: 26516712. 33. gaikwad ss, gheware a, kamatagi l, pasumarthy s, pawar v, fatangare m. dental caries and its relationship to malocclusion in permanent dentition among 12-15 year old school going children. j int oral health. 2014; 6(5):27-30. pmid: 25395789. 34. gilchrist f, marshman z, deery c, rodd hd. the impact of dental caries on children and young people: what they have to say? int j paediatr dent. 2015 prevalence of malocclusion in brazilian quilombola adolescents http://dentistry3000.pitt.edu vol 10 no 1 (2022) doi 10.5195/d3000.2022.155 sep;25(5):327-38. pmid: 26153526. 35. traebert e, nazário ac, nunes rd, margreiter s, pereira kcr, costa sxs, et al. prevalence of sleep bruxism and association with oral health conditions in schoolchildren in a municipality in southern brazil. pesqui bras odontopediatria clín integr. 2020; 20:e0019. 36. zakirulla m, alshehri ad, hudaybi ah, fageeh sn, alghothimi aa, ali mg, et al. oral habits: prevalence and effects on occlusion among 7 to 13 years old school children in aseer, saudi arabia. pesqui bras odontopediatria clín integr. 2020; 20:e0005. microsoft word 69 2017.docx   vol  5,  no  1  (2017)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2017.69           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     potential  inheritance  patterns  of  a  prothrombin  gene  mutation  in   a  23-­‐year-­‐old  female  and  ethical  considerations  of  a  positive   diagnosis:  a  case  report   hannah  brand1      1university  of  pi.sburgh,  school  of  dental  medicine abstract   background:  prothrombin,  also  called  factor  ii,  is  a  blood  clo4ng  protein  found  in  all  individuals  that  is  necessary  to   form  blood  clots.  in  most  individuals,  a  balance  between  bleeding  and  blood  clot  forma7on  occurs.  however,  in  indi-­‐ viduals  with  a  muta-on  in  the  prothrombin  gene,  the  balance  is  disrupted  due  to  excess  produc-on  of  prothrombin   which  leads  to  an  increase  in  blood  clot  forma2on  (1).   inherited  predisposi2ons  to  blood  clot  forma2on  are  termed   hereditary   thrombophilia.  prothrombin  g20210a  muta2ons  are  one  of   the  most   common  hereditary   gene  associa-­‐ !ons.  case  descrip+on:  this  report  examines  the  case  of  a  23-­‐year-­‐old  female  who  has  tested  posi/ve  for  the  pro-­‐ thrombin   gene  muta.on.   the   individual   has   an   extensive   history   of   blood   clots   including   8   deep   vein   thromboses   (dvts),  4  pulmonary  embolisms  with  one  complete  infarc;on,  4  superficial  clots,  and  a  miscarried  pregnancy  a?ribut-­‐ ed  to  her  thrombophilia.  the  individual  has  a  significant  family  history  of  the  muta9on  and  takes  coumadin  daily  for   preven&on  of  further  clots.  prac%cal  and  ethical  implica%ons:  despite  a  strong  familial  history  of  blood  clots  and  re-­‐ lated  hospitaliza-ons,  the  parents  of  the  case  individual  do  not  want  to  get  their  other  children  tested  for  the  muta-­‐ !on  for  fear  that  the  children  will  be  denied  insurance  coverage  in  their  futures.  the  case  individual  will  likely  con!n-­‐ ue  to  be  on  blood  thinners  indefinitely.  this  will  affect  many  aspects  of  her  life,  including  dental  treatment,  as  she  will   be  at  an  increased  risk  for  bleeding.     cita%on:  brand,  h.   (2017)  poten&al   inheritance   pa#erns   of   a   prothrombin   gene   muta2on   in   a   23-­‐year-­‐old  female  and  ethical  considera0ons  of   a   posi've   diagnosis:   a   case   report.   den$stry   3000.  1:a001  doi:10.5195/d3000.2017.69   received:    may  4,  2017   accepted:    may  9,  2017   published:    june  9,  2017   copyright:  ©2017  brand,  h.  this   is  an  open  ac-­‐ cess  ar!cle   licensed  under  a  crea!ve  commons   a"ribu%on  work  4.0  united  states  license.   email:  hnb16@pi).edu   introduction   having  a  mutation  in  a   blood  clotting  gene  such  as  pro-­‐ thrombin  g20210a,  means  that  an   individual  has  a  higher  tendency  to   form  blood  clots.  the  prothrombin   mutation  is  found  in  approximate-­‐ ly  2%  of  caucasians  in  the  united   states  and  is  the  second  most   common  inherited  form  of  throm-­‐ bophilia  (2-­‐4).  given  the  rarity  of   the  disease,  most  cases  are  inher-­‐ ited  as  heterozygous  prothrombin   mutations  as  it  is  not  likely  that   both  parents  would  possess  the   mutation,  and  in  heterozygous   cases,  only  one  copy  of  the  muta-­‐ tion  is  needed.  homozygous  muta-­‐ tions  are  thought  to  be  more  se-­‐ vere  than  heterozygous.  being   heterozygous  for  the  mutation  in-­‐ creases  the  risk  of  a  blood  clot  by   2-­‐3  times,  versus  homozygous   where  the  risk  is  increased  by  as   much  as  50  times  (3).     the  most  common  compli-­‐ cation  of  having  a  prothrombin   gene  mutation  is  the  increased  risk   for  formation  of  a  deep  venous   thrombosis,  or  dvt.    typically,   these  clots  form  in  the  leg,  but   have  the  potential  of  traveling   through  the  bloodstream  to  reach   the  lungs,  known  as  a  pulmonary   embolism,  or  to  the  brain,  a  cere-­‐ bral  embolism  (5).    women  with   clotting  disorders  should  be  aware   that  oral  contraceptives,  hormone   therapy,  and  pregnancy  all  in-­‐ crease  the  risk  of  blood  clot  for-­‐ mation  (1).  the  prothrombin  mu-­‐ tation  has  been  suggested  to  lead   to  increased  pregnancy  complica-­‐ tions  as  well,  including  preeclamp-­‐ sia,  placental  abruption,  pregnan-­‐ cy  loss  in  all  trimesters,  and  still-­‐ birth  (1).  other  factors  that  in-­‐ crease  the  risk  of  clot  formation   are  smoking,  being  overweight  or   obese,  being  immobile  for  long    poten&al  inheritance  pa+erns  of  a  prothrombin  gene  muta3on  in  a  23-­‐year-­‐old  female  and  ethical  considera0ons  of  a  posi0ve  diagnosis:  a  case  report   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.69    http://dentistry3000.pitt.edu   periods  of  time,  and  infrequent   exercise  habits  (3).     while  refraining  from  the   aforementioned  risk  factors  can   sometimes  be  enough  to  prevent   the  formation  of  a  clot,  some  indi-­‐ viduals  with  the  prothrombin  mu-­‐ tation  still  develop  clots.  warfarin,   an  antiplatelet  agent,  is  commonly   used  to  treat  these  patients.  war-­‐ farin  works  by  preventing  the   proper  functioning  of  vitamin  k   during  the  formation  of  clots  (2,   6).  it  is  important  for  health  care   providers,  including  dentists,  to   take  medications  such  as  warfarin   into  consideration  when  treating   patients  as  they  may  be  at  an  in-­‐ creased  risk  for  bleeding  (7).   the  goal  of  this  paper  is  to   report  the  case  of  a  23-­‐year-­‐old   female  with  an  extensive  history   of  clotting  issues  stemming  from  a   prothrombin  gene  mutation.  this   paper  will  cover  the  biological  and   ethical  concerns  related  to  a  posi-­‐ tive  diagnosis  of  a  clotting  disor-­‐ der.   case  presentation:     this  report  examines  the   case  of  a  23-­‐year-­‐old  female,   “anne”  (the  individual’s  name  has   been  changed  to  protect  the  indi-­‐ vidual’s  identity).  anne  has  a  per-­‐ sonal  and  familial  history  of  blood   clots.  anne  has  had  8  dvts,  4  pul-­‐ monary  embolisms  with  one  com-­‐ plete  infarction,  4  superficial  clots,   and  delivered  a  stillborn  baby  at  5   months.  doctors  attributed  the   miscarriage  to  her  thrombophilia.     anne’s  father  is  caucasian   and  her  mother  is  half  japanese   and  half  caucasian.  her  paternal   grandmother  and  grandmother’s   two  sisters  all  had  a  history  of   hospitalizations  due  to  blood  clots.   her  father’s  sister  tested  positive   as  having  the  mutation  and  has   also  been  hospitalized  for  dvts   and  pulmonary  embolisms.  anne   reported  that  her  father  tested   negative  for  the  mutation  and  that   her  father  was  not  a  carrier,  but  it   is  likely  that  he  was  a  heterozy-­‐ gous  carrier  for  the  trait.  anne’s   aunt  with  a  history  of  blood  clots   and  anne  herself  tested  positive   as  homozygous  recessive  for  the   trait.  given  that  many  individuals   who  are  heterozygous  for  the  trait   never  form  blood  clots  and  that   heterozygous  individuals  are  only   at  2-­‐3  times  higher  risk,  it  is  plau-­‐ sible  to  think  that  anne’s  parents   are  carriers  of  the  trait.  the  exten-­‐ sive  history  of  blood  clots  and  em-­‐ bolisms  in  anne’s  family  suggest   that  anne  is  homozygous  for  the   trait,  as  increased  likelihood  of   developing  clots,  up  to  50  times,   and  increased  severity  are  report-­‐ ed  with  homozygous  cases  (3).  a   hypothetical  pedigree  has  been   proposed  (see  figure  1).   discussion:   being  diagnosed  with  a  clotting   disorder  like  a  prothrombin  muta-­‐ tion  has  many  implications  that   can  last  a  lifetime.  individuals  with   such  mutations  are  at  increased   risk  for  clotting  without  medica-­‐ tion,  and  are  at  increased  risk  for   bleeding  if  on  blood  thinners  such   as  warfarin  (2).  because  factors   such  as  smoking  and  hormonal   birth  control  significantly  increase   one’s  risk  for  developing  a  clot,  it   is  advised  that  individuals  with   clotting  disorders  abstain  from   them.     anne  has  3  siblings  from   the  same  parents.  it  is  possible   that  her  siblings  may  also  carry  the   mutation.  anne  has  an  older    poten&al  inheritance  pa+erns  of  a  prothrombin  gene  muta3on  in  a  23-­‐year-­‐old  female  and  ethical  considera0ons  of  a  posi0ve  diagnosis:  a  case  report   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.69    http://dentistry3000.pitt.edu   brother,  26,  who  is  on  his  own   health  insurance  plan.  her  two   younger  sisters,  16  and  12,  are  still   on  their  parents’  health  insurance   plan.  anne’s  dad  is  unsure  wheth-­‐ er  he  wants  to  have  the  younger   daughters  tested  for  the  mutation.   while  he  feels  it  would  be  benefi-­‐ cial  to  know  if  they  have  the  muta-­‐ tion  to  better  watch  for  symptoms   and  avoid  risk  factors,  he  fears  a   positive  diagnosis  may  lead  to  a   lifetime  of  insurance  denial  and   anxiety  related  to  the  condition.   the  genetic  information  nondis-­‐ crimination  act  (gina)  prohibits   employers  and  health  care  provid-­‐ ers  from  discriminating  against   individuals  because  of  genetic  in-­‐ formation  (8).  it  is  unknown  if  the   father  is  unaware  of  this  act,  is   afraid  that  this  regulation  could  be   overturned  in  the  future,  or  fears   that  employers  and  providers  do   not  always  abide  by  this  law.     when  seeking  medical  care   and  attention,  patients  with  clot-­‐ ting  disorders  should  alert  their   providers  of  their  condition  and  of   the  medications  they  are  taking.   dentists  treating  patients  on  war-­‐ farin  should  be  careful  to  watch   for  excessive  bleeding,  though   medication  adjustments  are  rarely   warranted  (2).  one  could  also   raise  the  question  of  if  a  patient   presents  to  a  dentist  with  a  clot-­‐ ting  disorder  and  a  positive  history   of  smoking  and/or  hormonal  birth   control,  is  it  the  obligation  of  the   dentist  to  advise  the  patients  of   the  risks  for  increased  clotting   when  taking  such  substances?   based  on  current  literature  report-­‐ ing  the  significantly  heightened   risks  of  clotting  with  these  agents,   it  is  reasonable  to  think  that  a   dentist  should  bare  this  responsi-­‐ bility.  the  dentist  should  also   counsel  the  parents  on  the  pros   and  cons  of  having  the  other  sib-­‐ lings  tested  given  the  potential   severity  of  the  disease,  while  ulti-­‐ mately  leaving  the  decision  to  the   family.     conclusion     anne’s  medical  and  familial   history  of  blood  clots  show  the   need  for  awareness  of  this  disor-­‐ der  and  the  importance  of  under-­‐ standing  modes  of  genetic  inher-­‐ itance  for  determining  the  risk  for   other  family  members.  individuals   with  clotting  disorders  should  be   advised  to  abstain  from  factors   that  increase  the  risk  for  clotting,   including  smoking  and  hormonal   birth  control.  while  it  may  be  con-­‐ sidered  beneficial  to  know  one’s   status  regarding  the  mutation  to   increase  awareness  and  avoid   triggering  factors,  the  current   state  of  health  care  policy  in  the   united  states  may  discourage  in-­‐ dividuals  from  getting  tested  for   fear  of  insurance  denial  or  em-­‐ ployment  discrimination.  it  is  up  to   the  individual  and  their  family  to   make  these  decisions,  but  regard-­‐ less  of  the  decision  made,  dentists   and  health  care  providers  have   professional  and  ethical  obliga-­‐ tions  to  be  aware  of  and  prepared   for  any  potential  complications   that  may  arise  during  treatment.   references   1. prothrombin  20210  mutation   (factor  ii  mutation);  varga   ea,  moll  s;  circulation;  2004,   july;110(3):e15-­‐8.  pmid:   15262854.   2. yagiela,  dowd,  johnson,  et  al   (2011)  pharmacology  and   therapeutics  for  dentistry.   st.  louis:  mosby  elsevier.  504   p.     3. lentz,  sr;  university  of  iowa   hospitals  and  clinics;  2005.   [cited  2017  mar  15].  availa-­‐ ble  from   https://www.healthcare.uio wa.edu/labs/lentz/informati on_for_patients/pdf/prothr om-­‐ bin%20gene%20mutation% 20brochure.pdf   4. homozygotes  for  prothrom-­‐ bin  gene  20210  a  allele  in  a   thrombophilic  family  without   clinical  manifestations  of  ve-­‐ nous  thromboembolism;  sou-­‐ to  jc,  mateo  j,  soria  jm,  et  al.;   haematologica;  1999;   84(7):627-­‐32.  pmid:   10406905.   5. genetics  home  reference;   national  institute  of  health;   u.s.  national  library  of  medi-­‐ cine;  2008.  [cited  2017  mar   26].  available  from   https://ghr.nlm.nih.gov/cond ition/prothrombin-­‐ thrombophilia#inheritance    poten&al  inheritance  pa+erns  of  a  prothrombin  gene  muta3on  in  a  23-­‐year-­‐old  female  and  ethical  considera0ons  of  a  posi0ve  diagnosis:  a  case  report   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.69    http://dentistry3000.pitt.edu   6. anticoagulants;  the  johns   hopkins  lupus  center;  2017.   [cited  2017  mar  26].  availa-­‐ ble  from   https://www.hopkinslupus.o rg/lupus-­‐ treatment/common-­‐ medications-­‐ conditions/anticoagulants/   7. anticoagulant  and  antiplate-­‐ let  medications  and  dental   procedures;  the  american   dental  association;  2015.   [cited  2017  mar  28]  available   from   http://www.ada.org/en/me mber-­‐center/oral-­‐health-­‐ topics/anticoagulant-­‐ antiplatelet-­‐medications-­‐ and-­‐dental-­‐   8. genetic  information  nondis-­‐ crimination  act;  u.s.  depart-­‐ ment  of  health  and  human   services;  2017.  [cited  2017   mar  28].  available  from   https://www.hhs.gov/hipaa/ for-­‐professionals/special-­‐ topics/genetic-­‐ information/index.html       comparative evaluation of dimensional stability and flexural strength of different light cured tray materials with self-cured tray material: an in-vitro study vol 10 no 1 (2022) doi 10.5195/d3000.2022.179 http://dentistry3000.pitt.edu comparative evaluation of dimensional stability and flexural strength of different light cured tray materials with selfcured tray material: an in-vitro study taha attarwala1, ramandeep dugal1, aamir godil1, sanaa wadwan1, pallavi madanshetty2, arshi kazi1 1 m.a.rangoonwala college of dental sciences and research centre, india, department of prosthodontics. 2 rural dental college, pravara institute of medical sciences, india, department of prosthodontics. abstract objectives: to carry out a comparative evaluation of dimensional stability and flexural strength of different light cured tray materials with self-cured tray material as control. materials and methods: the study was carried out in four groups based on the different available tray materials. group a (n=30) – light cured resin (individo lux-voco™ gmbh germany); group b (n=30)light cured resin (profibase-voco™ gmbh germany); group c (n=30)light cured resin (plaque photo-willmann and pein™ gmbh germany); group d (n=30)control group-self cured resin (asian special instant tray material asian acrylates™, mumbai, india). a digital micro-meter was used to measure the length of each specimen first at 1 hour, 24 hour and 48 hour and three readings were taken up to 0.001 decimal. the values for flexural strength of each specimen were measured using the 3-point bending technique with the help of a universal testing machine. the mean values of dimensional stability and flexural strength were calculated by the one-way anova and tukey’s post-hoc test for multiple group`s comparison. results the mean change in length (in mm) of light cured resins samples were significantly lower than self-cured resin samples at all three time intervals. the flexural strength values of light cured resin samples-group a (84.46±13.32 n/mm2), b (83.43±14.52 n/mm2) and c (86.80±14.73 n/mm2) was significantly higher than the self-cured resin samples group d (41.29±7.93 n/mm2) conclusions: light cured tray materials are more dimensionally stable and have a higher flexural strength compared to self-cured tray materials. keywords: custom trays; light cured resins; dimensional stability; flexural strength. citation: attarwala, t, et al. (2022) comparative evaluation of dimensional stability and flexural strength of different light cured tray materials with self-cured tray material: an in-vitro study? 1:a001 doi:10.5195/d3000.2022.179 received: april, 30, 2021 accepted: july, 13, 2021 published: march, 7, 2022 copyright: ©2022 attarwala, t, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: tahaattarwala@gmail.com http://dentistry3000.pitt.edu/ comparative evaluation of dimensional stability and flexural strength of different light cured tray materials with self-cured tray material: an in-vitro study vol 10 no 1 (2022) doi 10.5195/d3000.2022.179 http://dentistry3000.pitt.edu introduction a dimensionally accurate impression is of primary importance for a precise fitting dental prosthesis. according to the glossary of prosthodontic terms 9, a custom tray is defined as “an individualized impression tray made from a cast recovered from a preliminary impression. it is used in making a final impression.1-2 ideal requirements for a custom tray material are that it must have dimensional and volumetric stability in air and in a moist environment over a period of time, good rigidity (high modulus of elasticity), the impression material must adequately adhere to the tray and should control the thickness of layer of impression material.3 several materials are available commercially for the construction of custom trays such as poly methyl methacrylate (pmma), shellac, thermoform sheets, light cured acrylic resin, polycaprolactone materials and printable thermoplastic resins. shellac was a popular tray and temporary denture base material in the twentieth century. however, basker et al (1976) and wilson et al (1987) reported that it is brittle, breaks easily and is difficult to trim and make the periphery smooth and future heating is likely to cause distortion. its use as a special tray material was discontinued as poly methyl methacrylate gained popularity.4 chemically cured (cc) / autopolymerising pmma resin is used most commonly for the fabrication of custom trays due to its convenience, cost efficiency, ready availability and favourable mechanical properties5. however, acrylic resins have also shown to be cytotoxic as a result of substances that leach from the resin. the primary cause is residual monomer, resulting from incomplete conversion of monomers into polymer; which has the potential to cause irritation, inflammation and an allergic response of the oral mucosa6. a residual monomer content of about 2-6% has been reported in self-cured resins. despite the fact that poly methyl methacrylate has low solubility in water, remaining monomer may spread into the oral mucosa. various researches have shown that the volumetric shrinkage of chemically cured resins is 7%, along with this there is also some linear shrinkage which affects the dimensional stability. these resins also absorb water slowly over a period of time, which acts as a plasticizer further affecting their dimensional stability.3 the flexural strength of acrylic resins depend on numerous factors such as polymer molecular weight and bead size, residual monomer level, plasticizer composition, cross–linking agent, internal porosity of the polymer matrix and action of chemical agents. one drawback of pmma resins during clinical service is its ability to fracture, especially when large undercuts are present and any type of fracture is a time consuming and costly problem besides being inconvenient for patients and clinicians.7 taking into consideration all the drawbacks related to pmma resins, several newer light cured resins were introduced in the early 1980s and emerged as one of the promising materials for custom tray fabrication in removable and fixed prosthodontics. the lightcuring resin is composed of urethane dimethacrylate (udma) matrix and a small amount of silica which adjusts flow characteristics of this material8. although they are in routine use in current clinical practice, their physical properties and dimensional accuracy are not sufficiently reported in current literature. hence, in the present study, the dimensional stability of three different commercially available light cured tray materials was http://dentistry3000.pitt.edu/ comparative evaluation of dimensional stability and flexural strength of different light cured tray materials with self-cured tray material: an in-vitro study vol 10 no 1 (2022) doi 10.5195/d3000.2022.179 http://dentistry3000.pitt.edu evaluated at 1 hour, 24 hour and 48 hour time intervals and compared to self-cured tray material which was taken as the control. moreover, since custom trays should exhibit an adequate stiffness and fracture resistance in all clinical situations; flexural strength was also evaluated for these materials. the null hypothesis of this study is that there is no difference between the dimensional stability and flexural strength of the test light cure and autopolymerising custom tray materials. the alternate hypothesis is that light cure tray materials have better dimensional stability and flexural strength as compared to autopolymerising custom tray materials. material and methods the study was divided into four groups as follows: group alight cured resin (individo lux-voco™ gmbh germany) / group b light cured resin (profibase-voco™ gmbh germany) / group c light cured resin (plaque photo-willmann and pein™ gmbh germany) / group d self cured resin (asian special instant tray material asian acrylates™, mumbai, india) 1. fabrication of template: a perspex sheet was used to prepare the template by laser cutting and engraving using rdworks v7 software. the customised template was designed in the desired dimensions of 2mm thickness, 20 mm length and 4 mm width according to the guidelines of the american society of testing materials3. this planning was then transferred to a laser cutting machine (co2 laser, trilok lasers pune, india). the templates were laser cut with 5 mm extra space on each side. the cover of the template was made by placing an uncut perspex sheet on top of the accurately prepared template. a total of 60 templates were prepared. the sample size was determined by power analysis with a confidence interval of 95% and p value <0.05 was considered as statistically significant. 2. fabrication of light cured and self-cured specimens: a thin layer of petroleum jelly was applied on the customised template as a separating medium prior to preparation of specimens. a sheet of light cured tray material was cut and adapted into the template. the entire assembly was then placed into a light curing unitbre. lux power unit 2 at 230 v, 50 hz and 28 w and was allowed to cure for 3 minutes. once set, the template was disassembled and the specimens of the desired 2x20x4 mm dimensions were retrieved. the specimens were then separately placed back into the light curing unit for an additional 3 minutes to allow complete polymerization of the material. the specimens were checked for any irregularities and discrepancies or errors in dimensions and those with visible defects were discarded and replaced. the specimens were stored in a dry environment (figure 1). fig 1: preparation of samples using perspex template similarly, the self-cured specimens were prepared by mixing the polymer and monomer according to the manufacturer`s instructions (3:1 v/v or 2.5:1 w/w) in a silicon cup and added to the perspex template. the specimens were retrieved after complete polymerization and stored in a dry environment. total number of specimens prepared in the study (sample size): http://dentistry3000.pitt.edu/ comparative evaluation of dimensional stability and flexural strength of different light cured tray materials with self-cured tray material: an in-vitro study vol 10 no 1 (2022) doi 10.5195/d3000.2022.179 http://dentistry3000.pitt.edu 15 specimens were prepared for each group for measuring dimensional stability (n=60) and 15 specimens for each group for measuring flexural strength (n=60). 3. testing of specimens: a. dimensional stability: dimensional stability of prepared samples was evaluated at 1 hour, 24 hours and 48 hours respectively. a digital micro-meter was used to measure the length of each specimen first at 1 hour and three readings were taken up to 0.001 decimal. the same procedure was followed at 24 hours and 48 hours respectively. average of the three readings was made and this average was subtracted from the initial length of 20 mm to determine change in length and get the dimensional stability at 1, 24 and 48 hours respectively. change in length ∆l = l1 l2 l1 = initial length l2 = measured length b. flexural strength: specimens were subjected to a single compressive load using the 3-point bending technique with the help of a universal testing machine, star testing system, india. model no. sts-248 accuracy of machine ± 1%, cross head speed 0.5mm/min. (figure 2) fig 2: measurement of flexural strength using universal testing machine a centrally located load was applied until the specimens fractured. the maximum force (in n) was recorded and presented by software program attached to the universal testing machine. this maximum force was used to calculate the flexural strength in mpa using the following formula: fs = 3fl 2bd2 f = the maximum force registered during testing l= length of the support span b = width of specimen d = thickness of specimen results a total of 60 specimens were prepared and evaluated for dimensional stability and flexural strength. (15 samples per group for each test) the inter group comparison of dimensional stability between each of the four test groups was done by anova with tukey’s post-hoc test for multiple groups comparison. all p values< 0.05 were considered statistically significant. the mean change in length (mm) for group a (0.09±0.026), b (0.09±0.028) and c (0.09±0.026) was noted to be significantly lower than group d (0.39±0.035) at time interval of 1hr. similarly for the time interval of 24 hoursgroup a (0.11±0.02), b (0.10±0.02) and c (0.10±0.02) was significantly lower than group d (0.43±0.03). as well as for 48 hour intervalgroup a (0.12±0.02), b (0.11±0.01) and c (0.12±0.02) was significantly lower than group d (0.44±0.03). the inter group comparison of flexural strength between each of the four test groups was done by anova with tukey’s post-hoc test for multiple groups comparison. the p-value corresponding to the f-statistic of one-way anova is lower than 0.05, suggesting that the one or more treatments are significantly different. the mean flexural strength of group a (84.46±13.32 n/mm2), b (83.43±14.52 n/mm2) and c (86.80±14.73 n/mm2) was noted to be significantly higher compared to group d (41.29±7.93 n/mm2) (tables 1.1 and 1.2) http://dentistry3000.pitt.edu/ comparative evaluation of dimensional stability and flexural strength of different light cured tray materials with self-cured tray material: an in-vitro study vol 10 no 1 (2022) doi 10.5195/d3000.2022.179 http://dentistry3000.pitt.edu table 1.1: inter group comparison of the four test groups with respect to the flexural strength in n/mm2: groups mean (n/mm2) standard deviation individo lux (group a) 84.46 13.12 profibase (group b) 83.43 14.52 plaque photo (group c) 86.80 14.73 self-cure (group d) 41.29 7.93 table 1.2: tukey hsd test: product difference 95% confidence interval (upper and lower limits) p value inference a vs c 2.34 -10.10 to 14.78 0.95 not significant a vs b 1.03 -13.47 to 11.41 0.99 not significant a vs d -43.17 -55.61 to -30.72 0.000 the mean of self-cure was significantly lower c vs b -3.37 -15.81 to 9.07 0.88 not significant c vs d -45.51 -57.95 to -33.06 0.0000 the mean of self-cure was significantly lower b vs d -42.14 -54.58 to -29.69 0.0000 the mean of self-cure was significantly lower http://dentistry3000.pitt.edu/ comparative evaluation of dimensional stability and flexural strength of different light cured tray materials with self-cured tray material: an in-vitro study vol 10 no 1 (2022) doi 10.5195/d3000.2022.179 http://dentistry3000.pitt.edu discussion the null hypothesis is not accepted as there is a difference between the dimensional stability and flexural strength of the test light cure and autopolymerising custom tray materials. accuracy of an impression is the fundamental aspect of prosthodontic therapy. numerous factors governing the precision of these impressions and subsequent casts include: type of impression trays, correct manipulation of impression materials, the method used to make the impression, the materials used for making casts and proper time of cast fabrication. among the enlisted factors, the type of impression tray used plays a significant role in the fit of the final prosthesis.8-9 custom trays and stock trays have been widely used for making dental impressions. thonthammact et al9 noted that accurate casts can be made with either stock or custom trays. however, using a custom tray represents a dentist`s best efforts to obtain exact replication of the teeth and tissues. it exemplifies rigidity and uniform thickness of the impression material as recommended by several other authors. therefore, any materials used to make custom trays must be dimensionally stable over time. autopolymerising acrylic resins have been used for the fabrication of custom trays, but concerns about the exposure of dental personnel to acrylic resin monomer have been expressed. distortion from polymerization shrinkage and residual stress relaxation makes them a less than ideal material for custom trays. research has suggested that custom trays should be fabricated at least 24 hours before impressions are made, although some investigators have suggested different periods (40 minutes to 9 hours) between making resin trays and using them, to allow the material to become relatively dimensionally stable.10 hamza et al11 found that autopolymerising resin continues to shrink, with significant dimensional change occurring for up to 180 days. light cured resins have gained popularity as a custom tray material in the past few years and hence has been the centre of research question in this study. several studies have focussed on the properties of the light cured resins and numerous advantages have been identified when compared to the self-cured resins, such as lack of offensive odour; improved working time; excellent dimensional and volume stability; sufficient rigidity and stiffness; ease of manipulation and the immersion in disinfectants with no effect on the physical or mechanical properties of this material.9 its disadvantages include needs a special light-curing unit, and difficulty in trimming and finishing. a perspex template was constructed and used for the fabrication of the specimens of equal dimensions.11 this is preferred to machining the specimens since it has been reported by neihart et al12 that grinding may weaken the specimen by creating multiple cracks and surface irregularities which can initiate the fracture process. specimens can be fabricated using a metal, plastic or perspex template. since, in the present study the specimens had to be placed in a light-curing unit for polymerization, a light transmitting template was required. thus, in this study, perspex material was used to prepare the template. when fabricating self-cured specimens, correct powder and liquid ratios must be adhered to decrease any source of error. the ratio of powder to liquid (3:1 v/v or 2.5:1 w/w) by measurement as http://dentistry3000.pitt.edu/ comparative evaluation of dimensional stability and flexural strength of different light cured tray materials with self-cured tray material: an in-vitro study vol 10 no 1 (2022) doi 10.5195/d3000.2022.179 http://dentistry3000.pitt.edu instructed by manufacturers was used. however, this presented a relative technique sensitive as well as time consuming step as the specimens fabricated without adhering to the recommended powder to liquid ratio would result in them becoming dry and brittle. the light cured materials on the other hand was less technique sensitive to fabricate as it comes in a wafer of uniform thickness and can be cut to desired size until cured in the polymerisation unit. in this study, the change in length of the four study groups was measured at 1, 24 and 48 hours respectively with the help of a digital micro-meter, since it is accurate, easy to use and readily available. the inter group comparison of change in length between each of the four groups at the three time intervals was done by anova with post-hoc test (tukey hsd) for multiple groups comparison. the light cure materials displayed greater dimensional stability at all three time intervals compared to the self-cure material (table 2.1). between the light cured materials, there was no significant difference in the dimensional stability at all three intervals which is in accordance with the studies conducted by breeding et al13 and wirz et al14. table 2.1: inter group comparison of change in length of the four test groups at different intervals: treatment individo lux (group a) (n=15) profibase (group b) (n=15) plaque photo (group c) (n=15) self-cure (group d) (n=15) interval 1h 24h 48h 1h 24h 48h 1h 24h 48h 1h 24h 48h mean change in length (mm) 0.09 0.11 0.12 0.09 0.10 0.11 0.09 0.10 0.12 0.39 0.43 0.44 sample sd 0.026 0.02 0.02 0.028 0.02 0.01 0.026 0.02 0.02 0.035 0.03 0.03 http://dentistry3000.pitt.edu/ comparative evaluation of dimensional stability and flexural strength of different light cured tray materials with self-cured tray material: an in-vitro study vol 10 no 1 (2022) doi 10.5195/d3000.2022.179 http://dentistry3000.pitt.edu intra group comparison (table 2.2) showed that the light cured groups a and b did not show any significant difference in the three time intervals whereas group c showed a statistical difference between 1 and 48 hour time interval. for group d the mean change in length of the samples after 1 hour (0.39±0.03 mm) was significantly lower as compared to after 24 hours (0.43±0.03 mm) and 48 hours (0.44±0.03 mm) respectively. no difference in the mean change in length between 24 (0.43±0.03 mm) and 48 hours (0.44±0.03 mm) was seen. table 2.2: intra group comparison of change in length of the four test groups at different intervals using tukey hsd test: these results show that the self-cured resins when being kept in a dry environment undergo dimensional changes up to 24 hours and thus must be stored for at least 24 hours before they become dimensionally stable. thus a waiting period may be recommended between fabrication and use of the tray clinically. this is in agreement with various studies by wirz et al14, rueda et al15, burton et al16 and anderson et al17. custom tray materials also must not permanently deform during the impression making procedure when the filled custom tray is inserted intra orally or as the completed impression is retrieved from the oral cavity.14the tray materials should be able to withstand and resist the force required to remove a completed impression from the oral cavity and the resulting internal stresses. the mean of flexural strength value of group a (84.46±13.32 n/mm2) was significantly higher compared to group d (41.29±7.93 n/mm2). similarly mean flexural strength of group b (83.43±14.52 n/mm2) and group c (86.80±14.73 n/mm2) were significantly higher as compared to the control group d (41.29±7.93 n/mm2). the lower flexural strength values of the self-cured resins may be due to the presence of residual monomer (2-6%) and a lesser interpenetrating polymer network due to its short curing time which could affect the strength. the light cured tray materials may exhibit a higher fracture resistance and can withstand higher forces due to cross-linking agents though this cannot be emphatically stated. the extreme hardness has been labelled a disadvantage by smith et al18 as it makes it more difficult to grind. khan and greets3 reported that the light cured tray materials have a significantly higher resistance against bending as compared to the selfcured material. in the present study, among the light cured groups, plaque photo™ (group c) showed the highest flexural strength values (86.80±14.73 n/mm2), it may be partly attributed to the filler particle size and volume fraction. flexural strength values may also be affected by the curing light intensity, time of curing and temperature. treatment pair tukey hsd (p-value) 1h 24h 48h a vs b 0.86 0.89 0.84 a vs c 0.89 0.67 0.89 a vs d 0.001 0.001 0.001 b vs c 0.89 0.89 0.88 b vs d 0.001 0.001 0.001 c vs d 0.001 0.001 0.001 http://dentistry3000.pitt.edu/ comparative evaluation of dimensional stability and flexural strength of different light cured tray materials with self-cured tray material: an in-vitro study vol 10 no 1 (2022) doi 10.5195/d3000.2022.179 http://dentistry3000.pitt.edu use of light cured materials as a routine custom tray material can be advocated not only due to them exhibiting acceptable strength and dimensional stability over the conventional and routinely used self-cured materials, but also due to the relative absence of any unreacted monomer after their polymerisation and their ease of manipulation. therefore, the use of light cured tray materials can be recommended over autopolymerising resins for fabrication of custom trays in routine clinical practice. limitations of the study in-vitro nature of the study the samples do not replicate the exact shape and anatomy of the custom tray. conclusions within the limitations of the study, the following conclusions were derived: a) the three different light cured tray materials showed better dimensional stability compared to the self-cured tray material at all the three time intervals of 1 hour, 24 hours and 48 hours respectively. between the light cured materials, plaque photo™ (group c) showed a significant difference in dimensional stability between the 1 hour and 48 hour time interval. b) the three light cured tray materials showed greater flexural strength as compared to the selfcured tray material. between the light cured materials, plaque photo™ (group c) showed the highest flexural strength, however it was not statistically significant when compared to individo lux™ (group a) and profibase™ (group b). this highlights that the light cured tray materials are suitable custom tray materials and provide a viable alternative to the self-cured material. these results, should however be validated in in-vivo conditions before clinical use. competing interests the authors declare that they have no competing interests. references 1. payne ja, pereira bp. bond strength of three nonaqueous elastomeric impression materials to a light-activated resin tray. international journal of prosthodontics. 1992 jan 1;5(1).pmid: 1520444 2. ferro kj, morgano sm, driscoll cf, freilich ma, guckes ad, knoernschild kl, mcgarry tj, twain m. the glossary of prosthodontic terms. 3. khan sb, geerts g. determining the dimensional stability, fracture toughness and flexural strength of light-cured acrylic resin custom tray material. european journal of prosthodontics and restorative dentistry. 2009 jun 1;17(2):67. pmid: 19645307 4. azouka a, huggett r, harrison a. the production of shellac and its general and dental uses: a review. j oral rehabil. 1993 jul;20(4):393-400. https://doi.org/10.1111/j.13652842.1993.tb01623.x 5. pagniano rp, scheid rc, clowson rl, dagefoerde ro, zardiackas ld. linear dimensional change of acrylic resins used in the fabrication of custom trays. journal of prosthetic dentistry. 1982 mar 1;47(3):279-83. https://doi.org/10.1016/00223913(82)90157-3 6. jorge jh, giampaolo et, machado al, vergani ce. cytotoxicity of denture base acrylic resins: a literature review. j. prosthet. dent. 2003 aug 1;90(2):190-3. https://doi.org/10.1016/s00223913(03)00349-4 7. asli hn, moradian s, asli mn. comparison of transverse http://dentistry3000.pitt.edu/ comparative evaluation of dimensional stability and flexural strength of different light cured tray materials with self-cured tray material: an in-vitro study vol 10 no 1 (2022) doi 10.5195/d3000.2022.179 http://dentistry3000.pitt.edu strength of three different types of heat cured resin acrylics. biosci biotechnol res commu. 2017 apr 1;10(2):248-51. 8. scott a, egner w, gawkrodger dj, hatton pv, sherriff m, van noort r, yeoman c, grummitt j. the national survey of adverse reactions to dental materials in the uk: a preliminary study by the uk adverse reactions reporting project. british dental journal. 2004 apr;196(8):471. https://doi.org/10.1038/sj.bdj.481 1176 9. thongthammachat s, moore bk, barco mt, hovijitra s, brown dt, andres cj. dimensional accuracy of dental casts: influence of tray material, impression material, and time. journal of prosthodontics. 2002 jun;11(2):98108. https://doi.org/10.1053/jopr.2002 .125192 10. valderhaug j, fløystrand f. dimensional stability of elastomeric impression materials in custom-made and stock trays. j. prosthet. dent. 1984 oct 1;52(4):514-7. https://doi.org/10.1016/00223913(84)90336-6 11. hamza ta, rosenstiel sf, elhosary mm, ibraheem rm. the effect of fiber reinforcement on the fracture toughness and flexural strength of provisional restorative resins. j. prosthet. dent. 2004 mar 1;91(3):258-64. https://doi.org/10.1016/j.prosden t.2004.01.005 12. neihart tr, li sh, flinton rj. measuring fracture toughness of high-impact poly (methyl methacrylate) with the short rod method. j. prosthet. dent. 1988 aug 1;60(2):249-53. https://doi.org/10.1016/00223913(88)90325-3 13. breeding lc, dixon dl, moseley jp. custom impression trays: part i—mechanical properties. j. prosthet. dent. 1994 jan 1;71(1):31-4. https://doi.org/10.1016/00223913(94)90252-6 14. wirz j, jaeger k, schmidli f. light-polymerized materials for custom impression trays. international journal of prosthodontics. 1990 jan 1;3(1).pmid: 2196894 15. rueda lj, sy-muñoz jt, naylor wp, goodacre cj, swartz ml. the effect of using custom or stock trays on the accuracy of gypsum casts. international journal of prosthodontics. 1996 jul 1;9(4).pmid: 8957875 16. burton jf, hood ja, plunkett dj, johnson ss. the effects of disposable and custommade impression trays on the accuracy of impressions. journal of dentistry. 1989 jun 1;17(3):121-3. https://doi.org/10.1016/03005712(89)90104-8 17. anderson gc, schulte jk, arnold tg. dimensional stability of injection and conventional processing of denture base acrylic resin. journal of prosthetic dentistry. 1988 sep 1;60(3):394-8. https://doi.org/10.1016/00223913(88)90292-2 18. smith pw, richmond r, mccord jf. prosthodontics: the design and use of special trays in prosthodontics: guidelines to improve clinical effectiveness. br dent j. 1999 oct;187(8):423. https://doi.org/10.1038/sj.bdj.480 0295 http://dentistry3000.pitt.edu/ microsoft word 99 2020_manuscript.docx vol 8, no 1 (2020) issn 2167-8677 (online) doi 10.5195/d3000.2020.99 http://dentistry3000.pitt.edu tobacco use cessation efforts among private dental practitioners of delhi, india abhishek mehta 1, gurkiran kaur 2, rishabh popli 3, suchi khurana 4 1 department of public health dentistry, faculty of dentistry, jamia millia islamia, new delhi, india. 2 department of oral pathology and microbiology, pdm dental college and research institute, pdm university, bahadurgarh, haryana, india. 3 pdm dental college and research institute, pdm university, bahadurgarh, haryana, india. 4 department of public health dentistry, pdm dental college and research institute, pdm university, bahadurgarh, haryana, india abstract introduc)on: tobacco use remains the single largest cause of premature death around the world. mo8va8ng tobacco users through behavioral counseling is the mainstay for achieving tobacco abs8nence. research evidence suggests a larger role of dental professionals in mo8va8ng tobacco users to quit. the efforts of private dental prac88oners in providing tobacco cessa8on interven8ons (tci) in their clinics need to be measured and acknowledged. methods: a cross-sec8onal survey based on self-administered ques8onnaire with private dental prac88oners was carried out in the state of delhi, india. the main objec8ves of the survey were to assess the current prac8ces and the barriers experienced in providing tci by the par8cipa8ng den8sts. sta8s8cal analysis was conducted to find out the associa8on between independent variables and the responses to ques8ons on tobacco cessa8on. results: the mean age and years of prac8ce of 297 par8cipa8ng den8sts was 37.2 (sd 9.8) and 11.9 (sd 9.5) years respec8vely. most of them were graduate (n=204, 68.7%). only 17.2% (n=51) of the par8cipa8ng den8sts had undergone any training in providing tci. most of the den8sts felt that the lack of proper training (n=146, 49.2%) and resistance by pa8ents (n=185, 62.5%) are the main barriers in providing tci. younger den8sts (<35 years) performed significantly be[er than older den8sts in many parameters pertaining to a\tude and prac8ces of tci in their clinics. conclusion: this is a pioneer survey conducted on the issue of tci provided by private dental prac88oners of delhi. the study results show that around 2/3rds of the par8cipa8ng private dental prac88oners were providing tci to their pa8ents but there are certain barriers for its proper implementa8on. therefore, there is an urgent need to remove these selfreported barriers and build self-confidence among the den8sts for ins8tu8ng effec8ve tci in their clinics. key words: tobacco use cessa8on, den8sts, ques8onnaire, nico8ne replacement, india. cita)on: mehta a, et al. (2020) tobacco use cessa)on efforts among private dental prac))oners of delhi. den8stry 3000. 1:a001 doi:10.5195/d3000.2020.99 received: april 4, 2020 accepted: may 14, 2020 published: july 23, 2020 copyright: ©2020 mehta a, et al. this is an open access ar8cle licensed under a crea8ve commons a[ribu8on work 4.0 united states license. email: amehta@jmi.ac.in introduction consuming tobacco increases the risk of several oral and general health problems such as cancer of oral, lung and pharyngeal region, cardiovascular problems and periodontal diseases [1]. it is estimated that by the year 2030, tobacco will be the leading cause of mortality in human population worldwide [2]. india is home to the second highest tobacco consumers in the world, with 267 million active tobacco users and more than one million tobacco related deaths occurring each year [3]. these numbers suggest we are in the second stage of tobacco epidemic [4]. a person can quit the habit of tobacco consumption with self-motivation and professional help. tobacco cessation programmes are gaining acceptance worldwide as they are found to be an effective preventive measure [5,6]. tobacco cessation interventions (tci) provided by physicians and dental care professionals have shown improvement in tobacco abstinence rates [7,8]. in spite of the strong evidence, it was observed in the second global adult tobacco survey tobacco use cessa*on efforts among private dental prac**oners of delhi vol 8 no 1 (2020) doi 10.5195/d3000.2020.99 http://dentistry3000.pitt.edu (gats-2) india, only 54.5% smokers were asked regarding their tobacco use status and 48.8% were advised to quit tobacco by a health care professional (hcp) [9]. there is an urgent need to provide accessible and universally available tobacco cessation services for indian population. dental professionals are role models and leaders who can affect the attitude of people in a community. dentists are usually the first person to become aware of an individual’s tobacco habit during the course of routine dental check-up. dental clinic is an excellent venue for providing tci to the visiting patients. dentists can show visible evidence of the ill effects of tobacco on the oral cavity to their patients, which can motivate them to quit tobacco. smoking cessation program run by the dentists in usa and uk had achieved quit rates of 10-20% [6,7]. in spite of these facts, most of the dentists don’t provide tci services to their patients. even in developed countries like usa and canada, half of the dentists don’t advise their patient to quit tobacco [2,10]. there are several reasons reported for this finding such as lack of time, reimbursement or training and resistance by patient [10]. india has 274154 registered dentists [11] and a dentist population ratio of 1:10,571 [12]. this large health care workforce can be trained and utilized to provide tci to individual patients and community. most of the dentists in india are involved in private practice of their own or as an employee. very few dentists are working in government or private dental colleges. therefore, if these private dental practitioners are able to provide effective tci to their patients it can have an enormous public health impact. efforts are being made at government and dental associations’ level to increase the role of dental professionals in the fight against the tobacco epidemic in our country [13,14,15]. as per our knowledge only three studies [16,17,18] have been conducted to assess the tobacco cessation efforts of private dental practitioners in india and no study is reported from delhi which is capital of india. therefore, a cross-sectional study was planned with the following objectives: 1. to assess the level of tci provided by private dental prac^^oners in delhi state. 2. to find out the barriers incurred in providing these interven^ons. methods study sample the sampling frame for this study was all the dentists working in a private dental clinic in delhi state. as per available records from dental council of india, there are 14366 registered dentists in delhi [11]. list of dentists with their phone number or email id was available from local ida branch. sample size for this survey was calculated using epinfo software. required sample size was 316 (at 95% confidence level and 5% error margin), if we considered 70% of the dentists in delhi are practicing tci in their clinics. we contacted 330 dentists for participating in this study but only 297 dentists agreed for the same. participating dentists were recruited using multiple methods e.g. personal visit, telephonically, by visiting conferences or continuing dental education programs organized by state dental associations, and through email. every effort was made to give equal representation to both the genders as well as dentists practicing in urban or rural areas. eligibility criteria dentist practicing in a dental clinic in delhi and willing to participate in the study. ethical clearance an approval was obtained from institutional ethical committee before commencement of data collection (letter no. pdm/iec/01/2019). assessment tool a structured, self –administered questionnaire was prepared to record necessary information. the questionnaire was divided into five domains, starting with recording baseline information of the participating dentists, assessing their attitude towards tci, practice of 5as (ask-advice-assess-assist-arrange) approach in their clinic, different strategies utilized to motivate their patients to quit tobacco and finally, tobacco use cessa*on efforts among private dental prac**oners of delhi vol 8 no 1 (2020) doi 10.5195/d3000.2020.99 http://dentistry3000.pitt.edu self-reported roadblocks/barriers in providing these tci. the face and content validity of the questionnaire was evaluated by sending it to five experts working in the field of tobacco cessation. these experts were asked to rate each question in order to assess its suitability for this study. based on the feedback received from the experts necessary changes were made in the questionnaire. this questionnaire was then pilot tested on 20 private dental practitioners and their responses and suggestions were recorded. after these steps, the questionnaire was finalized for the main survey. reliability of the questionnaire was assessed using cronbach alpha test (alpha = 0.8) by repeating it to ten previous study participants. data collection the data were collected by single investigator (rp). randomly selected dentists were recruited using multiple methods by the investigator e.g. telephonically, by visiting conferences or continuing dental education programs organized by state dental associations, and through email. the dentists were explained the study objectives and were asked to provide signed informed consent for participation in the study. in order to achieve a higher response rate, and wherever possible, the investigator personally visited the clinic of the participating dentists on a mutually consented time and date. the filled form were collected on the same day (preferably) or within 2 days of submission. those dentists who agreed to fill an online form were sent a prepared google form of the questionnaire. after receiving the filled form, the investigator checked the form to make sure all the questions are answered by the participating dentist(s). data collection was completed within two months (june and july 2019). statistical analysis the data was analyzed using statistical package for the social sciences version 21.0 (ibm corp., armonk, ny, usa). frequency distribution tables were prepared to present overall and subgroups data. chisquare test was used to analyze the statistical differences between dichotomous independent variables such as number of years of practice, age and gender of the participating dentist with regards to responses to various sections of questionnaire (dependent variables). probability value (p)< 0.05 was considered as statistically significant. results baseline characteristics of the study population we contacted 330 dentists to participate in this study, of which, 297 (155 males and 142 females) responded and gave their consent. therefore, the response rate was 90%. baseline characteristics of study participants showed that slightly more than half (51.2%) of the participating dentists were in age bracket of 35 years or above, and were into private practice from last 8 years or more (n=153, 51.5%). around two-thirds of them (n=204, 68.7%) were graduate and only few had done post-graduation (n=93, 31.3%). as the numbers of dentists with graduate degree were far more than those with post-graduation, it was decided that no further comparison for outcome variables will be done between these two groups (table 1). table 1. baseline characterisdcs of pardcipating dendsts. attitude of participating dentists towards tobacco cessation most of the participating dentists were aware of the fact that tci can be provided in a dental clinic (n=265, 89.2%) and 87.5% thought tci in dental clinic can be effective. thirty six percent of the participating dentists (n=107) felt their patients expect them to provide tci but only 51 (17.2%) had undergone any formal training to learn these services or interventions. majority of dentists were educating their patients regarding ill effects of tobacco on general and oral health (n=293, 98.7%). bivariate analysis using chi-square test showed that a significantly tobacco use cessa*on efforts among private dental prac**oners of delhi vol 8 no 1 (2020) doi 10.5195/d3000.2020.99 http://dentistry3000.pitt.edu higher awareness towards providing tci in dental office was seen in dentists in older age group (>35 years) and among females. significantly more dentists in older age group, those with more years of practice (>9 years) and females practitioners believed that their patients expect them to provide tci in their clinic (p<0.05). on the question of effectiveness of tci in dental office, significantly more female dentists felt that such interventions are an effective tobacco control measure when compared to male dentists (p<0.05) (table2). practicing 5as approach two-thirds (n=196, 66%) of the participating dentists were asking most of their patients (more than 75%) about their tobacco use status; rest 34% (n=101) were asking from less than 75% of their total patients. almost all of the participating dentists (n=290, 98.6%) were advising their patients (who were consuming tobacco) to quit the habit and more than 3/4th (n=226,76.1%) were assessing their patients’ intention to quit as well as assisting them in quitting tobacco (n=235, 79.1%). less than one-third of the participating dentists were helping their patients in setting a quit tobacco date (n= 88, 29.6%) and even lesser number were arranging for a follow-up appointment after the quit date (n= 45, 15.2%). table 2. pardcipadng dendsts’ responses to quesdons related to providing tobacco cessadon services in dental office. *sta-s-cally significant difference p<0.05, chi square test tobacco use cessa*on efforts among private dental prac**oners of delhi vol 8 no 1 (2020) doi 10.5195/d3000.2020.99 http://dentistry3000.pitt.edu female dentists’ response was significantly more positive compared to the males for awareness of tci provided in dental office, assessing their patient‘s intention to quit tobacco and arranging for follow up after quit date (p<0.05). significantly more dentists in the older age group and those who had more than eight years of practice were asking most of their patients (>75%) regarding their tobacco use addiction. dentist of older group (>36 years) and female dentists were assessing their patients’ intention to quit tobacco in significantly higher frequency when compared to dentists of younger age (<35 years) and males respectively (p<0.05). there was no difference in any of these dichotomous groups about arranging follow up appointment after quit date (table 3). strategies for helping patients to quit tobacco prescribing nicotine replacement therapy (nrt) was the most common strategy of the participating dentists in helping their patients achieve tobacco abstinence (n=153, 51.5%). providing self-help educational material (n=122, 41.1%) was second most common strategy followed by referring the patient to a tobacco cessation centre (n=72, 24.2%). very few dentists were prescribing drugs such as bupropion or varnecline (n=19) or providing tobacco quit line number table 3. responses of the pardcipadng dendsts to quesdons related to 5as approach of tobacco cessadon. *sta-s-cally significant difference p<0.05, chi square test. tobacco use cessa*on efforts among private dental prac**oners of delhi vol 8 no 1 (2020) doi 10.5195/d3000.2020.99 http://dentistry3000.pitt.edu to their patients. younger dentists reported to have provided educational materials to their patients in significantly higher frequency than older dentists (table 4). there was no significant difference in gender, age and years of practice groups regarding implementation of any other tobacco cessation strategies. self-reported barrier to providing tci to a dental patient resistance by patient to receive tci was the biggest barrier reported by the participating dentists (n=185). female dentists and those who had practiced dentistry for more than eight years reported this as a barrier significantly more than compared to male dentists and dentists with less than eight years of practice respectively. lack of proper training in providing tobacco cessation and amount of time consumed were the other common barriers reported by the participating dentists. significantly more dentists of older age and more years of practice felt lack of training as a barrier as compared to their comparative groups. lack of time to provide tci to their patients was significantly a more important barrier for the male dentists and dentists with less than eight years of practice. less than fifty dentists perceived lack of reimbursement, non-familiarity with the available referral options and fear of losing patients as a barrier to tci in dental clinic. no significant difference was observed between the different groups for these three barriers (table 5). discussion the purpose of the present study was to evaluate the knowledge, attitude and practices of private dental practitioners of delhi in implementing tci in their dental office. self-reported barriers encountered during these interventions were also recorded. the results of the study show most of the participating dentists believed in the effectiveness of tci in dental office and many of them were implementing four (ask-advice-assess-assist) out of five tasks (except “arrange”) of 5as approach recommended for tobacco cessation. the major lacunae (identified through this study) table 4. strategies udlized by the pardcipadng dendsts for helping their padents to quit tobacco. *sta-s-cally significant difference p<0.05, chi square test tobacco use cessa*on efforts among private dental prac**oners of delhi vol 8 no 1 (2020) doi 10.5195/d3000.2020.99 http://dentistry3000.pitt.edu hindering the proper implementation of tci by the participating dentists were lack of formal training, most of the dentists didn’t think that patients expect them to provide tci, and there was lack of proper followup mechanism of tobacco users in the dental office. tobacco treatment guidelines developed by government of india [4] had suggested strategies for tobacco cessation in the form of 5as (if person is willing to quit tobacco) or 5rs approach (if person is not willing to quit tobacco) [7]. these guidelines are research-based and accepted globally [19]. a typical tci involves counseling session of approximately 5-8 minutes to each tobacco user using 5as approach. if this is not possible (due to lack of time) a brief intervention session (approx. 2-3 minutes) that requires health care professionals (hcp) to ask every patient regarding their tobacco status, advise them on quitting tobacco and assess their willingness to quit (3as) [2,20]. in our study, only 66% participating dentists were asking the majority of (more than 75%) their patients about tobacco consumption status. these numbers are quiet similar to those reported among dentists of saudi arabia and kuwait [21], united kingdom [22], iran [23] and netherlands [24] but higher than that reported in canada [10] and udaipur, india [25] and lesser than that reported in pakistan [26], modinagar, india [16], nigeria [27], yemen [28] and united states of america [29]. asking about tobacco consumption status is the foremost step a hcp must perform towards identifying patients who are addicted to tobacco. hence there is need to improve the awareness of all hcp to ask each and every patient regarding their tobacco history. in our study, most (97.6%) of the participating dentists were advising tobacco users to quit. this percentage was much higher than that reported by similar studies around the world [10,16,21,23,25,26,27,29]. it is an encouraging finding since research had shown even a brief advice by hcp could motivate patient to quit or at least reduce tobacco consumption [30]. around 3/4th of the study participants (76.1%) were table 5. barriers perceived by pardcipadng dendsts in providing tci to their padents. *sta-s-cally significant difference p<0.05, **p<0.001, chi square test tobacco use cessa*on efforts among private dental prac**oners of delhi vol 8 no 1 (2020) doi 10.5195/d3000.2020.99 http://dentistry3000.pitt.edu assessing their patients’ intention to quit tobacco. these figures are much higher than that reported by similar studies [10,23,25,27]. as per gats-2 india less than 15% of tobacco users were planning to quit in next one year and even less than 10% in next one month [9]. these figures are suggestive of failure on part of health care services to motivate tobacco users to quit the habit. obtaining information on patient’ intention to quit helps dental professionals to evaluate tobacco user motivation to quit tobacco. along with setting a quit date, intention to quit has been found to be associated with higher tobacco abstinence rates [20]. assisting the tobacco users in quitting the habit requires time and commitment from health care professionals. in present study, more than 3/4th (79.1%,n=235) of the participating dentists had reported to assist their patients in achieving tobacco abstinence. however, when specifically asked about the strategies for assistance, less than 30% of the participating dentists were helping their patients to set a quit date and even fewer (15.2%) stated that they were arranging a follow-up appointment for tobacco users. these numbers are much less than that is reported by studies done in canada [10], us [29] and nigeria [27] but higher than studies done in pakistan [26], iran [23] and udaipur, india [31]. these observations reflect a lack of long term commitment of participating dentists to make sure their patients are free of tobacco addiction. it is a widely known fact that tobacco users require multiple attempts before they finally quit [32]. therefore, if a health care professional is not providing an option of future follow up it will ultimately lead to poor abstinence rate among its patients. follow-up appointments are also extremely important for dentists to reinforce their health education message and for patients for sharing problems they are facing during the process of tobacco abstinence. patients must be reminded repeatedly regarding immediate and long-term benefits of quitting tobacco [33]. most common strategies utilized by participating dentists to help their patients quit tobacco were prescribing nrt, providing self-education material and referring to a tobacco cessation centre. frequency of self-reported prescription of nrt to their patients was much higher in our study sample compared to similar studies [10,16,22,26,29]. prescribing nrt is an effective method of replacing nicotine obtained from direct tobacco products and has shown to increase the chances of tobacco abstinence rate up to 50-70% [34]. in our study, we tried to find out the barriers impeding implementation of tci by the participating dentists. “resistance by patients” was the most common barrier reported by the dentists. studies done among dentists of european union [35] and canada [10] had found this factor as one of the main barrier. “lack of adequate training” in providing tci was another important barrier reported. dentists without proper knowledge and training will lack confidence in conducting tci in their office and hence would not be able to motivate their patient to quit tobacco. similar results were observed by studies conducted in different parts of the world [10,16,27]. “lack of time” to provide tci was the third most common barrier reported by the dentists in our study. many studies had reported this as one of the most common barrier [10,18,21,22,25,27]. dental treatment is inherently laborious and requires more time for a single patient or procedure when compared to a general physician. the dentists must be educated and motivated towards the rewards and responsibilities of providing tci. in the present study, lack of monetary benefit from tci was considered as a barrier by only 42 (14.1%) dentists. this figure is quiet lower when compared to other studies around the world [10,22,27] and india [18,19] except for one indian study [16]. tci in dental office is usually not a reimbursable service. although efforts should be directed to include it as a chargeable service but more importantly dentists must be educated that tobacco cessation activities can help them build a reputation with their patients and neighborhood community. bivariate comparative analysis of the study participants shows younger dentists (<35 years of age) had better awareness and selftobacco use cessa*on efforts among private dental prac**oners of delhi vol 8 no 1 (2020) doi 10.5195/d3000.2020.99 http://dentistry3000.pitt.edu belief regarding provision of tci in their clinic. dentist of younger age group performed significantly better in implementing ask, advice and assist tasks of 5as approach to their patients. they were providing self-help education materials in higher frequency as compared to dentists above 35 years of age. finally, the younger dentists didn’t perceive lack of training as a barrier in providing tci when compared to older dentists. possible explanation for such observations could be better awareness of younger generation of dentists regarding importance of providing tci to their patients. it could be due to changes in dental school’s curriculum with compulsory lectures as well as hands on training for dental students on tobacco cessation [36]. establishment of tobacco cessation centre is now mandatory for all dental colleges in india [13]. this initiative will increase practical knowledge and confidence of graduates in providing tci to their patients once they come out of dental college and open there clinic. there has been better availability of training in form of continuing education and workshops on tobacco cessation which helps private practitioners to update their knowledge on tobacco cessation. limitations and strengths the present study is not without limitations and therefore its results should be interpreted keeping them in mind. this study is limited to private dentists practicing in delhi state. therefore, these results are not representative of dental professionals of the whole country. second, the findings of the study are based on self-reported answers where individuals might report good knowledge of a particular topic without undergoing assessment on its practical implementation part. for example, most dentists are aware and understand the importance of tobacco cessation but whether they are actually implementing it in their dental office cannot be assessed with a questionnaire study. such self –reporting studies are also prone to recall bias. third, is the issue of selection bias in the study sample, as dentists interested and motivated towards tci may get enrolled for this study. we feel chances of selection bias is less in our sample as refusal rate (not to participate in the study) is just ten percent and every attempt was made to recruit dentists regardless of their past experience in providing tci. fourth, as this study is cross sectional in design therefore it’s not suitable to draw any causal relationships. finally, we didn’t assess participating dentists own habit of tobacco use. it has been reported that dentists who smoke tobacco are less keen on engaging their patients for tci [21,28]. conclusions the results of our study present an overview of the current scenario of tobacco cessation efforts made by private dental practitioners of delhi and barriers experienced by them. efforts should be made to eliminate these barriers and increase the practical knowledge of dentists on providing tci to their patients. efficient and skill-based training provided through changes in dental curriculum, conducting workshops and online courses could help in increasing tobacco abstinence among indian population. acknowledgments this study was selected by indian council of medical research for funding under their studentship scheme (icmr-sts 2019). conceptualization: am. data curation: am, gk, rp, sk. formal analysis: am, sk. funding acquisition: rp, sk. methodology: am, gk, sk. project administration: am, gk, sk, rp. visualization: am, gk, rp, sk. writing – original draft: am, gk. writing – review & editing: am, gk, rp, sk. the authors have no conflicts of interest to declare for this study. references 1. global, regional, and na^onal disability-adjusted life-years (dalys) for 359 diseases and injuries and healthy life expectancy (hale) for 195 countries and territories, 1990-2017: a systema^c analysis for the global burden of disease study 2017. gbd 2017 dalys and hale collaborators.lancet. 2018 nov 10;392(10159):18591922. doi: 10.1016/s0140tobacco use cessa*on efforts among private dental prac**oners of delhi vol 8 no 1 (2020) doi 10.5195/d3000.2020.99 http://dentistry3000.pitt.edu 6736(18)323353.pmid: 30415748 2. den^stry's role in tobacco control. tomar sl.j am dent assoc. 2001 nov;132 suppl:30s35s. doi: 10.14219/jada.archive.2001.0386.pmid: 118 03650 3. a quasi-experimental interven^on to assess the effec^veness of a physician-delivered tobacco cessa^on interven^on in india : a detailed study protocol. panda r, health p, gaurav k, health p, pa^ s, mathur mr. sage open med. 2017;5:1-8. doi: 10.1177/20503121176 97173. pmid: 28540045 4. ministry of health and family welfare government of india. tobacco dependence treatment guidelines.; 2011. 5. physician advice for smoking cessa^on. stead lf, buitrago d, preciado n, sanchez g, hartmann-boyce j, lancaster t.cochrane database syst rev. 2013 may 31;2013(5):cd000165. doi: 10.1002/14651858.cd0001 65.pub4.pmid: 23728631 6. effec^veness of tobacco counseling in the dental office. warnakulasuriya s.j dent educ. 2002 sep;66(9):107987.pmid: 12374269 7. tobacco cessa^on in dental sewngs: research findings and future direc^ons. gordon js, lichtenstein e, severson hh, andrews ja.drug alcohol rev. 2006 jan;25(1):27-37. doi: 10.1080/09595230500459 495.pmid: 16492575 8. interven^ons for tobacco cessa^on in the dental set^ng. carr ab, ebbert j.version 2. cochrane database syst rev. 2012 jun 13;2012(6):cd005084. doi: 10.1002/14651858.cd0050 84.pub3.pmid: 22696348 9. ministry of health and family welfare government of india. global adult tobacco survey (gats)-2 india. hxps://www.mohfw.gov.in /sites/default/files/gats-2 factsheet.pdf 10. tobacco use cessa^on services provided by den^sts and dental hygienists in manitoba: part 1. influence of prac^^oner demographics and psychosocial factors. brothwell dj, gelskey sc.j can dent assoc. 2008 dec;74(10):905.pmid: 191 26359 11. dental council of india. total registered den^sts. accessed january 14, 2019. hxp://www.dciindia.org.in /den^stregistered.aspx 12. dental manpower planning in india: current scenario and future projec^ons for the year 2020. vundavalli s.int dent j. 2014 apr;64(2):62-7. doi: 10.1111/idj.12063. epub 2013 nov 4.pmid: 24180215 13. government of india. establishment of tobacco cessakon centers in dental insktutes : an integrated apporach in india operakonal guidelines 2018.; 2018. 14. role of dental ins^tu^ons in tobacco cessa^on in india: current status and future prospects. mohanty vr, rajesh gr, aruna ds.asian pac j cancer prev. 2013;14(4):267380. doi: 10.7314/apjcp.2013.14.4.2 673.pmid: 23725194 15. world health organiza^on. effec^ve implementa^on of the who framework conven^on on tobacco control through the mpower policy package. tfi newsl. 2009;2(2):1-8. 16. tobacco cessa^on counselling prac^ces and awtude among the den^st and the dental auxiliaries of urban and rural areas of modinagar, india. singla a, paxhi b, singh k, jain s, vashishtha v, kundu h, malhi r, pandita v.j clin tobacco use cessa*on efforts among private dental prac**oners of delhi vol 8 no 1 (2020) doi 10.5195/d3000.2020.99 http://dentistry3000.pitt.edu diagn res. 2014 sep;8(9):zc15-8. doi: 10.7860/jcdr/2014/9250. 4799. epub 2014 sep 20.pmid: 25386513 17. tobacco cessa^on and counseling: a den^sts' perspec^ve in bhopal city, madhya pradesh. amit s, bhambal a, saxena v, basha s, saxena s, vanka a.indian j dent res. 2011 may-jun;22(3):400-3. doi: 10.4103/09709290.87061.pmid: 220485 79 18. addressing tobacco control in dental prac^ce: a survey of den^sts' knowledge, awtudes and behaviours in india. chandrashekar j, manjunath bc, unnikrishnan m.oral health prev dent. 2011;9(3):2439.pmid: 22068180 19. knowledge, awtude and prac^ce regarding tobacco cessa^on among indian den^sts. shaheen s, reddy s, doshi d, reddy p, kulkarni s.oral health prev dent. 2015;13(5):427-34. doi: 10.3290/j.ohpd.a33924.pm id: 25789360 20. factors determining inten^on to quit tobacco: exploring pa^ent responses visiting public health facili^es in india. panda r, venkatesan s, persai d, trivedi m, mathur mr.tob induc dis. 2014 jan 20;12(1):1. doi: 10.1186/1617-9625-121.pmid: 24444137 21. tobacco cessa^on support among den^sts: a crosssec^onal survey in saudi arabia and kuwait. alajmi b, abu-hammad o, al-sharrad a, dar-odeh n.tob prev cessat. 2017 aug 1;3:121. doi: 10.18332/tpc/75795. ecollec^on 2017.pmid: 32432195 22. tobacco cessa^on ac^vi^es of uk den^sts in primary care: signs of improvement. johnson nw, lowe jc, warnakulasuriya ka.br dent j. 2006 jan 28;200(2):85-9. doi: 10.1038/sj.bdj.4813148.p mid: 16444223 23. awtude and prac^ces among den^sts and senior dental students in iran toward tobacco cessa^on as an effort to prevent oral cancer. razavi sm, zolfaghari b, doost me, tahani b.asian pac j cancer prev. 2015;16(1):333-8. doi: 10.7314/apjcp.2015.16.1.3 33.pmid: 25640375 24. facilita^ng smoking cessa^on in pa^ents who smoke: a large-scale cross-sec^onal comparison of fourteen groups of healthcare providers. meijer e, van der kleij rmjj, chavannes nh.bmc health serv res. 2019 oct 25;19(1):750. doi: 10.1186/s12913-019-4527x.pmid: 31653215 25. awtudes, prac^ces and perceived barriers in smoking cessa^on among den^sts of udaipur city, rajasthan, india. bhat n, jyothirmai-reddy j, gohil m, khatri m, ladha m, sharma m.addict health. 2014 winter;6(1-2):7380.pmid: 25140220 26. farrukh u, shakeel s. den^sts’ prac^ce and perceived barriers towards smoking cessa^on and interven^on in karachi, pakistan. j pharm care heal syst. 2016;03(01):1-5. 27. tobacco cessation services and related challenges among dentists in southwest nigeria. dedeke aa, popoola oa, adebiyi ao, asuzu mc.ann ib postgrad med. 2018 dec;16(2):125130.pmid: 31217769 28. tobacco cessa^on counseling: awtudes and prac^ces among yemeni dental professionals. al-maweri sa, al-soneidar wa, almaqtari a, hunaish a, al-sufyani g, halboub e.j cancer educ. 2018 oct;33(5):1088-1093. doi: 10.1007/s13187-017-12129.pmid: 28324226 29. den^sts' awtudes, behaviors, and barriers related to tobacco use cessa*on efforts among private dental prac**oners of delhi vol 8 no 1 (2020) doi 10.5195/d3000.2020.99 http://dentistry3000.pitt.edu tobacco-use cessa^on in the dental sewng. prakash p, belek mg, grimes b, silverstein s, meckstroth r, heckman b, weintraub ja, gansky sa, walsh mm.j public health dent. 2013 spring;73(2):94102. doi: 10.1111/j.17527325.2012.00347.x. epub 2012 jun 26.pmid: 22731618 30. guidance for commissioners on the cost effec^veness of smoking cessa^on interven^ons. health educa^onal authority. parrox s, godfrey c, raw m, west r, mcneill a.thorax. 1998 dec;53 suppl 5 pt 2(suppl 5):s138.pmid: 10226676 31. assessment of knowledge, awtude, behaviour and interpersonal factors related to the use of tobacco among youth of udaipur city, rajasthan, india: a cross-sec^onal study. multani s, reddy jj, bhat n, sharma a.addict health. 2012 summer-autumn;4(34):142-50.pmid: 24494149 32. trea^ng tobacco use and dependence: 2008 update u.s. public health service clinical prac^ce guideline execu^ve summary. 2008 phs guideline update panel, liaisons, and staff. respir care. 2008. pmid: 18807274 33. world health organisa^on (who). the role of health professionals in tobacco control. world heal organ. published online 2005:138. hxp://www.who.int/tobac co/resources/publica^ons/ wntd/2005/bookle{inal_2 0april.pdf 34. nico^ne replacement therapy for smoking cessa^on. stead lf, perera r, bullen c, mant d, hartmannboyce j, cahill k, lancaster t.cochrane database syst rev. 2012 nov 14;11:cd000146. doi: 10.1002/14651858.cd0001 46.pub4.pmid: 23152200 35. tobacco and oral health: at^tudes and opinions of european den^sts; a report of the eu working group on tobacco and oral health. allard rh.int dent j. 2000 apr;50(2):99-102. doi: 10.1002/j.1875595x.2000.tb00806.x.pmid : 10945189 36. dental council of india. bds course regulakons.; 2007. accessed december 30, 2019. hxp://www.dciindia.gov.in /rule_regula^on/revised_ bds_course_regula^on_2 007.pdf vol 2, no 1 (2014) issn 2167-8677 (online) doi 10.5195/d3000.2014.28 http://dentistry3000.pitt.edu new articles in this journal are licensed under a creative commons attribution 4.0 united states license. this journal is published by the university library system, university of pittsburgh as part of its d-scribe digital publishing program and is cosponored by the university of pittsburgh press. sustainability is success alexandre r. vieira editor in chief, dentistry 3000 department of oral biology, university of pittsburgh, school of dental medicine, pittsburgh, pa, usa citation: vieira ar. (2014) sustainability is success. dentistry 3000 1:a006 doi: 10.5195/d3000.2014.28 published: december 18, 2014 copyright: ©2014 vieira. this is an open-access article licensed under a creative commons attribution work 4.0 united states license. email: arv11@pitt.edu this is volume 2 of dentistry 3000. this means the journal survived its first year and the inherited challenges to sustain its activities over time. the journal has received contributions from investigators based in several countries, and authors have enjoyed timely peer-reviews and publication without any publishing costs. both volumes of the journal include topics of craniofacial development, craniofacial disorders, and endodontics. we are in the process of indexing the journal in medline and pubmed, and some authors have deposited their papers in pubmed central, which allows for these papers to be found in the pubmed website search tools. dentistry 3000 is a journal with the modern concept of open access to the world using a format that dates from the time libraries in universities would keep scientific journals. this philosophy allows for papers to be published based on their merit and not on what the potential interest a given article will generate among the readership that pays for membership fees. we are looking forward to receiving your future contributions to dentistry 3000 in the many years to come. http://dentistry3000.pitt.edu/ http://www.library.pitt.edu/ http://www.pitt.edu/ http://www.library.pitt.edu/articles/digpubtype/index.html http://upress.pitt.edu/ microsoft word azizi proof dec 4.docx   vol  2,  no  1  (2014)   issn  2167-­‐8677  (online)   doi  10.5195/d3000/2014.24         http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.   a  gas  adsorption  porosimetry  analysis  of  portland  cement  prepared   by  compaction  vs.  compaction  with  indirect  ultrasonic  agitation   nader  azizi1,  yltze  p.  cubas2,  maria  f.  orellana3     1dds,  msc,  private  prac.ce,  new  york  city,  usa   2dds.  post-­‐doctoral  fellow,  department  of  orofacial  sciences,  school  of  den4stry,  university  of  california,  san  francisco,  usa   3dds,  msc,  phd.  director  predoctoral  orthodon4c  program,  department  of  orofacial  sciences,  division  of  orthodon4cs,  school  of  den&stry,  university  of  california,   san  francisco,  usa abstract     background:  mineral   trioxide   aggregate   (mta),   is   commonly   used   in   endodon9c   and   re-­‐ stora&ve  procedures.  objec&ve:  our  objec&ve  was  to  introduce  gas  adsorp&on  porosimetry   as  a  viable  method  for  evalua1on  of  general  porosity  and  specific  pore  characteris1cs  of  set   portland  cement  used  in  subs1tute  for  mta,  to  inves1gate  the  effect  of  two  different  obtu-­‐ ra#on  methods  (compac#on  and  compac#on   in  conjunc#on  with   indirect  ultrasonic  agita-­‐ !on  of  the  cement  paste),  and  to  evaluate  the  correla.on  between  the  specific  pore  char-­‐ acteris(cs   to   compressive   strength   in   general.  material   and   methods:   portland   cement   samples  were  prepared  and  divided  into  two  groups  based  on  compac4on  techniques.  an   ini#al   stereomicroscopic   evalua*on  was   done   to   assess   any   differences   in   appearance   of   pores  randomly  selected  from  either  of  the  two  experimental  groups.  specific  pore  charac-­‐ teris&cs  and  compressive  strength  werequan&fied  by  a  gas  adsorp&on  porosimeter  and  an   instron  universal  tes(ng  machine.  a  two-­‐tailed  student  t-­‐test  was  used  for  sta,s,cal  com-­‐ parison   of   data,   and   a   regression   analysis  was   done   to   evaluate   the   correla6on  between   each  specific  pore  characteris-c  and  compressive  strength  in  general.  results:  the  gas  po-­‐ rosimetry  method   provided  measurable   values   rela2ng   to   specific   pore   characteris2cs   of   portland   cement.   the   stereomicroscopy   evalua6on   revealed  marked  differences  between   samples  from  the  two  groups,  namely  visibly  larger  pores  both  on  the  outside  surface  and   in  cross-­‐sec$ons  of  specimens  prepared  by  the  indirect  ultrasonic  ac$va$on  method.  con-­‐ clusions:   gas   adsorp*on  porosimetry   is   a   feasible  method   for   evalua*on  of   specific   pore   characteris)cs  of  portland  cement  and  poten)ally  other  dental  materials  as  well.       cita%on:  azizi  n,  cubas  y,  orellana  m.  (2014)  a  gas  ad-­‐ sorp%on  porosimetry   analysis   of   portland   cement  pre-­‐ pared   by   compac,on   vs.   compac,on   with   indirect   ul-­‐ trasonic   agita+on.   den+stry   3000.   1:a001   doi:10.5195/d3000.2014.24   received:  march  25,  2014   accepted:  october  15,  2014   published:  november  3,  2014   copyright:   ©2014   azizi   et   al.   this   is   an   open   access   ar#cle   licensed  under  a  crea#ve  commons  a2ribu#on   work  4.0  united  states  license.   email:  orellanam@den,stry.ucsf.edu       introduc!on     the   key   requirement   for   the   strength  and  stability  of  hydraulic  cement  is   that   the   hydrates   formed   on   immediate   reaction   with   water   should   be   essentially   insoluble  in  water.    mineral  trioxide  aggre-­‐ gate  (mta)  is  an  inorganic  hydraulic  cement   used   as   a   restorative  material   in   dentistry.     portland  cement  is  reported  to  be  the  main   component   of   this   inorganic   hydraulic   ce-­‐ ment.   similar   to   other   inorganic   cements,   mta   can   set   and   harden   in  moist   environ-­‐ ments   [1],   a   property  which  makes   it   ideal   for   use   in   dentistry.     in   addition,   mta   has   been   shown   to   provide   a   predictable   seal   against  leakage  in  bacterial  and  dye  leakage   studies   [2-­‐4].     the   calcium  hydroxide   com-­‐ ponent   released   during   its   hydration   reac-­‐ tion   provides   alkalinity   to   this   material,   which  may  account  for  both  its  antibacterial   and   hard-­‐tissue   growth   induction   proper-­‐ ties  [5,6]     mta   placement   technique   affects   some  of  the  structural  and  material  proper-­‐ ties  of   the   set   cement   [7,8].     these  proper-­‐ ties   range   from   the   cement’s   adaptation   to   its   container’s   walls,   to   changes   in   its   re-­‐ sistance  to  leakage  and  variations  in  its  sur-­‐ face   and   internal   porosity.   it   is   now   well   accepted   that   pores   decrease   the  mechani-­‐ cal  properties  of  cement  [9].    therefore,  any   technique   that   would   potentially   affect   the   porosity   of   mta   may   similarly   affect   its   mechanical  properties.  selection  of  handling   and  placement  methods,  which  optimize  the   physical   and   mechanical   properties   of   the   cement,  may  prove  bene/icial.    for  instance,   commonly   observed   .lexural   weakness   of   cements   has   been   related   to   presence   of   large  voids  in  a  set  material.  the  removal  of   macro-­‐defects  during  preparation  has  been   shown   to   increase   the   )lexural   strength   of   hydraulic  cements  [10].    similar  effects  may   potentially   be   expected   in   respect   to   com-­‐ pressive  strength.     pores   in   hydraulic   cements,   such   as   mta,   may   stem   from   a   number   of   sources.     they   could   be   either   internal   in   the  source,  such  as  those  created  as  a  result   of  the  material’s  hydration  reaction,  or  they   could   have   an   external   source.     internal   sources   of   porosity   may   have   a   profound   impact   on   its   mechanical   properties   as      a  gas  adsorp)on  porosimetry  analysis  of  portland  cement  prepared  by  compac)on  vs.  compac)on  with  indirect  ultrasonic  agita)on   vol  2,  no  1  (2014)        doi  10.5195/d3000/2014.24    http://dentistry3000.pitt.edu   2   figure  1:    best  &it  lines  through  intercepts  of  speci/ic  pore  characteristics  and  compressive  strength  of  samples.    a,b.  graphs  show  a  slight   positive  correlation.  c.  graph  shows  a  slight  negative  correlation.     powder-­‐to-­‐water   is   the   most   important   parameter   in*luencing   the   porosity   of   hy-­‐ draulic   cements   [11].   selection  of   the  pow-­‐ der-­‐to-­‐water  ratio  would  affect  the  porosity   of  a  cement  mix  due  to  its  effect  on  the  size   and  distribution  of  its  capillary-­‐pores  which   are   inevitably   formed  during   the  hydration   reactions.     similarly,   external   sources   of   porosity  may  also  in-luence  some  structural   properties   of   restorative   cements.     for   ex-­‐ ample,   direct   ultrasonic   activation   of   an   mta  cement  paste  during  its  placement  has   been   linked   to   entrapment   of   larger   sized   air-­‐bubbles   in   the   set   specimen   [12]   possi-­‐ bly  due  to  a  proposed  “whipping”  motion  of   the  ultrasonic  delivery  device.    these  larger   pores,  similar  to  those  affecting  the  (lexural   strength   of   the  material,   may   affect   on   the   compressive  strength  of  the  material.         although  mta  is  traditionally  used   in   low   pressure   bearing   areas,   its   use   in   high  pressure  bearing  areas  may  potentially   be   indicated   (i.e.   as   bases   under   other   re-­‐ storative  dental  materials).    some  have  pro-­‐ posed   the  use  of  portland  cement  as  an   in-­‐ expensive  alternative   to  other  more  expen-­‐ sive  dental  restorative  materials  for  restora-­‐ tion   of   coronal   defects   [13],   with   potential   uses   in   economically   challenged   areas.     when   used   in   such   critical   locations,   both   mta   and   portland   cement   must   withstand   compressive   forces   that   could   challenge   their   clinical   longevity   through   time.   other   factors   affecting   porosity   such   as   pore   size   distributions,  microcracks,  interface,  among   others   are   also   important   in   determining   the  mechanical   properties   of   cement  mate-­‐ rials  [14,  15].    however,  porosity  which  can   be   semi-­‐empirically   and   concisely   used   to   describe   the   relationship   between   strength   and   microstructure   of   porous   material   is   still   being   studied   [16,   17].   porosity   has   been   shown   to   display   an   inverse   relation-­‐ ship   to   compressive   strength,   with   reduc-­‐ tion  of  porosity  leading  to  increases  in  com-­‐ pressive  strength  of  a  solid  but  porous  ma-­‐ terial.       gas  adsorption  has  been  one  of  the   most  popular  techniques  used  for  the  study   of   pore   structure   in   materials   that   contain   micropores  and  mesopores,  or  mainly  pores   in   cement   pastes   with   radii   between   1nm   and   approximately   60nm.     therefore,   gas   adsorption  technique  can  mainly  character-­‐ ize   gel   pores   and   small   and  medium   capil-­‐ lary   pores.   gas   adsorption   methods   are   based   on   measurements   of   the   amount   of   gas  adsorbed  on  the  surface  of  a  powder  on   the   monomolecular   depth   of   the   particle’s   surface   [18].     when   a   porous   solid   is   ex-­‐ posed   to   gas   of   a   certain   volume  and  pres-­‐ sure,   it   begins   to   adsorb   the   gas  molecules   on   its   outside   surface   and   inside   its   pores.     the   amount   of   gas   adsorbed  during   an   ad-­‐ sorption  experiment  can  then  be  calculated   by   either   a   gravimeric,   volumetric,   or   ther-­‐ mal   conductivity   measurement   method.     from   the   amount   of   gas   adsorbed   and   the   corresponding   relative   pressure   recorded   during   the   experiment,   a   great   number   of   methods   are   developed   for   analysis   which   include   methods   for   determining   the   pore   volume,   pore   size   distribution,   and   the   brunauer-­‐emmett-­‐teller   (bet)   speci.ic   surface   area   of   the   material.   surface   area   measurements   are   the   most   widely   used   means  for  characterization  of  porous  mate-­‐ rials   .   since   the   surface   corresponds   to   the   roughness   of   the   particle   and   its   porous   interior,   gas   adsorption   is   the   preferred   technique  [19,20].     we   investigated   the   effect   of   two   common   clinically   practiced   placement   techniques   on   porosity   and   compressive   strength   of   portland   cement   in   an   in   vitro   model.    we   compared   the   porosity   of   sam-­‐ ples   of   portland   cement   placed   by   either   compaction  alone   (group  1)  or   compaction   in  conjunction  with   indirect  ultrasonic  acti-­‐ vation  of  the  cement  paste  during  its  place-­‐ ment  (group  2).    initially,  stereomicroscopic   inspections   of   samples   were   made   in   an   attempt   to   make   visual   comparisons   be-­‐ tween  specimens  selected  from  either  of  the   two   experimental   groups.   later,   a   gas   ad-­‐ sorption  porosity  method  was  used  to  quan-­‐ tify   and   compare   few   speci1ic   pore   charac-­‐ teristics   of   the   cement   samples   –   namely   their   bet   speci-ic   pore   surface   area,   pore   volume,   and   pore   size   distribution-­‐   from   both   groups.   compressive   strengths   of   samples   were   quanti/ied   using   an   instron   universal  material  testing  machine.         materials  and  methods     in  a  clean  glass  dappen  dish,  1.0  g   of  portland  cement  was  mixed  with  0.32  ml   of   deionized   water   using   a   metal   mixing   spatula.     care   was   taken   to   gently  mix   the   two   components   to   avoid   introduction   of   large   air   bubbles   into   the   cement   paste.     mixing   was   done   for   30   seconds   until   the   material   took   a   consistency   which   allowed   for   easy   transfer   and   placement   of   its   ali-­‐ quots   by   an   amalgam   carrier   into   metallic   moulds.   then   each   sample   followed   treat-­‐ ment  by  either  of  the  two  methods  of  obtu-­‐ ration.  in  group  1,  8  samples  were  obturat-­‐ ed   into  the  molds  using  a   ten  second  appli-­‐ cation   of   an   equal   and   constant   amount   of   vertical   force   to   the   cement   paste.     a   3ive   pound  weight  placed  on  top  of  a  piston  con-­‐ nected   to   the   metallic   obturator   delivered   the   vertical   component   of   the   obturation   force.    in  group  2,  8  samples  were  prepared   similarly,   with   the   difference   of   indirect   ultrasonic   energy.     the   tip   of   a   vibrating   ultrasonic   instrument   (buc-­‐2)   was   placed   along  an  area  about  one   inch  above   the   tip   of   the   obturator   to   transfer   a   10   second   burst   of   ultrasonic   energy   to   the   metallic   instrument.     the   obturator,   in   turn,   propa-­‐ gated   the   ultrasonic   energy   to   the   paste   along  with  delivering  its  vertical  component   force  of  compaction.  a  gridded  plastic  sepa-­‐ rator  was  placed  over  a  thin  pool  of  water  at   the  bottom  of   the  container  with  the  molds   placed   on   top.   the   plastic   separator   was   used   to   prevent   the   samples   from   directly   coming  in  contact  with  the  water  reservoir,   while   allowing   them   to   set   at   nearly   100%   a b ca b c    a  gas  adsorp)on  porosimetry  analysis  of  portland  cement  prepared  by  compac)on  vs.  compac)on  with  indirect  ultrasonic  agita)on   vol  2,  no  1  (2014)        doi  10.5195/d3000/2014.24    http://dentistry3000.pitt.edu   3   figure  2:  samples  of  set  cement  placed  side  by  side  un-­‐ der  5x    magni&ication.    note  the  presence  of  visible  pores   on   the   surface   of   specimen   from   the   indirect   ultrasonic   activation  group  (a);    slight  variation  in  color  marks  the   border   between   aliquots   of   cement   placed   to   make   up   figure  3:  at  10x  magni,ication,  large  internal  pores  (air-­‐bubbles)   could  be  seen   in   cross   sections  of  samples   from   the   indirect  ul-­‐ trasonic  activation  group  (a);  transition  areas  between  aliquots   of  cement  present  as  “watered-­‐down,  weak  fault-­‐lines.     humidity   and   at   room   temperature.     after   one   week,   setting   of   samples   was   visually   con$irmed.     samples  were   then  pushed   out   of  each  mold  and  prepared  for  porosity  and   compressive  strength  evaluations.    random-­‐ ly   selected   samples   from   each   group   were   prepared   for   stereomicroscopic   evaluation   (5x,  10x,  and  20x  magni,ications.)    the  ex-­‐ ternal   surfaces   of   samples   were   initially   evaluated   for   presence   of   visibly   large   sur-­‐ face   pores.     then,   samples   were   ground   down  to  make  half-­‐cylinders  of  samples  and   exposing   their   internal   appearance.     stereomicroscopic   evaluations   of   internal   surfaces   were   also   done   for   presence   of   notably  visible  pores.   gas  adsorption  porosimetry  evaluation    samples   were   sent   to   micrometrics   (mi-­‐ cromeritics,  norfolk,  ga,  usa.)  and  evaluat-­‐ ed   after   varying   durations   of   setting   time.     specimens   were   de-­‐gassed   at   40°c   for   16   hours   then   placed   in   a   sample   tube   and   heated  under   vacuum  or   .lowing   gas   to   re-­‐ move   contaminants   on   the   surfaces   of   the   samples.   the   sample   tube  was   then   placed   in   the   analysis   port   of   a   2420   accelerated   area  and  porosimetry  system  for  automatic   analysis.   the   krypton   adsorption   isotherm   was  recorded  at  120  k.         compressive  strength  analysis      cylindrical   samples   from   each   experimental   group   were   carefully   placed   on  their  vertical  axes  to  stand  perpendicular   to   the   two   horizontal   plates   of   the   instron   testing   machine.     the   mobile   upper   plate   was   set   to   advance   at   a   uniform   speed   of   1mm   per   second   to   deliver   the   crushing   vertical   forces   to   the   samples.     increasing   vertical   forces  were  applied  to  each  sample   until   structural   failure   was   achieved,   at   which  point   this  maximum  weight  was  rec-­‐ orded.   statistical  analsysis     statistical   analysis   comparing   the   average  values  of   speci/ic  pore   characteris-­‐ tics   and   compressive   strength   of   samples   was  done  using  a  two-­‐tailed  student’s  t-­‐test.     correlation   and   regression   tests  were  used   to   look   for   any   relationship   between   each   speci&ic   pore   characteristic   (bet   speci,ic   surface   area   of   pores,   pore   volume,   and   pore   size   distribution)   and   compressive   strength  in  general.     results     in   table   1,   we   ob-­‐ served   an   apparent   trend   in   bet   pore   surface   area   and   a   trend   in   pore   volume   of   sam-­‐ ples   in  group  1,   however;   the   difference   in   values   are   not   statistically  signi#icant.     additionally,   there   is   an   ap-­‐ parent  trend  in  pore  size  and  a   trend   in  compressive  strength   of  samples  in  group  2,  but  the   difference  in  values  is  not  sta-­‐ tistically  signi(icant.   regression   and   correla-­‐ tion  analysis   compressive   strength   vs.   bet   speci!ic   pore  surface     this   revealed   a   positive   regression   with   a   correlation   coef!icient   of   +0.65.   this   shows   that   as   the   bet   pore   surface   area   of  samples  increased,  so  did   the  compressive  strength  of   the  samples  (figure  1a).   compressive   strength   vs.   pore  volume     a   positive   regres-­‐ sion   with   a   correlation   coef!icient  of  +0.64  shows   that   if   the  pore  volume  of   samples   increased,   so   did   the   compressive   strength   of   the   samples   (figure   1b).     compressive   strength   vs.   pore   size   dis-­‐ tribution     a  negative  regression  with  a  corre-­‐ lation   coef!icient   of   -­‐0.42   shows   that   if   the   pore   size   of   samples   increased,   the   com-­‐ pressive   strength   of   samples   tends   to   de-­‐ crease  (figure  1c).   stereomicroscopy     5x   magni!ications   of   randomly   selected   samples   from   each   group   were   placed   side   by   side.     initially   we   noticed   visible   differences   in   the   surface   appear-­‐ ance   of   the   two   cylindrical   specimens.   the   sample   prepared   by   vertical   compaction   force   alone   presented   a   more   uniform   ap-­‐ pearance  of  its  outside  surface  area  with  no   obvious   large  porosities   or   transition   lines.     however,  this  specimen  presents  numerous   larger  porosities  and  a  number  of  transition   lines  which   correspond   to   the   junction   be-­‐ tween  aliquots  of  cement  paste  place  on  top   of  each  other  during  the  segmental  compac-­‐ tion  of  the  cement.     the  same  side  by  side  comparison   of  cross  sections  of   samples  were  arranged   as   before,   but   at   10x  magni+ication   reveals   similar   arrangement   of   larger   pore   sizes   and  transition   lines  present   throughout   the   interior   bulk   of   the   sample   prepared   by   compaction   in   conjunction   with   ultrasonic   activation  (figure  3a).  a  closer  look  at  tran-­‐ sition   lines  visualized   at   25x  magni2ication   reveals   weak   faults   created   between   ali-­‐ quots   of   cement   samples   placed   by   the   combined  compaction  and  ultrasonic  activa-­‐ tion  method  (figure  3b).     discussion     in   this   study,   we   introduced   gas   adsorption   porosimetry   as   an   acceptable   method   for   evaluation   and   distinction   of   speci&ic   pore   characteristics   of   a   dental   re-­‐ storative   material,   namely   its   bet   speci4ic   surface   area,   pore   volume   and   pore   size   distribution.     this   method   of   analysis   gen-­‐ erates   quantitative   data   with   values   that   re#lect  on  those  speci#ic  pore  characteristics   of  solid  but  porous  materials.    according  to      a  gas  adsorp)on  porosimetry  analysis  of  portland  cement  prepared  by  compac)on  vs.  compac)on  with  indirect  ultrasonic  agita)on   vol  2,  no  1  (2014)        doi  10.5195/d3000/2014.24    http://dentistry3000.pitt.edu   4   ying-­‐zi  et  al   “gas  adsorption  porosimetry   is   suitable   for   the   materials   with   apertures   smaller   than  50nm  or   the  powder  with   the   particle  size  smaller  than  100nm  [20].”  our   data  showed  that  gas  adsorption  porosime-­‐ try   could   be   used   for   comparative   evalua-­‐ tion   and   statistical   analysis   of   pore   charac-­‐ teristics  of  different  dental  materials   in  ad-­‐ dition   to   evaluating   the   effects   of   chemical   or  physical  manipulations  on  the  materials’   speci&ic  pore  characteristics.  lee  at  al  stated   that   gas   adsorption   technique   gives   the   largest   amount   of   information   for   the   po-­‐ rous  structure  of  solids  [21].     when  comparing   the  pore  charac-­‐ teristics   of   samples   placed   by   either   of   the   two   tested   placement  methods,   our   overall   preliminary  results   show  no  signi2icant  dif-­‐ ferences   between   the  bet   pore   surface   ar-­‐ ea,  pore  volume,   and  pore   size  distribution   of   samples   from   either   of   the   two   groups   however;   these  data  displayed  a  number  of   observed   trends   which   indicate   a   positive   effect   on   compaction   in   conjunction   with   indirect  ultrasonic  technique.    the  numbers   of  specimens   tested   in   this  study  were   lim-­‐ ited   due   to   the   high   cost   of   a   larger   scale   analysis.     based   on   the   limited   number   of   samples  tested,  eight  samples  per  group,  no   statistically   signi+icant   differences   in   either   the  average  bet  speci0ic   surface  area,  pore   volume  or  pore  size  distribution  of  samples   from   either   of   the   two   groups   may   be   re-­‐ ported.    however,  from  this  data  trends  may   be  observed.    first,  our  data  showed  a  trend   toward  larger  average  surface  area  of  pores   in   those   samples   prepared   by   compaction   alone.    in  contrast,  there  was  a  trend  toward   smaller   average   surface   area   of   pores   in   those   samples   prepared   by   compaction   in   conjunction  with  ultrasonic  activation  of  the   paste   during   its   placement.     second,   we   observed   similar   results  with   samples   pre-­‐ pared  by  compaction  alone  showing  a  trend   toward   larger   average   pore   volumes   com-­‐ pared   to   those   prepared   by   the   indirect   ultrasonic   activation   method.     lastly,   this   data  revealed  a  trend  which  showed  smaller   average   pore   size   distributions   in   samples   prepared  by  compaction  compared  to  those   made   using   the   indirect   ultrasonic   activa-­‐ tion  method.         surface   and   cross   sectional   stere-­‐ omicroscopic   inspection   of   the   samples   from  each  group   led  us   to  a  number  of   ini-­‐ tial   impressions  related  to  structural  differ-­‐ ences  between   the   two.     it   appeared   that   a   sample  prepared  by  compaction  alone  had  a   more   uniform   outer   surface     which   was   devoid   of   any   noticeably   large   sized   air-­‐ entrapped   pores   that   were   more   readily   observed  on  the  outer  surface  of  the  sample   prepared   by   the   indirect   ultrasonic   activa-­‐ tion   of   the   paste   during   its   placement.     a   similar   pattern   was   also   observed   in   mid-­‐ line  cross  sectional  preparations  of  samples   from   both   groups.     these   cross   sectional   preparations   also   revealed   the   presence   of   more   numerous   air-­‐entrapped   pores   in   samples   prepared   by   ultrasonic   activation.     similar  observations  have  also  been  report-­‐ ed   in   other   investigations  where   a   “direct”   activation   of   the   cement   through   insertion   of  the  vibrating  ultrasonic  tip  into  the  paste   is   thought   to   create   a   “whip-­‐ ping”  motion  in  the  paste,  lead-­‐ ing  to  introduction  of  larger  air   bubbles   into   the   paste   [14].   another   explanation   for   this   observation  may   be   related   to   a  phenomenon  of   the  action  of   ultrasonically   vibrating   in-­‐ struments   in   liquid  media,   call   “acoustic   cavitation   [22]”.   this   phenomenon   may   potentially   introduce  larger  bubbles  into  a   more  viscous  medium,   such   as   that   of   our   portland   cement   paste  specimens.         another   noteworthy   observation   that   was   made   only  in  the  sample  prepared  by   the   indirect   ultrasonic   activa-­‐ tion  was   the   presence   of   hori-­‐ zontal   demarcation   lines   be-­‐ tween   aliquots   of   cement   placed  during  obturation  of  the   mold.    a   plausible   explanation   for   formation  of  such  demarcations  may  be   that  the  agitation  provided  by  the  ultrasonic   energy  may  have  potentially  forced  the  solid   particles   of   portland   cement   to   stack-­‐up   more   closely   against   each   other   by   forcing   any   unbound   water   out   of   the   spaces   be-­‐ tween  those  particles,  and  essentially,  caus-­‐ ing  sedimentation  of  the  solid  entities  in  the   water-­‐cement   paste  mixture.   other   reports   have   shown   that   a   critical   increase   in   the   liquid   component   of   hydraulic   cement   would   lead   to  a  decrease   in   the  strength  of   the  material.     these   “watered-­‐down”   inter-­‐ faces  may  ultimately  present  as  those  weak   “fault-­‐lines”   seen   in   the   indirect   ultrasoni-­‐ cally   prepared   samples   in   our   magni!ied   images.       the   presence   of   visually   larger   pores   in   samples   placed  by   the   indirect   ul-­‐ trasonic  activation  method  concur  with  our   report   of   larger   values   for   average   pore   sizes   along   with   the   expected   correspond-­‐ ingly   smaller   average   pore   volume   and   av-­‐ erage   pore   surface   area   of   samples   pre-­‐ pared  with   the  aid  of  an   indirect  ultrasonic   activation   method.     such   .indings   are   in   contrast   to  possibly  more   sedimentation  of   solid  portland  cement  particles  in  the  paste   prepared  by  the  indirect  ultrasonic  method.     this  conclusion  was  based  on  the  pooling  of   water   observed   on   top   of   each   aliquot   of   cement   paste   placed   to   make   up   a   whole   sample.    this  would  suggest  that  sa   mples   prepared   by   the   indirect   ultrasonic   activation   method   would   expectedly   have   smaller  pores  sizes,  while  presenting  larger   pore   surface   areas   and   pore   volumes.     yet   this  was  not   supported  by  our  quantitative   data.    we  believe  that  the  indirect  ultrasonic   activation   creates   more   densely   packed   specimens   with   smaller   pores   and   poten-­‐ tially   larger   pore   surface   areas   and   pore   volumes.    the   larger   air  bubbles,   as   visual-­‐ ized   in   stereomicroscopic   evaluations,  may   be   outliers   that   skew   the   data   enough   to   support   results   reporting   larger   average   pore  size  distributions  and  smaller  average   pore   surface   areas   and   pore   volumes   that   contradict   the   visually   observed   signs   of   sedimentation   in   the   indirectly   activated   ultrasonic  method.    excluding  these  outliers   we  report  that  samples  prepared  by  indirect   ultrasonic   activation   are   indeed   more   densely   packed   at   a   more   microstructural   level   when   compared   to   those   placed   by   compaction  alone.    such  extrapolations  may   be   further   supported   when   comparisons   between   data   re*lecting   the   average   com-­‐ pressive   strength   of   samples   from   the   two   groups  is  made.     our   data   showed   that   the   place-­‐ ment   method   did   not   have   a   statistically   signi%icant   effect   on   the   average   compres-­‐ sive   strength   of   the   samples   from   the   two   groups;  however,  there  was  a  trend  toward   samples   in   the   ultrasonically   activated   group  having  a  higher  average  compressive   strength   values   than   those   samples   placed   table  1:  average  values  of  group  1  and  group  2.     group  1   group  2   p-­‐value   bet  speci!-­‐ ic  pore  sur-­‐ face   6.175m2/g   sd=3.0   5.172m2/g   sd=4.072   0.762   pore  vol-­‐ ume   0.0257m3/g   sd=0.02   0.0241m3/g   sd=4.072   0.922   pore  size   distribu-­‐ tion   239.886  å   sd=144.27   318.09  å   sd=162.27   0.4984   compres-­‐ sive   strength   88.667kg   sd=24.8   92.0kg     sd=  34.7   0.894      a  gas  adsorp)on  porosimetry  analysis  of  portland  cement  prepared  by  compac)on  vs.  compac)on  with  indirect  ultrasonic  agita.on   vol  2,  no  1  (2014)        doi  10.5195/d3000/2014.24    http://dentistry3000.pitt.edu   5   by   compaction   alone.     presence   of   few   air-­‐ bubbles  may   be   compensated   by   the  more   densely  packed  cement  particles  at  a  micro-­‐ structural   level   in   the   indirect   ultrasonic   method.    this  density  may  be  related  to  the   observed   higher   average   compressive   strength  of  samples  from  this  group.     conclusions     gas   adsorption   porosimetry   is   a   method   that   can   be   potentially   used   for   evaluation  of  speci/ic  pore  characteristics  of   dental   restorative   materials.     larger   scale   studies  are  needed  to  evaluate  the  superior-­‐ ity   of   one   method   of   placement   over   the   other.     references     1. evaluation  of  setting  properties  and   retention  characteristics  of  mineral   trioxide  aggregate  when  used  as  a   furcation  perforation  repair  mate-­‐ rial.  sluyk  sr,  moon  pc,  hartwell  gr.   j  endod.  1998  nov;24(11):768-­‐71.   pmid:  9855831.     2. a  comparison  of  mta,  super-­‐eba,   composite  and  amalgam  as  root-­‐end   filling  materials  using  a  bacterial  mi-­‐ croleakage  model.  adamo,  hl,  bu-­‐ ruiana  r,  schertzer  l,  boyland  rj.  in-­‐ tendod  j.  1999  may;32(3):197-­‐203.   pmid:  10530207.     3. leakage  evaluation  of  root  end  fill-­‐ ing  materials  using  endotoxin.  tang   hm,  torabinejad  m,  kettering  jd.  j   endod.  2002  jan;28(1):5-­‐7.  pmid:   11806652.     4. dye  leakage  of  four  root  end  filling   materials:  effects  of  blood  contami-­‐ nation.  torabinejad  m,  higa  rk,   mckendry  dj,  pitt  ford  tr.j  endod.   1994  apr;20(4):159-­‐63.  pmid:   8035153.     5. antibacterialeffects  of  some   rootendfillingmaterials.  torabinejad   m,  hong  cu,  pitt  ford  tr,  kettering   jd.  j  endod.  1995  aug;21(8):403-­‐6.   pmid:  7595152.     6. reaction  of  rat  connective  tissue  to   implanted  dentin  tubes  filled  with  a   white  mineral  trioxide  aggregate.   holland  r1,  souza  vd,  nery  mj,  far-­‐ acojúnior  im,  bernabé  pf,  otoboni-­‐ filho  ja,  dezanjúnior  e.  braz  dent  j.   2002;13(1):23-­‐6.  pmid:  11870957.     7. a  comparison  of  laterallycon-­‐ densedgutta-­‐percha,  thermoplasti-­‐ cizedgutta-­‐percha,  and  mineral  tri-­‐ oxide  aggregate  as  root  canal  filling   materials.  bogen  g.  j  endod.  2004   dec;30(12):826.  pmid:  15564859.     8. a  quantitative  comparison  of  the  fill   density  of  mta  produced  by  two   placement  techniques.  yeung  p1,   liewehr  fr,  moon  pc.  j  endod.  2006   may;32(5):456-­‐9.  pmid:  16631848.     9. porosity-­‐dependence  of  effec   10. tive  mechanical  properties  of  pore-­‐ solid  composite  materials.  pal  r.  j   com-­‐pos  mater.  2005   jun;39(13):1147-­‐1158.     11. flexural  strength  and  porosity  of  ce-­‐ ments.  birchall  j,  howard  aj,  kendall   k.  nature.  1981  jan;289:388-­‐390     12. bye  gc  (1999).  portland  cement,   2ndedition:  composition,  production   and  properties.  united  king-­‐ dom:thomas  telford  ltd.248  p.     13. placement  of  mineral  trioxide  aggre-­‐ gate  using  two  different  techniques.   aminoshariae  a,  hartwell  gr,  moon   pc.  j  endod.  2003  oct;29(10):679-­‐ 82.  pmid:  14606796.     14. comparative  chemical  study  of  mta   and  portland  cements.  oliveira  mg,   xavier  cb,  demarco  ff,  pinheiro  al,   costa  at,  pozza  dh.  braz  dent  j.   2007  18(1):3-­‐7.  pmid:  17639192.     15. microstructure  and  strength  of  hy-­‐ drated  cement.feldman  rf,   beaudoin  jj.  cemconcr  res.  1976   may;6(3):398-­‐400.     16. mehta  pk  (1993).  concrete:  struc-­‐ ture,  properties,  and  materials.  new   york:  prentice  hall.  704  p.     17. relationship  between  pore  struc-­‐ ture  and  mechanical  properties  of   ordinary  concrete  under  bending  fa-­‐ tigue.zhang  b.  cem  and  concr  res.   1998  may;28(5):699–711.     18. compressive  strength  and  pore   structure  of  high-­‐performance  con-­‐ crete  after  exposure  to  high  temper-­‐ ature  up  to  800c.chan  y,  luo  x,  sun   w.  cemconcr  res.  2000  30(2):247-­‐ 251.     19. asthana  r,  kumar  a,  dahotre  n   (2006).  materials  processing  and-­‐ manufacturing  science,  first  edition.   united  kingdom:  butterworth-­‐ heinemann.  656  p.     20. aligizaki  kk  (2005).  pore  structure  of   cement-­‐based  materials:  testing,   interpretation  and  requirements.   boca  raton:  crc  press,  llc.  432  p.     21. determination  of  pore  size  distribu-­‐ tion  and  surface  area  of  several  ma-­‐ terials  using  mercury  porosimetry   and  gas  adsorption.  tian  y,  chen  ke-­‐ fu.  china  pulp  and  paper.  2004-­‐04.     22. the  characterization  of  porous  sol-­‐ ids  from  gas  adsorption  measure-­‐ ments.lee  ck,  chiang  ast,  tsay  cs.   key  eng  mater.  1996  115:21-­‐44.     23. ultrasonic  debridement  of  root  ca-­‐ nals:  acoustic  cavitation  and  its  rele-­‐ vance.  1988.  ahmad  m,  pitt  ford  tr,   crum  la,  walton  aj.  intendod  j.   2009  may;42(5):391-­‐8.  pmid:   19356172.   microsoft word 76 2017.docx   vol  5,  no  1  (2017)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2017.76           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     assessment  of  dentofacial  variations  in  monozygotic  twins   victor  tu1      1university  of  pi.sburgh,  school  of  dental  medicine abstract   with  dentofacial   development  being  a  mul4factorial   process,  we   face   the  ques1on  of  whether   gene#c  or   environ-­‐ mental   factors  play  a   larger  role   in  specific  phenotypic   traits.  the  use  of   twin  studies  allows  one  to  employ  gene/c   controls   to   the   study   such   that  one   can   focus   solely  on   traits   that  may  be  more  environmentally  determined.   this   study  involves  retrospec0ve  as  well  as  current  analy0cal  measurements  between  a  set  of  monozygo0c  twins  to  assess   gene$c  and  outside  influences  on  facial  and  intraoral  development.  while  similari$es  were  acknowledged,  we  found   differences   in  measurements  of  the  facial  thirds,  the  congenital  presence  of  mandibular  third  molars,  and  the  pres-­‐ ence  of  mandibular  tori.   cita%on:  tu,  v.  (2017)  assessment  of  dentofacial   varia%ons  in  monozygo%c  twins.  den$stry  3000.   1:a001  doi:10.5195/d3000.2017.76   received:    may  22,  2017   accepted:    may  26,  2017   published:    june  16,  2017   copyright:  ©2017  tu,  v.  this   is   an  open  access   ar!cle   licensed   under   a   crea!ve   commons   a!ribu%on  work  4.0  united  states  license.   email:  vwt2@pi(.edu   introduction   dentofacial  development  is   a  multifactorial  phenomenon  that   relies  on  the  interactions  between   multiple  genes,  environmental   contributions  and  epigenetic  fac-­‐ tors  that  all  come  together  like   pieces  of  a  puzzle  to  yield  a  defini-­‐ tive  set  of  phenotypic  attributes.   much  of  the  studies  on  dentofacial   characteristics  and  pathologies   have  been  focused  on  the  contrib-­‐ uting  role  of  genetics  in  the  hopes   of  being  able  to  utilize  it  as  a  tool   in  diagnosis  and  treatment  plan-­‐ ning.  this  has  led  to  the  confirma-­‐ tion  that  genetic  inheritance  is  the   controlling  factor  in  facial  for-­‐ mation  through  craniometrical   and  cephalometric  studies  of  facial   similarities.  furthermore,  similari-­‐ ties  in  craniofacial  bones  and  pro-­‐ files  are  observed  when  cephalo-­‐ grams  of  siblings  are  superim-­‐ posed  on  that  of  their  parents   [5,6].  however,  the  use  of   monozygotic  twin  studies  has  also   helped  to  elucidate  how  heredi-­‐ tary  and  environmental  factors   mesh  with  one  another  to  create   the  final  physical  result.  this  is  at-­‐ tributed  to  the  reasoning  that  any   differences  observed  in  a  physical   feature  between  monozygotic   twins  that  are  genetically  identical   implies  that  environmental  factors   may  play  a  significant  role  in  that   aspect  of  a  person  compared  to   the  other  more  genetically  con-­‐ trolled  phenotypes.  past  studies   between  monozygotic  adult  twins   have  uncovered  differences  in   measurements  of  the  anterior   cranial  base,  man-­‐ dibular  body   length,  total  facial   height,  and  lower   facial  height  [1].     further-­‐ more,  another   study  on  dental   arch  forms  and  size   demonstrated  that   the  effect  from  en-­‐ vironmental  factors   were  more  at  play   for  this  part  of  development  ra-­‐ ther  than  genetics  alone  [2].  an-­‐ other  study  on  dental  arch  forms   and  the  structure  of  individual   teeth  of  several  monozygotic  twin   pairs  showed  that  twins  are  not   necessarily  always  occlusally  iden-­‐ tical  [4].    an  analysis  on  42  pairs  of   twins  found  hereditary  factors  to   be  responsible  for  only  40%  of  the   total  skeletal  and  dental  features   that  caused  malocclusion  with  the   genetic  component  being  more  of   a  determining  factor  for  skeletal   features  than  dental  features  [3].   assessment  of  dentofacial  varia1ons  in  monozygo1c  twins   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.76    http://dentistry3000.pitt.edu   however,  genes  that  regulate  faci-­‐ al  proportions  and  jaw  relation-­‐ ships  may  still  play  an  indirect  role   in  determining  occlusion.     regardless,  occlusal  rela-­‐ tions  seem  to  be  more  effected  by   the  environment.  as  with  any  mul-­‐ tifactorial  process,  there  is  debate   in  terms  of  whether  genetic  or  en-­‐ vironmental  factors  are  weighted   more  in  terms  of  the  final  out-­‐ come.  twin  studies  offer  the   chance  to  gain  insight  into  wheth-­‐ er  there  are  other  factors  at  play   in  terms  of  development  when   genetic  factors  are  controlled  for.     subjects  and  methods   this  study  involves  retro-­‐ spective  sources  and  current  facial   measurements  to  gauge  differ-­‐ ences  in  dentofacial  development   between  twin  a  and  twin  b.  in  ad-­‐ dition  to  past  photographs  (figure   1),  current  facial  measurements   were  taken  to  help  assess  differ-­‐ ences  in  a  quantitative  way  (table   1).  while  most  of  the  measure-­‐ ments  were  self-­‐explanatory,  the   first  facial  third  was  taken  as  the   distance  between  the  trichion  and   the  glabella.  the  middle  third  is   the  distance  from  the  glabella  to   the  subnasale.  finally,  the  lower   third  was  measured  from  the  sub-­‐ nasale  to  the  menton.  twins  a  and   b  are  currently  under  invisalign   treatment.  patient  treatment  dia-­‐ grams  from  the  start  of  treatment   are  shown  in  figure  2  to  provide  a   snapshot  of  the  occlusion  before   treatment.  past  bitewing  radio-­‐ graphs  combined  with  current  in-­‐ traoral  pictures  were  taken  to  as-­‐ sess  the  oral  condition  (figure  3).   results  and  discussion   dentofacial  development  is   a  multifactorial  process  but  it  is   unknown  how  involved  environ-­‐ mental  factors  are  in  particular   aspects  of  the  final  outcome.  as-­‐ sessing  the  photos  in  figure  1,  one   should  be  able  to  detect  subtle   differences  already  between  twin   a  and  twin  b  after  birth.  over  time   as  these  twins  mature  in  growth   and  development,  these  differ-­‐ ences  are  magnified.  to  assess   these  measurements  in  a  quanti-­‐ tative  fashion,  facial  measure-­‐ ments  were  taken  between  twin  a   and  twin  b,  which  presents  largely   a  general  consensus  in  facial  out-­‐ come.  of  greatest  interest  is  the   difference  in  measurement  in  the   middle  and  lower  thirds  of  the   face,  where  one  finds  the  greatest   amount  of  dissimilarity.  one  plau-­‐ sible  explanation  is  based  on  the   growth  pattern  of  the  lower  half   of  the  face.  lower  facial  develop-­‐ ment  occurs  at  relatively  fewer   growth  sites  and  over  a  more  ex-­‐ tended  period  of  time  compared   to  the  upper  half  of  the  face  [1].   the  form  and  size  of  the  upper   facial  region  is  dependent  on  nu-­‐ merous  elements  that  interact  in  a   more  complex  set  of  pathways  [1].   with  less  growth  sites  on  the  low-­‐ er  half  of  the  face,  one  can  ex-­‐ trapolate  that  genetic  factors  are   less  involved  in  lower  facial  devel-­‐ opment,  which  opens  the  door  for   environmental  factors  to  leave  a   greater  impact  in  facial  develop-­‐ ment  of  the  lower  half.     assessment  of  dentofacial  varia1ons  in  monozygo1c  twins   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.76    http://dentistry3000.pitt.edu   despite  the  fact  that  previ-­‐ ous  studies  have  found  that  envi-­‐ ronment  contributes  significantly   to  the  development  of  arch  form   and  size  and  dental  occlusion,   there  are  still  a  lot  of  occlusal  simi-­‐ larities  between  twin  a  and  b.  de-­‐ spite  the  assumption  that  genetics   is  less  involved  in  the  lower  half  of   the  face,  both  twins  present  with   shifted  midlines  yet  maintain  a   class  i  occlusal  relationship.  the   only  striking  dissimilarities  that   can  be  observed  is  that  there  is   spacing  between  the  mandibular   anterior  teeth  of  twin  b  whereas   that  space  is  closed  in  twin  a  with   the  cervical  necks  of  the  mandibu-­‐ lar  incisors  tipped  more  mesially.   the  gingival  margins  also  appear   to  be  more  rounded  in  twin  a  and   more  pointed  in  twin  b.  environ-­‐ mental  factors  such   as  differences  in   tooth  brushing   technique  is  a  plau-­‐ sible  explanation   for  this.   assessing   the  bitewing  radio-­‐ graphs  between   the  two  siblings,   the  presence  of   existing  restora-­‐ tions  was  used  to   determine  caries   susceptibility.   based  on  the  radi-­‐ ographs,  both  sib-­‐ lings  have  three   restorations  with   twin  a  having  res-­‐ torations  on  num-­‐ bers  2  (mo),  19   (o),  and  20  (endo-­‐ dontic  treatment).  twin  b  has  res-­‐ torations  on  numbers  3  (o),  19   (o),  and  31  (o).  as  the  presenta-­‐ tion  of  carious  lesions  follows  a   multifactorial  causation  that  relies   on  the  balance  between  the  host   (protective  mechanisms  like  sali-­‐ vary  flow  rate)  and  environmental   factors  (diet  and  indigenous  bacte-­‐ rial  flora),  it  would   not  be  a  surprise  if   the  twins  differed  in   caries  presentation   as  genetic  similari-­‐ ties  alone  cannot   determine  caries   risk.  the  similarities   seen  in  this  case  can   be  attributed  to   growing  in  the  same   household  with   similar  habits  in  di-­‐ eting  and  oral  hygiene.  this  may   not  be  the  case  if  the  siblings  were   separated  at  birth.  it  is  also  intri-­‐ guing  to  note  that  the  twins  pre-­‐ sent  with  differences  in  mandibu-­‐ lar  third  molar  formation.  while   twin  a  retained  his  third  molars  to   the  age  of  23,  twin  b  is  congenital-­‐ ly  missing  number  32.  also  as  a   child,  twin  b’s  number  18  was  se-­‐ verely  broken  down  by  caries  that   it  was  extracted  and  number  17   was  allowed  to  erupt  into  its   place.  moreover,  the  root  tips  on   number  17  are  divergent  com-­‐ pared  to  twin  b’s,  which  is  notice-­‐ ably  convergent  apically.  this  sup-­‐ ports  the  idea  that  third  molars   are  very  susceptible  to  develop-­‐ mental  variation  despite  identical   genetics,  which  places  importance   on  environmental  influences.  in-­‐ traoral  pictures  in  figure  4  further   reveal  that  twin  a  possesses  a   mandibular  torus  behind  number   21.  twin  b,  on  the  other  hand,   possesses  bilateral  mandibular  tori   behind  both  premolars  (#21  and   #27).  mandibular  tori  are  com-­‐ monly  present  early  in  adult  life   and  are  associated  with  several   factors  including  bruxism,  local   stresses,  and  genetic  influences.  in   assessment  of  dentofacial  varia1ons  in  monozygo1c  twins   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.76    http://dentistry3000.pitt.edu   this  case,  the  unilateral  mandibu-­‐ lar  torus  may  indicate  that  twins  a   isn’t  occluding  evenly  if  he  is  brux-­‐ ing  on  a  normal  basis.     when  comparing  twins   where  genetic  factors  are  con-­‐ trolled  for,  one  can  shift  the  focus   towards  other  factors  that  may   cause  dissimilarities  between   identical  siblings,  which  includes   epigenetic  factors,  maternal  varia-­‐ bles,  and  intrauterine  forces.  epi-­‐ gentics  refers  to  dna  methylation,   acetylation,  phosphorylation,   ubiquitination,  sumoylation  and   histone  modifications  that  reversi-­‐ bly  alter  dna  structure  without   changes  to  the  dna  sequence.   these  changes  accumulate  over   time  as  they  are  transmitted   through  mitosis.  while  de  novo   methylation  by  dna  methyltrans-­‐ ferase  targets  cytosine  bases   without  apparent  specificity,  dele-­‐ tion  of  dna  methyltransferase   genes  (dmnt1)  in  mice  causes   global  demethylation  and  apopto-­‐ sis  [7].  this  shows  how  there  is  a   certain  randomness  that  can  have   far  reaching  effects,  which  may   lead  to  phenotypic  differences  be-­‐ tween  twins.  the  earlier  the  zy-­‐ gote  splits,  the  sooner  twins  de-­‐ velop  independently,  with  even   the  slightest  dna  modification   having  the  potential  to  generate   drastic  developmental  conse-­‐ quences  in  later  periods.  while  the   etiology  of  epigenetic  changes   needs  further  study,  there  is  a   connection  between  methylation   and  chemical  exposure,  which  may   translate  to  an  attempt  at  envi-­‐ ronmental  adaptation  [8].  a  study   to  assess  the  extent  of  dna  meth-­‐ ylation  and  histone  acetylation  in   the  genomes  of  40  pairs  of   monozygotic  twins  found  that  epi-­‐ genetic  profiles  were  almost  iden-­‐ tical  in  65%  of  the  twin  pairs  and   significant  differences  in  the  re-­‐ maining  35%  of  twin  pairs  [9].  ac-­‐ cordingly,  the  amount  of  epigenet-­‐ ic  differences  was  directly  corre-­‐ lated  to  the  age  of  the  twins  and   the  amount  of  time  co-­‐twins  spent   apart.  as  such,  epigenetics  may   play  a  huge  role  in  why  twins  a   and  b  start  to  look  progressively   different  as  they  age  in  figure  1.   intrauterine  factors  have   also  been  suspected  to  play  a  role   in  phenotypic  differences  between   twins,  especially  in  regards  to  in-­‐ trauterine  special  constraint,   which  are  common  during  twin   fetal  development  and  may  also   lead  to  deviations  in  facial  devel-­‐ opment  between  twins.  this  is  de-­‐ picted  in  one  study  in  which  re-­‐ straint  stress  was  applied  to  a   group  of  pregnant  rat  study  mod-­‐ els  to  determine  the  extent  of  any   changes  in  craniofacial  growth   patterns  in  rat  offspring  that  in-­‐ trauterine  stresses  elicited  [10].   the  prenatally  stressed  group  ex-­‐ perienced  increases  in  anterior   cranial  base  length  and  viscer-­‐ ocranium  measures  with  back-­‐ ward  rotation  of  the  midface  and   decreased  flattening  of  the  cranial   vault.  in  this  case  prenatal  chronic   stress  ultimately  promoted  endo-­‐ chondral  growth  in  the  cranial   base  and  nasal  septum.     finally,  maternal  variables   also  play  a  key  role  in  difference  in   dentofacial  development.  the  ef-­‐ fects  from  maternal  exposure  to   nitrosatable  drugs,  fertility  treat-­‐ ments,  and  nutrition  intake  such   as  folic  acid  intake  may  lead  to  ep-­‐ igenetic  changes  that  are  distrib-­‐ uted  unequally  to  developing   twins  in  utero,  potentially  affect-­‐ ing  dentofacial  formation.  folic   acid  consumption  for  example  af-­‐ fects  neural  tube  closure,  which  in   turn  impacts  brain  and  craniofacial   structures  later  on  in  development   [11].   in  conclusion,  this  study   brings  attention  to  the  genetic,   epigenetic,  as  well  as  the  envi-­‐ ronmental  impacts  on  develop-­‐ ment  and  allows  one  to  fully  ap-­‐ preciate  the  multifactorial  forces   of  this  process.  this  can  aid  in  pa-­‐ tient  education  in  terms  of  what   can  and  can’t  be  definitively   passed  down  to  the  offspring.   while  genetics  are  often  the  prime   suspect  in  rationalizing  phenotypic   manifestations,  it  is  important  for   patients  to  be  aware  of  other  envi-­‐ ronmental  factors  that  occur  after   conception  that  may  also  ulti-­‐ mately  influence  dentofacial   presentations.     references   1.  horowitz  sl,  osborne  rh,  de-­‐ george  fv.  a  cephalometric  study   of  craniofacial  variation  in  adult   twins.  angle  orthod.  1960;30:1–5.   2.  cassidy  km,  harris  ef,  tolley  ea,   keim  rg.  genetic  influence  on   dental  arch  form  in  orthodontic   patients.  angle  orthod.  1998   oct;68(5):445-­‐54.  pubmed  pmid:   9770103.   3.  lundström  a.  nature  versus   nurture  in  dento-­‐facial  variation.   assessment  of  dentofacial  varia1ons  in  monozygo1c  twins   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.76    http://dentistry3000.pitt.edu   eur  j  orthod.  1984  may;6(2):77-­‐ 91.  pubmed  pmid:  6587973.   4.  sognnaes  rf,  rawson  rd,  gratt   bm,  nguyen  nb.  computer  com-­‐ parison  of  bitemark  patterns  in   identical  twins.  j  am  dent  assoc.   1982  sep;105(3):449-­‐51.  pubmed   pmid:  6957451.   5.  curtner  rm.  predetermination   of  the  adult  face.  am  j  or-­‐ thod.  1953;39:201–217   6.  hunter  ws.  a  study  of  the  in-­‐ heritance  of  craniofacial  charac-­‐ teristics  as  seen  in  lateral  cephalo-­‐ grams  of  72  like-­‐sexed  twins.  rep   congr  eur  orthod  soc.1965;41:59-­‐ 70.  pubmed  pmid:  5222375.   7.  singh  sm,  murphy  b,  o'reilly  r.   epigenetic  contributors  to  the  dis-­‐ cordance  of  monozygotic  twins.   clin  genet.  2002  aug;62(2):97-­‐ 103.  review.  pubmed   pmid:12220446.   8.  poulsen  p,  esteller  m,  vaag  a,   fraga  mf.  the  epigenetic  basis  of   twin  discordance  in  age-­‐related   diseases.  pediatr  res.  2007   may;61(5  pt  2):38r-­‐42r.review.   pubmed  pmid:  17413848.   9.  fraga  mf,  ballestar  e,  paz  mf,   ropero  s,  setien  f,  ballestar  ml,   heine-­‐suñer  d,  cigudosa  jc,  uri-­‐ oste  m,  benitez  j,  boix-­‐chornet  m,   sanchez-­‐aguilera  a,  ling  c,  carls-­‐ son  e,  poulsen  p,  vaag  a,  stephan   z,  spector  td,  wu  yz,  plass  c,  es-­‐ teller  m.  epigenetic  differences   arise  during  the  lifetime  of   monozygotic  twins.  proc  natl  acad   sci  u  s  a.  2005  jul   26;102(30):10604-­‐9.  pubmed   pmid:  16009939;  pubmed  central   pmcid:  pmc1174919.   10.  aminabadi  na,  behroozian  a,   talatahari  e,  samiei  m,  sadigh-­‐ eteghad  s,  shirazi  s.  does  prenatal   restraint  stress  change  the  cranio-­‐ facial  growth  pattern  of  rat  off-­‐ spring?  eur  j  oral  sci.  2016   feb;124(1):17-­‐25.  doi:   10.1111/eos.12230.  pubmed   pmid:  26620628.   11.  kappen  c.  modeling  anterior   development  in  mice:  diet  as   modulator  of  risk  for  neural  tube   defects.  am  j  med  genet  c  semin   med  genet.  2013   nov;163c(4):333-­‐56.  doi:   10.1002/ajmg.c.31380.  review.   pubmed  pmid:  24124024;  pub-­‐ med  central  pmcid:   pmc4149464.     evalua&on of tongue volume and airway volume in skeletal class i and class ii pa&ents using cone beam tomographya cross sec&onal study vol 11 no 1 (2023) doi 10.5195/d3000.2023.245 h#p://den*stry3000.pi#.edu evaluation of tongue volume and airway volume in skeletal class i and class ii patients using cone beam computed tomographya cross sectional study jim thomas abraham1, roshan m sagarkar2, sharanya sabrish2, prashantha govinakovi shivamurthy3, silju ma<hew2, nagaraju rakesh4 1faculty of dental sciences, m s ramaiah university of applied sciences, msrit post, m s r nagar, bengaluru, karnataka, india. pin 560054 2department of orthodontics, faculty of dental sciences, m s ramaiah university of applied sciences, msrit post, m s r nagar, bengaluru, karnataka, india. pin 560054 3head of department of orthodontics, faculty of dental sciences, m s ramaiah university of applied sciences, msrit post, m s r nagar, bengaluru, karnataka, india. pin 560054 4department of oral medicine and radiology, faculty of dental sciences, m s ramaiah university of applied sciences, msrit post, m s r nagar, bengaluru, karnataka, india. pin 560054 abstract background: the volumetric rela&onship between the tongue and the oral cavity is an important factor in tooth alignment and occlusion. the aim of this study was to evaluate the difference in tongue and airway volume in skeletal class i and class ii malocclusion. materials and methods: the cbct data which comprised of 30 skeletal class i and 30 skeletal class ii was stored in dicom format. tongue volume and airway volume was analyzed with the help of 3d dolphin sokware. values obtained were subjected to sta&s&cal analysis with help of spss. results: skeletal class i pamern had more tongue volume with a mean of 45.32 mm3 than skeletal class ii pamern with mean value of 40.27 mm3 with p value of <0.001. skeletal class i pamern has more airway volume with a mean of 15058.6 mm3 than pa&ents with skeletal class ii pamern with mean value of 12458.8 mm3 with a p value of 0.003. a p value of 0.001 indicated that there is a strong correla&on between the tongue volume and airway volume in both skeletal class i and skeletal class ii subjects. conclusion: the volume of tongue and airway was greater in skeletal class i malocclusion compared to skeletal class ii malocclusion. there exists a posi&ve correla&on between the tongue volume and airway volume in both the skeletal pamerns. keywords: airway volume; class i skeletal malocclusion; class ii skeletal malocclusion; dolphin sokware; tongue volume cita9on: thomas abraham, j et al. (2023) evalua9on of tongue volume and airway volume in skeletal class i and class ii pa9ents using cone beam computed tomographya cross sec9onal study den9stry 3000. 1:a001 doi:10.5195/d3000.2023.245 received: october 5, 2021 accepted: april 4, 2023 published: october 13, 2023 copyright: ©2023 thomas abraham, j et al. this is an open access ar9cle licensed under a crea9ve commons avribu9on work 4.0 united states license. email: drsharanyaortho@gmail.com introduction according to the equilibrium theory, teeth alignment is influenced by the tongue and the lip [1]. the tongue is a powerful muscular organ which is centrally located within the oral cavity and has the ability to affect the position of the teeth and the surrounding structures [2]. abnormality of either function or position of tongue can lead to change in the surrounding dentoalveolar http://dentistry3000.pitt.edu/ evalua&on of tongue volume and airway volume in skeletal class i and class ii pa&ents using cone beam tomographya cross sec&onal study vol 11 no 1 (2023) doi 10.5195/d3000.2023.245 h#p://den*stry3000.pi#.edu structures and skeleton. in cases of mandibular prognathism, the tongue occupies the floor of the mouth, and can be bulky, hypotensive, and projected [3]. on the other hand, when mandibular retrognathism occurs, with reduction in the anteroposterior space, the usual tongue position shows a lowered apex with dorsum more elevated, which can be located between the dental arches in case there is a concomitant maxillary retrusion [4]. respiratory function and the upper airway are very important for orthodontic diagnosis and treatment planning as varied breathing function could influence facial growth and morphology [3,5]. oropharyngeal and nasopharyngeal structures play a role in the development of the dentofacial complex [5]. according to the functional-matrix hypothesis proposed by melvin moss, soft-tissue units guide the hard tissues to an extent. it has also been suggested that an increase in the volume of soft tissues induces osteogenic reaction at the growth site of the bone [5]. patient exhibiting class ii skeletal base tend to have smaller airway volume, but the tongue volume remains a topic of debate. there are 2 schools of thought regarding this issue. the first is that the tongue adapts to the retrognathic mandible, which leads to the posterior positioning of tongue as well as the inferior and posterior displacement of the hyoid bone. but studies have shown that although the hyoid bone adapts to the tongue position, there is always a tendency for the hyoid bone to relapse to a superior position which eventually can cause narrowing of airway leading to obstructive sleep apnea in class ii skeletal base. the other concept regarding the same is that the mandible adapts to the highvolume tongue which eventually leads to increase in the inter molar distance in class ii skeletal base [6]. this debate has been longstanding. hence determining the size of the tongue in different facial morphological variants can be useful [3]. dentistry has witnessed tremendous advances and with these advances, the need for use of precise diagnostic tools, especially imaging methods, has become mandatory. cone beam ct technology (cbct) has emerged as a potential alternative to magnetic resonance imaging (mri) and ct scanning to obtain complete, fast, and detailed images at a relatively modest cost. the advantages of cbct include lower radiation dose which causes less radiation hazards to the patients [7]. hence the present study was done to evaluate the tongue volume in skeletal class i and class ii subjects and its association with the airway volume using cbct. material and methods this cross-sectional study conducted using cone beam computed tomography scans of 60 patients. the scans were obtained from the patient archives from oral d scan center and from the department of oral medicine and radiology fds, ruas. the study protocol was approved by the ethics review committee of ramaiah university of applied sciences. the research project protocol number was ec2020/ug/075 and approval date was 01/09/2020. the sample size was estimated using the g power software v. 3.1.9.2. considering the effect size to be 80%, power of the study at 85% and the margin of the error at 5%, the sample size needed was calculated to be 60. the scans which were selected for the study were of those patients whose cbcts were made placing the patient in natural head position during the image acquiring procedure with the lips and tongue relaxed so that the borders delineating the tongue shape could be obtained. the patients whose scans were used from the institute archives had all given informed consent for their scans to be used for research purposes. care was taken to avoid any involuntary movements like swallowing, which would have caused kinetic defects in cbct slices. all the scans in the archives were made using the same machine and using carestream software (carestream 9300) with parameters of 6.3 ma, 90 kvp, and 300 microns resolution with full field of view (fov) of 17x13.5 cms. the age of the patients ranged from 1420 years, and this included 39 females and 21 males. http://dentistry3000.pitt.edu/ evalua&on of tongue volume and airway volume in skeletal class i and class ii pa&ents using cone beam tomographya cross sec&onal study vol 11 no 1 (2023) doi 10.5195/d3000.2023.245 h#p://den*stry3000.pi#.edu inclusion criteria applied for the study were: cbct of male or female patients in the age group ranging between 1420 years, no history of orthodontic treatment or facial surgery, cbct was taken with patient biting in centric occlusion and no enlarged tonsil or adenoids present. the exclusion criteria applied were: cbct with bite block placed, congenital anomalies like cleft lip and palate. patients were categorized into skeletal class i and class ii based on following cephalometric parameters[8, 9]: anb value: anb angle ranging from 20-40 were classified as class i skeletal pattern and anb value greater than 40 were classified as class ii skeletal pattern [8]. witt’s appraisal: ao ahead of bo greater than 2mm was classified as class ii skeletal pattern [9]; angle’s molar relation; overjet: overjet of 24mm was considered as skeletal class i pattern and greater than 4 mm was considered as skeletal class ii pattern. 60 cbcts (30 class i, 30 class ii) which fulfilled all the inclusion and exclusion criteria, were selected for the study. the cbct data which comprised of 30 skeletal class i and 30 skeletal class ii was transferred to a computer which stored the data in the digital imaging and communications in medicine (dicom) format. a 3d data and 3d coordinate system were constructed using dolphin 3d software. (dolphin software, version-11.96, dolphin imaging & management solutions, chatsworth, ca) the tongue volume determination was carried out as follows. landmarks were considered both in lateral and transverse plane to delineate the borders of the tongue from the adjacent structures. once the image in the lateral view was cropped it was then assessed in the transverse view to obtain the final 3d image. the obtained cropped 3d image of the tongue was subjected to volumetric analysis using 3d dolphin software (version 11.96) in the computer. the volume of the tongue was hence displayed in cubic millimeters by the software. the boundaries considered in the lateral view aspect were (figure 1) anterior point: incisal point of maxillary incisor, superiorly: ans – pns plane, postero-superiorly: pns point, postero-inferiorly: at the junction of c 2 and c 3, inferiorly: cej level of second molar to first premolar. the boundaries considered in the transverse plane are: (figure 2) laterally: borders delineating the alveolar bone and the soft tissue, posteriorly: borders delineating the soft tissue and the airway pathway (figure 3). the airway volume determination was carried out as follows. to obtain airway volume using dolphin 3d, it was required to define the outline of the airway. seed points were placed in the target airway. seed points denote densities which represents the airway. the volume of the airway was displayed in cubic millimeters. the boundaries of the airway: superiorsuperior tip of nasopharyngeal airway, anterior border-vertical plane through posterior nasal spine, perpendicular to the sagittal plane, posterior border-posterior wall of the pharynx, inferior borderplane tangent to the most medial projection which is in the caudal aspect of the third cervical vertebrae at right angles to the sagittal plane. figure 1. boundaries marked for cropping to obtain the borders of the tongue from lateral view aspect (a); image obtained after cropping (b). figure 2. boundaries delineating alveolar & bone airway from the tongue (a); cropped 3d image (b). figure 3. boundaries delineating the airway from the adjacent structures. http://dentistry3000.pitt.edu/ evalua&on of tongue volume and airway volume in skeletal class i and class ii pa&ents using cone beam tomographya cross sec&onal study vol 11 no 1 (2023) doi 10.5195/d3000.2023.245 h#p://den*stry3000.pi#.edu spss (spss inc., chicago v 18.5) software packages were used for data entry and analysis respectively. the results were averaged (mean + standard deviation) for continuous data and number and percentage was used for dichotomous data. the student ‘t’ test was used to determine if there was a statistical difference. pearson correlation coefficients were calculated to determine if there was any correlation between the tongue volume and the airway volume. results the current study consisted of 60 cbct samples of skeletal class i and class ii subjects. among the 60 samples, the percentage distribution was 68.3% females and 31.7% males as shown in figure 4. table 1 depicts the distribution of males and females in skeletal class i and class ii and there was no statistically significant difference seen. table 2 shows that the mean age of males and females in skeletal class i case was 17.4 years and 16.9 years respectively. p value was 0.448 which indicates statistically insignificant difference. the mean age of males and females in skeletal class ii cases were 17.7 years and 18.3 years respectively. p value was 0.420 which indicated statistically insignificant difference. table 3 depicts the comparison of mean values of tongue volume in skeletal class i and class ii pattern. patients with skeletal class i pattern had a significant more tongue volume with a mean of 45.32 mm3 compared to patients with skeletal class ii pattern who had mean value of 40.27mm3 respectively with p value of <0.001. table 4 shows the comparison of mean values of airway volume between skeletal class i and class ii pattern. patients with skeletal class i pattern have large airway volume (with a mean of 15058.6 mm3) compared to patients with skeletal class ii pattern (with mean value of 12458.8mm3) respectively with p value of 0.003. table 5 shows the correlation between tongue volume and airway volume in skeletal class i and class ii pattern. the p value of 0.001 indicates that there is a strong correlation between the tongue volume and airway volume in skeletal class i and skeletal class ii subjects. also, r value of 0.4596 depicted strong positive correlation between tongue volume and airway volume in skeletal class i subjects. the r value of 0.3211 depicted strong positive correlation between tongue volume and airway volume in skeletal class ii subjects. skeletal pattern gender n mean age sd min. max. t value p value class 1 male 9 17.4 2.404 15 21 0.592 0.448 female 21 16.9 1.682 15 20 total 30 17.0 1.903 15 21 class 2 male 10 17.7 2.312 14 21 0.670 0.420 female 20 18.3 1.658 14 21 total 30 18.1 1.882 14 21 skeletal pattern gender total c2 value p value male female class 1 9 21 30 0.077 0.781 30.0% 70.0% 100.0% class 2 10 20 30 33.3% 66.7% 100.0% total 19 41 60 31.7% 68.3% 100.0% figure 4. pie diagram representing gender distribution among the study samples. table 1: gender distribution amongst the individual skeletal pattern and its significance. table 2: table showing age distribution in skeletal class i and class ii subjects. http://dentistry3000.pitt.edu/ evalua&on of tongue volume and airway volume in skeletal class i and class ii pa&ents using cone beam tomographya cross sec&onal study vol 11 no 1 (2023) doi 10.5195/d3000.2023.245 h#p://den*stry3000.pi#.edu skeletal pattern n me an sd median min. max. mean diff se ‘t’ value p value class 1 30 45. 32 2.5 17 45.40 39.83 49.46 5.052 0.545 9.270 <0.00 1 class 2 30 40. 27 1.6 04 40.25 37.54 42.92 skeletal pattern n mean sd median min. max. mean diff se ‘t’ value p value class 1 30 15058. 6 3960.46 7 14,241.0 9569 23367 2599.7 836.623 3.107 0.003 class 2 30 12458. 8 2304.96 1 12,716.0 8150 16834 skeletal pattern airway volume class 1 tongue volume correlation 0.678 p value <0.001 class 2 tongue volume correlation 0.567 p value 0.001 discussion the present study was done to evaluate the tongue volume and airway volume in skeletal class i and class ii malocclusion and also to find if there is a correlation between the tongue volume and airway volume in the respective skeletal pattern. with the advent of imaging techniques like cbct, diagnosis and treatment planning is greatly supplemented, thus making 3d analysis of maxillofacial skeleton and soft tissues possible [10]. the usual method of evaluating the tongue volume was magnetic resonance imaging (mri), since mri is the gold standard for measuring soft tissues. 3d dolphin software is considered as the gold standard method for evaluating the airway volume [11].. currently numerous imaging software’s are available for evaluating the tongue volume and the airway volume. mimics (materialize interactive medical image control system) is an image processing software for 3d design and modeling. it is used to create 3d surface models from stacks of 2d image data. weissheimer et al compared 6 imaging software’s and concluded that mimics, dolphin3d, osiri x and itksnap were similar and more accurate than in vivo dental and on demand for upper airway assessment [12]. in the present study, the anb angle was used to identify the skeletal pattern of the samples. using anb alone for identification of skeletal pattern is unreliable as it is subjective to many variables like nasion area morphology, vertical dimension of the face, anterior cranial base inclination, and inclination of the jaws. using only anb would have table 3: comparison of mean values of tongue volume in skeletal class i and class ii pattern. table 4: comparison of mean values of airway volume between skeletal class i and class ii pattern. table 5: correlation between tongue volume and airway volume in skeletal class i and class ii pattern. http://dentistry3000.pitt.edu/ evalua&on of tongue volume and airway volume in skeletal class i and class ii pa&ents using cone beam tomographya cross sec&onal study vol 11 no 1 (2023) doi 10.5195/d3000.2023.245 h#p://den*stry3000.pi#.edu confounded the study, as the location of point a and point b will have an impact on the angle. thus, in addition to anb values, witt’s appraisal and molar relationship were also taken into consideration for identifying the skeletal pattern [13]. the age group of the sample consisted of both adolescents and adults, despite the fact that airway volume varies with age. kollias et al had performed a long term follow up study and had concluded that between 20 to 50 years of age, there is a gradual decrease of airway dimension noted both behind the tongue as well as and posterior to the soft palate [14]. in other studies, it was reported that pharyngeal structures continue to grow rapidly until 13 years of age and a quiescent period was noted between the ages of 14 to 18 years which meant that the variation of airway size of this age group was minimal [15, 16]. thus, in this study, age was standardized by taking samples with ages ranging from 14 to 20 years to minimize variation due to growth. the volume of tongue is determined by factors such as size of mandibular arch, maxillary width, vertical facial height and combined vertical and horizontal position of chin and symphysis. it has been an accepted fact that increases in volume of soft tissue induces increase in osteogenesis at the growth site of the bone. earlier the volume of tongue was measured with the help of lateral cephalogram which basically determines the 2-dimensional size of the tongue. lui et al evaluated the volume of tongue with the help of contrast agent such as barium [17]. vig and cohen compared sagittal area of the tongue to that sagittal area of the oral cavity with the help of bony landmarks [18]. few other studies have evaluated the size of the tongue by direct measurement but those were not able to measure the posterior limit of the tongue. bandy and hunter measured the volume of tongue by water displacement method [19]. in the present study, the tongue volume was measured with defined landmarks based on a previous study conducted by liegeois et al.[20] in our study the tongue volume was measured with the help of 3d dolphin software. studies have proved that dolphin imaging was effective in measurements and had better prediction compared to the other imaging software [7]. the influence of oral function on growth has been a topic of discussion in orthodontics for decades. initial studies related to airway were started in the early 1970s. since then, it has been a subject of great interest for the orthodontist to evaluate any correlation associated with airway and structures associated with it, especially the tongue because of its close proximity [21]. in this study the pharyngeal airway was considered as a whole with defined landmarks instead of dividing it into upper middle and lower compartments. this was done to ensure ease of computing and to avoid errors in volume determination by adding more landmarks to the study. this was done in accordance with the study by kyung min oh [22]. in this study the volume of airway was greater in skeletal class i malocclusion. this was in accordance with the studies conducted by palamo et al who proved that the oropharyngeal and nasopharyngeal airway volumes of class ii patients were smaller when compared with class i and class iii patients [23]. similarly another study conducted by deepthi et al had proved that there was a negative correlation between airway volume with anb angle, implying that class ii skeletal base patients had lesser airway volume [24]. studies conducted by frieties et all had shown that there was no statistically significant difference in airway volume in different malocclusion but there was statistically significant difference in patients exhibiting vertical and horizontal growth pattern where vertical growth pattern had lesser volume of airway as compared to horizontal [25]. it was also observed that the position of the mandible with respect to the cranial base had an effect on the pharyngeal airway volume. this may be due to the fact that if the class ii skeletal pattern is caused by the mandible being retrognathic, the overall oropharyngeal airway space will be reduced. in the current study, the distinction has not been made based http://dentistry3000.pitt.edu/ evalua&on of tongue volume and airway volume in skeletal class i and class ii pa&ents using cone beam tomographya cross sec&onal study vol 11 no 1 (2023) doi 10.5195/d3000.2023.245 h#p://den*stry3000.pi#.edu on whether the skeletal class ii pattern is caused by mandibular retrognathism of by maxillary prognathism. the degree of jaw discrepancy was relatively less in the selected samples as the anb value of the selected class ii samples ranged between 50-70. hence the effect of significant mandibular retro position requires further investigation as it might have further influence on airway morphology and volume. in the present study the ‘r’ value of 0.4596 depicts strong positive correlation between tongue volume and airway volume in skeletal class i subjects, similarly ‘r’ value of 0.3211 indicates strong positive correlation between tongue volume and airway volume in skeletal class ii subjects. there was not much literature found comparing the correlation between tongue volume and airway volume in skeletal class i and skeletal class ii. in this study it was found there was a statistically significant difference between the tongue volume in skeletal class i and skeletal class ii malocclusion indicating that the tongue volume was greater in skeletal class i malocclusion compared to skeletal class ii malocclusion. this was in agreement with a previous study’s findings [26]. in another study it was shown that that there was no statistical difference between size of the tongue and airway volume in relation to different skeletal pattern [27]. this was in contradiction to the present study where the volume of tongue and airway was greater in skeletal class i samples compared to skeletal class ii samples. these limitations of this study were that the sample did not have an equal proportion of males and females and was limited with respect to age range considered. the future prospects for research are to conduct a similar study in a larger sample size, to include different age brackets and compare between them, and to compare between sexes. conclusion the present study was concluded that the mean tongue volume (45.32mm3) and mean airway volume (15058.5mm3) were greater in samples exhibiting skeletal class i malocclusion compared to class ii samples. a strong positive correlation exists between tongue volume and airway volume in skeletal class i and skeletal class ii subjects. references 1. proffit wr. equilibrium theory revisited: factors influencing posi@on of the teeth. angle orthodon-st; 48. epub ahead of print 1978. doi: 10.1043/00033219(1978)048<0175:etrfip>2. 0.co;2. 2. verma s, agrawal d, prabhat k, et al. a cephalometric evalua@on of tongue from the rest posi@on to centric occlusion in the subjects with class ii division 1 malocclusion and class i normal occlusion. j orthod sci; 1. epub ahead of print 2012. doi: 10.4103/2278-0203.99758. 3. aby dm, sagarkar rm, mathew s, et al. comparison of airway morphology and volume in skeletal class i and class ii pa@ents using cone-beam computed tomography: a crosssec@onal study. world journal of den-stry; 11. epub ahead of print 2020. doi: 10.5005/jpjournals-10015-1754. 4. paolantonio eg, ludovici n, saccomanno s, et al. associa@on between oral habits, mouth breathing and malocclusion in italian preschoolers. eur j paediatr dent; 20. epub ahead of print 2019. doi: 10.23804/ejpd.2019.20.03.07. 5. liu zj, shcherbatyy v, gu g, et al. effects of tongue volume reduc@on on craniofacial growth: a longitudinal study on orofacial skeletons and dental arches. arch oral biol; 53. epub ahead of print 2008. doi: 10.1016/j.archoralbio.2008.05.0 10. 6. indriksone i, jakobsone g. the influence of craniofacial morphology on the upper airway dimensions. angle orthodon-st; 85. epub ahead of print 2015. doi: 10.2319/061014-418.1. 7. primozic j, farčnik f, perineh g, et al. the associa@on of tongue posture with the dentoalveolar maxillary and mandibular morphology in class iii malocclusion: a controlled study. eur j orthod; 35. epub ahead of print 2013. doi: 10.1093/ejo/cjs015. 8. steiner cc. cephalometrics for you and me. am j orthod; 39. epub ahead of print 1953. doi: 10.1016/0002-9416(53)90082-7. 9. jacobson a. applica@on of the ‘wits’ appraisal. am j orthod; 70. epub ahead of print 1976. doi: 10.1016/s00029416(76)90318-3. 10. alves m, bara@eri c, nojima li, et al. three-dimensional http://dentistry3000.pitt.edu/ evalua&on of tongue volume and airway volume in skeletal class i and class ii pa&ents using cone beam tomographya cross sec&onal study vol 11 no 1 (2023) doi 10.5195/d3000.2023.245 h#p://den*stry3000.pi#.edu assessment of pharyngeal airway in nasaland mouthbreathing children. int j pediatr otorhinolaryngol; 75. epub ahead of print 2011. doi: 10.1016/j.ijporl.2011.06.019. 11. chandra pk, godfrey k. assessment and predictability of anb angle. aust orthod j; 11. 12. jeans wd, fernando dcj, maw ar, et al. a longitudinal study of the growth of the nasopharynx and its contents in normal children. bri-sh journal of radiology; 54. epub ahead of print 1981. doi: 10.1259/00071285-54-638-117. 13. handelman cs, osborne g. growth of the nasopharynx and adenoid development from one to eighteen years. angle orthodon-st; 46. epub ahead of print 1976. doi: 10.1043/00033219(1976)0462.0.co;2. 14. chiang cc, jeffres mn, miller a, et al. three-dimensional airway evalua@on in 387 subjects from one university orthodon@c clinic using cone beam computed tomography. angle orthodon-st; 82. epub ahead of print 2012. doi: 10.2319/122811-801.1. 15. bandy he, hunter ws. tongue volume and the mandibular den@@on. am j orthod; 56. epub ahead of print 1969. doi: 10.1016/0002-9416(69)90230-9. 16. vig ps, cohen am. the size of the human tongue shadow in different mandibular postures. britjorthodont; 1. epub ahead of print 1974. doi: 10.1179/bjo.1.2.41. 17. lowe aa, takada k, yamagata y, et al. dentoskeletal and tongue soo-@ssue correlates: a cephalometric analysis of rest posi@on. am j orthod; 88. epub ahead of print 1985. doi: 10.1016/0002-9416(85)90133-2. 18. liégeois f, albert a, limme m. comparison between tongue volume from magne@c resonance images and tongue area from profile cephalograms. eur j orthod; 32. epub ahead of print 2010. doi: 10.1093/ejo/cjp105. 19. tourné lpm. growth of the pharynx and its physiologic implica@ons. american journal of orthodon-cs and dentofacial orthopedics; 99. epub ahead of print 1991. doi: 10.1016/08895406(91)70115-d. 20. ceylan i, oktay h. a study on the pharyngeal size in different skeletal parerns. american journal of orthodon-cs and dentofacial orthopedics; 108. epub ahead of print 1995. doi: 10.1016/s0889-5406(95)700684. 21. aboudara c, nielsen i, huang jc, et al. comparison of airway space with conven@onal lateral headfilms and 3-dimensional reconstruc@on from cone-beam computed tomography. american journal of orthodon-cs and dentofacial orthopedics; 135. epub ahead of print 2009. doi: 10.1016/j.ajodo.2007.04.043. 22. el h, palomo jm. threedimensional evalua@on of upper airway following rapid maxillary expansion a cbct study. angle orthodon-st; 84. epub ahead of print 2014. doi: 10.2319/012313-71.1. 23. paul d, varma s, ajith v. airway in class i and class ii skeletal parern: a computed tomography study. contemp clin dent; 6. epub ahead of print 2015. doi: 10.4103/0976237x.161856. 24. yoo e, murakami s, takada k, et al. tongue volume in human female adults with mandibular prognathism. j dent res; 75. epub ahead of print 1996. doi: 10.1177/0022034596075012070 1. 25. lee rww, sutherland k, chan asl, et al. rela@onship between surface facial dimensions and upper airway structures in obstruc@ve sleep apnea. sleep; 33. epub ahead of print 2010. doi: 10.1093/sleep/33.9.1249. 26. kim yj, hong js, hwang yi, et al. three-dimensional analysis of pharyngeal airway in preadolescent children with different anteroposterior skeletal parerns. american journal of orthodon-cs and dentofacial orthopedics; 137. epub ahead of print 2010. doi: 10.1016/j.ajodo.2009.10.025. 27. chauhan a, autar r, pradhan k, et al. comparison of pharyngeal airway dimension, tongue and hyoid bone posi@on based on anb angle. natl j maxillofac surg; 6. epub ahead of print 2015. doi: 10.4103/09755950.168237. http://dentistry3000.pitt.edu/ vol 7, no 1 (2019) issn 2167-8677 (online) doi 10.5195/d3000.2019.85 http://dentistry3000.pitt.edu systematic literature review of emergency department physicians’ confidence to treat dental pain and the frequency with which they prescribe pain medications: considerations for improved outcomes sara barna1, jean o’donnell1, marnie oakley1 1university of pittsburgh school of dental medicine, pittsburgh, pa, usa abstract opioid analgesics, when taken as prescribed, are effective therapeutic options that provide pain relief for moderate to severe pain. the use of opioids in the treatment of pain has been increasing in the u.s. at an alarming rate, possibly contributing to the simultaneous rise in opioid abuse. emergency departments play a major role in managing patients who present in pain, with approximately 10% of all opioid analgesic prescriptions written in hospital emergency departments. it is estimated that dental pain patients represent between 0.3-4% of the overall patient emergency department workload. yet, the literature suggests that many of these physicians may not have sufficient training in handling dentofacial emergencies. the goal of this study was to systematically review the available literature on the topics of: 1) emergency department physicians’ training related to treating dental pain patients, and 2) the frequency in which they prescribe opioid medications to these patients. methods: a systematic literature review was conducted among publications from 1985-2014 in the databases pubmed, ovid, and science citation index. the following search terms were used in this systematic literature review in order to identify the available literature of interest: “opioid and dental and emergency departments,” “dental pain and drug abuse,” “er physicians and dental pain,” “er physicians and drug abuse,” and “dental pain and emergency departments.” publications in any language or country were considered, as well as editorials and commentaries. findings: a total of 769 publications were identified. seventeen publications met the criteria for inclusion. eight studies commented on the emergency department physicians’ perceived “lack of training” in handling dentofacial emergencies and found that the majority of this group did not feel comfortable in managing dental patients. nine studies assessed the frequency in which physicians prescribe pain medications to dental patients. within these 9 studies, 5 specifically reported that between 29.6% and 81% of dental patients treated, received an “opioid” or a “narcotic” upon discharge. the remaining 4 studies in this group instead used non-specific terms that included “prescription medications,” “analgesics,” “pain medicine,” and “pharmacotherapy” to describe their findings and did not particularly report opioid prescribing trends. of those publications rejected, 9 addressed the topic of dental pain patients presenting to non-dental providers, but did not include data that met the criteria related to emergency department prescribing frequency or physician training. conclusions: this review of the literature suggests that emergency department physicians’ training level in treating dentofacial pain is less than ideal. it also confirms that individuals presenting to emergency departments with dental pain are a subset of the population of patients who are prescribed opioids as an analgesic. coupling these results with the increased use of opioids in this country, dentists are in a key position to collaborate with emergency department physicians to help positively affect change. to further justify this approach, research agendas must carefully monitor prescribing patterns for dentofacial pain in the emergency department that are specific to opioid use, carefully excluding other non-narcotic analgesics. should comparable outcomes of data related to the same topic in other non-dental settings exist, additional areas in medicine that may benefit from this partnership may also be identified. moving forward, this interprofessional team approach may include a presence in medical school and residency program curricula so that alternative treatment options for addressing dental pain patients can be presented that consider the increased prescribing trends of opioids. citation: barna s, et al. (2019) systematic literature review of emergency department physicians’ confidence to treat dental pain and the frequency with which they prescribe pain medications: considerations for improved outcomes. dentistry 3000. 1:a001 doi:10.5195/d3000.2019.85 received: august 17, 2018 accepted: september 18, 2018 published: june 26, 2019 copyright: ©2019 barna s, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: barnas1589@gmail.com introduction opioid analgesics, when taken as prescribed, are effective therapeutic options that provide adequate pain relief for moderate to severe pain. prescription opioids are essential pain management tools; however, balancing their adverse effects and abuse potential with appropriate pain relief can be challenging (1). recently, the increase in the prescribing of opioids for chronic non-malignant pain has increased accessibility, and most likely has contributed to the growing trend of nonmedical use as well as nonfatal and fatal overdoses (2). according to the center for disease control and prevention (cdc) latest policy report, it is estimated that nearly 3 out of 4 prescription drug overdoses are caused by opioid pain relievers (3). in 2010, approximately 7.0 million people were current users of psychotherapeutic (pain relievers, tranquilizers, stimulants, http://dentistry3000.pitt.edu/ systematic literature review of emergency department physicians’ confidence to treat dental pain and the frequency with which they prescribe pain medications: considerations for improved outcomes vol 7 no 1 (2019) doi 10.5195/d3000.2019.85 http://dentistry3000.pitt.edu sedatives) drugs taken nonmedically--nearly 3 percent of the u.s. population. (4). more than three out of four people who misuse prescription pain relievers use drugs prescribed to someone else (3). these trends continue to generate great concern among providers and government officials alike. it has been estimated that approximately 10% of opioid analgesic prescriptions for people 20-39 years of age are written in hospital emergency departments (5). these emergency departments play an important role in the health care system and act as the “provider of last resort” for millions of americans who lack adequate access to health care professionals, including dentists (4). and, although short-term analgesics are critical for acute pain management, long-term use of these medications creates a potential for substance abuse and drug-seeking behavior (6). many patients lack access to appropriate dental care due to barriers such as practice hours and lack of dental insurance (7). as a result, these patients often turn to medical providers when they experience dental pain (7). several studies have shown that there has been a substantial increase in prescribing pain medications by non-dental providers (6, 8). in 2010, there were approximately 129.8 million emergency department visits, and it is estimated that dentofacial emergencies represent between 0.3% and 4.0% of the overall patient emergency department workload (9, 7, 5). given these trends, emergency department providers should be equipped to diagnose, provide basic treatment, and ensure appropriate follow-up care for dental problems. patients receiving limited and focused dental care in this setting could potentially decrease the need for prescribed opioid analgesics. however, the literature suggests that emergency department physicians do not feel confident in managing dentofacial emergencies and are interested in additional education (9, 10, 7). these data suggest that emergency department providers may require more extensive training in alternative treatment procedures such as local anesthetic administration, nerve blocks or immobilization in order to respond to emergency department visits of dentofacial origin. this paper systematically reviewed the available literature that relates to emergency department physicians’ confidence in treating dental pain patients and the frequency with which they prescribe pain medication for these patients. results are summarized and suggestions for improving outcomes are discussed. methods the literature review by the author (smb) included a search of pubmed, ovid and science citation index databases using the keywords “dental pain and drug abuse,” “er physicians and dental pain,” “er physicians and drug abuse,” and “dental pain and emergency departments” for studies published from 1985-2014. the following search terms were used because they provided the greatest number of publications pertaining to the topic. publications, editorials, and commentaries in any language or country regarding dental pain patients and the emergency department were considered. the editorials and commentaries that were included pertained information that was relevant to the topic, strengthening the (discussion) that emergency physicians may not feel confident handling dentofacial emergencies. in july of 2014, a total of 769 publications were identified. these publications were then reviewed and a total of seventeen publications were selected based on relevance of the title and abstract. results publications that commented on the emergency department physicians’ clinical training related to treating dental pain patients: eight studies commented on emergency department physicians’ perceived “lack of training” in handling dentofacial emergencies. the studies that had surveyed the physicians found that the majority of them did not feel comfortable in managing http://dentistry3000.pitt.edu/ systematic literature review of emergency department physicians’ confidence to treat dental pain and the frequency with which they prescribe pain medications: considerations for improved outcomes vol 7 no 1 (2019) doi 10.5195/d3000.2019.85 http://dentistry3000.pitt.edu dental patients (7,9). of these 8 articles, however, only 2 articles reported statistical evidence where emergency department physicians did not feel confident in managing dentofacial emergencies. the other 6 articles stated that emergency department physicians lack training on treating dental pain patients, but did not survey the physicians in order to gather statistical evidence. the author of this systematic review (smb) checked the references of these 8 articles in order to verify whether a referenced source had conducted a study and provided statistical evidence. trivedy et al. surveyed 103 emergency department physicians in various london hospitals as well others in the uk. the majority of participants (76.5%) did not receive any clinical training in handling dental emergencies and indicated that they do not feel confident in managing certain dentofacial emergencies presenting to the emergency department. skapetis et al. mentioned how “medical personnel lack both knowledge and training in dental emergencies.” furthermore, skapetis et al. referenced a research article that surveyed 102 emergency medical personnel and found that 52% had no previous training in “examination of the mouth.” both of these studies were conducted outside of the united states. the other 6 publications did not survey physicians in order to determine if they were confident in treating dental pain patients in the emergency departments. rather, they referenced other studies that surveyed physicians and/or referenced one another for the purpose of emphasizing the fact that emergency department physicians do not feel confident in handling dentofacial emergencies. publications that assessed the frequency in which emergency department physicians prescribed pain medication for dental patients: nine studies assessed the frequency in which physicians prescribe pain medications to dental patients. within these 9 studies, 5 specifically reported that between 29.6% and 81% of dental patients treated received an “opioid” or a “narcotic” upon discharge. the remaining 4 studies in this group instead used nonspecific terms that included “prescription medications,” “analgesics,” “pain medicine,” and “pharmacotherapy” to describe their findings and did not particularly report opioid prescribing trends. of those publications rejected in this review, 9 addressed the topic of dental pain patients presenting to non-dental providers, but did not include data that met the criteria related to emergency department prescribing frequency or physician training. these studies mentioned information such as: techniques on how to manage dental patients in the emergency department, the national drug abuse problem, how dentists can help address the issue, and some of the more common dental emergencies (figure 1). notably, according to the studies in figure 1, emergency department physicians prescribe analgesics and/or opioids at variable frequencies. this could be contributed to factors such as the location of the study, the time frame, data collection methods, or the type of intervention that was conducted at each individual site. for example, lewis et al looked at a 4-year period from 1997 to 2000 and found that approximately 72.4% of dental pain patients in the united states were prescribed an analgesic at the emergency department. these authors used data from the center of disease control and prevention. however, okunseri et al looked at the time frame between 1997-2000 and 2003-2007 and found that dental pain patients in the emergency department were prescribed opioid analgesics only 43% of the time. these authors used data from the national hospital ambulatory medical care survey. limitations there were limitations to this review that should be noted. the authors of the reviewed http://dentistry3000.pitt.edu/ systematic literature review of emergency department physicians’ confidence to treat dental pain and the frequency with which they prescribe pain medications: considerations for improved outcomes vol 7 no 1 (2019) doi 10.5195/d3000.2019.85 http://dentistry3000.pitt.edu publications varied in their description of “pain medication,” therefore, the studies were not consistent in vocabulary used. the different terminology used without specifically stating the type of medication prescribed could result in an incorrect estimate of the frequency of prescribing pain medication (to include opioids) to dental pain patients in the emergency department. additional studies with consistent definitions of pain medications are needed to fully understand the scope of the problem. there were few studies that met the criteria to be included in the systematic review, suggesting that more research is this area is warranted. also, the time frame of the studies varied, which could account for the difference in the prescribing frequencies. discussion prescription drug abuse is a national epidemic and dentists as well as physicians are in key positions to help address this pertinent health care issue. the outcome of this systematic review reveals that there is an increasing trend in emergency department visits for dental issues, and that these patients are frequently treated with prescriptions for antibiotics and/or pain medication (15, 25). the underlying dental issue may never be addressed in any setting due to issues such as cost, access, insurance coverage, or dental treatment limitations. the following are suggestions targeted to improve oral health treatment of patients who present with dental pain to the emergency department. these suggestions will also help address the national drug abuse epidemic, while still achieving adequate pain relief for dental patients presenting to emergency departments. one suggestion is to improve the oral health clinical training in medical school and residency programs to include other treatment options, such as administering local anesthetics through infiltration and/or nerve block techniques. the results from a national united states survey in 2009 found that most medical schools only offered 1-2 hours (28.4%), or 3-4 hours (30.7%) of oral health curriculum during a student’s four years of training. furthermore, the same study notes that 1 in 10 schools offered no oral health curriculum (10.2%) (30). currently, there are no studies that have evaluated the application of hands-on clinical training to physicians in administering local anesthetics to dental pain patients in the emergency department. administering local anesthetics coupled with prescribing nonopioid medications to dental pain patients in the emergency department can provide adequate relief until the patient can be evaluated by a dentist in a nonemergent setting at a later time. this technique may result in a decrease in prescribing opioids in the emergency department, yet still achieve effective pain relief for these dental pain patients (2). another suggestion is to implement prescribing guidelines into the emergency departments to assist emergency department physicians in treating dental pain patients. some studies found that implementing prescribing guidelines drastically reduced the amount of opioids prescribed for patients presenting to the emergency department in pain (1, 13). ma et al found that access to guidelines reduced narcotic prescriptions by 20.1%. gugelmann et al implemented a multifaceted educational program into 2 urban emergency departments, and the relative opioid discharge orders decreased by 39.6% and 56.2% for the primary and affiliated hospital, respectively. these guidelines highlight recent literature that supports using high doses of nonsteroidal anti-inflammatory drugs (nsaids) as an equally effective modality of care for patients with dental pain (2). in summary, the literature suggests that the rate of prescribing pain medications to dental pain patients in the emergency department has been increasing (8, 6). a center for disease control and prevention http://dentistry3000.pitt.edu/ systematic literature review of emergency department physicians’ confidence to treat dental pain and the frequency with which they prescribe pain medications: considerations for improved outcomes vol 7 no 1 (2019) doi 10.5195/d3000.2019.85 http://dentistry3000.pitt.edu (cdc) report from 2010 found that analgesics were the most mentioned medication class (i.e. medication given in the emergency department or prescribed at discharge) in the united states emergency departments at approximately 34.9%. this number included narcotic and non-narcotic analgesics as well as nsaids (national hospital ambulatory medical care survey: 2010 emergency department summary tables). providing additional education to emergency department physicians may help bridge the gap in care for patients who present with dental pain. through additional education, heightened awareness and/or guidelines for prescribing, physicians may be better equipped to address underlying dental problems of patients who present to emergency departments with dental pain. acknowledgements s.b. was supported by the university of pittsburgh school of dental medicine dean’s summer research program. references 1. multidisciplinary intervention decreases the use of opioid medication discharge packs from 2 urban eds. gugelmann h, shofer fs, meisel zf, perrone j.am j emerg med. 2013 sep;31(9):1343-8. doi: 10.1016/j.ajem.2013.06.002 2. prevention of prescription opioid abuse: the role of the dentist. 3. denisco rc, kenna ga, o'neil mg, kulich rj, moore pa, kane wt, mehta nr, hersh ev, katz np. j am dent assoc. 2011 jul;142(7):800-10. 4. policy impact: prescription painkiller overdoses. centers for disease control and prevention. policy impact: prescription painkiller overdoses. http://www.cdc.gov/homeandr ecreationalsafety/rxbrief/ accessed august 4, 2014. 5. national institute of drug abuse. topics in brief: prescription drug abuse. http://www.drugabuse.gov/pu blications/topics-inbrief/prescription-drug-abuse. accessed august 4, 2014. 6. centers for disease control and prevention. addressing prescription drug abuse in the united states. 2013;1-36. http://www.cdc.gov/homeand recreationalsafety/pdf/hhs_pr escription_drug_abuse_report_ 09.2013.pdf. accessed august 4, 2014. 7. medications prescribed in emergency departments for nontraumatic dental condition visits in the united states. okunseri c, okunseri e, thorpe jm, xiang q, szabo a. med care. 2012 jun;50(6):508-12. doi: 10.1097/mlr.0b013e318245a5 75. 8. managing dental emergencies: a descriptive study of the effects of a multimodal educational intervention for primary care providers at six months. skapetis t, gerzina t, hu w. bmc med educ. 2012 oct 30;12:103. doi: 10.1186/1472-6920-12103. 9. prescription of opioid and nonopioid analgesics for dental care in emergency departments: findings from the national hospital ambulatory medical care survey. okunseri c, okunseri e, xiang q, thorpe jm, szabo a. j public health dent. 2014 fall;74(4):283-92. doi: 10.1111/jphd.12055. 10. the attitudes and awareness of emergency department (ed) physicians towards the management of common dentofacial emergencies. trivedy c, kodate n, ross a, alrawi h, jaiganesh t, harris t, anderson je. dent traumatol. 2012 apr;28(2):121-6. doi: 10.1111/j.16009657.2011.01050.x. 11. expanding the physician's role in addressing the oral health of adults. cohen la. am j public health. 2013 mar;103(3):40812. doi: 10.2105/ajph.2012.300990 12. [11] reducing the burden of dental patients on the busy hospital emergency department. mccormick ap, abubaker ao, laskin dm, gonzales ms, garland s. j oral maxillofac surg. 2013 mar;71(3):475-8. doi: 10.1016/j.joms.2012.08.023 13. dental complaints in emergency departments: a national perspective. lewis c, lynch h, johnston b. ann emerg med. 2003 jul;42(1):93-9. 14. effect of education and guidelines for treatment of uncomplicated dental pain on patient and provider behavior. ma m, lindsell cj, jauch ec, pancioli am. ann emerg med. 2004 oct;44(4):323-9. http://dentistry3000.pitt.edu/ systematic literature review of emergency department physicians’ confidence to treat dental pain and the frequency with which they prescribe pain medications: considerations for improved outcomes vol 7 no 1 (2019) doi 10.5195/d3000.2019.85 http://dentistry3000.pitt.edu 15. emergency department visits for dental care of nontraumatic origin. quin onez c, gibson d, jokovic a, locker d. community dent oral epidemiol. 2009 aug;37(4):366-71. doi: 10.1111/j.16000528.2009.00476.x. 16. visiting the emergency department for dental problems: trends in utilization, 2001 to 2008. lee hh, lewis cw, saltzman b, starks h. am j public health. 2012 nov;102(11):e7783. doi: 10.2105/ajph.2012.300965. emergency dental anesthesia blocks. daymude ml, hilliard mw. adv emerg nurs j. 2007;29(2):172-179. 17. emergency department bane-dental pain used to obtain narcotics. hupp jr. j oral maxillofac surg. 2013 dec;71(12):2009-10. doi: 10.1016/j.joms.2013.09.028 18. doctor, my tooth hurts: the costs of incomplete dental care in the emergency room. davis ee, deinard as, maï ga ew. j public health dent. 2010 summer;70(3):205-10. doi: 10.1111/j.17527325.2010.00166.x. 19. the root of the problem. mckenna m. ann emerg med. 2010;55(6):17-19. 20. dental emergencies presenting to a general hospital emergency department in hobart, australia. verma s, chambers i. aust dent j. 2014 sep;59(3):329-33. doi: 10.1111/adj.12202. 21. managing dental pain in the emergency department: dental disparities with practice implications. dowling evans d, gisness c. adv emerg nurs j. 2013 apr-jun;35(2):95-102. doi: 10.1097/tme.0b013e31828f70 1e. 22. cohen l, bonito a, eicheldinger c et al. comparison of patient visits to emergency departments, physician offices, and dental offices for dental problems and injuries. j public health dent. 2011;71:13-22. 23. visits to non-dentist health care providers for dental problems. cohen la, manski rj. fam med. 2006 sep;38(8):556-64. 24. pain and prescription monitoring programs in the emergency department. todd kh. ann emerg med. 2010 jul;56(1):24-6. doi: 10.1016/j.annemergmed.2010. 02.025. 25. open wide! dental settings are an untapped resource for substance misuse screening and brief intervention. mcree b. addiction. 2012 jul;107(7):1197-8. doi: 10.1111/j.13600443.2012.03777.x. 26. dental visits to a north carolina emergency department: a painful problem. hocker mb, villani jj, borawski jb, evans cs, nelson sm, gerardo cj, limkakeng at. n c med j. 2012 sep-oct;73(5):346-51. 27. dentistry in the emergency department. gibson de, verono aa. j emerg med. 1987;5(1):3544. 28. common dental emergencies. evaluation and management for emergency physicians. klokkevold p. emerg med clin north am. 1989 feb;7(1):29-63. 29. community-wide emergency department visits by patients suspected of drug-seeking behavior. zechnich ad, hedges jr. acad emerg med. 1996 apr;3(4):312-7. 30. teaching oral health in u.s. medical schools: results of a national survey. ferullo a, silk h, savageau ja. acad med. 2011 feb;86(2):226-30. doi: 10.1097/acm.0b013e3182045a 51 31. a performance improvement prescribing guideline reduces opioid prescriptions for emergency department dental pain patients. fox tr, li j, stevens s, tippie t. ann emerg med. 2013 sep;62(3):237-40. doi: 10.1016/j.annemergmed.2012. 11.020 http://dentistry3000.pitt.edu/ microsoft word kaufer proof 4-8.docx   vol  3,  no  1  (2015)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2015.33           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.   agenesis  of  all  third  molars  in  two  half  siblings   jamie  a.  kaufer1       1  university  of  pittsburgh  school  of  dental  medicine,  pittsburgh  pa,  usa     abstract     tooth  agenesis   is  one  of   the  most   common  dental   anomalies   and   is   influenced  by   factors  including  pa0ent  gene#cs.  although  there  are  several  specific  genes  associated  with   certain  pa*erns  of  agenesis,  there  does  not  seem  to  be  a  gene4c  pa*ern  iden4fied  to  date   that   is   associated  with   isolated   complete   third  molar   agenesis.   this   report   presents   two   half-­‐siblings  who  both  express  complete  agenesis  of  third  molars  despite  the  fact  that  their   shared  parent  does  not  express   the   same  phenotype.   the   case  discussion   focuses  on  ad-­‐ dressing   the   poten-al   gene-c   possibili-es   including   autosomal   dominant   inheritance   and   variable   expressivity   of   a  muta!on.     there   remains   an   uncertainty   to   the   exact   gene   in-­‐ volved.  poten+al  op+ons  include  wnt10a  and  pax9.  the  affected  individuals  are  not  need-­‐ ing  extrac+ons  or  experiencing  complica+ons  due  to  the  presence  of  third  molars.    howev-­‐ er,  the  concern  remains  that  there  is  the  poten.al  for  more  severe  expression  of  the  muta-­‐ !on  in  future  genera!ons.     cita%on:  kaufer   ja.   (2015)  agenesis   of   all   third   molars   in   two   half   siblings.   den$stry   3000.   1:a001  doi:10.5195/d3000.2015.33   received:  april  4,  2015   accepted:    april    8,  2015   published:    april  24,  2015   copyright:  ©2015  kaufer  ja.  this  is  an  open  ac-­‐ cess  ar!cle   licensed  under  a  crea!ve  commons   a"ribu%on  work  4.0  united  states  license.   email:  akl33@pi(.edu   introduction   the  agenesis  of  teeth  has  been  con-­‐ tinuously  reported  as  one  of  the   most  common  dental  abnormalities   in  humans.  the  classifications  of   missing  teeth  are  in  three  catego-­‐ ries:  hypodontia  is  defined  as  miss-­‐ ing  one  to  six  teeth,  oligodontia   meaning  missing  more  than  six   teeth,  and  anodontia  missing  all   teeth.  the  definitions  of  both  hypo-­‐ dontia  and  oligodontia,  however,   exclude  third  molars  (commonly   known  as  wisdom  teeth).  the  agen-­‐ esis  of  third  molars  is  a  very  com-­‐ mon,  if  not  the  most  common,  pat-­‐ tern  of  tooth  agenesis  [1].  many   genes  have  been  associated  with   tooth  development  [2].  certain   genes,  including  pax9,  msx1,  axin2,   eda,  edar,  edaradd,  and  wnt10a   have  been  associated  with  specific   patterns  of  tooth  loss.  however   most  instances  of  tooth  agenesis  are   not  associated  with  any  of  these   genes.  pax9  has  been  associated   with  third  molar  agenesis,  but  no   coding  mutations  in  that  gene  have   been  identified  that  explain  isolated   third  molar  agenesis  [3].  this  report   describes  a  case  of  third  molar   agenesis  between  two  half-­‐siblings   followed  by  a  discussion  of  poten-­‐ tial  genetic  influence.     case  report   a  24-­‐year  old  caucasian  female  re-­‐ ported  to  the  university  of  pitts-­‐ burgh  school  of  dental  medicine  for   routine  care.  upon  taking  radio-­‐ graphs,  it  was  observed  that  her   third  molars  were  not  present.  the   patient  reported  that  her  third  mo-­‐ lars  were  not  extracted  and  had   never  erupted.  when  asked  further,   the  patient  stated  that  her  half-­‐ brother,  15  years  old,  was  also   missing  all  of  his  third  molars,  and   that  all  parents  of  the  siblings  had  at   least  two  wisdom  teeth.  regarding   the  pedigree  (figure  1),  the  only   information  that  could  be  complet-­‐ ed  was  of  the  immediate  parents.   individual  2.5,  the  mother  of  indi-­‐ vidual  3.4,  had  two  wisdom  teeth,   both  in  the  maxillary  arch  and  fully   erupted.  individual  2.3,  the  mother   of  individual  3.3,  had  all  four  wis-­‐ dom  teeth  removed.  individual  2.4,   the  father  of  both  individuals  3.3   and  3.4,  had  three  third  molars  re-­‐ moved.  no  known  genetic  testing   for  any  individuals  in  the  family  re-­‐ garding  tooth  agenesis  has  been   completed.    it  should  be  noted  that   individual  3.3  is  missing  his  maxil-­‐ lary  first  premolars  as  well,  but  the-­‐    agenesis  of  all  third  molars  in  two  half  siblings   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.33    http://dentistry3000.pitt.edu   2   se  were  extracted  in  preparation  for   orthodontic  treatment.     discussion     in  most  research  regarding   tooth  agenesis,  third  molars  are  of-­‐ ten  excluded  from  the  studies.  it  is   for  that  reason  that  it  is  difficult  to   discuss  the  exact  potential  genetic   cause  of  agenesis  of  third  molars.   for  this  particular  case,  there  seems   to  be  some  history  of  missing  third   molars  for  individual  3.4  in  both  of   her  parents.  however,  neither  par-­‐ ent  is  missing  all  four  of  their  third   molars.  individual  3.3  has  only  one   parent,  individual  2.4,  who  is  miss-­‐ ing  any  third  molars.  however,  de-­‐ spite  the  difference  in  parental   presence  of  third  molars,  the  same   phenotype  resulted  in  individuals   3.3  and  3.4.     as  mentioned  previously,   individual  2.5  is  missing  both  third   molars  in  the  mandible,  which  sug-­‐ gests  a  particular  pattern  of  agene-­‐ sis  associated  with  a  genetic  influ-­‐ ence.  for  individual  3.4,  there  is  a   chance  that  given  one  parent  ex-­‐ presses  a  phenotype  of  partial   agenesis  that  her  phenotype  of   complete  agenesis  is  the  result  of  an   inherited  mutation  with  more  se-­‐ vere  expression.  as  for  individual   3.3,  there  could  be  a  chance  of  in-­‐ herited  mutation  from  individual   2.4  or  2.3,  or  a  spontaneous  muta-­‐ tion.     considering  individuals  3.3   and  3.4  have  the  same  phenotype   and  only  one  shared  parent,  there  is   also  the  potential  that  there  may  be   a  pattern  of  inheritance  in  this  fami-­‐ ly.  there  is  a  good  chance  that  this   case  follows  an  autosomal  dominant   pattern  of  inheritance.  using  the   pedigree  as  a  reference,  it  is  unlike-­‐ ly  that  whatever  genetic  mutation   took  place  is  x-­‐linked  because  there   is  not  a  preference  for  a  particular   biological  sex.  it  is  also  unlikely  that   this  mutation  followed  an  autoso-­‐ mal  recessive  pattern  of  inher-­‐ itance.  in  order  for  this  case  to  be   recessive,  multiple  people  in  the   family  would  be  carriers  of  the   gene,  which  would  be  very  unlikely.       there  is  also  the  question  of   which  genes  may  be  involved  in  the   agenesis  molars  in  this  case.   wnt10a  mutations  are  the  most   common  identifiable  cause  of  hypo-­‐ dontia.  pax9,  a  homeo-­‐ box  gene  on  chromosome   14,  has  also  been  associ-­‐ ated  with  preferential   agenesis  of  the  molars   due  to  haploinsufficiency.   haploinsufficiency  means   that  missing  one  copy  of   the  gene  decreased  the   amount  of  translated   protein,  therefore  in-­‐ creasing  the  chance  of   missing  teeth  [4].  anoth-­‐ er  possibility  for  these   two  individuals  is  varia-­‐ ble  expressivity  of  the   mutation  as  it  is  passed   down  to  further  genera-­‐ tions,  increasing  the   number  of  teeth  missing.   however,  because  a  genetic  test  for   this  condition  is  yet  to  be  devel-­‐ oped,  none  of  these  options  can  be   confirmed  with  complete  certainty.       there  could  be  some  impli-­‐ cations  both  in  terms  of  dentistry   and  the  future  of  those  affected.   agenesis  of  third  molars  could  be   beneficial  in  that  they  will  not  influ-­‐ ence  relapse  following  orthodontic   treatment,  or  put  the  patient  at  risk   of  impaction  and  need  for  oral  sur-­‐ gery.  there  may  not  be  any  disad-­‐ vantages  in  terms  of  dentistry,   however  if  these  genes  can  be   passed  on,  those  involved  may  have   some  considerations.  if  this  muta-­‐ tion  only  affects  the  third  molars,   there  would  be  little  to  worry  about   in  passing  down  the  mutation.   however,  if  this  mutation  has  the   potential  to  cause  other  teeth  to  be   missing,  it  may  be  important  for   those  affected  to  know.       conclusion     these  two  individuals  ex-­‐ hibit  a  very  common  dental  abnor-­‐ mality  that  is  most  likely  caused  by   an  autosomal  dominant  mutation.   there  is  little  reason  to  be  con-­‐    agenesis  of  all  third  molars  in  two  half  siblings   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.33    http://dentistry3000.pitt.edu   3   cerned  for  the  future  generations,   unless  there  is  also  an  association   with  impairment  of  overall  health.     references   1. genetic  basis  of  dental   agenesis-­‐-­‐molecular  genet-­‐ ics  patterning  clinical  den-­‐ tistry.  chhabra  n,  goswami   m,  chhabra  a.  med  oral   patol  oral  cir  bucal.  2014   mar  1;19(2):e112-­‐9.  re-­‐ view.  pmid:  24121910       2. the  genetic  basis  of  tooth   development  and  dental  de-­‐ fects.  thesleff  i.  am  j  med   genet  a.  2006  dec   1;140(23):2530-­‐5.  review.   pmid:  16838332     3. association  between  poly-­‐ morphism  in  the  promoter   region  (g/c-­‐915)   of  pax9  gene  and  third  mo-­‐ lar  agenesis.  bianch  fj,  de   oliveira  tf,  saito  cb,  peres   rc,  line  sr.  j  appl  oral  sci.   2007  oct;15(5):382-­‐6.   pmid:  19089165     4. vieira,  alexandre  r  (may   2012)  genetics  of  congeni-­‐ tal  tooth  agenesis.  els.  john   wiley  &  sons,   ltd:chichester.  doi:   10.1002/9780470015902.a 0023576     microsoft word morieira proof feb 11.docx   vol  3,  no  1  (2015)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2015.29           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.   profile  of  fluoride  release  from  a  nanohybrid  composite  resin   raquel  assed  bezerra  silva1,  fernanda  regina  ribeiro  santos1,  augusto  cesar  cropanese  spadaro2,  ana  cris)na  morseli  polizello2,  andiara  de   rossi1,  marilia  rodrigues  moreira1,  paulo  nelson-­‐filho1     1  department  of  pediatric  clinics,  preventive  and  community  dentistry,  school  of  dentistry  of  ribeirão  preto,  university  of  são  paulo,  ribeirão  preto,  sp,  brazil   2  department  of  physics  and  chemistry,  school  of  pharmaceutical  sciences  of  ribeirão  preto,  university  of  são  paulo,  ribeirão  preto,  sp,  brazil   abstract   the  aim  of   this   study  was   to   evaluate   in   vitro   the   amount   and  profile  of   fluoride   release   from  a  fluoride-­‐containing  nanohybrid  composite  resin  (tetric®  n-­‐ceram)  by  direct  poten1-­‐ ometry.  thirty  specimens  (5  mm  diameter  x  3  mm  high;  n=10/material)  were  made  of  tet-­‐ ric®  n-­‐ceram,  vitremer®  resin-­‐modified  glass  ionomer  cement  (rmgic)  (posi8ve  control)  or   filtek®   z350  nanofill   composite   resin   (nega8ve   control).   the   specimens  were   stored   indi-­‐ vidually  in  plas,c  tubes  containing  1  ml  of  ar,ficial  saliva  at  37°c,  which  was  daily  renewed   during  15  days.  at  each  renewal  of  saliva,  the  amount  of  fluoride  ions  released  in  the  solu-­‐ !on  was  measured  using  a  fluoride  ion-­‐selec%ve  electrode  with  ion  analyzer,  and  the  values   obtained  in  mv  were  converted  to  ppm  (µg/ml).  data  were  analyzed  sta=s=cally  by  anova   and  tukey’s  post-­‐hoc  test  at  a  significance  level  of  5%.  the  results  showed  that  the  resins   tetric®  n-­‐ceram  and  filtek®  z350  did  not  release  significant  amounts  of  fluoride  during  the   whole  period  of  evalua,on   (p>0.05).  only  vitremer®  released  significant  amounts  of  fluo-­‐ ride  ions  during  the  15  days  of  the  experiment,  with  greater  release  in  first  2  days  (p<0.05)   and  stabiliza%on  in  the  subsequent  days  (p>0.05).  in  conclusion,  the  nanohybrid  composite   resin  tetric®  n-­‐ceram  did  not  present  in  vitro  fluoride-­‐releasing  capacity  throughout  the  15   days  of  study.       cita%on:   silva   rab,   santos   frr,   spadoro   acc,   polizello  acm,  de  rossi  a,  moreira  mr,  nelson-­‐ filho  p.   (2015)  profile  of  fluoride  release  from  a   nanohybrid   composite   resin.   den$stry   3000.   1:a001  doi:10.5195/d3000.2015.29   received:  january  1,  2015   accepted:  february  2,  2015   published:  february  23,  2015   copyright:  ©2015  silva  et  al.  this  is  an  open  ac-­‐ cess  ar!cle   licensed  under  a  crea!ve  commons   a"ribu%on  work  4.0  united  states  license.   email:  marilia.moreira@ig.com.br     introduction       the   effect   of   !luoride   on   dental   caries   pre-­‐ vention  appears  to  be  dependent  of  its  con-­‐ stant   presence   at   adequate   levels   on   oral   environment,   which   may   interfere   in   the   dynamics  of  the  carious  lesion  by  a  decrease   in   demineralization   and   increase   in   remin-­‐ eralization   (1,2,3).   since   the  most   common   cause   of   restoration   failure   in   the   dental   clinic   is   the  occurrence  of   secondary  or   re-­‐ current   caries   around   the   restoration  mar-­‐ gins  (4,5),  the  incorporation  of  4luoride  into   restorative   materials   could   improve   the   success  of  restorative  treatment  (2,6,7).  the   intimate   contact   between   the   restoration   and   the   tooth   margins   may   also   prevent   penetration  of  cariogenic  bacteria  and  acids   and   facilitate   the   exchange  of   /luoride  with   the   hydroxyapatite   (8,9).   thus,   the   use   of   restorative  materials   that   not   only   release   !luoride,   but   also   have   adhesivion   to   tooth   structure   has   been   suggested   to   prevent   recurrent  caries  around  restorations  and  to   promote   remineralization   of   incipient   cari-­‐ ous   lesions   on   restored   surfaces9   or   even   on  adjacent  teeth  (10).       although   the   use   of   composite   resins   has   increased   considerably   during   the   last   years,   their   ability   to   release   .luo-­‐ ride   is   low  or   insigni,icant  when  compared   to   other   materials,   such   as   glass   ionomer   cements   (gics)   (11,12).   thus,   there   has   been  a  growing  interest  in  the  development   of   newer   composites   resins   with   ef/icient   !luoride   release   abilities   and   improved   physical  and  mechanical  properties,  such  as   lower  polymerization  shrinkage  (9,13,14).  a   novel   'luoride   containing   composite   resin   (tetric®   n-­‐ceram,   ivoclar   vivadent   ag,   schaan   -­‐   liechtenstein)   was   developed   based   on   nanotechnology,   aiming   to   pro-­‐ mote   low   shrinkage   and   shrinkage   stress,   high   level  of  radiopacity,   low  wear  and   fast   polish   ability,   and   high   gloss.   tetric®   n-­‐ ceram  is  composed  by  dimethacrylates  and   the  nano-­‐!illers  contain  barium  glass,  copol-­‐ ymers,   mixed   oxide   and   ytterbium   tri1luo-­‐ ride   (ybf2),  which,  according   the  manufac-­‐ turer,   was   added   to   provide   radiopacity,   and  simultaneously  release  /luoride.  ybf2  is   a  well  know  radiopaque  agent  used   for  de-­‐ tection  of  secondary  caries  or  imperfections   like  air  bubbles,  but   its   -luoride   ion  release   ability  in  association  with  nanohybrid  parti-­‐ cles   has   not   been   documented.   it   has   been   hypothesized   that   the   presence   of   nano-­‐ sized  'illers  offers  increased  surface  area  to   volume  and  could  provide  a  quicker  release   of  $luoride  (12).     since   the   )luoride   release   ef)icacy   of  restorative  materials  is  largely  dependent   on   their   composition   and   setting   reaction   and   nature   of   +luoride   incorporated,   we   conducted  an   in  vitro  study   to  evaluate  hy-­‐ pothesis   that   the   *luoride-­‐containing   nano-­‐ hybrid   composite   resin   (tetric®   n-­‐ceram)   can   promote   +luoride   release   compared   with   a   non)luoride-­‐releasing   material   (fil-­‐ tek®   z350)   and   high   1luoride-­‐releasing   material  (vitremer®).       material  and  methods      profile  of  fluoride  release  from  a  nanohybrid  composite  resin   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.29    http://dentistry3000.pitt.edu   2     the   materials   used   in   this   study   were   the   'luoride-­‐containing   nanohybrid   composite   resin   tetric   n-­‐ceram®   (ivoclar   vivadent   ag,   liechtenstein),   the   rmgic   vitremer®   (3m   espe,   st.   paul,   mn,   usa),   and   the   nano)ill   composite   resin   filtek   z350®   (3m   espe,   st.   paul,   mn,   usa).   the   materials   composition   and   manufacturer   are  summarized  (table  1).         thirty  specimens  (5  mm  diameter   and   3   mm   thickness)   were   prepared   from   the   three   materials   (n=10)   by   packing   the   material   into   a   custom-­‐made  polytetra,luo-­‐ roethylene  matrix.   the  matrix  was   2illed   at   room   temperature   (approximately   25°c)   and   the   materials   were   mixed   and   light-­‐ activated   according   to   the   manufacturers’   instructions.   the   mass   of   each   specimen   was  recorded  using  a  digital  precision  scale.   vitremer®  specimens  presented  an  average   mass  of  0.143  g,  while  tetric  n-­‐ceram®  and   filtek  z350®  specimens  presented  an  aver-­‐ age  mass  of  0.159  g  and  0.140  g,  respective-­‐ ly.       the   specimens   were   stored   indi-­‐ vidually  in  eppendorf  tubes  containing  1  ml   of  arti(icial  saliva  (school  of  pharmaceutical   sciences  of  ribeirão  preto,  university  of  são   paulo,  sp-­‐brazil)  at  37°c  during  15  days  and   were   daily   re-­‐immersed   in   new   tubes   con-­‐ taining   1   ml   of   fresh   arti0icial   saliva.   the   amount   of   )luoride   released   was   studied   every   24   hours   for   15   days.   each   sample   was  buffered  with  citrate  0.5  mol/l,  ph  5.5,   and   the   (luoride   measurements   were   per-­‐ formed  by  direct  potentiometry  with  an  ion-­‐ selective   electrode   (orion   2008;   thermo   electron   corporation,   orion   products,   bev-­‐ erly,   ma,   usa)15.   for   the   measurements,   0.5   ml   of   arti-icial   saliva   were   collected   from  the  plastic   tubes  containing  the  speci-­‐ mens   and  homogenized  with  0.5  ml  of   the   citrate  buffer,  and  the  resulting  solution  was   homogenized   again   right   before   the   meas-­‐ urement  of  *luoride  release.   the  mean  values  among  the  different  study   groups   were   compared   using   one-­‐way   anova   followed  by  multiple-­‐range  tukey’s   hsd   test   to   identify   the   signi0icant   groups   (p=0.05)   in   the   graphpad   prism   4.0   soft-­‐ ware   (graphpad   software,   inc.,   san   diego,   ca,  usa).  a  signi-icance  level  of  5%  was  set   for  all  analyses.     results       results  were   recorded   in  mv   and   converted   into   ppm.   for   such   purpose,   a   standard   curve   was   prepared   using   11   samples   of   solutions   with   different   +luo-­‐ ride   concentrations.   from   this   standard   curve,   the   results   in   mv   were   converted   to   ppm   and   com-­‐ pared   with   the   val-­‐ ues   obtained   from   the   standard   curve.   the   values   of   ,luo-­‐ ride   release   were   tabulated   as  mean   ±   standard   deviations.   as   shown   in   figure   1,   the   results   of   the   present   study   showed   that,   on   days   1   and   2,   the   mean   value   of   !luoride  release  of  vitremer®  was  the  high-­‐ est   (p<0.05)   and   then   !luoride   release   stabilized   in   the   subse-­‐ quent   days   (p>0.05).   tetric   n-­‐ ceram®   and  filtek   z350®   showed   no   de-­‐ tectable   amounts   of   $luo-­‐ ride   release   through-­‐ out   the   experi-­‐ mental  period  and  presented  no  statistically   signi%icant   differences   (p>0.05)   from   each   other.       discussion       the   advances   in   nanotechnology   have   allowed   the   development   of   novel   composite   resins   with   improved   physical   properties,   high   strength,   good   wear   re-­‐ sistance,   and  excellent  esthetics   (13).  how-­‐ ever,   few   composites   are   reported   to   in-­‐ clude  in  their  composition  0luoride  that  may   be   ef%iciently   released   to   oral   environment   by   ion   exchange   or   hydrolysis   (16).   it   has   been  recently  suggested  that  0luoride  incor-­‐ poration   into   materials   containing   nano   sized  'illers  could  favor  its  faster  release  by   a   higher   surface   area-­‐to-­‐volume   ratio12   nanocomposites  with   high   strength   can   be   promising  materials  if  they  have  the  capaci-­‐ ty   to   release   *luoride,   phosphate   and   calci-­‐ um  ions  for  the  precipitation  of  0luorapatite   (17).  however,  according  to  our  results,  the   nanohybrid   resin   containing   ybf2   3illers   (tetric®  n-­‐ceram)  did  not  present  a  signi0i-­‐ cant   release   of   ,luoride   during   15   days   of   experiment,   having   similar   results   to   those   of   the   negative   control   (a   conventional   hy-­‐ brid   composite).   our   results   con3irmed   those   of   previous   studies,   which   found   a   constant  low  level  of  -luoride  released  from   different  composite  resins  with  ybf2   !illers   in  their  composition  (18,19).       the  low  (luoride  release  from  cur-­‐ rent   commercially   available   composite   res-­‐ ins   has   been   associated   with   the   low   amount   of   )luoride   incorporated   into   these   materials  as  *illers,  low  solubility  of  ybf2  in   water   (2,10),   low  water   content  of   the  ma-­‐ terial,   and   composite   resin   permeability   table  1.  restorative  materials  used  in  this  study.   material   manufacturer   classi&ication   glass  ionomer  ce-­‐ ment  vitremer®   3m  dental  prod-­‐ ucts,  st  paul,  min-­‐ nesota,  united   states   glass  ionomer  cement   resin-­‐modi%ied  type  ii   composite  resin   tetric  n-­‐ceram®   ivoclar  vivadent,   são  paulo/sp,   brasil   nanohybrid  compo-­‐ site  resin   composite  resin   filtek  z350®   3m  dental  prod-­‐ ucts,  st  paul,  min-­‐ nesota,  united   states   nanohybrid  compo-­‐ site  resin   figure  1.  release  of  0luoride  ions  from  the  three  materials  at  each  day  of   the  experiment.      profile  of  fluoride  release  from  a  nanohybrid  composite  resin   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.29    http://dentistry3000.pitt.edu   3   (18).  in  addition,  due  to  the  low  solubility  of   ybf2   in  water,   it   is   postulated   that   the   re-­‐ charging   effect   of   ‘.luoride-­‐releasing’   com-­‐ posite   resins   containing   this   agent   may   simply   be   the   release   of   surface-­‐retained   !luoride   (2).   the   incorporation   of   !luoride   into   composite   resins   has   not   shown   any   bene$icial  effect  in  reducing  the  deminerali-­‐ zation   of   carious   lesions   in   roots   when   compared  with  gics(12,20).       aiming   to   solve   this   problems,   novel   nanocomposites   containing   alterna-­‐ tive   &luoride   sources   has   been   developed   (13,19,21).   one   alternative   are   composite   resins   containing   nanoparticles   of   calcium   phosphates  (caf2),  which  may  release  6luo-­‐ ride   ions   that  matched  or  exceeded  report-­‐ ed   releases   from   conventional   gics   and   rmgics   (21).   the   mechanical   properties   and   ion   release   of   these   composites   could   be   tailored   by   changing   the   nanoparticle   !iller  level  and  reinforcing  !iller  level.  these   nanocomposites   present   high   release   of   !luoride,   phosphate   and   calcium   ions   for   precipitation   of   ,luorapatite   and   inhibition   of   caries   together   with   good   mechanical   properties.       the   reason   for   the   relatively   high   !luoride   release   from   these   new   caf2-­‐ containing  nanocomposite  is  likely  the  small   size  and  hence   the  high   surface  area  of   the   nanoparticles19.   other   alternative   are   ex-­‐ perimental   composites  made   of   novel   1luo-­‐ ride-­‐releasing   monomers,   such   as   ternary   heavy-­‐metal-­‐!luoride   chelates,   which   have   been   developed   based   on   the   hypothesis   that   reduction   of   hydrophilic   monomers   and   improvement   of   photoinitiators   could   reduce   water   sorption   and   signi0icantly   increase   the   mechanical   properties   of   the   composite13.   in   the   study   by   ling   et   al.   (2009)   (13),   one   of   these   experimental   composites   showed   signi.icantly   higher   !luoride-­‐release   and   recharge   capabilities   than   commercial   -luoride-­‐releasing   compo-­‐ sites,   but   physical   and   mechanical   proper-­‐ ties   were   still   lower   than   those   of   most   commercial  composite  resins.   to  date,  conventional  gics  and  rmgics  are     still  considered  the  unique  materi-­‐ als   with   higher   ,luoride-­‐releasing   ability   and   may   be   clinically   indicated   to   restore   decayed  non-­‐biting  areas  in  high-­‐caries-­‐risk   patients.  several  studies  have  demonstrated   the   ef&icacy   of   these   materials   as   an   addi-­‐ tional  measure   for   preventing   occlusal   car-­‐ ies   lesions   (1,7,10,19,22)  by  means  of   their   ef#icient   #luoride   release   ability,   which   not   only  may  accelerate  mineral  deposition,  but   also  can  change  the  metabolic  activity  of  the   dental  plaque.       in  the  present  study,  vitremer®,  a   rmgic,  was  selected  as  the  positive  control   due   to   its  wide  use   in   restorative  dentistry   and   its  broad  clinical   indication.   in   fact,  we   found   that   vitremer®   had   a   signi2icant   re-­‐ lease   of   (luoride   ions,   especially   in   the   1st   and  2nd  days  of  evaluation.  our  results  are   in   agreement  with   those   of   a   number   of   in   vitro   studies   that   have   also   shown   higher   !luoride   release   in   the   !irst   24   hours   11,23,24),   with   gradual   decrease   after   48   hours   (12,21).   this   high   initial   4luoride   re-­‐ lease  from  gics  is  due  to  an  acid-­‐base  reac-­‐ tion,   with   the   amount   of   .luoride   released   proportional   to   the   concentration   of   .luo-­‐ ride   in   the  material.  this   is   responsible   for   the  phenomenon  know  as  the  “burst  effect,”   wherein   high   amounts   of   0luoride   are   re-­‐ leased   during   the   +irst   two   days   (17,20).   fluoride  release  declines  rapidly  during  the   !irst  week  and  stabilizes  after   three   to   four   weeks  (7,10,22)  as  a  result  of  the  high  initial   release   from   the   glass   particles   that   are   partially  dissolved  in  polyalkenoic  acid  dur-­‐ ing   the   setting   reaction   (18).   an   additional   property  of  vitremer®  is   its  high  microbial   growth   inhibition   potential   because   it   re-­‐ tains  the  true  characteristics  of  convention-­‐ al   gics,   unlike   .luoride-­‐containing   compo-­‐ site   resins,  which  do  not  have  antibacterial   activity   (25,26).   caries   lesions   adjacent   to   the   restoration   are   likely   to   take   from   sev-­‐ eral   months   to   several   years   to   develop.   thus,   'luoride   must   be   continuously   re-­‐ leased  from  dental  material  for  long  periods   to  help  preventing  the  development  of  new   caries  lesions.         despite   the   well   demonstrated   effects   of   in   vitro   ,luoride   release   on   the   saliva,   dental   plaque   and   hard   dental   tis-­‐ sues,  clinical  studies  have  shown  controver-­‐ sial  results  with  respect  to  the  actual  clinical   relevance   of   +luoride-­‐releasing  materials   in   preventing  or   inhibiting   the   secondary   car-­‐ ies   development   compared   with   materials   without   capacity   of   releasing   3luoride   ions   (7).  therefore  further  research  is  still  need-­‐ ed  for  the  development  of  novel  composites   resins   with   *luoride-­‐release   and   recharge   capabilities,   while   maintaining   their   physi-­‐ cal,   mechanical,   and   aesthetic   properties,   which   are   highly   desirable   to   improve   res-­‐ toration   longevity   in   high-­‐caries-­‐risk   pa-­‐ tients.       references     1.   effects   of   aged   fluoride-­‐containing   re-­‐ storative  materials   on   recurrent   root   car-­‐ ies.  hsu  ys,  donly  kj,  drake  dr  &  wefel   js.   journal   of   dental   research.   1998;     77(2)   418-­‐425.  pmid:  9465175   2.  fluoride-­‐containing  restorative  materials.   burke  fm,  ray  nj  &  mcconnell  rj.   interna-­‐ tional   dental   journal.   2006;     56(1)   33-­‐43.   pmid:  16515011   3.   oral   (luoride   reservoirs   and   the   preven-­‐ tion  of  dental  caries.  vogel  gl.    monographs   in   oral   science.     2011;   22   146-­‐157.   pmid:   21701197   4.   reasons   for   replacement   of   restorations   in   permanent   teeth   in   general   dental   prac-­‐ tice.  mjor   ia,  moorhead   je  &  dahl   je.   inter-­‐ national   dental   journal.   2000;    50(6)   361– 366.  pmid:  11197194   5.  an  overview  of  reasons  for  the  placement   and  replacement  of   restorations.  deligeorgi   v,   mjor   ia   &   wilson   nh.   primary   dental   care.  2001;  8(1)  5-­‐11.  pmid:  11405031   6.   prevention   of   in   vitro   secondary   caries   with   an   experimental   .luoride-­‐exchanging   resin.  zimmerman  bf,  rawls  hr  &  querens   ae.  journal  of  dental  research.  1984;  63(5)   689–692.  pmid:  6584474   7.   review   on   +luoride-­‐releasing   restorative   materials   -­‐   fluoride   release   and   uptake   characteristics,   antibacterial   activity   and   in#luence   on   caries   formation.   wiegand   a,   buchalla   w   &   attin   t.   dental   materials.   2007;    23(3)  343–362.  pmid:  16616773   8.   dependence   of   in   vitro   demineralization   of   apatite   and   remineralization   of   dental   enamel   on   (luoride   concentration.   feather-­‐ stone   jd,   glena   r,   shariati  m  &   shields   cp.   journal   of  dental   research.   1990;     69   620-­‐ 625.  pmid:  2312892   9.  fluoride  release  from  restorative  materi-­‐ als   and   its   effects   on   dentin   demineraliza-­‐ tion.  .  francci  c,  deaton  tg,  arnold  rr,  swift   ej  jr,  perdigão  j,  bawden  jw.  journal  of  den-­‐ tal  research.  1999;    78(10)  1647-­‐54.  pmid:   10520970   10.  effect  of  +luorides  from  various  restora-­‐ tive   materials   on   remineralization   of   adja-­‐ cent   tooth:   an   in   vitro   study.   baliga   ms   &   bhat   ss.   journal   of   indian   society   of   pedo-­‐ dontics  and  preventive  dentistry.  2010;    28   84-­‐90.  pmid:  20660973   11.   fluoride   release   from   glass–ionomer   cements,  compomers  and  resin  composites.   vermeersch  g,  leloup  g  &  vreven  j.  journal   of   oral   rehabilitation.   2001;     28   26-­‐32.   pmid:  11298906   12.   fluoride   release   from   a   new   glass-­‐ ionomer   cement.   neelakantan   p,   john   s,   anand  s,  sureshbabu  n  &  subbarao  c.  oper-­‐ ative   dentistry.   2011;     36(1)   80-­‐85.   pmid:   21488733   13.   novel   f-­‐releasing   composite   with   im-­‐ proved   mechanical   properties.   ling   l,   x   x,   choi   gy,   billodeaux  d,   guo  g  &  diwan  rm.   journal  of  dental  research.  2009;    88(1)  83-­‐ 88.  pmid:  19131323   15.   enamel   +luoride   concentrations   in   une-­‐ rupted   third   molars   and   the   in0luence   of      profile  of  fluoride  release  from  a  nanohybrid  composite  resin   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.29    http://dentistry3000.pitt.edu   4   !luoridated   water   on   caries   scores.   mes-­‐ triner  w  jr,  polizello  ac  &  spadaro  ac.  car-­‐ ies   research.   1996;   30(1)   83-­‐7.   pmid:   8850588   16-­‐rechargeability   of   0luoride   releasing   pit   and   %issure   sealants   and   restorative   resin   composites.  .  steinmetz  mj,  pruhs  rj,  brooks   jc,  dhuru  vb  &  post  ac.  american  journal  of   dentistry.   1997;     10(1)   36-­‐40.   pmid:   9545919   17.   do   low-­‐shrink   composites   reduce   polymerization   shrinkage   effects?   tantbirojn   d,   pfeifer   cs,   braga   rr   &   ver-­‐ sluis   a.     journal   of   dental   research.   2011;     90(5)  596-­‐601.  pmid:  21282725   18.  strength  and  .luoride  release  character-­‐ istics   of   a   calcium   ,luoride   based   dental   nanocomposite.  xu  hhk,  moreau  jl,  sun  l  &   chow   lc.   journal   of   biomaterials.   2008;     29(32)  4261-­‐4267.  pmid:  18708252   19.  fluoride  release  of  six  restorative  mate-­‐ rials   in   water   and   ph-­‐cycling   solution.   garcez   rm,   buzalaf   rm,   buzalaf   ma   &   araújo   pa.     journal   of   applied   of   oral   sci-­‐ ence.   2007;   15(5)   406-­‐411.   pmid:   19089169   20.   novel   caf2   nanocomposite   with   high   strength  and  fluoride  ion  release.  xu  hhk,   moreau  jl,  sun  l  &  chow  lc.  journal  of  den-­‐ tal  research.  2010;    89(7)  739-­‐745.  pmid:   21.   elevated   fluoride   products   enhance   remineralization   of   advanced   enamel   le-­‐ sions.   cate   jm,  buijs  mj,   chaussain  miller  c   &   exterkate   ram.   dental   research.   2008;     87(10)  943-­‐947.  pmid:  20439933   22.   compressive   strength,   2luoride   release   and   recharge   of   ,luoride-­‐releasing   materi-­‐ als.   xu  x  &  burgess   jo.  biomaterials.   2003;     24(14)  2451-­‐2461.  pmid:  12695072   23.   fluoride   release   and   recharge   from   different  materials  used  as  fissure  sealants.   bayrak  s,  tunc  es,  aksoy  a,  ertas  e,  guvenc   d  &  ozer   s.   european   journal   of   dentistry.   2010;    4(3)  245-­‐250.  pmid:  20613911   24.  fluoride  release  and  uptake  capacities  of   !luoride-­‐releasing   restorative   materials.   attar   n   &   turgut   md.   operative   dentistry.   2003;    28(4)  395-­‐402.  pmid:  12877425   25.  fluoride  release  and  antibacterial  activi-­‐ ty   of   selected   dental   materials.   marczuk-­‐ kolada  g,  jakoniuk  p,  mystkowska  j,  lukzaj-­‐ cepowicz   e,   waszkiel   d,   dabrowski   jr   &   leszczynska  k.  postȩpy  higieny  i  medycyny   doświadczalnej.   2006;     60   416-­‐420.   pmid:   18493226   26.  fluoride  content  and  recharge  ability  of   !ive   glassionomer   dental   materials.   mar-­‐ kovic   dl,   petrovic   bb   &   peric   to.   biomed   central  oral  health.  .  2008;    28(8)  21.  pmid:   18655734   27.   comparative   evaluation   of   3luoride   re-­‐ lease   from   prg   –   composites   and   com-­‐ pomer  on  application  of  topical  .luoride:  an   in-­‐vitro  study.  dhull  ks  &  nandlal  b.  journal   of   indian   society   of   pedodontics   and   pre-­‐ ventive   dentistry.   2009;     27(1)   27-­‐32.   pmid:  19414971       vol 1, no 1 (2013)  issn 2167‐8677 (online)  doi 10.5195/d3000.2013.11     http://dentistry3000.pitt.edu this work is licensed under a creative commons attribution 3.0 united states license.  this site is published by the university library system, university of pittsburgh as part of its d‐scribe digital publishing program and is cosponsored  by the university of pittsburgh press.  non‐syndromic oral clefts and risk of cancer: a systematic review daniela araújo veloso popoff1, mateus prates coelho2, daniella reis barbosa martelli1, rajiv saini3, ricardo della cole a4, hercílio martelli‐ júnior1   1 department of den stry, state university of montes claros, montes claros, minas gerais, brazil.  2 department of medicine, state university of montes claros, montes claros, minas gerais, brazil.  3 department of periodontology and oral implantology, rural dental college, ahmednagar, maharashtra, india.  4 department of oral diagnosis, piracicaba dental school, university of campinas, piracicaba, são paulo, brazil.  abstract objec ve: to discuss the risk of cancer among rela ves of individuals with cle  lip and pal‐ ate (cl/p), as well as the risk of cl/p among rela ves of individuals with cancer, since stud‐ ies currently published  have suggested  an increased risk of cancer among rela ves of indi‐ viduals with cle s.  methods: a systema c  literature review was carried out  in accordance with the cochrane  collabora on  group  protocol,  including  literature  search  strategy,  selec on  of  papers  through  the  inclusion and exclusion criteria, data extrac on and quality assessment. pub‐ med, scopus and isi ‐ web of science databases were systema cally searched using the fol‐ lowing search strings: “cle   lip and palate” and “cancer,” “oral cle s” and “cancer” and  “orofacial cle s” and “cancer.”  results: from 653 studies accessed, eight comprised the final sample: six inves ga ng cl/p  index cases and their family history of cancer and two inves ga ng individuals with cancer  and their family history for cl/p. the sample sizes were not homogeneous. oral cle s, the  type of cancer and the degree of kinship family were not categorized in all studies. leuke‐ mia, breast cancer and colon cancer were the most cited types, even as first‐ and second‐ degree rela ves.  conclusion: an increased risk of cancer among rela ves of individuals with cle s could not  be en rely confirmed. however,  studies with  this  specific purpose  suggest  that first‐ and  second‐degrees  rela ves of  individuals with  cle  have  some  types of  cancer more o en  than unexposed families, highligh ng that future studies should expand their samples to in‐ ves gate possible common molecular mechanisms  that allow  rela ng oral cle s and can‐ cer.  cita on: popoff dav, coelho mp, martelli drb, saini r,  cole a rd, and martelli‐junior h. (2013) non‐ syndromic oral cle s and risk of cancer: a systema c  review. den stry 3000 1:a002 doi:  10.5195/d3000.2013.11  received: june 24, 2013  accepted: august 12, 2013  published: august 23, 2013  copyright: ©2013 popoff et al. this is an open‐access  ar cle licensed under a crea ve commons a ribu on  3.0 united states license.  email: danielavelloso@yahoo.com.br introduc on orofacial malformations are the most common form of congenital anomalies in the world [1]. among the orofacial alterations, the most prevalent is the cleft lip with or without cleft palate (cl/p) [2-5], which may occur more commonly in an isolated and non-syndromic form as a speci ic phenotype or, more rarely, composing several associations or syndromes [6,7]. embryologically, clefts result from primary fusion defects of the craniofacial processes that form the primary and secondary palate in the irst intrauterine trimester [8]. the incidence of cl/p varies according to geographical location, racial and ethnic groups, environmental exposures, and socioeconomic status, affecting approximately 1/700 live births with wide variability across geographic origin. generally, asian and amerindian populations have the highest reported birth prevalence rates, often as high as 1/500, european-derived populations have intermediate prevalence rates at about 1/1000, and african-derived populations have the lowest prevalence rates at about 1/2500 [5,9,10]. according to vieira (2008) [7], the last decade was crucial to elucidate issues concerning the etiology of cl/p when compared to other defects observed at birth. as this is a multifactorial trait, environmental risk factors such as smoking, alcohol, parental age, medications, birth order, interpregnancy interval, and folic acid de iciency are listed as modi iers, and risk factor identi ication is the irst step to better understanding and to prevent such craniofacial changes [7,11,12]. however, current knowledge about the causes of cl/p points particularly towards genetic risk factors, and the study of the pathogenesis of cl/p has provided ample opportunities to identify candidate genes for this disorder and even link them to the occurrence of cancer in relatives of individuals born with cl/p, or even in these individuals themselves when at an adult age, supporting the hypothesis that common genetic factors may be present in both conditions [13]. although other genes that have   non‐syndromic oral cle s and risk of cancer: a systema c review  vol 1, no 1 (2013)    doi 10.5195/d3000.2013.11  http://dentistry3000.pitt.edu 2 not been explored yet can be involved in the both conditions, three genes are suspected to underlie such associations: fgf ibroblast signaling pathway, cdh1 epithelial cadherin and axin2 axis inhibition protein 2 [14]. colorectal and breast cancer are the most associated with mutations of these genes [2].thus, given the growing number of studies published currently suggesting a common etiology for cl/p and cancer and increased risk of cancer among relatives of cleft individuals [2,5,13-18], the current study aimed to discuss the issue through a systematic literature review in order to provide additional evidence of these genetic risks, which will aid in the development of strategies that target more aggressive screening programs and preventive chemical treatments. methods the present review was carried out in accordance with the cochrane collaboration group protocol for systematic reviews [19], including a literature search strategy, selection of papers through the inclusion and exclusion criteria, data extraction, and quality assessment. meta-analysis was not possible since selected studies did not observe the same variables, methods, participants, and outcomes, which prevented comparisons. literature search strategy our review was performed in february 2013 in order to obtain literature regarding cleft lip and/or palate and family history of cancer. pubmed, scopus, and isi web of science databases were systematically searched using two basic approaches: the search strings “cleft lip and palate” and “cancer,” “oral clefts” and “cancer,” and “orofacial clefts” and “cancer” for studies published up to that time. selection of papers through the inclusion and exclusion criteria the selection of papers is diagrammatically explained in figure 1. of the 653 studies originally found through the literature search strategy, 235 studies were excluded (in non-english language, with no full text available, and non-cross-sectional, prevalence, cohort, or case-control design), remaining with 418 papers. additional studies were found through the references of the potential studies retrieved. then, all studies involving oral clefts (associated or not with craniofacial syndromes or any anomalies) and cancer were selected. afterwards, only studies presenting information regarding risk of cancer in relatives of individuals born with non-syndromic oral clefts were included in the present study, as well as articles regarding the frequency of nonsyndromic oral clefts among relatives of individuals diagnosed with cancer. the following speci ic inclusion criteria were used to identify relevant articles: (1) index cases with non-syndromic cleft lip and/or palate, whose relatives were investigated for any type of cancer; (2) index cases with any type of cancer, whose relatives were investigated for non-syndromic cleft lip and/or palate; (3) index cases compared to healthy controls for risk assessment; (4) the degree of relationship between index cases and investigated family members is investigated in the study. all studies that included individuals with cleft lip and/or palate associated with other abnormalities, having syndromic causes or regarding mendelian traits were excluded. thus, eight studies were selected. (figure 1) data extraction title and abstract screening was performed by two reviewers (davp and hmj) who worked independently to identify potentially relevant papers for which full text publications were retrieved. if, however, there was any discrepancy of opinion, the reviewers reexamined the paper together and arrived at a joint inal decision. a standardized form was used to extract information, such as author and year of publication of the paper, origin of participants, study design, sample size, type of oral clefts, type of cancer, degree of kinship of family members and the association between non-syndromic oral clefts and cancer. quality assessment strobe methodology (strengthening the reporting of observational studies in epidemiology) [20], considered the guideline for an appropriate assessment of observational studies, was used to perform the quality assessment of the studies selected. the speci ic strobe items considered were inclusion and exclusion criteria, assessment of exposure and outcome, statistical methods, confounders, bias, and report limitation. results the initial database search identified 653 citations published between 1980 and 2013. after screening, 235 studies were excluded (72 in non-english language, 93 with no full text available, and 70 which type of study was other than those listed in the methodology), remaining with 418 papers. a second screening was performed on the remaining papers and other studies were excluded, this time for the following reasons: 51 articles in duplicity (found in more than one of the databases) and 351 articles that did not report non-syndromic cleft lip and/or palate and risk of cancer. in addition, ive papers listed in the references of the selected ones were included, resulting in 21 papers for the inal screening. figure 1: flowchart of the studies included and excluded   non‐syndromic oral cle s and risk of cancer: a systema c review  vol 1, no 1 (2013)    doi 10.5195/d3000.2013.11  http://dentistry3000.pitt.edu 3 table 2: most cited types of cancer among rela ves of cle  individuals and cancer index cases.  *the study specifically searched for this type of cancer only.  the full text of these papers was read and 13 were there after excluded for not reporting risk of cancer in relatives of individuals with non-syndromic cleft lip and/or palate, as well as for not reporting the frequency of non-syndromic cleft lip and/or palate in relatives of individuals diagnosed with cancer. thus, eight papers were selected for the systematic review. the characteristics of the eight selected papers are presented in table 1. three used a case-control design, two used a crosssectional/prevalence design and one was a retrospective cohort study. those reporting frequency of non-syndromic cleft lip and/or palate in relatives of individuals diagnosed with cancer also used a case-control design. the sample sizes and the population investigated varied considerably amongst the studies, not being homogeneous. from those investigating risk of cancer in relatives of individuals with non-syndromic cleft lip and/or palate, two were conducted in the united states, two in denmark, one in turkey, and one in latvia. regarding those reporting frequency of non-syndromic cleft lip and/or palate in relatives of individuals diagnosed with cancer, one was conducted in the united states and one in india (table 1). most of studies investigated all types of oral clefts. type of cancer and the degree were not categorized in all studies of kinship in the family. despite this, leukemia, testicular cancer, melanoma, colon-rectal cancer, lymphoma, and breast cancer were the most cited types of cancer (table 2) in both irst and second-degree relatives (table 1). the relationship between oral clefts and cancer within the studies was tested using chi-square and fisher’s exact tests [14, 18], logistic regression [2, 17], odds ratio [15, 21], risk ratio [13], and cox regression [5] (table 1). all eight studies were submitted to quality assessment using strobe guidelines. inclusion and exclusion criteria were described in all of them (table 3). the outcomes of two studies provide suggestive evidence that families with individuals diagnosed with some types of cancer report family history of cleft lip and/or palate more  frequently than families without cases   first author, year [reference] country of study study design exposure sample size (case/population) cleft classification type of cancer statistical test kinship with index cases outcome jindal,  2012  india  case‐control  individuals  diagnosed  with cancer  142 (85/57)  cl/p  leukemia  x2 and  fischer  exact  first‐degree  increased risk  taioli,  2010  united  states  case‐control  cancer  survivors  338 (168/170)  all  unspecified  logistic  regression    first/second‐ degree  increased risk  yildirim,  2012  turkey  case‐control  cleft  individuals  319 (158/161)  all  unspecified  logistic  regression  first‐degree  increased risk  menezes ,  2009  united  states  case‐control  cleft  individuals  168 (75/93)  unspecified  unspecified  x2 and  fischer  exact  not reported  increased risk  zhu,  2002  denmark  case‐control  cleft  individuals  41206 (45/41161)  unspecified  unspecified  odds ratio  first degree  increased risk  vieira,  2012  latvia  cross‐ sectional  cleft  individuals  89  unspecified  unspecified  risk ratio  first/second/ third‐degree  increased risk  dietz,  2012  denmark  retrospec‐ tive cohort  cleft  individuals  1809  all  breast  cancer  cox regression  first‐degree  no association  when all non‐ syndromic  cl/p were  analyzed  together    steinwachs,  2000  united  states  cross‐ sectional  cleft  individuals  196  cl/p and cl  unspecified  odds ratio  first/second‐ degree  no increased  risk  first author, year [reference] most commonly found types of cancer types of cancer with increased risk at index cases rela ves jindal, 2012 leukemia*    leukemia  taioli, 2010 breast, colon‐rectal, melanoma, tes cular  tes cular, melanoma, colon‐ rectal  yildirim, 2012 unspecified    unspecified  menezes, 2009 unspecified  unspecified  zhu, 2002 breast, melanoma of skin, tes s, cervix uteri,  brain and nervous system    lymphoma, leukemia  vieira, 2012 unspecified    unspecified  dietz, 2012 breast*    breast  steinwachs, 2000 breast, lung, leukemia, colon, uterine.  none found  table 1: main characteris cs of the eight selected papers.    non‐syndromic oral cle s and risk of cancer: a systema c review  vol 1, no 1 (2013)    doi 10.5195/d3000.2013.11  http://dentistry3000.pitt.edu 4 of cancer [2,18]. from studies investigating cancer in relatives of individuals with oral clefts, only two suggest that the irst and second-degree relatives of the individuals with clefts are not at an increased risk for cancer [5,21] . all studies de ined the exposure variables (table 1). although all studies had described potential bias/limitations, only three properly reported identi ication of bias/limitations (table 1). although all studies had described potential bias/limitations, only three properly reported identi ication of bias/limitations (table 4). discussion this systematic review assessed available studies suggesting a common etiology for non-syndromic oral clefts and cancer and increased risk of cancer among relatives of cleft individuals, as well as increased risk of non-syndromic oral clefts among relatives of individuals diagnosed with cancer, thereby providing additional evidence of these genetic risks, which could aid in the development of strategies that target more aggressive screening programs and preventive chemical treatments. our original intention was to integrate the results of included studies through a metaanalysis, however, given the differences in variables, methods, participants and outcomes among the studies, the most suitable and responsible was to address the issue through a systematic review. we demonstrated that there is a large amount of literature distinguishing nonsyndromic cleft lip and palate from those cases of clefts associated with other abnormalities, syndromes, or mendelian traits. our indings also demonstrated that the studies published agree that non-syndromic oral clefts are one of the most common human malformations, with an average prevalence of 1 per 700 or 1,000 live births [ 2,4,5,7,16,17,21-26], as well as that its incidence varies according to gender: 2:1 being the ratio of males to females for cleft lip and palate and 1:2 the approximate ratio of male to female for isolated cleft palate [1,10,16, 26,27]. other common information between the studies is the fact that unilateral clefts are more common than bilateral clefts, and of the unilateral cases of non-syndromic cleft lip and palate, left-sided cleft lips occur more frequently than right-sided cleft lips [ 4,5,,21,23,28,29]. many studies have also demonstrated that genetic factors may play a role in the cause of non-syndromic oral clefts in addition to certain environmental and/or stochastic factors, meaning that this malformation is a multifactorial trait [2,21,22,26,30,31]. similarly, studies have shown that cleft lip, with or without cleft palate, is entirely different from isolated cleft palate in both embryological and pathogenetic standpoints [32,33]. likewise, many of the studies report relationships between non-syndromic oral clefts and cancer or childhood cancer [14,18,25,34-41]. nonetheless, the studies that presented information regarding risk of cancer in relatives of individuals born with non-syndromic oral clefts or regarding the frequency of non-syndromic oral clefts first author, year [reference] inclusion criteria exclusion criteria jindal, 2012 all subjects were children being cared for at the general hospital of chandigarh, india. cases  were  identified  from  the  pediatric  leukemia  clinic  and  controls were  identified  randomly  from the allergy/immunology clinic.    children with lymphoma were excluded from the  case group and the ones with positive history for  cancer, from the control group.  taioli, 2010 case group was composed of patients who were  treated  for cancer  in  the past, who were  considered in remission for at least six months, and who were scheduled for an appointment  for  a  routine  checkup.  control  group was  composed  by  healthy  people  from  the  general  population who were 18 years of age or older.    not reported.  yildirim, 2012 all subjects were patients at the department of pedodontics clinic,  istanbul university, tur‐ key. case group was composed of  individuals born with any type of cl/p and controls were  individuals unrelated  to  them. case group members’  first‐degree  relatives were evaluated,  also.  case and control siblings were excluded.  menezes, 2009 all subjects were from white ancestry families from pittsburgh. cases were recruited from a  registry  maintained  by  the  cleft‐craniofacial  center,  children’s  hospital  of  pittsburgh  of  upmc.  not reported.  zhu, 2002 subjects were identified from the denmark’s central population registry by their kinship with  patients  diagnosed with  cancer  and  registered  at  the  cancer  registry.  they  also  ought  to  have been born between 1977 and 1995 in denmark to mothers with a danish citizenship.  patients whose  parents  had  other  children  be‐ fore  1977,  who  were  diagnosed  with  patent  ductus  arteriosus,  undescended  testis  and  hip  dislocation.  vieira, 2012 subjects were picked from the riga cleft lip and palate centre registry from the time period  of 1980‐2009.    not reported.  dietz, 2012 subjects were individuals with non‐syndromic cl/p registered at the danish facial cleft regis‐ ter.    individuals born after 1975 were excluded.  steinwachs, 2000 subjects ought to have been diagnosed with cleft lip with or without cleft palate and have no  known syndromal cause for the cleft.    not reported.  table 3: inclusion and exclusion criteria among the eight selected papers.    non‐syndromic oral cle s and risk of cancer: a systema c review  vol 1, no 1 (2013)    doi 10.5195/d3000.2013.11  http://dentistry3000.pitt.edu 5 table 4: poten al bias/limita ons and the author’s efforts to iden fy them among the eight selected papers. among relatives of individuals diagnosed with cancer, besides being scarce, vary greatly in their characteristics and designs, precluding a meta-analysis of the results found. in the current systematic review, both studies assessing the prevalence of cl/p in family members of cancer patients had a casecontrol design and suggest that this malformation is more frequent in families of cancer survivors in comparison with families of a population-based sample of controls. in the study of taioli et al. (2010) [2], the results were not statistically signi icant and the authors relate it to the sample size of the study and to the rarity of cl/p occurrence. in the second study, relating cl/p and childhood cancer, none of the children from the unaffected group had a positive family history of cleft lip and palate, while ive children with leukemia had a positive history of oral clefts [18]. in these studies, the population investigated, or their parents or guardians, answered standardized questionnaires with demographic information about the respondents and their family history of cl/p. it was a consensus between the authors that information on number of second-degree relatives is lacking for both, which could explain their indings for the irst-degree relatives, since the more distant the relative is, less information is known about him. in the study investigating adult individuals who survived cancer, the most frequent types of cancer were melanoma, testicular, breast, colon-rectal, and lymphoma. the authors state, however, that one limitation of the study was to include cancer survivors instead of newly diagnosed cancer patients, which could exclude highly fatal cancers [2]. despite the different characteristics of the populations investigated in the studies of taioli et al. (2010) and jindal and vieira (2012) [2,18], both suggest that shared genetic factors may explain an association between oral clefts and cancer. thus, investigating the relationship between malformations and malignancies becomes important as it is speculated that they might have common causes. endorsing that possibility, chalothorn et al. (2008) and küchler et al. (2013) [42,43] have previously proposed an association between tooth agenesis and cancer. in some cases, tooth agenesis and oral cleft may share the same genetic background. this review also accessed studies [5,1315,17,21] regarding risk of cancer in relatives of individuals born with nonsyndromic oral clefts. from the six studies found, three also had a case-control design and more cancer cases were reported among relatives of cleft individuals than by controls [14,15,17]. the most prevalent types of cancer have not been plainly identiied in all studies, which was a limitation of the studies declared by the authors. however, in those which it was possible, a clear predominance of colon cancer and leukemia cases was noticed, as well as some reports of breast cancer, lymphoma and brain cancer [14,15]. the most affected relatives were speci ied only in the study of zhu et al., (2002) [15], which exclusively investigated parents. a further limitation that was found is the fact that the speci ic data regarding the age of onset of cancer was not accessed, which makes it impossible to determine if family members of individuals with cleft developed cancer at earlier ages than the general population. a higher prevalence of cancer in family members of individuals born with cl/p was presented in a cross-sectional study with a latvian population, too [13]. the risk was calculated by dividing the prevalence of cancer in the target group by the prevalence of cancer in that population. it was demonstrated that this risk is three times higher in first author, year [reference] bias/limitations identification of bias/limitations jindal, 2012 information concerning illnesses on relatives more distant than the first‐degree  may not be so reliable.  not reported.  taioli, 2010 since it researched cancer survivors and not patients diagnosed with cancer per  se, the most fatal forms of cancer tend to have their association with cl/p not  reported accurately. also, an association between the type of cancer and cl/p  becomes impossible to be made in this case because of characteristic survivor‐ ships in each type.  not reported.  yildirim, 2012 as information regarding specific types of cancers was not available for all indi‐ viduals, an analysis by cancer types could not be done. also, the specific data  regarding the age of onset of cancer was not accessed, which made it impossible  to determine if family members of cleft individuals developed cancer at earlier  ages than the general population.  not reported.  menezes, 2009 the cancer types reported may not all have been related to the same causes.  also, as there was no access to data that specified the age of cancer develop‐ ment in those individuals’ family members, it was no possible to determine if  family members of cleft individuals developed cancer at earlier ages than the  general population.  proposes new research with increased sample size to  replicate findings and test the association between genetic  variants and specific types of cancer.  zhu, 2002 there may have been some confusion from the teratogenic effects of cancer  treatment in parents who had cancer previous to the child’s birth. the outcome  of previous births also could induce some bias as well as maternal age at birth  and sex of the child. lastly, the lack of data on stillbirths may have biased results  if fetal mortality differs between parents who will develop cancer and parents  who won’t.  it only used parents whose cancer diagnostic came up  after the birth of their indexed children. also, the children  analyzed were only firstborns and the age of the mother at  birth and the child’s sex were reported.  vieira, 2012 the population studied was composed mostly by individuals in their first years of  life, and because of that, the prevalence of pediatric or earlier onset types of  cancer was relatively increased when compared with the other types. the same  is true for their relatives, as they are most likely also relatively young.  not reported.  dietz, 2012 a left and right truncation in this study was observed, as no information can be  gotten neither previous nor post the period studied.  not reported.  steinwachs, 2000 there is known lack of accuracy in information provided by patient reports about  their family members.  proposes new and larger studies with sufficient power to  study only first‐degree relatives.    non‐syndromic oral cle s and risk of cancer: a systema c review  vol 1, no 1 (2013)    doi 10.5195/d3000.2013.11  http://dentistry3000.pitt.edu 6 irst and second-degree relatives and decreases to 1.5 times in third-degree relatives. similar to most available studies with the same proposal, the types of cancer were not consistently reported and, because of that, this study did not discriminate by cancer types. as well as in the studies addressing prevalence of cl/p in family members of cancer patients, indings from four [13-15,17] of six studies regarding risk of cancer in relatives of individuals born with non-syndromic oral clefts provide additional support for a common etiology between cl/p and cancer, suggesting that these families have a higher genetic load for cl/p, which also impacts their cancer risk. on the other hand, two other studies regarding risk of cancer in relatives of individuals with clefts could not conclude the same [5,21]. the irst one, performed by steinwachs et al., (2000) [21] was emphatic in denying an increased risk for cancer in the irst and second-degree relatives of individual affected with non-syndromic cl/p. the population investigated was composed of caucasians, hispanics, african americans, and asian individuals. the cancer data were analyzed in aggregate by type of cancer, by degree of relation to the index case, and by ethnicity. as a result, neither irst-degree relatives nor second-degree relatives of the index cases had a signi icantly increased risk for cancer. no particular ethnic group demonstrated an excessively increased risk for cancer. not even the most commonly reported cancers (lymphoproliferative cancers, female cancers, and lung cancer) were increased as a whole or in irst-degree relatives only. in the same way, in a recent retrospective cohort study [5] testing whether mothers and sisters of individuals born with cl/p have an increased risk for breast cancer, the authors were not able to con irm a general increase in this risk for breast cancer among the study population. however, some associations were revealed when cleft subtype was analyzed, and having a child with isolated cleft palate was associated with an increased risk of breast cancer for the mother, which may be biologically plausible due to the evidence that the different cleft types have different genetic origins [44,45]. thus, the hypothesis that parental cancer and congenital malformations in offspring may be correlated could not be entirely con irmed by the present systematic review. despite it being known that possible mechanisms at the basis of an association between cl/p and cancer are shared genetic factors, once polymorphisms in genes involved in cell–cell adhesion and cellmotility are associated with both cancer and cl/p [16,18], studies in this direction are very scarce and of those existing and most investigated all subtypes of oral clefts, which sets a limitation to ind out such correlation. there is agreement among almost every reviewed author [2, 14,17,18] that the investigated populations in their studies need to be expanded in order to avoid any random indings in multiple testing or the potential impact of type i error. it is also in agreement that future studies should explore the possibility that there is a preferential occurrence of speci ic types of cancer in families with individuals born with cl/p and test the hypothesis that common genetic and epigenetic mechanisms are playing a role in both conditions [14,17]. through this information, future studies may be better able to identify the causes of nonsyndromic orofacial clefts and ultimately to predict its occurrence and to facilitate genetic counseling of affected families [21]. another issue raised by dietz et al. (2012) is the fact that having a cleft leads to social marginalization and changes in lifestyle could predispose not only the individual, but also their family for cancer, which seems plausible from a standpoint of relationship between stress and cancer, as discussed in the literature. conflict of interest: there are no conflicts of interest to report. references 1. the evolu on of human gene c studies of cle   lip  and  cle   palate. marazita  l m.  annu  rev  genomics  hum  genet. 2012;13:263‐83.  epub  2012 jun 6. pmid:22703175 [pubmed ‐ indexed  for medline]  2. cle  lip and palate  in family members of can‐ cer  survivors.  taioli  e,  ragin  c,  robertson  l,  linkov  f,  thuman  le,  vieira  ar.  cancer  in‐ vest. 2010  nov;28(9):958‐62.  pmid:20569073  [pubmed ‐ indexed for medline]  3. iden fying  determinants  of  quality  of  life  of  children with cancer and childhood cancer sur‐ vivors: a systema c  review. klassen af, antho‐ ny sj, khan a, sung l, klaassen r. support care  cancer. 2011  sep;19(9):1275‐87.  epub  2011  may 25. pmid:21611865 [pubmed ‐ indexed for  medline]  4. parental  age  as  a  risk  for  non‐syndromic  oral  cle s:  a  meta‐analysis.  herkrath  apcq,  her‐ krath fj, rebelo mab, ve ore mv. j dent. 2012  jan;40(1):3‐14.epub  2011  oct  13.  pmid:22019990  [pubmed  ‐  indexed  for med‐ line]  5. risk  of  breast  cancer  in  females with  cle   lip  and palate. dietz a, pedersen da,  jacobsen r,  wehby gl, murray  jc, christensen k. ann epi‐ demiol. 2012  jan;22(1):37‐42.  epub  2011  oct  28.  pmid:  22037380    [pubmed  ‐  indexed  for  medline] .  6. prevalence  of  nonsyndromic  oral  cle s  in  a  reference hospital in the state of minas gerais,  brazil,  between  2000‐2005.  martelli‐junior  h,  porto lcvp, barbosa dr, bonan prf, freitas ab,  cole a  rd.  braz  oral  res. 2007  oct‐ dec;21(4):314‐7.  pmid:18060257  [pubmed  ‐  indexed for medline]  7. unraveling human cle  lip and palate research.  vieira  ar.  j  dent  res. 2008  feb;87(2):119‐25.  pmid:18218836  [pubmed  ‐  indexed  for med‐ line]  8. the  current  understanding  of  cle   lip malfor‐ ma ons.  wan a  n,  re nger  g.  facial  plast  surg. 2002  aug;18(3):147‐53.  pmid:12152133  [pubmed ‐ indexed for medline]  9. the  complex  gene cs  of  cle   lip  and  palate.  cobourne mt. eur  j orthod. 2004  feb;26(1):7‐ 16.  pmid:14994877  [pubmed  ‐  indexed  for  medline]   10. cle   lip  and  palate:  synthesizing  gene c  and  environmental  influences. dixon m, ml,   beaty  th,murray  jc.  nat  rev  genet. 2011  march; 12(3):  167–178.  pmid:21331089   [pubmed ‐ indexed for medline]   11. zeiger  js,  beaty  th  (2002) gene–environment  interac on and risk to oral cle s. in: wyszynski  df editors. cle   lip and palate:  from origin  to  treatment. new york: oxford university press;  pp. 283–289.  12. frequency  of  congenital  craniofacial  malfor‐ ma ons  in  a brazilian reference center. para‐ naiba lmr, miranda rt, ribeiro la, barros lm,  martelli‐júnior  h.  rev  bras  epidemi‐ ol. 2011;14:151‐160.  13. risk of cancer in rela ves of children born with  isolated cle  lip and palate.  vieira ar, khaliq s,  lace  b.  am  j  med  genet  a. 2012  jun;158a(6):1503‐4.  epub  2012  may  14.  pmid:22585460  [pubmed  ‐  indexed  for med‐ line]  14. axis  inhibi on protein 2, orofacial cle s and a  family  history  of  cancer. menezes  r, marazita  ml, goldstein mchenry t, cooper me, bardi k,  brandon c, letra a, mar n ra, vieira ar. j am  dent  assoc. 2009  jan;140(1):80‐4.  pmid:19119171  [pubmed  ‐  indexed  for med‐ line]  15. do parents of children with congenital malfor‐ ma ons  have  a  higher  cancer  risk?  a  na on‐ wide study  in denmark. zhu  jl, basso o, hasle  h,  winther  jf,  olsen  jh,  olsen  j.  br  j  can‐ cer. 2002  aug  27;87(5):524‐8.  pmid:12189550  [pubmed ‐ indexed for medline]   16. cle   lip/palate and cdh1/e‐cadherin muta on  in  families with  hereditary  diffuse  gastric  can‐ cer. frebourg t, oliveira c, hochain p, karam r,  manouvrier  s,  graziadio  c,  vekemans  m, hartmann  a, baert‐desurmont  s, alexandre  c, lejeune  dumoulin  s,marroni  c, mar n  c, castedo  s, love   m, winston  j, machado  jc, a é  t, jabs  ew, cai  j, pellerin  p, triboulet  jp, sco e m, le  pessot  f, hedouin  a, carneiro  f, blayau  m, seruca  r.  j  med  genet. 2006  feb;43(2):138‐42. epub 2005 apr 14.    non‐syndromic oral cle s and risk of cancer: a systema c review  vol 1, no 1 (2013)    doi 10.5195/d3000.2013.11  http://dentistry3000.pitt.edu 7 17. defining predictors of  cle   lip  and palate  risk.  yildirim, seymen f, deeley k, cooper me, vieira  ar.  j  dent  res. 2012  jun;91(6):556‐61.  epub  2012  apr  10.  pmid:22496123  [pubmed  ‐  in‐ dexed for medline]  18. family history of cle  lip and palate in subjects  diagnosed  with  leukemia.  jindal  a,  vieira  ar.  am  j  med  genet  a. 2012  mar;158a(3):678‐ 9.epub 2012 feb 2. pmid:22302642 [pubmed ‐  indexed for medline]  19. alderson  p,  green  s,  higgins  jpt,  editors.  cochrane reviewers’ handbook 4.2.2  [updated  march 2004]. in: the cochrane library, issue 1,  2004. chichester, uk: john wiley & sons, ltd.  20. the  strengthening  the  repor ng  of  observa‐ onal  studies  in  epidemiology  (strobe)  state‐ ment:  guidelines  for  repor ng  observa onal  studies. von elm e, altman dg, egger m, pocock  sj,  gøtzche  pc,  vanderbroucke  jp.  bmj. 2007  oct  20;335(7624):806‐8.  pmid:17947786  [pubmed ‐ indexed for medline]   21. nonsyndromic cle  lip and palate is not associ‐ ated with  cancer or other birth defects. stein‐ wachs ef, amos c, johnston d, mulliken j, stal  s,  hecht  jt  am  j  med  genet. 2000  jan  3;90(1):17‐24  pmid:10602112  [pubmed  ‐  in‐ dexed for medline]  22. major  hematological  diseases  associated  with  cle   lip  and  palate.  lin  ch,  lo  lj, wang ml,  chen yr, noordhoff ms. cle  palate craniofac  j. 2000  sep;37(5):512‐5.  pmid:11034036  [pubmed ‐ indexed for medline]  23. mossey  p,  li le  j  (2002)  epidemiology  of  oral  cle s: an interna onal perspec ve. in: wyszyn‐ ski df, editors. cle   lip and palate: from origin  to  treatment.  new  york:  oxford  university  press.pp 127‐158.   24. maternal use of an bio cs and the risk of oro‐ facial  cle s:  a  na onwide  cohort  study. møl‐ gaard‐nielsen,  hviid  a.  pharmacoepidemiol  drug  saf. 2012  mar;21(3):246‐53.  epub  2011  nov  28.  pmid:  22125260  [pubmed  ‐  indexed  for medline]  25. cancer  risk  in persons with oral cle   ‐ a popu‐ la on‐based  study  of  8,093  cases.  bille  c,  winther  jf, bautz a, murray  jc, olsen  j, chris‐ tensen  k.  am  j  epidemiol. 2005  jun  1;161(11):1047‐55. pmid:15901625  [pubmed  ‐  indexed for medline]   26. nevoid  basal  cell  carcionoma  syndrome  with  cle   lip  and  palate  associated with  the  novel  ptch  gene  muta ons.  sasaki  r,  saito  k,  watanabe  y,  yoshinaga  t,  fujii  k,  agawa  k,  miyashita  t,  ando  t,  akizuki  t.  j  hum  genet. 2009  jul;54(7):398‐402.  epub  2009  jun  12.  pmid:  19521425  [pubmed  ‐  indexed  for  medline]  27. cle   lip  and  palate  in  puerto  rico:  a  thirty‐ three  year  study.  oliver‐padilla  g,  mar nez‐ gonzález v. cle  palate j. 1986 jan;23(1):48‐57.  pmid:3455903  [pubmed  ‐  indexed  for  med‐ line]  28. incidence of  cle   lip,  cle  palate, and  cle   lip  and palate among races: a review. vanderas ap.  cle   palate  j. 1987  jul;24(3):216‐25.  pmid:3308178  [pubmed  ‐  indexed  for  med‐ line]  29. the  gene cs  of  isolated  orofacial  cle s:  from  genotypes to subphenotypes. jugessur a, farlie  pg,  kilpatrick n. oral  dis. 2009 oct;15(7):437‐ 53. epub 2009 jul 2. pmid:19583827 [pubmed ‐  indexed for medline]  30. hereditary  cle   lip/palate  and will  tumor:  a  rare associa on.  yu cc, wong  fh,  lo  lj, chen  yr.  cle   palate  craniofac  j. 2002  may;39(3):376‐9.  pmid:12019017  [pubmed  ‐  indexed for medline]  31. gene/environment  causes  of  cle   lip  and/or  palate.  murray  jc.  clin  genet. 2002  apr;61(4):248‐56.  pmid:12030886  [pubmed  ‐  indexed for medline]  32. cle  lip (+/‐ cle  palate) in danish twins, 1970‐ 1990.  christensen  k, fogh‐andersen  p.  am  j  med  genet. 1993  nov  1;47(6):910‐6.  pmid:8279491  [pubmed  ‐  indexed  for  med‐ line]  33. analysis of  family  incidence of  cle   lip  and/or  palate.  kot  m, kruk‐jeromini  j.  med  sci  monit. 2007  may;13  (5):cr231‐4.  pmid:17476195  [pubmed  ‐  indexed  for med‐ line]  34. a popula on‐based study of cancer incidence in  twins  and  in  children  with  congenital malfor‐ ma ons  or  low  birth  weight,  norway,  1967‐ 1980. windham  gc,  bjerkedal  t,  langmark  f.  am  j  epidemiol. 1985  jan;121(1):49‐56.  pmid:3155484  [pubmed  ‐  indexed  for  med‐ line]  35. increased  prevalence  of  minor  anomalies  in  childhood malignancy. mehes k, signer e, pluss  hj,  müller  hj, stalder  g.  eur  j  pediatr. 1985  sep;144(3):243‐54.  pmid:4054163  [pubmed  ‐  indexed for medline]  36. excess  of  mild  errors  of  morphogenesis  in  childhood  lymphoblas c  leukemia.  mehes  k,  kajtar  p,  sandor  g,  scheel‐walter  m, niethammer d. am  j med genet. 1998  jan  6;75(1):22‐7. pmid:9450852 [pubmed ‐ indexed  for medline]  37. maternal  and  perinatal  risk  factors  for  child‐ hood  leukemia.  zack m, adami ho, ericson a.  cancer  res. 1991  jul  15;51(14):3696‐701.  pmid:  2065325  [pubmed  ‐  indexed  for med‐ line]  38. risk of  childhood  cancer  for  infants with birth  defects, part  i: a  record‐linkage study, atlanta,  georgia, 1968‐1988. mili f, khoury mj, flanders  wd, greenberg rs. am  j epidemiol. 1993 mar  15;137(6):629‐38.  pmid:8470664  [pubmed  ‐  indexed for medline]  39. congenital  malforma ons  and  childhood  can‐ cer. nishi m, miyake h, takeda t,  hatae y. med  pediatr oncol. 2000;34:250–254.   40. increased  prevalence  of  minor  anomalies  in  children with hematologic malignancies. roga‐ novic j, radojcic‐badovinac a, ahel v. med pe‐ diatr  oncol.  2002  feb;38(2):128‐30.  pmid:11813182  [pubmed  ‐  indexed  for med‐ line].  41. infant leukemia and congenital abnormali es: a  children’s  oncology  group  study.  johnson  kj,  roesler ma, linabery am, hilden ja, davies sm,  ross  ja.  pediatr  blood  cancer. 2010  jul;55(1):95‐9.  pmid:20486175  [pubmed  ‐  in‐ dexed for medline]  42. hypodon a as a risk marker for epithelial ovari‐ an  cancer: a  case‐controlled  study. chalothorn  la, beeman cs, ebersole jl, kluemper gt, hicks  ep, kryscio rj, desimone cp, modesi  sc. j am  dent  assoc. 2008  feb;139(2):163‐9.   pmid:18245684  [pubmed  ‐  indexed  for med‐ line]  43. tooth agenesis associa on with self‐ reported family history of cancer.  küchler  ec, lips  a, tannure  pn, ho  b, costa  mc, granjeiro  jm, vieira  ar.  j  dent  res. 2013  feb;92(2):149‐55.  epub  2012  nov  20.  pmid:23169889  [pubmed  ‐  indexed  for med‐ line]  44. disrup on  of  an  ap‐2alpha  binding  site  in  an  irf6  enhancer  is  associated  with  cle   lip.  rahimov  f,  marazita  m,  visel  a,  cooper  m,  hitchler  m,  rubini  m,  domann  fe, govil  m, christensen  k, bille  c, melbye  m, jugessur  a, lie  rt, wilcox  aj,fitzpatrick  dr, green  ed, mossey  pa, li le  j, steegers‐theunissen  rp, pennacchio  la, schu e  bc, murray  jc. nat  genet. 2008 nov;40(11):1341‐7. epub 2008 oct  5.  pmid:18836445  [pubmed  ‐  indexed  for  medline].  45. a  cohort  study  of  recurrence  pa erns  among  more than 54,000 rela ves of oral cle  cases in  denmark:  support  for  the  mul factorial  threshold  model  of  inheritance.  grosen  d,  chevrier  c,  sky he  a,  bille  c,  mølsted  k,  sivertsen  a, murray  jc, christensen  k.  j  med  genet. 2010  mar;47(3):162‐8.epub  2009  sep  14.  pmid:19752161  [pubmed  ‐  indexed  for  medline]  vol 7, no 1 (2019) issn 2167-8677 (online) doi 10.5195/d3000.2019.88 http://dentistry3000.pitt.edu absence of history of oral cleft in first-degree relatives of patients with prostate cancer cláudia de alvarenga diniz fonseca,1,2 daniella reis barbosa martelli,3 ianná luana freitas almeida,2 galeno hassen sales,2 rodrigo soares de andrade,4 verônica oliveira dias,3 letízia monteiro de barros,5 hercílio martelli júnior1,3,5 1health science program, state university of montes claros, unimontes, minas gerais, brazil. 2medicine school, state university of montes claros, montes claros, minas gerais, brazil. 3dental school, state university of montes claros, montes claros, minas gerais, brazil. 4department of oral pathology, dental school, university of campinas, unicamp, piracicaba, são paulo state, brazil. 5center for rehabilitation of craniofacial anomalies, university of josé rosario vellano, alfenas, minas gerais, brazil. abstract objective: to evaluate the occurrence of nonsyndromic cleft lip and/or palate (nscl/p) in families of patients with prostate cancer (pc). study design: we conducted a case-control study involving a total of 748 individuals, 280 of which had pc, and 468 were free-cancer healthy individuals. the patients answered a questionnaire with basic demographic information and family history of nscl/p in firstdegree relatives. the information collected was stored in a database and analyzed by using the statistical program spss® 24.0 for windows (chicago, il, usa). in order to determine the association with nscl/p, chi-square and fisher’s exact test and odds ratio (or) with its 95% confidence interval (95% ci) for risk magnitude assessment. values with p<0.05 were considered statistically significant. results: of total patients with pc, 2 had a positive history of nscl/p. in the control group, 7 patients reported family history of nscl/p (1df chi-square, p=0.34; fisher´s exact test, p=0.49). the average age of the cases diagnosed with pc was 71.35±7.70 years, and control group was 64.42±9.67 years. conclusion: despite the limited population, the frequency of nscl/p was not significantly increased in the first-degree relatives of patients with pc. studies with larger samples and molecular analyses are needed to better understand the possible relationships in the etiology of cancer and nscl/p. citation: fonseca cad, et al. (2019) absence of history of oral cleft in first-degree relatives of patients with prostate cancer. dentistry 3000. 1:a001 doi:10.5195/d3000.2019.88 received: november 3, 2018 accepted: april 4, 2019 published: july 25, 2019 copyright: ©2019 fonseca cad, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: cdadiniz@gmail.com introduction prostate cancer (pc) is one of the most frequent cancers in men, and it is estimated to affect 1,618,000 men, and led to 366,000 deaths globally in 2015 (1). an american estimate published in 2017 showed that prostate, lung and bronchus, and colorectal cancers account for 42% of all cases in men, with prostate cancer alone accounting for almost 1 in 5 new diagnoses. for women, the 3 most common cancers are breast, lung, and colorectum, which collectively represent one-half of all cases; breast cancer alone accounts for 30% all new cancer diagnoses in women (2). in brazil, 68,220 new cases of pc are estimated for each year in 2018-2019. these values correspond to an estimated risk of 66.12 new cases for every 100 thousand men. pc is the most incident among men in all regions in brazil (3). http://dentistry3000.pitt.edu/ oral clefts in patients with prostate cancer. vol 7 no 1 (2019) doi 10.5195/d3000.2019.88 http://dentistry3000.pitt.edu nonsyndromic cleft lip and/or cleft palate [(nscl/p); omim #119530] is the most common craniofacial birth defect in humans with a global prevalence ranging from 1:500 to 1:2,500 live births (4). in brazil, the prevalence varies from 0.36 to 1.54 per 1,000 live births (5,6). about 70% of the cases occur as a nonsyndromic form (nscl/p), and the remaining 30% are associated with mendelian disorders or chromosomal, teratogenic and sporadic conditions (7). although there has been marked progress in identifying environmental and genetic risk factors associated with nscl/p, its etiology in most of the cases remains unclear (4,8). based on epidemiological features and embryologic timing, nscl/p are traditionally divided in nonsyndromic cleft lip (nscl), nonsyndromic cleft lip and palate (nsclp) and nonsyndromic cleft palate (nscp) (9). it has been proposed that cancer and congenital malformations such as nscl/p may occasionally have a common etiology. the underlying concept is that genes acting in normal development may also have an important role in malignancies (9). a study published in 2015 showed that having an oral cleft increases the risk for self-reported history of cancer in family members, and genetic variants in dlx1 and mmp3 are associated with oral cleft regardless of the history of cancer (10). over the past years, epidemiological studies have assessed the relationship between cancer and nscl/p in different populations [texas, usa (11), southeast asian (12), denmark (13), pittsburgh, usa (14), latvia (15), india (16), the netherlands (17), danish (18), turkey (19), brazil (20), france (21), brazil (8,22), poland (23), pakistan (24), brazil (25)], and the results are controversial. the finding of an increased risk of nscl/p in families with cancer case would strengthen the hypothesis that common genetic factors may play a role in both situations. thus, the aim of this study was to evaluate the frequency of nscl/p in first-degree families of patients with pc. materials and methods we conducted a case-control study. all patients with pc (case group; n=280) attended in a reference service in oncology, in minas gerais state, brazil, from june 2016 to december 2017 were evaluated. the following inclusion criteria were applied: having a diagnosis of pc and to be undergoing treatment at the institution. the sampling was performed by convenience, and the groups were matched concerning age. the control group included 468 free-cancer healthy individuals randomly obtained at the general clinics and at the university hospital. study participants signed an informed consent and answered in person a structured questionnaire with basic demographic information that included the following data on self-reported for analysis: age, skin color, history of nscl/p in first-degree relatives (mother, father, siblings and children) through self-identification by illustrative figures. the information collected was stored in a database and analyzed by using the statistical program spss® version 24.0 (statistical package for social sciences for windows, inc., chicago, il,usa). a descriptive and comparative statistical analysis was initially carried out with baseline characteristics among groups such as age, skin color, and history of nscl/p (first degree relatives). a binary logistic regression was used for statistical analysis of the color categorical variable. mannwhitney's test for unpaired samples was performed to compare the age averages between the groups. in order to determine the association with nscl/p, chi-square and fisher’s exact test and odds ratio (or) with its 95% confidence interval (95% ci) for risk magnitude assessment. values with p<0.05 were considered statistically significant. all subjects enrolled in this study lived in the same geographical area (northern of the state of minas gerais, brazil). thus, controls group presented demographic ethnic and sociocultural characteristics similar to the pc group. written informed consents were obtained, and the study was carried out with approval of the university human research ethics committee (# 436.750). http://dentistry3000.pitt.edu/ oral clefts in patients with prostate cancer. vol 7 no 1 (2019) doi 10.5195/d3000.2019.88 http://dentistry3000.pitt.edu results the average age of the cases diagnosed with pc was 71.35±7.70 years, and control group was 64.42±9.67 years (p=0,000). the majority of the participants de clared themselves to be brown or black, both in pc and in freecancer healthy individuals groups (79.3%, 72.6%, respectively). black subjects were 1.81 times more likely to have prostate cancer than white subjects, with a statistically significant difference (p=0.02; or:1.81; ci:1.17-2.80). nine out of 748 study participants had a positive history of nscl/p. among 280 patients with pc, 2 had a positive family history of nscl/p. in the control group, 7 patient reported family history of nscl/p (1df chi-square, p=0.34; fisher´s exact test, p=0.49) (table 1). with this sample size, the frequency of nscl/p as 1/1,000 live births and the average of the brazilian family size of 16 individuals (8), we would expect to identify approximately 4 or 5 cases of nscl/p in relatives of patient with pc (280x16/1,000) and 7 or 8 cases among relatives of the controls (468x16/1,000). thus, in this study nscl/p frequency was not significantly increased in firstdegree relatives of patients with pc. discussion cancer is a multifactorial disease in which both genetic and environmental factors play a significant role. preventive plans for decreasing cancer incidence include both the removal of environmental agents known to be carcinogens and the identification of cancer susceptibility gene polymorphism or mutations (12). cancer is a major public health problem in the world. it is currently the second leading cause of death, and approximately 14 million new cancer cases are reported worldwide (26). prostate cancer is the fourth most common cancer in both sexes combined and the second most common cancer in men. in 2012, estimates showed approximately 1.1 million new cases of pc, accounting for 15% of the cancers diagnosed in men (26). regarding mortality, global estimates of the globocan project, the international agency for research on cancer (iarc) of the world health organization (who), pointed 307,000 deaths in 2012; and the overall mortality rate for the same year was 7.8/100,000, representing 6.6% of the total male cancer mortality (26). in this research the frequency of nscl/p was not significantly increased in first-degree relatives of patients with pc. dietz et al. (2012) analyzed the risk of breast cancer in families with nscl/p, but they were unable to confirm a general increase of breast cancer risk among female subjects in families with clefts (18). martelli et al. (2014) analyzed the risk of nscl/p in families of women with breast cancer, but they were unable to confirm a general increase in the risk of nscl/p among families of women with breast cancer (8). on the other hand, menezes et al. (2009) showed a family history of cancer (colon, brain, leukemia, breast, prostate, skin, lung and liver) more often in relatives of nscl/p compared to the control group (14). taioli et al. (2010) evaluated the presence of nscl/p in family members of cancer patients. among cancer survivors with a family member with nscl/p, there was an apparent table 1. frequency of prostate cancer (pc) patients with positive and negative history of nonsyndromic cleft lip and/or palate (nscl/p) in first-degree relatives. family history of nscl/p pc group n (%) control group n (%) or ci 95% pvalue yes 2 (0.7) 7 (1.5) 2.11 0.435-10.232 0.34a 0.49b no 278 (99.3) 461 (98.5) achi-square test; bfisher’s exact test http://dentistry3000.pitt.edu/ oral clefts in patients with prostate cancer. vol 7 no 1 (2019) doi 10.5195/d3000.2019.88 http://dentistry3000.pitt.edu excess of testicular cancer and melanoma in comparison with the cancer survivors with no family history of nscl/p (27). jindal and vieira (2012) showed a correlation between acute lymphoblastic leukemia and nscl/p in a population from india. the occurrence of nscl/p was more frequent in families with acute lymphoblastic leukemia than in families without the diagnosis of this pediatric cancer (16). possible mechanisms at the basis of an association between nscl/p and cancer are shared genetic factors. the first study to characterize possible pleiotropic loci for nscl/p and cancer using large genomic datasets was published in 2016. suggestive evidence for a common genetic background was found for nscl/p and follicular lymphoma at 6p21.33, and for nscl/p and squamous cell carcinoma of the skin in 20q12 (28). the hypothesized association between cancer and nscl/p could be attributed to factors that have been suspected to be at the basis of these associations which are polymorphic variants in genes involved in cell-to-cell adhesion and cell motility (17,21). study involving mutation analysis in pc cells pointed inactivating homozygous mutations in the synj2 and clptm1 genes. disruption of nonsense-mediated mrna decay (nmd) pathway suggested that both genes carried nonsense mutations in lncap cells. the sequence analysis identifies homozygous deletions within mononucleotide repeats in the coding regions of both of these genes (29). recently, we studied the relationship between breast and gastric cancer and nscl/p. our results suggest that polymorphic variants in axin2 and cdh1 may be associated with nscl/p and reinforce the putative link between cancer and oral clefts (9). the finding of an increased risk of nscl/p in families of cancer patients would strengthen the hypothesis that common genetic and epigenetic mechanisms are playing a role in both conditions. some limitations should be considered in the present study, including the use of self-reported information and the memory bias associated with the study design (control case), reason to choose the history of nscl/p only in first degree relatives. the lack of ethnicity pairing may also be considered a potential limitation when taking into account the higher incidence of prostate cancer in black men worldwide. however, some studies consider the risk of prostate cancer to be similar among black and white men in brazil (30). our results are in accordance with a systematic review with metaanalysis which has demonstrated a higher prevalence of prostate cancer in black men compared to brazilian white or brown men (31). we believe that the complex ethnic mix in the brazilian population, which includes the contribution of european, african and native american ancestry, makes this kind of pairing difficult. a study on the genomic ancestry of individuals from different geographic regions of brazil reported considerable variability in the ancestry of color categories in different regions, most of which are manifested in brown and black individuals. for individuals classified as black, they observed considerable but highly discrepant levels of european descents, ranging from 29.3% to 53.9%, depending on the region (32). the reduced number of nscl/p history among first-degree relatives in both groups did not allow a comparative analysis of the mean between them. finally, the small size and type of sample involving individuals from hospitals may pose a risk of not representing the general population. conclusion our results conclude that the frequency of nscl/p was not significantly increased in the firstdegree relatives of patients with pc. studies with larger samples and molecular analyses are needed to better understand the possible relationships in the etiology of cancer and nscl/p. acknowledgments this work was supported by grants from the state of minas gerais research foundation-fapemig, minas gerais, brazil and the national council for scientific and technological development-cnpq, brasília, brazil. http://dentistry3000.pitt.edu/ oral clefts in patients with prostate cancer. vol 7 no 1 (2019) doi 10.5195/d3000.2019.88 http://dentistry3000.pitt.edu references 1. fitzmaurice c, allen c, barber r. global, regional, and national cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life-years for 32 cancer groups, 1990 to 2015: a systematic analysis for the global burden of disease study. jama oncol. 2017; (3):524-548. pmid: 27918777 2. siegel rl, miller kd, jemal a. cancer statistics, 2017. ca cancer j clin [internet]. 2017;67(1):7–30. pmid: 28055103 3. inca. the national cancer institute. cancer statistic. brazil:inca, 2017. 4. dixon mj, marazita ml, beaty th, murray jc. cleft lip and palate: understanding genetic and environmental influences. nat rev genet. 2011;(12):167-178. pmid: 21331089 5. martelli-junior h, porto lv, martelli dr, bonan pr, freitas ab, coletta rd. prevalence of nonsyndromic oral clefts in a reference hospital in the state of minas gerais, brazil, between 2000-2005. braz oral res. 2007;(21):314-317. pmid: 18060257 6. rodrigues k, sena mfd, roncalli ag, ferreira maf. prevalence of orofacial clefts and social factors in brazil. braz oral res. 2009;(23):38-42. pmid: 19488470 7. meng l, bian z, torensma r, von denhoff jw. biological mechanisms in palatogenesis and cleft palate. j dent res. 2009;(88):22–33. pmid: 19131313 8. martelli dr, vieira ar, fonseca at, coletta rd, soares pb, martelli-júnior h. risk of nonsyndromic cleft lip and palate in relatives of women with breast cancer. am j medgenet. 2014;(164):270-271. pmid: 24259269 9. machado ra, de freitas em, de aquino sn, martelli dr, swerts ms, reis sr, et al. clinical relevance of breast and gastric cancer-associated polymorphisms as potential susceptibility markers for oral clefts in the brazilian population. bmc med genetics. 2017;(5):18-39. pmid: 28376813 10. sabóia tm, reis mf, martins âmc, romanos hf, tannure pn, granjeiro jm, et al. dlx1 and mmp3 contribute to oral clefts with and without positive family history of cancer. arch oral biol. 2015;60(2):223–228. pmid: 25463899 11. steinwachs ef, amos c, johnston d, mulliken j, stal s, hecht jt. nonsyndromic cleft lip and palate is not associated with cancer or other birth defects. am j med genet. 2000;(90):17-24. pmid: 10602112 12. nishi m, miyake h, takeda t, hatae y. congenital malformations and childhood cancer. med pediatr oncol. 2000;(34):250-254. pmid: 10742060 13. zhu jl, basso o, hasle h, winther jf, olsen jh, olsen j. do parents of children with congenital malformations have a higher cancer risk? a nationwide study in denmark. br j cancer. 2002;(87):524-528. pmid: 12189550 14. menezes r, marazita ml, goldstein mchenry t, cooper me, bardi k, brandon c, et al. axis inhibition protein 2, orofacial clefts and a family history of cancer. j am dent assoc. 2009;(140):80-84. pmid: 19119171 15. vieira ar, khaliq s, lace b. risk of cancer in relatives of children born with isolated cleft lip and palate. am j med genet a. 2012;(158):1503-1504. pmid: 22585460 16. jindal a, vieira ar. family history of cleft lip and palate in subjects diagnosed with leukemia. am j med genet a. 2012;(158):678-679. pmid: 22302642 17. kluijt i, sijmons rh, hoogerbrugge n, plukker jt, de jong d, van krieken jh, et al. familial gastric cancer: guidelines for diagnosis, treatment and periodic surveillance. fam cancer. 2012;(11):363-369. pmid: 22388873 18. dietz a, pedersen da, jacobsen r, wehby gl, murray jc, christensen k. risk of breast cancer in families with cleft lip and palate. ann epidemiol. 2012;(22):37-42. pmid: 22037380 19. yildirim m, seymen f, deeley k, cooper me, vieira ar. defining predictors of cleft lip and palate risk. j dent res. 2012;(91):556-561. pmid: 22037380 20. lima ls, silvério mde o, swerts ms, aquino sn, martelli dr, martellijúnior h. frequency of cancer in firstdegree relatives of patients with cleft lip and/or palate in the brazilian population. braz dent j. 2013;(24):200-203. pmid: 23969906 21. benusiglio pr, caron o, consolino e, duvillard p, coulet f, blayau m, et al. cleft lip, cleft palate, hereditary diffuse gastric cancer and germline mutations in cdh1. int j cancer. 2013;(132): 2466-2470. pmid: 23124477 22. gonçalves e, martelli dr, coletta rd, vieira ar, caldeira ap, martellijúnior h. risk of leukemia in first degree relatives of patients with http://dentistry3000.pitt.edu/ http://www.ncbi.nlm.nih.gov/pubmed/21331089 http://www.ncbi.nlm.nih.gov/pubmed/21331089 http://www.ncbi.nlm.nih.gov/pubmed/21331089 http://www.ncbi.nlm.nih.gov/pubmed/21331089 https://www.ncbi.nlm.nih.gov/pubmed/?term=machado%20ra%5bauthor%5d&cauthor=true&cauthor_uid=28376813 https://www.ncbi.nlm.nih.gov/pubmed/?term=de%20freitas%20em%5bauthor%5d&cauthor=true&cauthor_uid=28376813 https://www.ncbi.nlm.nih.gov/pubmed/?term=de%20aquino%20sn%5bauthor%5d&cauthor=true&cauthor_uid=28376813 https://www.ncbi.nlm.nih.gov/pubmed/?term=de%20aquino%20sn%5bauthor%5d&cauthor=true&cauthor_uid=28376813 https://www.ncbi.nlm.nih.gov/pubmed/28376813 https://www.ncbi.nlm.nih.gov/pubmed/28376813 https://www.ncbi.nlm.nih.gov/pubmed/?term=dietz%20a%5bauthor%5d&cauthor=true&cauthor_uid=22037380 https://www.ncbi.nlm.nih.gov/pubmed/?term=dietz%20a%5bauthor%5d&cauthor=true&cauthor_uid=22037380 file:///c:/downloads/d&cauthor=true&cauthor_uid=22037380 file:///c:/downloads/d&cauthor=true&cauthor_uid=22037380 file:///c:/downloads/d&cauthor=true&cauthor_uid=22037380 file:///c:/downloads/d&cauthor=true&cauthor_uid=22037380 file:///c:/downloads/d&cauthor=true&cauthor_uid=22037380 file:///c:/downloads/d&cauthor=true&cauthor_uid=22037380 file:///c:/downloads/d&cauthor=true&cauthor_uid=22037380 file:///c:/downloads/d&cauthor=true&cauthor_uid=22037380 https://www.ncbi.nlm.nih.gov/pubmed/?term=lima%20ls%5bauthor%5d&cauthor=true&cauthor_uid=23969906 file:///c:/downloads/d&cauthor=true&cauthor_uid=23969906 file:///c:/downloads/d&cauthor=true&cauthor_uid=23969906 file:///c:/downloads/d&cauthor=true&cauthor_uid=23969906 file:///c:/downloads/d&cauthor=true&cauthor_uid=23969906 file:///c:/downloads/d&cauthor=true&cauthor_uid=23969906 file:///c:/downloads/d&cauthor=true&cauthor_uid=23969906 file:///c:/downloads/d&cauthor=true&cauthor_uid=23969906 file:///c:/downloads/d&cauthor=true&cauthor_uid=23969906 oral clefts in patients with prostate cancer. vol 7 no 1 (2019) doi 10.5195/d3000.2019.88 http://dentistry3000.pitt.edu nonsyndromic cleft lip and palate. braz oral res. 2014;(28):1-3. pmid: 25337933 23. hozyasz kk, mostowska a, wójcicki p, lasota a, offert b, balcerek a, et al. nucleotide variants of the cancer predisposing gene cdh1 and the risk of non-syndromic cleft lip with or without cleft palate. fam cancer. 2014;(13):415-421. pmid: 24838934 24. bui ah, ayub a, ahmed mk, taioli e, taub pj. association between cleft lip and/or cleft palate and family history of cancer: a case-control study. ann plast surg. 2018;(80):178181. pmid: 29389703 25. cardoso ef, martelli dr, machado ra, coletta rd, de souza jd, barbosa ft et al. nonsyndromic cleft lip and palate, gastric cancer and tooth agenesis. med oral patol oral cir bucal. 2018;(23):44-48. pmid: 29274157 26. ferlay j, soerjomataram i, dikshit r, eser s, mathers c, rebelo m, et al. cancer incidence and mortality worldwide: sources, methods and major patterns in globocan 2012. int j cancer. 2015;(136):359-386. pmid: 25220842 27. taioli e, ragin c, robertson l, linkov f, thurman n e, vieira a r. cleft lip and palate in family members of cancer survivors. cancer invest. 2010; 28(9): 958–962. pmid: 20569073 28. dunkhase e, ludwig ku, knapp m, skibola cf, figueiredo jc, julie f, et al. nonsyndromic cleft lip with or without cleft palate and cancer: evaluation of a possible common genetic background through the analysis of gwas data. genomics data. 2016;(10):22–9. pmid: 27630819 29. rossi mr, hawthorn l, platt j, burkhardt t, cowell jk, ionov y. identification of inactivating mutations in the jak1, synj2, and clptm1 genes in prostate cancer cells using inhibition of nonsense-mediated decay and microarray analysis. cancer genet cytogenet. 2005;161(2):97– 103. pmid: 16102578 30. barros ms, silva vr, santos gb, hughes a, silveira ma. prevalence of prostate adenocarcinoma according to race in an university hospital. int braz j urol. 2003;(29): 306-11. pmid: 15745553 31. romero fr, romero aw, de almeida rms, filho rt. the prevalence of prostate cancer in brazil is higher in black men than in white men: systematic review and metaanalysis. int braz j urol. 2012;38(4):440–7. pmid: 22951172 32. pena sdj, di pietro g, fuchshubermoraes m, genro jp, hutz mh, kehdy fsg et al. the genomic ancestry of individuals from different geographical regions of brazil is more uniform than expected. plos one. 2011; 6(2). pmcid: pmc3040205 http://dentistry3000.pitt.edu/ https://www.ncbi.nlm.nih.gov/pubmed/?term=bui%20ah%5bauthor%5d&cauthor=true&cauthor_uid=29389703 https://www.ncbi.nlm.nih.gov/pubmed/?term=ayub%20a%5bauthor%5d&cauthor=true&cauthor_uid=29389703 https://www.ncbi.nlm.nih.gov/pubmed/?term=ahmed%20mk%5bauthor%5d&cauthor=true&cauthor_uid=29389703 https://www.ncbi.nlm.nih.gov/pubmed/?term=taioli%20e%5bauthor%5d&cauthor=true&cauthor_uid=29389703 https://www.ncbi.nlm.nih.gov/pubmed/?term=taioli%20e%5bauthor%5d&cauthor=true&cauthor_uid=29389703 https://www.ncbi.nlm.nih.gov/pubmed/?term=taub%20pj%5bauthor%5d&cauthor=true&cauthor_uid=29389703 https://www.ncbi.nlm.nih.gov/pubmed/29389703 https://www.ncbi.nlm.nih.gov/pubmed/?term=cardoso%20ef%5bauthor%5d&cauthor=true&cauthor_uid=29274157 https://www.ncbi.nlm.nih.gov/pubmed/?term=martelli%20dr%5bauthor%5d&cauthor=true&cauthor_uid=29274157 https://www.ncbi.nlm.nih.gov/pubmed/?term=machado%20ra%5bauthor%5d&cauthor=true&cauthor_uid=29274157 https://www.ncbi.nlm.nih.gov/pubmed/?term=machado%20ra%5bauthor%5d&cauthor=true&cauthor_uid=29274157 https://www.ncbi.nlm.nih.gov/pubmed/?term=coletta%20rd%5bauthor%5d&cauthor=true&cauthor_uid=29274157 https://www.ncbi.nlm.nih.gov/pubmed/?term=de%20souza%20jd%5bauthor%5d&cauthor=true&cauthor_uid=29274157 https://www.ncbi.nlm.nih.gov/pubmed/?term=barbosa%20ft%5bauthor%5d&cauthor=true&cauthor_uid=29274157 https://www.ncbi.nlm.nih.gov/pubmed/?term=barbosa%20ft%5bauthor%5d&cauthor=true&cauthor_uid=29274157 immunohistochemical expression of cyclooxygenase 2 reflects the proliferative activity in the epithelium of odontogenic lesions vol 10 no 1 (2022) doi 10.5195/d3000.2022.205 http://dentistry3000.pitt.edu immunohistochemical expression of cyclooxygenase 2 reflects the proliferative activity in the epithelium of odontogenic lesions vani verma, chetana chandrashekar, raghu radhakrishnan, monica charlotte solomon department of oral pathology and microbiology manipal college of dental sciences, manipal, manipal academy of higher education manipal 576104, karnataka, india abstract purpose: odontogenic cysts and tumors comprise a major component of lesions of the oral and maxillofacial region. the pathogenesis of these lesions involves the interaction between the odontogenic epithelium and the ectomesenchyme. however, the clinical behavior of these biological entities is unpredictable. the aim of this study was to evaluate the role of cyclooxygenase 2 (cox-2) in the pathogenesis and prognostication of odontogenic lesions. materials and methods: in this study formalin-fixed paraffin-embedded tissue section of odontogenic keratocyst (n=10) dentigerous cyst (n=10), radicular cyst (n=10) and unicystic ameloblastoma (n=10) were immunohistochemically stained with cox-2 (ncl2-cox-24h12) and with ki 67 (ki-67 gm001) using standard staining protocols. the cytoplasmic expression of cox-2 in all the lesions was semi-quantitatively assessed. the pattern of expression of cox-2 among the different odontogenic lesions was statistical analyzed using the anova test and the chi-square test. results: all the 40 odontogenic lesions that were evaluated expressed cox-2 immunohistochemically. a high number of odontogenic epithelial cells expressed cox-2 in most of the odontogenic keratocyst, radicular cyst and unicystic ameloblastomas. the expression of cox-2 was significantly (p=0.036) higher in unicystic ameloblastomas and radicular cyst compared to that of odontogenic keratocyst and the dentigerous cyst. conclusion: the recognition that expression of cox-2 by odontogenic epithelial cells may indeed shed a new light on the biological mechanisms involved in the development of these benign yet aggressive lesions of the jaws. an insight into the molecular interactions occurring in the odontogenic epithelium will aid in better management of these lesions. citation: verma, et al. (2022). immunohistochemical expression of cylooxygenase 2 reflects the proliferative activity in the epithelium of odontogenic lesions. dentistry 3000. 1:a001 doi:10.5195/d3000.2022.205 received: july 12, 2021 accepted: september 30, 2021 published: january 10, 2022 copyright: ©2022 verma, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email:monica.charlotte@manipal.edu introduction odontogenic lesions are the most common benign lesions that occur within the jaw bones. these lesions are derived from the epithelium associated with the formation of the hard and soft tissues of the tooth. around 52.3– 70.7% of the cysts that are submitted to an oral pathology biopsy service are radicular cysts, 16.6-21.3% are dentigerous cyst 5.4% 17.4% are odontogenic keratocyst (5.4–17.4%) [1]. the radicular cyst also called the periapical cyst is the most common odontogenic cystic lesion. the world health organization classification of head and neck tumors describes radicular cyst as a cyst of inflammatory origin associated with non-vital teeth [2]. it arises due to activation of the cell rests of malassez that are present in the periodontal ligament due to an inflammatory stimulus. radicular cysts present as a swelling of the jaw and may be associated with pain/loosening of tooth, root resorption of the affected tooth and displacement of the adjacent teeth [3]. immunohistochemical expression of cyclooxygenase 2 reflects the proliferative activity in the epithelium of odontogenic lesions vol 10 no 1 (2022) doi 10.5195/d3000.2022.205 http://dentistry3000.pitt.edu the most common developmental odontogenic cysts are dentigerous cysts. dentigerous cyst is an odontogenic cyst that is attached to the cervical region of an unerupted tooth and envelops the crown [2]. this cyst arises due to the separation of the dental follicle from around the crown of unerupted teeth. the dentigerous cysts usually develop in relation to an impacted mandibular third molar tooth. long standing dentigerous cysts can cause root resorption, bone expansion and local destruction [4] the odontogenic keratocyst is an odontogenic cyst characterized by a thin regular lining of parakeratinized stratified squamous epithelium with palisading hyperchromatic basal cells. [2]. odontogenic keratocyst originates from the remnants of the dental lamina and exhibits typical cystic lining of 6 to 10 cell layer thickness. the odontogenic keratocyst is recognized for its intrinsic growth potential and its high rate of recurrence [5]. unicystic ameloblastoma is a variant of intraosseous ameloblastoma that occurs as a single cystic cavity with or without luminal proliferation [2]. unicystic ameloblastomas usually occur in younger individuals. although a variant of ameloblastoma it possesses a less aggressive growth pattern compared to a solid ameloblastoma. on histologic examination they show a typical ameloblastomatous epithelium lining part of the cyst cavity, with or without luminal and/or mural tumor proliferation [5, 6]. cyclooxygenase 2 is a 72 kda cytokine-inducible enzyme that is situated in the nuclear membrane and the rough endoplasmic reticulum of the human cell [7,8]. cox-2 is an important enzyme responsible for the synthesis of the prostanoids; prostaglandins, prostacyclin and thromboxane [9]. during inflammation, there is an abundance of cox-2 in macrophages and other inflammatory cells [10]. an upregulation of cox-2 is brought about by growth factors and inflammatory mediators such as lipopolysaccharides and tumor necrosis factors [8]. an over expression of cox-2 has been associated with cell proliferation, apoptosis, angiogenesis and enhanced invasiveness [11,12]. the expression of c0x-2 has been associated with precancerous lesion and with the malignant tumors of the head and neck regions.[13]. however, the role of cox-2 in the clinical behavior of odontogenic lesions is less explored [14, 15, 16,17]. the aim of this study was to evaluate the expression of cox-2 in odontogenic lesions and assess its role in the clinical behaviour of these lesions. material and methods this study was approved by the ethics committee of kasturba medical college manipal (108/2016). forty formalin fixed paraffin embedded tissue specimens; odontogenic keratocyst (n=10) dentigerous cyst (n=10) radicular cyst (n=10) and unicystic ameloblastomas (n=10) were retrieved from the archives of the department. five-micron thick sections were cut from each block and the sections were stained with hematoxylin and eosin. tissue sections were reviewed to confirm the diagnosis and to determine the adequacy of the tissue for immunohistochemical staining. immunohistochemical procedure four-micron thick sections were cut from the ffpe tissue blocks and were taken onto aminopropyl triethoxy silane (apes, sigma-aldrich co. st. louis, usa) coated slides. sections were deparaffinized through 3 changes of xylene and hydrated through descending grades of alcohol (100%, 95%, 70%). the endogenous peroxidase activity was quenched by incubating the sections in 3% h2o2 for 10 minutes. antigen retrieval was carried out using a domestic pressure cooker method with sodium citrate solution (ph 6.0). following antigen retrieval, the tissue sections were allowed to cool at room temperature for 20 minutes. to block the non-specific binding sites the section were treated with protein block (novacastra lieca biosystem, newcastle) for 5 mins. the sections were then incubated in mouse monoclonal cox -2 primary antibody (novacastratm mouse monoclonal antibody cyclooxygenase-2: ncl-cox-24h12) immunohistochemical expression of cyclooxygenase 2 reflects the proliferative activity in the epithelium of odontogenic lesions vol 10 no 1 (2022) doi 10.5195/d3000.2022.205 http://dentistry3000.pitt.edu at 1:1000 dilution (with tris buffer, ph 7.6) for 60 minutes at 37°c in a moist humidifying chamber. the antigen-antibody reaction was developed by incubating the section with secondary antibody (secondary antibody novolink polymer antimouse igg-poly-hrp) for 30 minutes at room temperature in a moist chamber. the antigen-antibody reaction was detected using the chromogen diaminobenzidine and its buffer. the sections were counterstained in mayer’s hematoxylin, were dehydrated through ascending grades of alcohol (70%, 95% and 100%) cleared in xylene and mounted with dibutylphthalate xylene (dpx). for positive control tissue sections of normal buccal mucosa and colon carcinoma was used. for a negative control, a tissue section was stained in the similar manner except that instead of the primary monoclonal antibody, the tris buffer was used. for ki-67 four-micron thick sections were cut from the ffpe tissue blocks (one each of dentigerous cyst, radicular cyst, odontogenic keratocyst and ameloblastoma) and were taken onto aminopropyl triethoxy silane (apes, sigma-aldrich co. st. louis, usa) coated slides. sections were deparaffinized through 3 changes of xylene and hydrated through descending grades of alcohol (100%, 95%, 70%). antigen retrieval was performed using tris edta buffer at ph 9.0, endogenous peroxide was neutralized by treating the sections with pre-diluted 3% hydrogen peroxide. following this, sections were incubated with pre diluted mouse monoclonal primary antibody ki -67 (clone ki-67 gm001, pathnsitu, usa) for 1 hour at room temperature in a moist humidifying chamber. then the sections were incubated with pre diluted primary target binder (polyexcel target binder, pathnsitu, usa) at room temperature for 10 minutes. slides were then incubated with secondary antibody pre diluted (polyexcel poly hrp, pathnsitu,usa), at room temperature for 10 minutes. the peroxidase activity was developed with diamino benzidine tetra hydrochloride (dab). finally, the sections were counter stained with mayer’s haematoxylin, dehydrated, cleared, and mounted with dibutylphthalate xylene (dpx). assessment of cox-2 immunostaining the expression of cox-2 was carried out in a semi-quantitative manner independently by two pathologists using a light microscope (olympus bx41). five high-power fields were observed for each case and the cells that showed a diffuse brown stain in the cytoplasm was considered positive for cox-2. the percentage of positive cells in the four fields was recorded, the mean percentage of positive cells in all the four fields was determined. the expression of cox-2 was scored as 0 (absence of the stain), 1 (<25% of the cells were positive) 2 (25-50% of the cells were positive), 3 (51-75% of positive cells), and 4 (75-100% of positive cells). the intensity of the expression of cox-2 was graded as 0 (absence of the expression), 1 (weak expression), 2 (moderate expression) and 3 (strong expression). the total score was obtained by adding the score of percentage of positive cells and the score of staining intensity as suggested by mendes et al (modified) [15]. a total score of 0 was given if there was complete absence of the expression of cox-2. and finally, the expression of cox-2 was regarded as low expression when the sum of the score for the percentage of positive cells and the intensity of the expression ranged from 1-4 and the expression of cox-2 was regarded as a high expression when the sum of the score for the percentage of positive cells and the intensity of the expression ranged from 5-7. statistical analysis all statistical analyses were performed using the statistical package for social sciences (spss) version 16.0. immunohistochemical expression of cyclooxygenase 2 reflects the proliferative activity in the epithelium of odontogenic lesions vol 10 no 1 (2022) doi 10.5195/d3000.2022.205 http://dentistry3000.pitt.edu descriptive analysis was carried out for patient characteristics. inter-observer variability was assessed using cohen’s kappa coefficient and was found to be of good reproducibility (0.92). the association between expression of cox-2 and the odontogenic lesions was assessed using a one-way anova analysis method and the chi square test. a p value of < 0.05 was regarded as statistically significant. results in this study the expression of cyclooxygenase-2 was evaluated in 40 cases of odontogenic cysts and tumors that included 10 cases of dentigerous cysts, 10 cases of radicular cyst, 10 cases of odontogenic keratocyst and 10 cases of unicystic ameloblastoma. the age of the patients ranged from 10 years to 71 years (mean age 31.5 years). among the patients 27 of them were males and 13 of them were females. the clinico pathological details of all the odontogenic lesions along with their cox-2 expression pattern are given in table 1. all the odontogenic lesions except one case of dentigerous cyst that were evaluated showed a mild severe expression of cox-2 (fig 1). among the dentigerous cysts in 4/10 (40%) cysts the mean percentage of odontogenic epithelial cells that expressed cox-2 ranged between 25%-50%. among the radicular cyst in 4/10 (40%) the mean percentage of cox-2 positive cells was above 75%. among the odontogenic keratocysts in 3/10 (30%) cases of percentage of cox-2 positive cells was above 75%. among the unicystic ameloblastomas in 3/10 (30%) cases of percentage of cox-2 positive cells was above 75% (fig 2). among the dentigerous cysts in 5/10 (50%) cases the intensity of cox-2 expression was that of a moderate expression. all the 10/10 (100%) cases of radicular cysts showed a moderate intensity of the expression of cox-2. among the odontogenic keratocysts in 9/10 (90%) cases showed a moderate intensity of the expression of the of cox-2. among the unicystic ameloblastomas in 6/10 (60%) cases showed a moderate intensity of the expression of cox-2 (fig 3) table 1. clinico-pathological details of the odontogenic lesions and their cox-2 expression profile immunohistochemical expression of cyclooxygenase 2 reflects the proliferative activity in the epithelium of odontogenic lesions vol 10 no 1 (2022) doi 10.5195/d3000.2022.205 http://dentistry3000.pitt.edu based on the overall expression of cox-2 in the odontogenic lesions, all the dentigerous cysts 10/10 (100%) cases, 6/10 (60%) of the radicular cysts, 7/10 (40%) of the odontogenic keratocysts and 4/10 (40%) of unicystic ameloblastoma showed a low expression of cox-2. while 4/10 (40%) of radicular cysts. 3/10 (30%) of the odontogenic keratocysts and 6/10 (60%) of unicystic ameloblastoma showed a high expression of cox-2 (fig 4). the comparison of the mean percentage of odontogenic epithelial cells expressing cox-2 is given in table 2. the radicular cyst showed a high expression of cox-2 compared to dentigerous cyst or odontogenic keratocyst (p=0.031) (table 3). among all the odontogenic lesions, unicystic ameloblastoma showed a high expression of cox-2 (p=0.036) (table 4). the comparison of the relative risk among all the odontogenic lesions is given in table 5. discussion cox-2, one of the isoforms of cyclooxygenase is a key enzyme which mediates the process of inflammation. an up regulation of this enhances the synthesis of prostanoids especially prostaglandin e [18, 19]. the cox-2/pge2 pathway, by enhancing cell survival and growth, has been suggested to play a role in suppression of apoptosis and enhancing cell survival and growth [20]. this study evaluated the influence that cox-2 imparts on the proliferative activity of odontogenic epithelial cells. dentigerous cysts develop due to accumulation of fluid (including glycosaminoglycan’s) between reduced enamel epithelium of dental follicle and crown of unerupted teeth. in this study most of the dentigerous cyst (9/10=90%) showed that 25%-50% of the reduced enamel epithelial cells expressed cox-2. about 50% of the cysts showed a moderate intensity of the expression of cox-2. in the comparative study by seyedmajidi et al only 5/15 (33.3%) of the dentigerous cyst expressed cox-2, but only one case showed a high expression for cox-2. in the study by fig 1. expression of cox-2 in a. dentigerous cyst, b. odontogenic keratocyst c. radicular cyst, d. unicystic ameloblastoma. fig 2. comparison of the percentage of cox-2 positive (score) odontogenic epithelial cells in different odontogenic lesions odontogenic lesion mean % of positive odontogenic cells mean difference std. error anova test 95% confidence interval lower bound upper bound dentigerous cyst 26.10±17.7 -30.600 10.683 .033* -59.37 -1.83 radicular cyst 56.70±23.6 dentigerous cyst 26.10±17.7 -22.400 10.683 .174 -51.17 6.37 odontogenic keratocyst 48.50±26.2 dentigerous cyst 26.10±17.7 -30.900 10.683 .031* -59.67 -2.13 unicystic ameloblastoma 57±26.9 immunohistochemical expression of cyclooxygenase 2 reflects the proliferative activity in the epithelium of odontogenic lesions vol 10 no 1 (2022) doi 10.5195/d3000.2022.205 http://dentistry3000.pitt.edu alsaegh et al 13/16 cases expressed cox-2 [16]. in the later stages of the clinical course of dentigerous cyst, bone resorption is a common feature. prostaglandin e 2 is a vital molecule that stimulates or inhibits bone metabolism and can increase the number of functionally active osteoclasts [21]. an upregulation of cox-2 in the cells of the reduced enamel epithelium will induce the synthesis pge2 and facilitate bone resorption. kumamoto et al demonstrated that sonic hedge hog (shh) signaling molecule is expressed by the odontogenic epithelial cells of the dentigerous cyst [22]. the shh pathway through activation of ras/raf/erk pathway can in turn induce the expression of cox-2 in these cysts [16, 23]. radicular cysts are categorized as inflammatory odontogenic cysts that occur as a sequel to dental caries. the infection in the periapical area stimulates the cell rests of malassez in the periapical region to proliferate and result in a cyst formation. in this study the expression of cox2 was seen in more than 75% of the odontogenic rests in 4 out of the 10 cysts that were evaluated. tsai et al in their study found that radicular cysts with more inflammation were associated with a high cox 2 expression. in their study they found that cox-2 was expressed by the odontogenic epithelial layer, in the subepithelial fibroblasts, macrophages and endothelial cells [24]. in healthy tissue cox-2 is not detected or detected in only in low levels and is not expressed by normal epithelial or normal odontogenic epithelial cells. growth factors, hormones and cytokines induce the expression of cox-2 [25, 26]. table 2. comparison of the mean % of positive odontogenic cells among the different odontogenic lesions odontogenic lesion mean % of positive odontogenic cells mean difference std. error anova test 95% confidence interval lower bound upper bound dentigerous cyst 26.10±17.7 -30.600 10.683 .033* -59.37 -1.83 radicular cyst 56.70±23.6 dentigerous cyst 26.10±17.7 -22.400 10.683 .174 -51.17 6.37 odontogenic keratocyst 48.50±26.2 dentigerous cyst 26.10±17.7 -30.900 10.683 .031* -59.67 -2.13 unicystic ameloblastoma 57±26.9 radicular cyst 56.70±23.6 8.200 10.683 .868 -20.57 36.97 odontogenic keratocyst 48.50±26.2 radicular cyst 56.70±23.6 -300 10.683 1.000 -29.07 28.47 unicystic ameloblastoma 57±26.9 odontogenic keratocyst 48.50±26.2 -8500 10.683 .856 -37.27 20.27 unicystic ameloblastoma 57±26.9 *significant association odontogenic lesion mean % of positive odontogenic cells mean difference std. error anova test 95% confidence interval lower bound upper bound dentigerous cyst 26.10±17.7 -30.600 10.683 .033* -59.37 -1.83 radicular cyst 56.70±23.6 dentigerous cyst 26.10±17.7 -22.400 10.683 .174 -51.17 6.37 odontogenic keratocyst 48.50±26.2 dentigerous cyst 26.10±17.7 -30.900 10.683 .031* -59.67 -2.13 unicystic ameloblastoma 57±26.9 radicular cyst 56.70±23.6 8.200 10.683 .868 -20.57 36.97 odontogenic keratocyst 48.50±26.2 radicular cyst 56.70±23.6 -300 10.683 1.000 -29.07 28.47 unicystic ameloblastoma 57±26.9 odontogenic keratocyst 48.50±26.2 -8500 10.683 .856 -37.27 20.27 unicystic ameloblastoma 57±26.9 fig 3. comparison of the intensity of the expression of cox-2 by the odontogenic epithelial cells in different odontogenic lesions immunohistochemical expression of cyclooxygenase 2 reflects the proliferative activity in the epithelium of odontogenic lesions vol 10 no 1 (2022) doi 10.5195/d3000.2022.205 http://dentistry3000.pitt.edu during the inflammatory process there is an abundance of chemical mediators including tumor necrosis factor and interlukin-1. interlukin 1 alpha has been found to increase the expression of cox-2 in fibroblasts [27]. when cox-2 expressed, it catalyzes the synthesis of prostaglandin from arachidonic acid [28]. odontogenic keratocysts have a reputation of being aggressive in its clinical behaviour. in this study the expression of cox-2 was seen in more than 75% of the odontogenic cystic lining cells in 3/10 (30%) odontogenic keratocysts. seyedmajidi, et al in their study found that all (15/15) of the odontogenic keratocyst that they evaluated, expressed cox-2, but only 5/15 (33%) cysts showed a strong expression of cox2.they suggested that the cox-2 is a major acting marker in the aggressive behavior of odontogenic keratocysts [16]. in a study by mendes et al all the 20 cases of odontogenic keratocysts that they evaluated expressed cox-2, and 18 of these 20 odontogenic keratocyst showed a strong expression of cox-2. they also found that the cysts that showed a showed a strong expression of cox-2 also expresses p53 and ki 67.cox-2 may contribute to the biological regulation of its epithelial lining [15]. in another study by kaczmarzyk et al 37/41 odontogenic keratocyst that they evaluated expressed cox-2. they also found that the expression of cox-2 correlated with the expression of bcl2 in these odontogenic keratocysts [17]. table 3. the association between the odontogenic cysts and the expression (total score) of cox-2 fig 4. comparison of the pattern of expression of cox-2 among the different odontogenic lesions. immunohistochemical expression of cyclooxygenase 2 reflects the proliferative activity in the epithelium of odontogenic lesions vol 10 no 1 (2022) doi 10.5195/d3000.2022.205 http://dentistry3000.pitt.edu among the unicystic ameloblastomas in 3/10 (33%) cases the expression of cox-2 was seen in more than 75% of the odontogenic epithelial cells. earlier, studies have evaluated the expression of cox-2 in solid ameloblastoma than in unicystic ameloblastomas[13,15]. in the study by seyedmajdi et al 3/15 of the ameloblastomas showed a high expression of cox-2. they found that the pattern of expression of cox-2 in the ameloblastomas was similar to that of odontogenic keratocysts. but there was a significant difference between the expression of cox-2 in ameloblastomas and dentigerous cysts [16]. to validate the role of cox-2 in proliferation of odontogenic epithelium, one case each of dentigerous cyst, radicular cyst, odontogenic keratocyst and unicystic ameloblastoma was immunohistochemically stained with ki-67. the case of unicystic ameloblastoma and the case of odontogenic keratocyst stained positive for ki-67, whereas ki 67 was not expressed by the radicular cyst and dentigerous cyst (figure 5). the association of cox-2 and ki-67 in these odontogenic lesions will be evaluated in future. this study along with other studies in literature strengthens the notion that cox-2 plays a vital role in confirming an aggressive nature to odontogenic lesions. as a limited number of odontogenic lesions were evaluated in this study, further studies on a larger cohort of odontogenic lesions and by evaluating the expression of cox-2 with other proliferative markers is necessary to determine the molecular mechanism by which cox-2 functions in the odontogenic epithelial cells. table 4. association of the expression of cox-2 (total score) among the different odontogenic lesions figure 5. a. dentigerous cyst (absence of expression of ki 67) b. odontogenic keratocyst (ki-67 expression in the basal and supra basal cells of the odontogenic epithelial lining) c. radicular cyst (no expression of ki 67). d. unicystic ameloblastoma (expression of ki-67 by the ameloblast-like cells) *significant association odontogenic lesion mean % of positive odontogenic cells mean difference std. error anova test 95% confidence interval lower bound upper bound dentigerous cyst 26.10±17.7 -30.600 10.683 .033* -59.37 -1.83 radicular cyst 56.70±23.6 dentigerous cyst 26.10±17.7 -22.400 10.683 .174 -51.17 6.37 odontogenic keratocyst 48.50±26.2 dentigerous cyst 26.10±17.7 -30.900 10.683 .031* -59.67 -2.13 unicystic ameloblastoma 57±26.9 immunohistochemical expression of cyclooxygenase 2 reflects the proliferative activity in the epithelium of odontogenic lesions vol 10 no 1 (2022) doi 10.5195/d3000.2022.205 http://dentistry3000.pitt.edu conclusion the present study evaluated the expression of cox-2 in the odontogenic lesions; dentigerous cyst, radicular cyst, odontogenic keratocyst and unicystic ameloblastoma. the expression of cox-2 was higher in radicular cyst, odontogenic keratocyst and unicystic ameloblastoma compared to dentigerous cyst. cox-2 being an inflammatory marker, showed a high expression among the radicular cysts. this study emphasizes the fact that cox-2 plays a vital role in biological behavior of odontogenic cysts and tumors. thus, incorporating antiinflammatory medicaments and chemopreventive inhibitors of cox-2 will prove to be beneficial in the management of radicular cysts, odontogenic keratocysts and unicystic ameloblastomas. conflicts of interest the authors deny any conflicts of interest in regards to the current study. acknowledgement dr aditi deshpande for providing us with cox-2 monoclonal antibody for the study. references 1. shear m, speight p, shear m. cysts of the oral and maxillofacial regions. oxford: blackwell pub.; 2007. 2. speight p. odontogenic and nonodontogenic developmental cysts. in: el -naggar ak, chan jkc, grandis jr, takata t, slootweg pj, eds. who classification of head and neck tumours, 4th edn. lyon: world health organization. 2017;234-242. 3. radicular cyst of the anterior maxilla: an insight into the most common inflammatory cyst of the jaws. koju s, chaurasia nk, marla v, niroula d, poudel p. j dent res rev 2019; 6: 26-29 https://www.jdrrrg/text.asp 4. does cytokine profiling of aspirate from jaw cysts and tumors have a role in diagnosis? kolokythas a, karas m, sarna t, flick w, miloro m j oral maxillofac surg. 2012 70(5):107080. doi: 10.1016/j.joms.2011.04.003 5. cysts of the orofacial region, shafer’s textbook of oral pathology 9th edition, adaptive editor sivapathasundaram, elsevier page no 72-5. 6. unicystic ameloblastoma: a clinicopathologic study of 33 chinese patients. li tj, wu yt, yu sf, yu gy. am j surg pathol. 2000 oct;24(10):1385-92. doi: 10.1097/00000478-200010000-00008 7. prostaglandin endoperoxidase h synthase (cyclooxygenases)-1 and 2. smith wl, garavito rm, dewitt dl. j biol chem 1996; 271: 3157-60. doi: 10.1074/jbc.271.52.33157 8. the cyclooxygenases. chandrasekharan nv and simmons dl. genome biol 2004; 5:241 doi: 10.1186/gb-2004-5-9-241 9. the role of cox-2 in intestinal inflammation and colorectal cancer.wang d, dubois rn. oncogene 2010; 29: 781-789. doi: 10.1038/onc.2009.421 table 5. association of the expression of cox-2 (total score) and risk ratio among the odontogenic lesions https://www.jdrrrg/text.asp immunohistochemical expression of cyclooxygenase 2 reflects the proliferative activity in the epithelium of odontogenic lesions vol 10 no 1 (2022) doi 10.5195/d3000.2022.205 http://dentistry3000.pitt.edu 10. inducible isoforms of cyclooxygenase and nitric oxide synthase in inflammation. van jr, mitchell ja, appelton i, tomlinson a, bishop -bailey d, croxtall x et al proc natl acad sci usa 1994; 91(16): 20462050. doi: 10.1073/pnas.91.6.2046 11. alteration in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxidase synthase 2. tsujii m, dubois rn. cell 1995; 83 (3): 493-501. doi: 10.1016/0092-8674(95)90127-2 12. cyclooxygenase regulates angiogenesis induced by colon cancer cells. tsujii m kawano s, tuji s sawaoka h, hori m, dubois rn. cell 1998; 93 (5); 705-716 doi: 10.1016/s0092-8674(00)81433-6 13. deregulated cyclooxygenase -2 expression in oral permalignant tissues. banerjee ag, gopalkrishnan vk, bhattacharya i, vishwanatha jk. mol cancer ther i 2002; 12651271. 14. odontogenic epithelial proliferation is correlated with cox-2 expression in dentigerous cyst and ameloblastoma. alsaegh ma, miyashita h, taniguchi t, zhu sr. experimental and therapeutic medicine 2017;13: 247-253. doi: 10.3892/etm.2016.3939 15. a comparative immubohistochemical analysis of cox-2, p53, and ki 67 expression in keratocystic odontogenic tumors. mendes ra carvalho jfc, van der waal i. oral surg oral med oral path oral radiol endod 2011; 111(3): 333339.doi: 10.1016/j.tripleo.2010.10.004 16. immunohistochemical analysis of cox-2 expression in dentigerous cyst, keratocystic odontogenic tumor and ameloblastoma: a comparative study. seyedmajidi m, shafaee s, siadati s, moghaddam ea, ghasemi n, bijani a et al.dent res j 2015;12(3):278-284. 17. investigation of clinicopathological parameters and expression of cox-2, bcl-2, pcna, and p53 in primary and recurrent sporadic odontogenic keratocyst. kaczmarzyk t, kiselowski k, koszowski r, rynkiewicz m, gawelek e, babiuch k et al. clin oral invest 2018; 22: 3097106. doi: 10.1007/s00784-018-24007 18. expression of cyclooxygenase-2 in prostate carcinoma. yoshikawa r, sano h, masuda c, kawamura m, tsubouchi y, chargui j et al cancer 2000;89(3):589-596. 19. cyclooxygenase-2 expression in endometrial cancer: correlation with microvessel count and expression of vascular endothelial growth factor and thymidine phosphorylase. fujiwaki r,iida k, kanasaki h, ozaki t, hata k, miyazaki k. human pathology 2002; 33(2):213-219. doi: 10.1053/hupa.2002.31292 20. modulation of apoptosis and bcl-2 expression by prostaglandin e2 in human colon cancer cells. sheng h, shao j, morrow jd, beauchamp rd, dubois rn. cancer res 1998; 58(2):362366 21. prostaglandin and bone: potential risks and benefits related to the use of nonsteroidal antiinflammatory drugs in clinical dentistry francon rn, teofilo jm, satin rb and lamano tj. oral sci 2008; 50:247-52. doi: 10.2334/josnusd.50.247 22. expression of sonic hedgehog (shh) signaling molecules in ameloblastomas. kumamoto h, ohki k, ooya k. j oral pathol med 2004; 33: 185-190.doi: 10.1111/j.09042512.2004.00070.x 23. p53 mediated induction of cox-2 counteracts p53 or genotoxic stress induced apoptosis. han ja, kim jl, ongusaha pp, hwang dh, ballou lr, mahale a et al. embo j 2002; 21:5635 5644. doi:10.1093/emboj/cdf591 24. immunohistochemical localization of cyclooxygenase-2 in radicular cysts. tsai ch, huang fm, yang lc, chou my, chang yc. int endod j. 2002 oct; 35(10):854-858. doi: 10.1046/j.13652591.2002.00584.x 25. cyclooxygenase in biology and disease. dubois rn, abramson sb, crofford l, gupta ra, simon ls, van de putte lb et al. faseb j. 1998 sep; 12(12):1063-1073. 26. update on oral lichen planus: etiopathogenesis and management. scully c, beyli m, ferreiro mc, ficarra g, gill y, griffiths m et al crit rev oral biol med 1998;9(1):86-122. doi: 10.1177/10454411980090010501 27. signaling pathways regulating il-1 alpha induced cox-2 expression. immunohistochemical expression of cyclooxygenase 2 reflects the proliferative activity in the epithelium of odontogenic lesions vol 10 no 1 (2022) doi 10.5195/d3000.2022.205 http://dentistry3000.pitt.edu ogata s, kubota y, yamashiro t, takeuchi h, ninomiya t, suyuma y et al. j dent res 2007; 86: 186-91. doi: 10.1177/154405910708600215 28. cyclooxygenase 2 and carcinogenesis. prescott sm, fitzpatrick fa, biochem biophys acta 2000; 1470:69-78.doi: 10.1016/s0304-419x(00)00006-8 influence of occlusal anatomy on acrylic resin cad/cam crowns fracture load and stress distribution vol 9 no 1 (2021) doi 10.5195/d3000.2021.118 http://dentistry3000.pitt.edu influence of occlusal anatomy on acrylic resin cad/cam crowns fracture load and stress distribution alexandre luiz souto borges1, aline lins de lima1, larissa mendes campaner1, marco antonio bottino1, amanda maria de oliveira dal piva1, joão paulo mendes tribst1 1 department of dental material and prosthodontics, são paulo state university, institute of science and technology – são josé dos campos, sp, brazil. abstract objectives: this study compared the influence of occlusal anatomy on acrylic cad/cam crowns fracture load and stress distribution. the null hypothesis was that there would be no difference between the provisional crowns fracture load and stress according to different occlusal anatomy. methods: a full-crown preparation was simulated using dentin analogue (g10, protec, são paulo, brazil) totaling 20 identical preparations. next, twenty acrylic crowns were milled using different occlusal design parameter (young or adult) available in the software database. the crowns were cemented (temp-bond, ne kerr dental, brea, ca, usa) and fractured using a compressive load (0.5 mm/min of cross-head speed). data were analyzed by using one-way anova and tukey tests (p< 0.05). a similar geometry was modeled and exported to the analysis software to perform a static structural analysis. the maximum principal stress was calculated using the finite element method with 300 n chewing load simulation. results: the occlusal anatomy significantly influenced the load-to-fracture (p<0.05). young design showed lower fracture load (1139±132 n) than adult design (2007±345 n). the tensile stress distribution showed a similar pattern for both groups however the highest stress peak was calculated for young design (76 mpa) in the occlusal surface. conclusion: the anatomy design with higher cusp angulation and occlusal sulcus more evident can increase the stress concentration and reduce the fracture load for acrylic resin cad/cam crowns. key words: finite element analysis; computer-aided design; crowns; acrylic resins; weight-bearing. citation: borges als, et al. (2021) influence of occlusal anatomy on acrylic resin cad/cam crowns fracture load and stress distribution. dentistry 3000. 1:a001 doi:10.5195/d3000.2021.118 received: october 10, 2020 accepted: february 2, 2021 published: may 6, 2021 copyright: ©2021 borges als, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: joao.tribst@gmail.com introduction temporary crowns are indicated to be used as an intermediate restoration between the time of tooth preparation and the final indirect restoration procedure [1,2]. there are two major approaches for fabrication of temporary crowns: direct (manual) and indirect methods (cad/cam) [1]. computer-aided design and computer-aided manufacturing (cad-cam) method allow the manufacture of acrylic crowns in pre-polymerized conditions, which improves mechanical properties, reduces discoloration, and increases the anatomy precision and reduces the marginal gap [3]. another advantage of polymerbased cad/cam materials is their good machinability [4]. the literature reports that the provisional cad/cam fixed dental prosthesis [5] and crown [1] exhibit a higher mechanical strength compared to directly fabricated, when manufactured with the same material [5]. despite the restorative technique, another factor that was strongly linked to fractures of dental restorations is their occlusal anatomy. for ceramic crowns, the http://dentistry3000.pitt.edu/ influence of occlusal anatomy on acrylic resin cad/cam crowns fracture load and stress distribution vol 9 no 1 (2021) doi 10.5195/d3000.2021.118 http://dentistry3000.pitt.edu cusp angles can influence the prosthesis’s resistance and the stress concentration [6,7]. however the occlusal geometry and and fissure radius are still inconclusive [7]. basically, crowns with thicker margins, smaller convergence angle and fully bonded are recommended for increasing fracture resistance final restorations [8]. this can be justified because a greater crown thickness and cusp angle resulted in a higher crack initiation load [9]. however, these information were not available for acrylic resin cad/cam crowns. based on this, the aim of the current study was to investigate the influence of different occlusal anatomy patterns on the fracture behavior of acrylic resin full-crown restorations. the null hypothesis was that there would be no difference between the provisional crowns fracture load and stress according to different occlusal anatomy. methods twenty (20) crown preparations were machined in epoxy resin (g10, protec, são paulo, brazil) using cad/cam (computer aided design/computer aided machine) technology. the use of this dentin analogue has been previous validated in literature [10,11], and its usage allows the samples standardization with a homogeneous substrate, reducing the bias associated with the complexity of mechanical properties in natural human teeth. the occlusal and axial walls were uniformly reduced (1.5 mm). the preparations presented rounded internal angles, chamfer preparation, and total occlusal convergence of 20°. the abutments were cleaned in an ultrasonic bath with distilled water for 5 min and embedded in selfcured polyurethane resin (25 mm ø x 20 mm height) up to 1.5 mm bellow the chamfer [10]. polyether (impregum f, 3m espe) was used to simulate the periodontal ligament [12]. according to the literature, the root embedment using polyether material as periodontal ligament simulator have a significant effect on fracture resistance and can modify the root fracture modes [13]. while the present study was not expecting to observe root fractures before the provisional crown fracture, the periodontal ligament simulation was performed to allow a proper load dissipation. a thin layer of titanium dioxidebased powder was sprayed onto each abutment for scanning (ineos blue, inlab sw4.2, sirona, benshein, germany) and subsequent crown design. for that, the cerec inlab (sirona for dental systems, benshelm, germany) database was used to provide the restoration anatomy information , and two shapes of crowns were obtained: ‘‘adult’’ figure 1. sample preparation. a) prepared abutment tooth, b) digital impression in cad software, c) buccal view from adult design crown, d) buccal view from young design crown, e) occlusal view from adult design crown and f) occlusal view from young design. http://dentistry3000.pitt.edu/ influence of occlusal anatomy on acrylic resin cad/cam crowns fracture load and stress distribution vol 9 no 1 (2021) doi 10.5195/d3000.2021.118 http://dentistry3000.pitt.edu (ad plane cusp) and ‘‘young’’ (y high cusp heights) (figure. 1). the cad/cam temporary acrylic crowns (vita zahnfabrick, bad säckingen, germany) were randomly allocated in groups according to the anatomy shape (n=10). the g10 preparations were etched with 10% hydrofluoric acid (hf) for 60 s, washed with air/water jet for 30 s, and then dried [14]. the intaglio surface of each crown was air-abraded with 50 µm alumina particles at a pressure of 20 bar from a 10 mm distance for 50 s in each face with a constant particle incidence angle at 45º. the cement (temp-bond, ne kerr dental, brea, ca, usa) was manipulated in 1:1 ratio and applied onto the intaglio surface of the crowns, which were then placed on the respective abutment and kept in a 750g weight for 5 min. after the working time, the excess cement was carefully removed (figure 2). the cemented restorations were stored in 37 °c distilled water for 24 h before the fracture test. each crown was submitted to single load to fracture test (load cell of 1000 kgf and 0.5 mm/min of cross-head speed) in a universal testing machine (dl-1000, emic, são josé dos pinhais, brazil). the data was recorded as the mean load value (in n) of both groups. the maximum load to fracture data were statistically analyzed using one-way analysis of variance (anova) and the post-hoc tukey multiple range tests (α=0.05) [15]. finite element analysis a three-dimensional (3d) model of an upper first molar was used to evaluate the tensile stress of the resin crown. the model containing root, pulp chamber, periodontal ligament, medullary and cortical bones was imported to rhinoceros cad software (version 5.0 sr8, mcneel north america, seattle, usa). for each group (young and adult), the anatomy previous provided by the database during the sample preparations was exported as stl file to the cad software and converted to nurbs using a reverse engineering plugin. the volumetric solids were imported to the analysis software (ansys 16.0, ansys inc., houston, usa) in step (standard for the exchange of product model data) format. a mesh convergence test (10%) determined 331538 nodes and 223955 tetrahedral elements for the young design; and 3252248 nodes and 197852 elements for the adult design preparation (figure 3). the contacts were considered perfectly bonded. the static structural mechanical analysis was performed loading the groups with 300 n distributed at contact areas and the base constrained figure 2. luting procedure in a) crown sitting with digital pressure and b) excess cement removal. http://dentistry3000.pitt.edu/ influence of occlusal anatomy on acrylic resin cad/cam crowns fracture load and stress distribution vol 9 no 1 (2021) doi 10.5195/d3000.2021.118 http://dentistry3000.pitt.edu following the experimental setup [16]. the maximum principal stress criterion was used for analysis of the tensile stress concentration. results were plotted in stress maps and the peaks recorded for quantitative comparison. table 1 shows the material properties used for the finite element analysis. results one-way anova showed that crown design influenced on the fracture load (f = 55.19, p < 0.001). for adult design the load to fracture average was 2007±345 n and for young design the load to fracture average was 1139±132 n. the young design concentrated more stress compared to adult design restorations (figure 4). the higher the cusp angle, the higher the stress concentration in the crown’s intaglio surface and occlusal surface. the tensile stress peak values are 76 mpa for young design and 35 mpa for adult design. the stress concentration regions for both groups suggest that the failure origin will be located in the occlusal sulcus. discussion the goal of this study was to evaluate the effect of cad/cam acrylic crowns occlusal anatomy on the fracture behavior; it was observed that the design influenced the crowns’ fracture load and stress concentration, leading to rejection of the study’s hypothesis. figure 3. boundary conditions simulated in finite element analysis. a) mesh division, b) occlusal load contact points and c) fixed support. table 1. material properties used for the finite element analysis. *characterization done by an acoustic excitation pulse. materials elastic modulus (gpa)* poisson ratio* polyurethane 3.60 0.30 polyether 0.05 0.45 dentin analogue 17.90 0.32 cement 5.50 0.30 acrylic resin 6.00 0.30 http://dentistry3000.pitt.edu/ influence of occlusal anatomy on acrylic resin cad/cam crowns fracture load and stress distribution vol 9 no 1 (2021) doi 10.5195/d3000.2021.118 http://dentistry3000.pitt.edu provisional crowns are expected to have sufficient resistance against bite forces. a previous study [17] investigated the maximum human bite forces; for female they found a bite force ranging from 108.9 n to 834.6 n (mean of 424.2) and from 262.8 n to 999.3 n (mean of 587.2) for male. in our study, the fracture load of the acrylic crown had a value above the average bite strength values for both genders. a previous study [18] reported that there is no significant difference for the fracture resistance between provisional crowns using the chamfer preparation with a width ranging from 0.6 mm to 1 mm. the present study complements this information suggesting that the failure will start in the occlusal surface and the anatomy should be more relevant for the maximum fracture load on acrylic resin cad/cam crowns. the most important role that a provisional restoration plays is to stabilize and protect the existing tooth structure after tooth preparation. besides, temporary crowns also act as interim prosthesis in cases where treatment gets delayed as in orthodontics or implants. therefore, temporary materials are required to be tough and fracture resistant [19]. based on this, the present study suggests that the use of young design should be careful indicated since it presents higher stress concentration and lower load to fracture values, suggesting a higher failure possibility. despite the fitting accuracy of cad-cam restorations, an ideal occlusal surface is difficult to design, since it demands determining the dynamic relation figure 4. maximum principal stress maps results for the restoration with a-c) adult design and d-f) young design. http://dentistry3000.pitt.edu/ influence of occlusal anatomy on acrylic resin cad/cam crowns fracture load and stress distribution vol 9 no 1 (2021) doi 10.5195/d3000.2021.118 http://dentistry3000.pitt.edu between the dental arches, even if the cad database already present a generic restoration anatomy [20]. in this sense, the use of young or adult morphology can be used as a predominant factor to create the occlusal shape, however the contact points should be based in the antagonist tooth and individualized for each case. a previous report [21], found that full-crowns made using dental database with the antagonist design technique had a significantly smaller occlusal vertical discrepancy than those made with only correlation design technique; and that the use of the dental database with antagonist design provides the most accurate technique. the present study complements this report suggesting that the acrylic crown anatomy should be based in the patient`s occlusion also and not only in database anatomy. the major difference between both crown’s design simulated in the present study was the cusp angle and occlusal sulcus. despite both designs share the same clinical indication and can be manufactured for the same clinical case, the young design presents a more detailed anatomy. the explanation for the worst biomechanical behavior for the young design is because the cusps of posterior teeth substantial increased the bending and flexural loads during functional and parafunctional occlusal movements. these applied loads can theoretically generate a horizontal force component which tends to open up the principal sulcus space increasing the tensile stress concentration and subsequent creating cracks at the bottom of the fissure [22,23]. the present study corroborates with this statement since the numerical simulation showed an increased stress magnitude when the young anatomy was used. a previous finite element study suggest that the cusp angle can modify the loads incidence for provisional endocrowns restored molars [23]. the same indication can be performed for the present study considering only the fracture load and stress concentration for acrylic resin crowns. a 3d computer model can be used to simulate various conditions with numerous simulation parameters [24], however destructive mechanical tests, such as fracture load tests, are important for biomechanical analysis of tooth and dental restorative materials, as they enhance understanding of the behavior of teeth in high loading situations [25]. usually, areas of high stress concentration eventual failures are more expected to occur [26] and both methodologies can be associated to perform a properly investigation from the fracture mechanics [27]. in conventional acrylic resin restorations, the cross-linking agent may lead to variation in the material performance [28]. the results of the present study support the use of cad/cam, which may reduce voids in the composite, allowing for controlled polymerization and a more homogeneous matrix. according to an in vitro study [9] any surface irregularity including the sharp occlusal notch can be considered as a discontinuity in the microstructure of the material which can predispose the crown to crack initiation. factor that can be reduced using an adult design tooth anatomy. however, reducing the cusp angle and simplifying the tooth anatomy, can negatively affect the patient’s masticatory efficiency and muscular activity when used for artificial tooth in removable partial dentures [24,29]. the present study suggests, however that the use of simplified anatomical tooth can be positive for temporary crowns, in a shortterm period of use until the final restoration can be cemented. http://dentistry3000.pitt.edu/ influence of occlusal anatomy on acrylic resin cad/cam crowns fracture load and stress distribution vol 9 no 1 (2021) doi 10.5195/d3000.2021.118 http://dentistry3000.pitt.edu as strengths of the present methodologies, the fracture load is an important method employed to evaluate the ultimate strength of restored posterior teeth during compressive load fracture test [30]. this mechanical test is usually performed associating the influence of different factors that can affect simultaneously the restoration success [30,31]. in addition, the finite element method is extensively applied in dental materials mechanical assessment, allowing the simulation of different structures that compose the dental restoration [2,6,10,16,22-27]. this numerical approach allows the stress maps visualization and quantitative measurements of stress peaks, indicating with acceptable precision the failure area of possible fracture origin [23,32]. as study’s limitations is important to relate that a single compressive load to failure not replicate all of the clinical loads to which the restoration is exposed. the thermocycling also can reduce the bond strength between a cemented restoration increase the failure susceptibility [32,33]. the isotropic material simulated in the finite element study cannot be found in the clinics and patient’s occlusion can modify the load application pattern [32]. it is also important to note that, for acrylic resin, incomplete polymerization with residual monomer unreacted promotes high degree of water absorption, deterioration of intrinsic pigments and dissolution of material components [29,33] and further studies with mechanical aging simulation should also be performed. conclusion chairside digital workflow should be performed aiming to improve the indirect restoration mechanical response, however an anatomy design with higher cusp angulation and more evident occlusal sulcus can increase the stress concentration and reduce the fracture load for acrylic resin cad/cam crowns. acknowledgments the authors would like to thank são paulo research foundation (fapesp) with the grants nº 2013/14831-1 and nº 2014/006684. references 1. abdullah ao, tsitrou ea, pollington s. comparative in vitro evaluation of cad/cam vs conventional provisional crowns. j appl oral sci 2016; 24(3):25863. 2. rodrigues va, dal piva ao, yamaguchi ca, borges al, mukai mk, tribst jp. effect of framework type on survival probability of implant-supported temporary crowns: an in vitro study. j clin exp dent 2020; 12(5):433-9. 3. reeponmaha t, angwaravong o, angwarawong t. comparison of fracture strength after thermo-mechanical aging between provisional crowns made with cad/cam and conventional method. j adv prosthodont 2020; 12(4):218-24. 4. winter a, schurig a, rasche e, rösner f, kanus l, schmitter m. the flexural strength of cad/cam polymer crowns and the effect of artificial ageing on the fracture resistance of cad/cam polymer and ceramic single crowns. j mater sci mater med 2019; 31(1):9. 5. alt v, hannig m, wöstmann b, balkenhol m. fracture strength of temporary fixed partial dentures: cad/cam versus directly fabricated restorations. dent mater 2011; 27(4):339-47. 6. bowley jf, ichim ip, kieser ja, swain mv: fea evaluation of the resistance form of a premolar crown. j prosthodont 2013; 22(4):304-12. http://dentistry3000.pitt.edu/ influence of occlusal anatomy on acrylic resin cad/cam crowns fracture load and stress distribution vol 9 no 1 (2021) doi 10.5195/d3000.2021.118 http://dentistry3000.pitt.edu 7. sornsuwan t, swain mv. influence of occlusal geometry on ceramic crown fracture; role of cusp angle and fissure radius. j mech behav biomed mater 2011; 4(7):1057-66. 8. zhang z, sornsuwan t, rungsiyakull c, li w, li q, swain mv. effects of design parameters on fracture resistance of glass simulated dental crowns. dent mater 2016; 32(3):373-84. 9. shahmoradi m, wan b, zhang z, wilson t, swain m, li q. monolithic crowns fracture analysis: the effect of material properties, cusp angle and crown thickness. dent mater 2020; 36(8):1038-51. 10. dal piva ao, tribst jp, borges al, de melo rm, bottino ma. influence of substrate design for in vitro mechanical testing. j clin exp dent 2019; 1(11):119-25. 11. kelly jr, rungruanganunt p, hunter b, vailati f. development of a clinically validated bulk failure test for ceramic crowns. j prosthet dent. 2010 oct;104(4):228-38. 12. souza ac, xavier ta, platt ja, borges al. effect of base and inlay restorative material on the stress distribution and fracture resistance of weakened premolars. oper dent 2015; 40(4):158-66. 13. soares cj, pizi ec, fonseca rb, martins lr. influence of root embedment material and periodontal ligament simulation on fracture resistance tests. braz oral res. 2005 jan-mar;19(1):116. doi: 10.1590/s180683242005000100003. pmid: 16229350. 14. tribst jpm, monteiro jb, venturini ab, pereira gkr, bottino ma, melo rm, valandro lf. fatigue failure load of resinbonded simplified lithium disilicate glass-ceramic restorations: effect of ceramic conditioning methods. j adhes dent. 2019;21(4):373-381. 15. tribst jp, dal piva ao, madruga cf, valera mc, bresciani e, bottino ma, de melo rm. the impact of restorative material and ceramic thickness on cad\cam endocrowns. j clin exp dent. 2019; 1;11(11):969-977. 16. penteado mm, tribst jpm, dal piva am, ausiello p, zarone f, garcia-godoy f, borges al. mechanical behavior of conceptual posterior dental crowns with functional elasticity gradient. am j dent. 2019; 32(4):165-168. 17. calderon pds, kogawa em, lauris jrp, conti pcr. the influence of gender and bruxism on the human maximum bite force. j appl oral sci 2006;14(6):448-53. 18. karaman t, eser b, altintas e, atala mh. evaluation of the effects of finish line type and width on the fracture strength of provisional crowns. odontology 2020; 1-6. 19. dureja i, yadav b, malhotra p, dabas n, bhargava a, pahwa r. a comparative evaluation of vertical marginal fit of provisional crowns fabricated by computer-aided design/computer-aided manufacturing technique and direct (intraoral technique) and flexural strength of the materials: an in vitro study. j indian prosthodont soc 2018; 18(4):314-20. 20. muric a, gokcen röhlig b, ongul d, evlioglu g. comparing the precision of reproducibility of computer-aided occlusal design to conventional methods. j prosthodont res 2019; 63(1):110-4. 21. fasbinder dj, poticny dj. accuracy of occlusal contacts for crowns with chairside cad/cam techniques. int j comput dent 2010; 13(4):303-16. 22. wan b, shahmoradi m, zhang z, shibata y, sarrafpour b, swain m, li q. modelling of stress distribution and fracture in dental occlusal fissures. scientific reports 2019; 9(1):1-10. http://dentistry3000.pitt.edu/ influence of occlusal anatomy on acrylic resin cad/cam crowns fracture load and stress distribution vol 9 no 1 (2021) doi 10.5195/d3000.2021.118 http://dentistry3000.pitt.edu 23. tribst jpm, borges als, silvaconcílio lr, bottino ma, özcan m. effect of restorative material on mechanical response of provisional endocrowns: a 3d— fea study. materials 2021; 14(3):649. 24. syam s, soegiharto bm, siregar e. stress distribution of maxillary anterior retraction using miniscrew with anterior hook on continuous wire (3d simulation finite element analysis). j int dent medical res 2018; 11(3):791-798. 25. archangelo kc, penteado mm, dal piva amdo, nogueira as, tribst jpm, borges a ls. stress distribution and failure load of multilayer and monolayer glass ceramic. braz dent scii. 2019; 22(1), 17-22. 26. costa vl, tribst jp, borges al. influence of the occlusal contacts in formation of abfraction lesions in the upper premolar. braz dent sci. 2017; 20:115–123. 27. de andrade gs, tribst jpm, orozco ei, augusto mg, bottino ma, borges al, anami lc, saavedra gd. influence of different post-endodontic restorations on the fatigue survival and biomechanical behavior of central incisors. am j dent. 2020; 33(5):227-234. 28. saisadan d, manimaran p, meenapriya pk. in vitro comparative evaluation of mechanical properties of temporary restorative materials used in fixed partial denture. j pharm bioallied sci. 2016 oct;8(suppl 1):s105-s109. 29. türp jc, greene cs, strub jr. dental occlusion: a critical reflection on past, present and future concepts. j oral rehabil 2008; 35(6):446-53. 30. silva gr, silva nr, soares pv, costa ar, fernandes-neto aj, soares cj. influence of different load application devices on fracture resistance of restored premolars. braz dent j. 2012;23(5):484-9. 31. ausiello p, gloria a, maietta s, watts dc, martorelli m. stress distributions for hybrid composite endodontic post designs with and without a ferrule: fea study. polymers (basel). 2020 aug 16;12(8):1836. 32. tribst jpm, dal piva amdo, lopes gc, borges als, bottino ma, özcan m, et al. biaxial flexural strength and weilbull characteristics of adhesively luted hybrid and reinforced cad/cam materials to dentin: effect of self-etching ceramic primer versus hydrofluoric acid etching. j adhes sci technol. 2020; 34(12):1253-68 33. reeponmaha t, angwaravong o, angwarawong t. comparison of fracture strength after thermo-mechanical aging between provisional crowns made with cad/cam and conventional method. j adv prosthodont. 2020 aug;12(4):218-224. http://dentistry3000.pitt.edu/ the relationship between dental fear and cooperation of children during dental treatments with their parents’ general health vol 9 no 1 (2021) doi 10.5195/d3000.2021.121 http://dentistry3000.pitt.edu the relationship between dental fear and cooperation of children during dental treatments with their parents’ general health seyedeh hediyeh daneshvar 1*, saeedeh azizi 2 1assistant professor, dental sciences research center, department of pediatric dentistry, school of dentistry, guilan university of medical sciences, rasht, iran 2dentistry student, school of dentistry, guilan university of medical sciences, rasht, iran abstract objective: dental fear is a common phenomenon in children and parents can have a key role in the development of child's dental anxiety. there is moderate evidence to support the relationship between the parental general health and children’s dental fear. the aim of this study was to investigate the relationship between dental fear and cooperation of children during dental treatment with the general health of parents. methods: this cross-sectional descriptive study was done on 130 children aged 6 to 12 at the department of pediatric dentistry, guilan university of medical sciences from may to september 2020.the personality traits and general health of parents were assessed by using the general health questionnaire (ghq-28). the frankl's behavior rating scale and children's fear survey schedule dental subscale (cfss-ds) were used to respectively assess the degree of cooperation during dental practices and children’s dental fear. statistical approaches included t-test, chi-square and pearson linear correlation. a significant level of differences was taken as p<0.05. results: 69.3 % of children behaved positively and definitely positively during dental treatment. 56 boys and 74 girls with mean age of 9.13 ±2.02 years participated in this study. there was a significant correlation between the children's age with dental fear and cooperation level (p<0.001). there was no significant difference in dental fear score (p=0.63) and cooperation level (p=0.99) between boys and girls. there was not statistically significant relationship between the general health of the parents and the level of children's cooperation and dental fear during dental treatment (p>0.001). conclusion: there was not relationship between parent's general health with children's dental fear and cooperation. keywords: fear; behavior; health; dentistry. citation: daneshvar sh, et al. (2021) the relationship between dental fear and cooperation of children during dental treatments with their parents’ general health dentistry 3000. 1:a001 doi:10.5195/d3000.2021.121 received: november 19, 2020 accepted: january 22, 2021 published: june 25, 2021 copyright: ©2021 daneshvar sh, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email:hedyehdaneshvar@gmail.com introduction dental fear and anxiety (dfa) in children is a major issue affecting their oral health and behavior management and prevent children from cooperating well during dental treatment [1, 2]. dental fear is a common problem that develops mostly in childhood and affects people of all ages [3]. children with dfa often try all means to miss or postpone dental treatment, resulting in deterioration of their oral status which is not only distressing for the child and family but is also associated with poor oral health and an increased demand to more complex interventions [3,4]. thus, the frequency of dental pain and unpleasant dental experiences is greater among uncooperative children with dfa in comparison to cooperative children [5]. to offer better dental care, the prevalence and causes of dental fear and anxiety should not be overlooked in clinical practice, especially in children. strategies for the evaluation, prevention and control of dental fear should be implemented to http://dentistry3000.pitt.edu/ the relationship between dental fear and cooperation of children during dental treatments with their parents’ general health vol 9 no 1 (2021) doi 10.5195/d3000.2021.121 http://dentistry3000.pitt.edu allow better treatment for children and adolescents [2]. the etiology of dental fear in children is multifactorial and has been related to age, gender, personality, increased general fears, past dental experiences, parental dental fear, ethnicity and culture, socioeconomic status and dental office environment [6]. it is better to have information about the influence of parental general health on children's behavior in clinical situations. parents can provide an environment for children’s psychosocial growth which could shape the children’s behaviors. they can transmit feelings of fear and anxiety to their children [1]. many children are likely to internalize their parents’ values, attitudes through modeling and parents with dfa may transfer their fearful feelings to their children, causing a negative impact on their cooperation [3]. there is not enough evidence to support the relationship between parental and child dfa [5]. this study investigated the association between dental fear and cooperation of children during dental treatment with the general health of parents. material and methods ethical aspects this cross-sectional descriptive study was approved by guilan university of medical sciences (ir.gums.rec.1399.080) in 2020/5/27. sample this study was done on 130 children aged 6-12, seeking dental treatment at the department of pediatric dentistry, guilan university of medical sciences from may to september 2020. children with severe systemic diseases, physical or psychological disabilities were excluded. questionnaire a single researcher (pedodontist) explained the objectives and procedures to the parents. parents, who agreed to participate, signed a statement of informed consent and filled out general health questionnaire (ghq) which assessed theirs' general health and personality traits [7]. the same dentist interviewed with the parents and filled out the questionnaire. the parents have time enough to answer the questions. this questionnaire comprises of 28 questions, each with a four-point likret scale (0-1-2-3) for responses “not at all”, “no more than usual”, “rathermore than usual” and “much more than usual”. this questionnaire includes four subscales: social signs, anxiety and insomnia, social dysfunction and depression. each subscale includes seven questions and the score ranges from 0-21. general health of parents with 0-9 score are defined optimal, 10-15 is considered marginal and 1621 is defined as damaged health [7]. fear and cooperation evaluation fear and cooperation level of all children was evaluated in the first appointment during dental treatment by the same examiner (pedodontist). the children's fear survey schedule dental subscale (cfss-ds) with good reliability and validity, developed by cuthbert and melamed in 1982 was used to evaluate the children’s dental fear. cfss-ds consists of 15 items about different aspects of the dental situation. for each item, the response ranges 1–5, from “not afraid at all” to “very much afraid”. the total score ranges from 15 to 75. children with a total cfss-ds score below 32 are defined as non-fearful; 32–39 is considered moderate fearful and >39 is defined as fearful [8]. degree of cooperation of children during dental practices was assessed using frankl's behavior rating scale (definitely negative-1, negative-2, positive-3, definitely positive-4). definitely negative: refusal of treatment, crying forcefully, fearful, or any other overt evidence of extreme negativism. negative: reluctant to accept treatment, uncooperative, some evidence of negative attitude but not pronounced (sullen, withdrawn). positive: acceptance of treatment, at times cautious, willingness to comply with the dentist, at times with reservation, but patient follows the dentist’s directions cooperatively. http://dentistry3000.pitt.edu/ the relationship between dental fear and cooperation of children during dental treatments with their parents’ general health vol 9 no 1 (2021) doi 10.5195/d3000.2021.121 http://dentistry3000.pitt.edu definitely positive: good rapport with the dentist, interested in the dental procedures, laughing and enjoying [9]. statistical analysis the data obtained on the questionnaires were analyzed with the aid of the statistical package for the social sciences (spss®, version24.0, chicago,il, usa). comparing the dental fear and cooperation level of children among girls and boys was done using t-test. pearson correlation was used for evaluating the correlation between the children's age with dental fear. the relationship between the general health of the parents and the children's dental fear and cooperation was determined using the chi-square. results with p <0.05 were considered statistically significant. results 130 children, 56 boys and 74 girls, 612 years of age (average 9.13 ±2.02 years) participated in this study. 50.8% (66) of parents were male and 49.2 %(64) were female with 22-58 year old (average 39.24 ±5.56 years). according to cfss-ds, 68.5 % of the children were considered non-fearful, 12.3% were moderate and 19.2 % were fearful. there was no significant difference in dental fear score between boys and girls (t-test, p=0.63). cooperation of 20.8%, 48.5%, 28.5% and 2.2 % of children were definitely positive, positive, negative and definitely negative respectively, according to frankl's behavior rating scale. there was no significant difference in cooperation level between boys and girls (t-test, p=0.99) there was a significant correlation between the children's age with dental fear (pearson correlation, p<0.001), so that the dental fear decrease with increasing age. the personality traits and general health of parents, assessed by general health questionnaire (ghq-28) has been shown in table 1. there was not statistically significant relationship between the general health of the parents and the children's dental fear according to cfss-ds (chi-square, p>0.05) table 2. there was not a significant association between the parental general health and the cooperation level of children during dental practices according to frankl's behavior rating scale (chi-square, p>0.05) table 3. discussion this study explored the relationship between children’s dental fear and level of cooperation during dental practices with parental general table 1. frequency distribution of parental general health by four subscales variables score n (%) social signs 0-9 13(10) 10-15 75(57.7) 16-21 42(32.3) anxiety and insomnia 0-9 17(13.1) 10-15 57(43.8) 16-21 56(43.1) social dysfunction 0-9 9(6.9) 10-15 89(48.5) 16-21 32(24.6) depression 0-9 4(3.1) 10-15 38(29.2) 16-21 88(67.7) http://dentistry3000.pitt.edu/ the relationship between dental fear and cooperation of children during dental treatments with their parents’ general health vol 9 no 1 (2021) doi 10.5195/d3000.2021.121 http://dentistry3000.pitt.edu health. in contrast to the assumption of many people, the results showed that parental general health is not associated with children’s dental fear and cooperation. dental fear is more complex than can be explained by a single contributing factor and dentists must consider this issue to gain a better understanding of pediatric patients and their parents [2, 10]. there was no difference in dental fear and anxiety between genders which is in agreement with study by kyritsi ma et al [11] in a population of greek children in athens and study by raj s et al [8] in india, although some studies [12-14] reported that dental fear was seen more frequently in girls than boys. this difference can be related to different culture, social beliefs and biological origin [11]. in the present study, the dental fear of children decreased with increasing age which can be related to losing the fear of the unknown with maturation of children. some studies [15-17] reported that fear scores increased with age, likely due to the unpleasant past experiences and greater number of invasive treatments. similar to this study, in a study by wu l and gao x in hong kong, children’s dfa was not associated with parents’ dfa or parenting styles. parents’ and child’s dfa were measured by using the corah dental anxiety scale (cdas) and children fear survey schedule dental subscale (cfss–ds) respectively [1]. raadal m et al examined 5-11 year old us urban children from lowincome families. dental fear of children was evaluated by cfss-ds questionnaire and behavioral problems and personality traits were evaluated by parent report on the child behavior checklist (cbcl). there was not relationship between dental anxiety and personality traits in this population [18]. salem et al examined the relationship between dental fear and concomitant factors in children aged 3-6. results showed that general anxiety of iranian parents had no effect on the child’s dental anxiety, which is in the favor of conditioning theory which emphasizes more on personal experiences rather than parental influence [12, 14, 19]. it is often thought that parental dental fear is associated with children’s dental fear. such association was supported by a study conducted in the madrid table 2. frequency distribution of parental general health by children's dental fear variables score non-fearful n (%) moderate fearful n (%) fearful n (%) *pvalue social signs 0-9 (61.6(8 (15.4)2 (23)3 0.78 10-15 (72)54 (9.3 )7 (18.7)14 16-21 (64.3)27 (16.7)7 (19)8 anxiety and insomnia 0-9 (53)9 (17.6)3 (29.4)5 0.61 10-15 (73.4)42 (7.8 )6 (15.8)9 16-21 (67.8)38 (12.5)7 (19.8)11 social dysfunction 0-9 (88.8)8 (11.2)1 (0)0 0.46 10-15 (70)62 (11.2)01 (18.8)17 16-21 (60)19 (15.6)5 (24.4)18 depression 0-9 (100 )4 (0)0 (0)0 0.74 10-15 (65.7)25 (13.1)5 (21.2)8 16-21 (68.1)60 (12.5)11 (19.4)17 *p-value was based on chi square (p<0.05) http://dentistry3000.pitt.edu/ the relationship between dental fear and cooperation of children during dental treatments with their parents’ general health vol 9 no 1 (2021) doi 10.5195/d3000.2021.121 http://dentistry3000.pitt.edu (spain) by lara a et al which used cfss-ds questionnaire for evaluating the dental fear of schoolchildren and their parents. it was shown that family member's levels of dental fear are significantly correlated with children's dental fear [20]. findings of this study suggest that the influence of parents on the development of children’s dental fear is not as straightforward as previously suspected. as dental fear in children may lead to a disruptive behavior and postpone the dental treatment, it is imperative to identify the possible negative factors and make necessary efforts to prepare children for dental visits. this study was done on children in iran and the results cannot be directly generalized to all other populations, although some useful information can be drawn especially for populations with similar cultures [1]. the age range of 6 to 12 years old may be a limitation of this study, once younger or older children may have other results. conclusion there was not relationship between parent's general health and personality traits with children's dental fear and their level of cooperation during dental practices. there was no significant difference in cooperation level between the genders and the dental fear decreased with increasing age. acknowledgements we would like to thank department of pediatric dentistry, school of dentistry, guilan university of medical sciences for their great support. conflicts of interest the authors deny any conflicts of interest in regards to the current study. references table 3. frequency distribution of parental general health by cooperation level of children variables score definitel y negative n(%) negative n(%) positive n(%) definitely positive n(%) *pvalue social signs 0-9 0 (0) (54)7 5(38.4) 1(7.6) 0.21 10-15 (1.3)1 (28)21 39(52.1) 14(18.6) 16-21 (4)2 (21.5)9 19(45.2) 12(29.3) anxiety and insomnia 0-9 (0)0 (41.1)7 7(41.1) 3(17.8) 0.12 10-15 (0)0 (35)20 28(50) 9(15) 16-21 (5.3)3 (18.8)10 28(52.8) 15(23.1) social dysfunction 0-9 (0)0 (33.3)3 5(55.5) 1(11.2) 0.87 10-15 (3)3 (30)26 42(47.1) 18(19.9) 16-21 (0)0 (25)8 16(50) 8(25) depression 0-9 (0)0 (25)1 3(75) 0(0) 0.26 10-15 (2)1 (34.2)13 21(55.2) 3(8.6) 16-21 (2)2 (26.1)23 39(44.7) 24(27.2) *p-value was based on chi square (p<0.05) http://dentistry3000.pitt.edu/ the relationship between dental fear and cooperation of children during dental treatments with their parents’ general health vol 9 no 1 (2021) doi 10.5195/d3000.2021.121 http://dentistry3000.pitt.edu 1. children’s dental fear and anxiety: exploring family related factors. wu l, gao x. bm c oral health.2018 dec 1;18(1):100.doi: 10.1186/s12903-0180553-z 2. the relationship between dental anxiety in children, adolescents and their parents at dental environment. assunção cm, losso em, andreatini r, de menezes jv. j indian soc pedod prev dent. 2013 jul 1;31(3):175.doi:10.4103/09704388.117977. 3. dental fear and anxiety in children and adolescents: qualitative study using youtube. gao x, hamzah sh, yiu ck, mcgrath c, king nm. j med internet res. 2013;15:e29.doi:10.2196/.jmir.2290. 4. understanding children’s dental anxiety and psychological approaches to its reduction. porritt j, marshman z, rodd hd. int j paediatr dent.2012;22:397-405. doi:10.1111/j.1365263x.201101208.x. 5. empirical evidence of the relationship between parental and child dental fear: a structured review and metaanalysis. themessl-huber m, freeman r, humphris g, macgillivray s, terzi n int j paediatr dent. 2010;20:83101.doi:10.1111/j.1365x.2009.00998. x. 6. cross-cultural aspects of fears in african children and adolescents.ingman ka, ollendick th, akande a. behav res ther. 1999 apr;37(4):337345.doi:10.1016/s00057976(98)00108-9. 7. the general health questionnaire28 (ghq-28) as an outcome measurement in a randomized controlled trial in a norwegian stroke population. hjelle eg, bragstad lk, zucknick m, kirkevold m, thommessen b, sveen u. bmc psychology. 2019 dec 1;7(1):18.doi:10.1186/s40359-0190293-0. 8. evaluation of dental fear in children during dental visit using children's fear survey schedule-dental subscale. raj s, agarwal m, aradhya k, konde s, nagakishore v. int j clin pediatr dent. 2013 jan;6(1):12.doi:10.5005/jp-journals10005-1178. 9. identifying dental anxiety in children’s drawings and correlating it with frankl’s behavior rating scale. mathur j, diwanji a, sarvaiya b, sharma d. int j clin pediatr dent. 2017 jan;10(1):24.doi:10.5005/jpjournals-10005-1401. 10. dental anxiety in children: a review of the contributing factors.alasmari aa, aldossari gs, mohammed s. children. 2018 apr 1;5:7.doi:10.7860/jcdr/2018/350 81.11379. 11. parental attitudes and perceptions affecting children’s dental behaviour in greek population. a clinical study. kyritsi ma, dimou g, lygidakis na. europ arch paediatr dent. 2009 jan 1;10(1):2932.doi:10.1007/bf03262664 12. a clinical study of child dental anxiety. townend e, dimigen g, fung d. behav res ther. 2000 jan1;38(1):3146.doi:10.1016/s000 5-7967(98)00205-8. 13. dental fear/anxiety and dental behaviour management problems in children and adolescents: a review of prevalence and concomitant psychological factors. klingberg g, broberg ag. int j paediatr dent. 2007 nov;17(6):391406.doi:10.1111/j.1365263x.2007.00872.x. 14. dental fear and concomitant factors in 3-6 year-old children.salem k, kousha m, anissian a, shahabi a. j dent res dent clin, dent prospects. 2012;6(2):70.doi:10.5681/jodd.2012. 015. 15. prevalence of dental anxiety among 5to 8-year-old taiwanese children. lee cy, chang yy, huang st. j public health dent 2007;67:3641.doi:10.1111/j.17527325.2007.00006.x. 16. dental anxiety and temperament in 15-year olds. stenebrand a, wide boman u, hakeberg m. acta odontol scand. 2013;71:1521.doi:10.3109/00016357.2011.6450 68. http://dentistry3000.pitt.edu/ the relationship between dental fear and cooperation of children during dental treatments with their parents’ general health vol 9 no 1 (2021) doi 10.5195/d3000.2021.121 http://dentistry3000.pitt.edu 17. dental fear and oral health and family characteristics of finnish children. rantavuori k, lahti s, hausen h, seppä l, kärkkäinen s. acta odontol scand. 2004;62:20713.doi:10.1080/00016350410001586. 18. the prevalence of dental anxiety in children from low-income families and its relationship to personality traits. raadal m, milgrom p, weinstein p, mancl l, cauce am. j dent res. 1995 aug;74(8):143943.doi:10.1177/00220345950740080 201. 19. childhood dental fear in the netherlands: prevalence and normative data. ten berge m, veerkamp js, hoogstraten j, prins pj. community dent oral epidemiol.2002 apr;30(2):101-7.doi:10.1034/j.16000528.2002.300203.x.. 20. emotional contagion of dental fear to children: the fathers’ mediating role in parental transfer of fear.lara a, crego a, romero-maroto m. int j paediatr dent.2012 sep;22(5):324-30.doi:10.1111/j.136526x.2011.01200.x. http://dentistry3000.pitt.edu/ stress analysis of over-denture abutments treated with two obturation techniques vol 9 no 1 (2021) doi 10.5195/d3000.2021.156 http://dentistry3000.pitt.edu stress analysis of over-denture abutments treated with two obturation techniques ahmed n. elsherbini1, mohamed m nagy2, nancy n elsherbini3 1 prosthodontics department, modern sciences and arts university, cairo, egypt 2 endodontics department, ain shams university, cairo, egypt 3 prosthodontics department, cairo university, cairo, egypt abstract aim: evaluation of stresses induced on over-denture abutments endodonticaly treated with two different obturation techniques. materials and methods: 4 pesudo-realistic models were fabricated with two remaining canines as abutments. one canine was treated with lateral obturation technique and the other with vertical obturation. an over-denture was fabricated on the model. then strain gauges were attached on the coronal one third of the root buccally and lingually. 50 n were applied on the middle of the model using universal testing machine and readings were collected. results: from the collected data the lateral obturation showed 85.25±104.901 µε and the vertical obturation showed 4.875±3.555 µε with significance difference of p<0.05 between the groups. conclusion: it can be concluded that the abutments treated with vertical obturation techniques reduced the stresses induced to the supporting structures when compared with the lateral condensation technique. keywords: over denture; abutments; root canal obturation; endodontic obturation technique; stress analysisdentists. citation: elsherbini a, et al. (2021) stress analysis of over-denture abutments treated with two obturation techniques. dentistry 3000. 1:a001 doi:10.5195/d3000.2021.156 received: march 01, 2021 accepted: july 19, 2021 published: august 18, 2021 copyright: ©2021 elsherbini a, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: ahmed_elsherbini@live.com introduction over-denture is one of the basis treatment options, over-dentures can be either tooth supported or implant supported. in an overdenture the abutments support the prosthesis and share in bearing the forces induced by the masticatory forces. forces induced can have severe complications if their values are above the threshold of the abutments and the surrounding structures. such complications include loss of the abutments, peri-apical lesions, periodontal diseases, and fracture of the abutments eittinger and qian 1. over-denture designs there are different types of preparations of the abutments in tooth over-dentures: when crown-root ratio is favorable telescopic crown is usually used in over-denture cases as it provides support and retention, besides to the advantage of directing forces along long axis of the abutments. abutments usually do not need endodontic treatment elsherbini2. the conventional over-denture with bare root dome shaped preparation with an amalgam plug is one of the mostly advocated over-dentures. usually two abutments bilaterally positioned are indicated in the conventional over-denture and the abutments are prepared to be 2-3mm from the gingival level. in this type of over-dentures endodontic treatment for the abutments are required. it’s usually indicated with unfavorable crown-root ratio. a modification of the bare dome shaped root is a metal coping covering the root, proving adequate support and http://dentistry3000.pitt.edu/ stress analysis of over-denture abutments treated with two obturation techniques vol 9 no 1 (2021) doi 10.5195/d3000.2021.156 http://dentistry3000.pitt.edu protecting the abutment from falling forces. the height of the abutments in the conventional type is 2-3mm from gingival margin. abutments with metal coping showed high susceptibility of gingival inflammation, and root caries, percentage of metal copings loss were high, however the incident of the mobility of the abutments recorded was low elsherbini2, chabara et al. 3. another form is the over-denture retained with attachments; numerous attachments have been used with varying success rates, some have proved high performers and improved patients’ quality of life. however they had some drawbacks, as wear of retention by time, high maintenance requirements, fracture of the attachment, in addition to their high costs. attachments can be used with either tooth and implant supported over-denture nicolovska et al. 4. overdentures with short coping and the ones with attachments showed no difference in the preservation of the remaining teeth schuha et al.5. in a systematic review of implants, the use of different attachments designs in over-dentures didn’t seem to have an effect on the marginal bone loss around the abutments ceherli et al.6. endodontic treatment the abutment apparatus bearing the load consists of the tooth structure, periodontal ligament, and the endodontic treatment. cold lateral condensation has been always the golden standard in endodontic obturation. however, it has been found that this technique depends on the root canal sealer to fill any discrepancies within the main canal or the presence of any accessory anatomy nguyen7. moreover, lack of adaptation, spreader tracts, and voids, have been reported weller et al. 8 et.al. additionally, compaction forces during lateral condensation might cause detrimental stresses to the surrounding root canal dentin soros et al. 9. thermoplasticized, injectable gutta-percha has been found to be significantly superior to lateral condensation in terms of better adaptation to the threedimensional root canal anatomy with minimal voids budd et al. 10 and minimum stresses during obturation procedure telli et al. 11. stresses induced during obturation might have future effect on root dentin affecting the long term survival of the tooth specially when used as an abutment liable to extra undue stresses. what about changing the obturation technique in the endodontic treatment of the abutments in the conventional over-denture?! material and methods a pseudorealistic model, with two natural canines as the remaining abutments was selected for the study. for the reliability of the study four models were fabricated, 8 canines were selected with the same height and stored in saline, to have same environment for all models. in each model, of the two canines, one was treated with vertical obturation technique, the other the canines was treated with lateral obturation technique. the ridge for all models was covered with pseudo mucosa of 3mm thickness. endodontic treatment of the abutments a total of 8 sound mandibular canines of average length 25.25±0.95 mm were used. access cavity was done using a suitable sized-round bur and refined using a diamond stone with rounded end. the working length was determined by introducing a size 10 k-file (mani inc.) in the canal until the tip of the file became visible at the major apical foramen under a dental operative http://dentistry3000.pitt.edu/ stress analysis of over-denture abutments treated with two obturation techniques vol 9 no 1 (2021) doi 10.5195/d3000.2021.156 http://dentistry3000.pitt.edu microscope (seiler iq) at x10 magnification. the rubber stopper was then carefully adjusted to the reference point and the distance between the rubber stopper and the file tip was measured approximately to the nearest 0.5 mm. the canal was checked using a size 15 k-file to ensure the initial canal size before canal enlargement. the canal was enlarged using protaper next dentsply sirona in the following sequence; x1 (0.17/0.04), x2 (0.25/0.06), x3 (0.30/0.07), x4 (0.40/0.06), and x5 (0.50/0.06) according to the manufacturer recommendation using a continuous rotation movement at 300 rpm and 2 ncm powered by the x-smart plus endodontic motor dentsply sirona up to the working length. patency was checked after the use of each file using a size 10 k-file. during instrumentation, the canal was irrigated using 20 ml of 2.6% sodium hypochlorite, which was used between each file. after instrumentation, the canal was irrigated with 5 ml 17% edta and a final rinse with 5 ml 2.6% naocl. canals were then dried with size #50 absorbent paper points and stored in an incubator at 37◦c and 100% humidity until obturation. 4 canines were obturated using lateral condensation technique whereas the other 4 canines were obturated using vertical compaction technique. for lateral condensation, a standardized gutta-percha master point size #50, 2% taper meta biomed was selected, checked and introduced into the root canal to full working length. ah plus sealer dentsply de trey gmbh, was mixed according to the manufacturer's recommendations. the master cone was then coated with a sealer and introduced into the root canal until the working length was reached. lateral condensation was performed using standardized finger spreader size #30 (mani inc.). gutta percha points size #25 was used as accessory cones until the whole canal was filled. excess gutta percha was trimmed using hot burnisher till the orifice of the canal. the access was then sealed using composite resin 3m espe. regarding vertical condensation, a master gutta-percha cone size 50, 4% taper (meta biomed) was fitted 0.5 mm short of working length. the gutta-percha cone was coated with ah plus sealer and inserted into the root canal, and down-packing was performed with a heated suitable sized plugger at 220 °c e and q plus system, meta dental corp. using the continuous wave of condensation technique. backfilling was completed with warm guttapercha from (e and q plus system obturation unit. excess filling material was removed at the level of the orifice. the access was then sealed using composite resin 3m espe. subsequently, all teeth were radiographed in bucco-lingual and mesio-distal directions to evaluate the quality of filling for adequacy. teeth were then stored in an incubator at 37◦c and 100% humidity until further use. fabrication of the models a wax replica of the model required was prepared, stone replica of the canines were inserted in the wax model, surrounded by 1mm thickness of aluminum foil to act pseudo periodontium. putty index was taken of the stone canines to register their position. stone replica was used to avoid any distortion to the obturation by the heat used during acrylic processing. model was flasked, wax elimination, packing of the clear acrylic resin vertex, and processing was done. the model was deflasked and finished. the stone canines and aluminum foil were removed; the natural teeth with vertical obturation were placed in the putty index and guided to its position. tissue mimic genesis material was injected in the sockets of the http://dentistry3000.pitt.edu/ stress analysis of over-denture abutments treated with two obturation techniques vol 9 no 1 (2021) doi 10.5195/d3000.2021.156 http://dentistry3000.pitt.edu canines. preparation of the canines was done, reducing the clinical crown to 3 mm from the gingival margin. afterwards a vacuum stent was made of the model to record the current situation. the acrylic ridge was reduced by 3 mm to make a space for the pseudo mucosa; silicon adhesive was sprayed on the prepared areas. the vacuum stent was filled with tissue mimic material and placed back on the model. the tissue mimic formed the pseudo mucosa on the model. the same exact procedure was done for the other 3 models. testing procedure strain gauge was attached on the buccal and lingual surfaces of the coronal one third of the roots of the abutments, and placed on the universal testing machine. a metal rod was placed horizontally on the premolarmolar area, and a 50 n load was applied on the middle of the metal rod and readings were collected. the same procedure was repeated for the 4 models. a total of 80 readings were collected from each canine in each model. statistical analysis was performed by independent student’s t-test using spss 20.0 (spss inc.). a significant difference was observed when p<0.05 fig.1 (a-b). results table 1 showing mean values of micro strain (µε) induced in teeth obturated with both techniques. *p<0.05 is considered significant statistical analysis showed that strain induced within teeth obturated using lateral condensation technique was significantly higher than strain induced in teeth obturated by vertical compaction. figure 1a strain gauge placed on the coronal one third of the abutments roots figure 1b over-denture placed on the pseudo realistic model with strain gauges http://dentistry3000.pitt.edu/ stress analysis of over-denture abutments treated with two obturation techniques vol 9 no 1 (2021) doi 10.5195/d3000.2021.156 http://dentistry3000.pitt.edu * p<0.05 is considered significant discussion a load of 50 n was applied on the over-denture and the abutments supporting it, and data was interpreted. our results showed that strain induced within the teeth obturated by lateral condensation was significantly higher than that induced in teeth obturated by vertical compaction. this might be due to the latent influence of lateral compaction forces on the root canal dentin. the potential presence of preexisting microcracks from either pre-existing or iatrogenic origin could be added to that stresses induced during lateral condensation forces to cause the propagation of such cracks reaching full-thickness fracture of the root dentin. this is in agreement with brosh et al 12 and shemesh et al. 13, who showed that microcracks were induced in the root dentin by rotary instrumentation in addition to stresses induced during lateral compaction resulting in vertical root fracture. another possible cause is the higher volumetric portion of set cement in cases of lateral condensation in comparison to vertical condensation which is in full agreement with wu et al 14 and hugh et al 15. the lower modulus of elasticity of the set cement does not favor the formation of a mechanically homogenous unit with the radicular dentin williams et al. 16. the inability of the root canal sealer to increase the root resistance to fracture might be attributed to the limited creeping ability of the sealer inserted into radicular canal that resulting in failures at the sealer/dentin interface tay et al.17. more over, the bonding ability of resin sealer may also be influenced by the very high c-factor of root canals that leads to failure in the bonding ability of the filling material tay et al.18. conclusion within the limitation of the study it can be concluded that the abutments treated with vertical obturation techniques reduced the stresses induced to the supporting structures when compared with the lateral condensation technique. less forces transmitted means less reliability of fracture of the abutments and thus better prognosis of the tooth supported over-denture. based on the previous results it can be recommended that during the fabrication of conventional overdenture supported by non-vital teeth, that vertical obturation technique should be advocated during the endodontic treatment of the abutments. acknowledgment all authors declare that the research was done by self-funding, no any other source of funding was received. all authors contributed to conception, design, data acquisition and interpretation, performed all statistical analyses, drafted and vertical compaction lateral compaction 4.875±3.555 85.25±104.901* table 1. showing mean values of micro strain (µε) induced in teeth obturated with both techniques. http://dentistry3000.pitt.edu/ stress analysis of over-denture abutments treated with two obturation techniques vol 9 no 1 (2021) doi 10.5195/d3000.2021.156 http://dentistry3000.pitt.edu critically revised the manuscript. “all authors gave their final approval and agree to be accountable for all aspects of the work.” conflicts of interest all authors declare that there is no conflict of interest in this research references 1ettinger rl, qian f. abutment tooth loss in patients with overdentures. j am dent assoc. 2004 jun;135(6):739-46 2elsherbini an. mini-poll coping as an alternative attachment in tooth supported over-denture prosthesis. egyptian dental journal. 2019 oct 1;65(4-october (fixed prosthodontics, dental materials, conservative dentistry & endodontics)):3671-5. 3chhabra a, chhabra n, jain a, kabi d. overdenture prostheses with metal copings: a retrospective analysis of survival and prosthodontic complications. journal of prosthodontics. 2019 oct;28(8):876-82. 4nikolovska j, petrovski d, petricevic n, kapusevska b, korunoska-stevkovska v. overdentures on implants for better quality of life among the fully edentulous patients–case reports. prilozi. 2015 dec 1;36(2):225-34. 5schuh c, skupien ja, pereiracenci t, boscato n. five-year of tooth-supported overdenture as prosthetic solution for elderly patients: a case series. revista odonto ciência. 2014;29(1). 6cehreli mc, karasoy d, kökat am, akça k, eckert s. a systematic review of marginal bone loss around implants retaining or supporting overdentures. int j oral maxillofac implants. 2010 mar-apr;25(2):266-77. 7nguyen tn. obturation of the root canal system. pathways of the pulp. 1994;6:219-71. 8weller rn, kimbrough wf, anderson rw. a comparison of thermoplastic obturation techniques: adaptation to the canal walls. journal of endodontics. 1997 nov 1;23(11):703-6. 9soros c, zinelis s, lambrianidis t, palaghias g. spreader load required for vertical root fracture during lateral compaction ex vivo: evaluation of periodontal simulation and fracture load information. oral surgery, oral medicine, oral pathology, oral radiology, and endodontology. 2008 aug 1;106(2):e64-70. 10budd cs, weller rn, kulild jc. a comparison of thermoplasticized injectable gutta-percha obturation techniques. journal of endodontics. 1991 jun 1;17(6):260-4. 11telli c, gülkan p, günel h. a critical reevaluation of stresses generated during vertical and lateral condensation of guttapercha in the root canal. dental traumatology. 1994 feb;10(1):10. 12brosh t, metzger z, pilo r. circumferential root strains generated during lateral compaction with stainless steel vs. nickel-titanium finger spreaders. european journal of oral sciences. 2018 dec;126(6):518-25. 13shemesh h, bier ca, wu mk, tanomaru-filho m, wesselink pr. the effects of canal preparation and filling on the incidence of dentinal defects. international endodontic journal. 2009 mar;42(3):208-13. 14wu mk, özok ar, wesselink pr. sealer distribution in root canals obturated by three techniques. international endodontic journal. 2000 jul;33(4):340-5. 15hugh cl, walton re, facer sr. evaluation of intracanal sealer distribution with 5 different obturation techniques. quintessence international. 2005 oct 1;36(9). 16williams c, loushine rj, weller rn, pashley dh, tay fr. a comparison of cohesive strength and stiffness of resilon and guttapercha. journal of endodontics. 2006 jun 1;32(6):553-5. http://dentistry3000.pitt.edu/ stress analysis of over-denture abutments treated with two obturation techniques vol 9 no 1 (2021) doi 10.5195/d3000.2021.156 http://dentistry3000.pitt.edu 17tay fr, hiraishi n, pashley dh, loushine rj, weller rn, gillespie wt, doyle md. bondability of resilon to a methacrylate-based root canal sealer. journal of endodontics. 2006 feb 1;32(2):133-7. 18tay fr, pashley dh. monoblocks in root canals: a hypothetical or a tangible goal. journal of endodontics. 2007 apr 1;33(4):391-8. http://dentistry3000.pitt.edu/ microsoft word d3000 2017.docx   vol  5,  no  1  (2017)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2017.59           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     oral  mucosal  lesions  in  patients  with  pemphigus  and  pemphigoid   skin  diseases:  a  cross  sectional  study  from  southern  india     arvind  babu  rajendra  santosh1,    venkat  ramana  reddy  baddam2,  chigurupa(  anuradha  3,  chandrashekar  poosarla2      1den$stry  programme,  faculty  of  medical  sciences,  the  university  of  the  west  indies,  mona,  jamaica,  west  indies.    2department  of  oral  and  maxillofacial  pathology,  sibar  ins'tute  of  dental  sciences,  guntur,  andhra  pradesh,  india.   3department  of  oral  and  maxillofacial  pathology,  panineeya  ins*tute  of  dental  sciences  and  research  centre,  hyderabad,  india.   abstract   objec&ve:to  assess  the  prevalence  of  oral  mucosal  lesions  in  pa3ents  with  pemphigus  and  pemphigoid  diseases  from   southern  india.  design  and  methods:  a  cross-­‐sec$onal  hospital  based  study  was  conducted  from  august  2010  to  july   2011.  pa(ents  with  confirmed  pemphigus  and  pemphigoid  skin  disease  were  selected  and  informed  to  par(cipate  in   the  study.  oral  examina2on  of  all  par2cipants  were  done  to  document  site  and  type  of  oral  manifesta3on  and  diag-­‐ nos$c  procedures  such  as  histopathological  and  immunofluorescence  methods  were  performed  to  confirm  the  diag-­‐ nosis.  demographic  details  such  as  age,  gender  and  occupa5on  were  also  documented.  the  results  of  the  study  were   analyzed  by  spss  so.ware  version  19.0  (armonk,  ny)  and  presented  as  descripbve  stabsbcs.  results:  sixty  percent  of   the  pa'ents  exhibited  oral  mucosal  manifesta'ons.  a  higher  female  (86.66%)  predilec'on  of  autoimmune  blistering   disease  was  observed  in  the  study.  a  slightly  higher  number  of  pemphigoid  pa8ents  (53.33%,  16  out  of  30)  were  re-­‐ ported  than  pemphigus  (46.66%,  14  out  of  30  cases).  the  most  common  subtypes  of  pemphigus  is  pemphigus  vulgaris   71%  (10  out  of  14)  among  pemphigus,  and  bullous  pemphigoid  87.5%(14  out  of  16)  among  pemphigoid.  buccal  muco-­‐ sa   (92.85%)   is   the  most  common  site   in  pemphigus  pa7ents,  where  as  hard  palate   (12.5%)   is  mostly  commonly   re-­‐ ported   site   in  pemphigoid  pa/ents.   ini/al   involvement  of  oral  'ssue   in  disease  process  was  observed   in  78.57%  of   pemphigus,  and  12.5%  of  pemphigoid  pa5ents.  conclusion:  oral  mucosal   lesions  are  more   frequently  associated   in   pemphigus  pa+ents.  oral  mucosal  lesions  are  the  ini+al  site  of  disease  process  in  pemphigus  pa+ents.  the  significance   of  diagnosis  of  oral  lesions  at  earlier  stage  of  disease,  specifically  pemphigus  may  help  in  early  interven7on  of  disease   and  help  to  reduce  the  morbidity  and  mortality  state.  the  study  emphasizes  mul6disciplinary  approach  in  diagnosis   and  management  of  both  pemphigus  and  pemphigoid.       cita%on:  santosh,  et  al.  (2017)     oral  mucosal  lesions  in  pa/ents  with  pemphigus   and  pemphigoid  skin  diseases:  a  cross  sec1onal   study  from  southern  india.   den$stry  3000.  1:a001   doi:10.5195/d3000.2017.59   received:    august  12,  2016   accepted:    february  14,  2017   published:    march  28,  2017   copyright:  ©2017  santosh,  et  al.  this  is  an  open   access   ar!cle   licensed   under   a   crea!ve   com-­‐ mons   a"ribu"on   work   4.0   united   states   li-­‐ cense.   email:  arvindbabu2001@gmail.com   introduction   mucocutaneous  are  a   group  of  disorders  primarily  ob-­‐ served  in  dermatology  practice.   oral  mucosal  manifestations  may   be  an  initial  presentation,  most   important  clinical  symptom,  or   often  times  it  is  the  only  sign  of   such  disease  process.  in  most  in-­‐ stances,  the  mucocutaneous  dis-­‐ orders  are  known  to  affect  the   skin  and  mucus  membranes,  with   severe  clinical  manifestations  af-­‐ fecting  the  oral  mucosal  mem-­‐ branes.  the  oral  presentations  of   such  mucocutaneous  are  primarily   as  fluid  filled  blister  such  as  vesi-­‐ cle,  bullae,  erosive  or  ulcerative   conditions.1  early  identification  of   oral  manifestation  and  correlation   with  dermatological  condition  may   help  in  diagnosis,  treatment  plan-­‐ ning  and  prognosis.     pemphigus  is  one  of  the   few  potentially  fatal  autoimmune   diseases  affecting  predominantly   skin  and  oral  mucosa.  it  is  an  auto-­‐ immune  disease  characterized  by   the  clinical  presentation  of  fluid   filled  blisters,  and  raw  eroded  sur-­‐ faces  when  the  blister  burst  open.   the  disease  had  been  considered   to  be  associated  with  the  autoan-­‐ tibodies  (igg)  and  complement   factor  (c3)  against  the  deso-­‐ mosome  structure  at  the  intercel-­‐ lular  components  of  epithelium   and  epidermis.  the  autoimmune   process  that  destructs  the  inter-­‐ cellular  proteins  such  as  desmo-­‐ somes  is  histologically  termed  as   acantholysis.  the  acantholysis  of   epithelial  or  epidermal  cells  are   responsible  for  the  resultant  cleft   formation  above  the  basal  layer.   epithelial  or  epidermal  blisters  or   bullous  presentation  are  the  hall-­‐ mark  of  this  disease,  however  ero-­‐ sions  or  ulcers  over  the  mucosal   membranes  and  skin  or  epidermis   are  also  classical  clinical  presenta-­‐ tion  of  this  disease  process.2  the   most  commonly  oral  sites  are  pal-­‐ ate,  labial  mucosa,  buccal  mucosa,   ventral  surface  of  tongue,  and  gin-­‐ oral  mucosal  lesions  in  pa/ents  with  pemphigus  and  pemphigoid  skin  diseases:  a  cross  sec/onal  study  from  southern  india     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.59    http://dentistry3000.pitt.edu   2   gival.  pemphigus  includes  three   primary  clinically  recognizable   subsets  such  as  pemphigus  vulgar-­‐ is,  pemphigus  foliaceus,  and  para-­‐ neoplastic  pemphigus.3  histologi-­‐ cally  the  lesion  is  identified  based   on  the  supra  basal  or  intra  epithe-­‐ lial  cleft  formation,  with  acantho-­‐ lytic  or  tzanck  cells.  clinical  and   histopathological  findings  are  usu-­‐ ally  confirmed  by  direct  or  indirect   immunofluorescence  methods.1   pemphigoid  is  an  autoimmune   disease  characterized  by  the  ve-­‐ siculo-­‐bullous  manifestations  over   skin  and  mucosal  surfaces.  the   two  distinct  clinical  forms  of  this   disease  includes  bullous  and  cica-­‐ tricial  pemphigoid.4    the  disease   process  is  known  to  be  mediated   by  autoantibodies  directed  against   components  of  basement  mem-­‐ brane  zone.  the  disease  is  clinical-­‐ ly  recognized  as  bullous  lesions,   which  may  rupture  to  produce  ul-­‐ cers.  the  healing  is  usually  accom-­‐ panied  by  scaring  in  cicatricial  type   of  pemphigoid,  and  not  in  bullous   forms.  histologically  the  lesion  is   identified  based  on  the  sub-­‐basal   cleft  formation.  clinical  and  histo-­‐ pathological  findings  are  usually   confirmed  by  direct  or  indirect   immunofluorescence  methods.1,5     the  main  aim  of  the  present  study   is  to  assess  the  prevalence  of  oral   mucosal  lesions  in  patients  with   pemphigus  and  pemphigoid  dis-­‐ eases  from  southern  india.     methods     the  study  was  approved  by   ethics  committee/institutional  re-­‐ view  board  of  the  sibar  institute   of  dental  sciences,  guntur.  the   study  obtained  verbal  consent   from  all  participants  involved;  also   ethics  committee/institutional  re-­‐ view  board  of  the  sibar  institute   of  dental  sciences  approval  was   received  on  the  consent  form.  the   present  study  has  been  conducted   in  full  accordance  with  the  world   medical  association  declaration  of   helsinki.     sampling,  inclusion  and  exclusion  crite-­‐ ria     the  study  was  conducted   at  the  dermatology  department  at   government  general  hospital,   guntur,  andhra  pradesh,  india   from  the  period  august  2010,  to   july  2011.  all  the  patients  with   pemphigus  or  pemphigoid  disease   were  invited  to  participate  in  the   present  study.  the  participants   who  provided  the  consent  for  clin-­‐ ical  evaluation,  and  histopatholog-­‐ ical  diagnostic  procedures  were   included  in  the  study.  the  confi-­‐ dentiality  of  the  patients’  clinical,   histological,  and  photographic  de-­‐ tails  was  maintained.  the  study   was  conducted  by  a  qualified  den-­‐ tal  surgeon,  and  institutional  eth-­‐ ics  approval  was  received.  during   the  clinical  examination,  patients’   socio-­‐demographic  details  such  as   age,  gender,  occupation,  marital   status,  residential  address,  and   other  oral  hygiene  habits,  primary   site  of  disease  manifestations,  dis-­‐ ease  symptoms,  and  other  under-­‐ oral  mucosal  lesions  in  pa/ents  with  pemphigus  and  pemphigoid  skin  diseases:  a  cross  sec/onal  study  from  southern  india     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.59    http://dentistry3000.pitt.edu   3   lying  medical  conditions  (if  any)   were  obtained.  the  patients’  con-­‐ ditions  such  as  pemphigus  and   pemphigoid  were  diagnosed  by   consultant  dermatologist  of  the   hospital,  and  dental  surgeon  per-­‐ formed  oral  examination  and  bi-­‐ opsy  procedure  for  histological   and  immunofluorescence  exami-­‐ nation.  examining  dental  surgeon   performed  excisional  biopsy  on  all   the  study  participants.  tissue  was   anesthetized  using  lignocaine  hcl   2%  and  biopsy  procedure  was   done  using  scalpel  blade  no.  15.   the  details  of  the  clinical  presen-­‐ tation  of  skin  and  oral  mucosa   were  recorded  in  the  microsoft   excel  data  sheet  for  software   analysis.  the  final  histopathologi-­‐ cal  diagnosis  of  the  oral  biopsies   was  given  by  an  expert  oral  and   maxillofacial  pathologist  at  sibar   institute  of  dental  sciences,  gun-­‐ tur,  andhra  pradesh,  india.       histopathological  examination  (hema-­‐ toxylin  and  eosin  stain;  and  immunoflu-­‐ orescence)     oral  tissue  biopsies  were   taken  from  the  lesional  areas.  the   tissue  was  fixed  in  formalin  solu-­‐ tion  and  eventually  embedded  in   paraffin  wax.  the  microtomed  tis-­‐ sue  sections  were  statined  with   hematoxylin  and  eosin  (h  and  e)   and  examined  using  bright  field   microscope.  to  evaluate  the  pres-­‐ ence  of  autoantigens  in  the  oral   tissue  sections,  an  immunofluo-­‐ rescence  procedure  was  conduct-­‐ ed  for  the  part  of  biopsied  speci-­‐ men.  the  tissues  for  immunofluo-­‐ rescence  procedure  were  stored  in   michel’s  medium.  immunofluores-­‐ cence  methods  for  detection  of   auto-­‐antibodies  in  the  tissue  sec-­‐ tions  were  performed  with   thin(less  than  6  micrometer)  cryo-­‐ stat  sections.  the  sections  were   washed  with  0.1m  phosphate   buffered  saline  with  three  changes   over  a  period  of  thirty  minutes.   excess  of  the  solution  were   drained  and   covered  with   diluted  conju-­‐ gate  and  al-­‐ lowed  to  react   for  thirty   minutes  at   room  temper-­‐ ature.  then   sections  were   mounted  by   using  1,4-­‐diazabicyclooctance   dabco  solution.  the  preparations   were  examined  using  fluorescent   microscope.     clinical  and  histopathologi-­‐ cal  data  were  documented  in  the   prescribed  forms.  data  on  clinical   and  histopathological  details  were   documented  in  microsoft  2007   excel  spreadsheets,  followed  by   tabulations  of  various  parameters.   the  descriptive  statistical  analysis   using  spss  software  version  19.0   (armonk,  ny)  was  done  for   presentation  of  study  results.     results   age,  gender  and  occupation  de-­‐ mographics  of  the  study  participants   a  total  of  30  patients  were   examined  and  consisted  of  male  4   (13.33%)  and  female  26  (86.66%)   with  pemphigus  or  pemphigoid   had  participated  in  the  present   study  during  the  period  of  august   2011  to  july  2012  (figure  1a).  in   pemphigus,  1  male  (7.14%)  and  13   female  (92.85%)  patients  were  ob-­‐ served  (figure  1b).  whereas  in   pemphigoid,  3  male  (18.75%)  and   13  female  (81.25%)  were  reported   (figure  1c).  female  predilection   was  observed  in  both  pemphigus   and  pemphigoid.  the  patients  in   oral  mucosal  lesions  in  pa/ents  with  pemphigus  and  pemphigoid  skin  diseases:  a  cross  sec/onal  study  from  southern  india     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.59    http://dentistry3000.pitt.edu   4   the  total  study  ranged  between   21-­‐70  years.  the  mean  age  of  the   pemphigus  patients  were  42  years,   and  pemphigoid  was  48  years.   (figure  2)  analysis  of  the  partici-­‐ pants  occupation  showed  farmers   were  11(36.6%),  unemployed   9(30%),  drivers  4(13.33%)  and  self   employed  or  business  6(20%).   (figure  3).     prevalence  of  clinical  sub  types  and  oral   mucosal  involvement  in  pemphigus  and   pemphigoid     the  analysis  of  the  clinical   subtypes  of  pemphigus  and  pem-­‐ phigus  in  the  study  population  re-­‐ vealed  that,  pemphigus  vulgaris   10(71.42%),  pemphigus  vegetans  2   (14.28%),  and  pemphigus  foliaceus   2(14.28%);  and  bullous  pemphi-­‐ goid  14(87.5%),  and  cicatricial   pemphigoid  2(12.5%)  was  ob-­‐ served.  (figure  4)  a  total  of   53.33%  (16  out  of  30)  patients   showed  oral  mucosal  manifesta-­‐ tions.  higher  prevalence  of  oral   mucosal  lesions  were  observed  in   pemphigus  14(100%),  and  in  pem-­‐ phigoid  2(12.5%)  (figure  5)     initial  site  involvement  of  skin  versus   oral  mucosa  in  pemphigus  and  pemphi-­‐ goid   a  to-­‐ tal  of  60%   (18  out  of   30)  patients   has  skin  as  a   initial  site  of   manifesta-­‐ tion,  and   40%  (12  out   of  30)  as   oral  mucosa   as  a  initial   site  of  manifestation  of  diseases.  a   higher  frequency  of  oral  mucosa   as  a  initial  site  of  manifestation   was  observed  in  pemphigus  pa-­‐ tients  11(78.57%);  and  pemphi-­‐ goid  1(6.25%)  (figure  6)     oral  mucosal  site  geography  in  pemphi-­‐ gus  and  pemphigoid   buccal  mucosa  was  the   most  common  site  for  pemphigus   13(92.85%),  and  hard  palate  was   most  common  site  among  patients   with  pemphigoid  2  (12.5%).  most   of  the  patients  had  presented  the   oral  lesions  with  more  than  one   site.  (figure  7)   immunofluorescence  characterization  in   pemphigus  and  pemphigoid   in  pemphigus,  direct  im-­‐ munofluores-­‐ cence  method   demonstrated   the  presence  of   igg  and  c3   components.  all   the  patients   (100%)  showed   the  positivity  for   igg  autoanti-­‐ body,  and  8  out   of  13  (61.53%)   patients  showed   c3  component  positivity.  whereas   in  pemphigoid,  direct  immunoflu-­‐ orescent  method  demonstrated   the  positivity  for  igg  and  c3  com-­‐ ponents  in  basement  membrane   zones  of  two  oral  tissue  speci-­‐ mens.  (figure  8)     mortality  in  pemphigus  and  pemphigoid     mortality  rate  of  the  pem-­‐ phigus  in  the  present  study  is  be   14.28%  (2  out  of  14),  a  patient   with  pemphigus  foliaceus  and  an-­‐ other  patient  with  pemphigus   vegetans.  no  death  observed  in   pemphigoid  patients  during  study   period.     discussion   pemphigus  and  pemphi-­‐ goid  are  considered  to  be  a  pre-­‐ dominantly  a  dermatological  con-­‐ dition.  oral  manifestations  of  the-­‐ se  diseases  provides  a  great   chance  to  dental  surgeons  to  iden-­‐ tify  the  disease  at  earlier  stage,   and  help  in  early  intervention  and   eventually  for  a  better  prognosis.6   the  present  study  showed  a  high-­‐ er  prevalence  of  pemphigus  and   pemphigoid  in  females.  similar   report  was  published  in  a  study   from  mexico.7  the  present  study   oral  mucosal  lesions  in  pa/ents  with  pemphigus  and  pemphigoid  skin  diseases:  a  cross  sec/onal  study  from  southern  india     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.59    http://dentistry3000.pitt.edu   5   showed  a  higher  prevalence   among  farmers  and  laborers,  and   our  results  were  consistent  with   previously  published  study  from   kerala  population  of  india.8  the   peak  age  of  pemphigus  and  pem-­‐ phigoid  disease  in  the  present   study  has  fall  between  the  31-­‐40   years,  with  the  mean  age  of  42   years  for  pemphigus  and  48  years   for  pemphigoid.  ajith  kumar  etal.,   in  their  study  reported  that  inci-­‐ dence  of  pemphigus  is  higher  in   the  age  group  between  40-­‐50   years  from  thrissur  district  of  ker-­‐ ala,  india.9           the  prevalence  of  pemphi-­‐ gus  in  the  present  study  is  46.66%,   with  higher  prevalence  of  pemphi-­‐ gus  vulgaris  (71.42%)  among  the   various  clinical  subtypes  of  pem-­‐ phigus,  which  is  followed  by  pem-­‐ phigus  vegetans  (14.28%)  and   pemphigus  foliaceus  (14.28%).   however,  the  present  study  had   not  reported  any  paraneoplastic   pemphigus.  mortality  rate  of  the   pemphigus  in  the  present  study  is   considered  to  be  14.28%  (2  out  of   13),  a  patient  with  pemphigus  foli-­‐ aceus  and  another  patient  with   pemphigus  vegetans  had  died  due   to  the  severity  of  the  disease.  lan-­‐ gan  et  al  in  2008,  mentioned  that   mortality  rate  of  pemphigus  may   range  as  4.8%  and  54%.10  oral  mu-­‐ cosal  manifestation  (fluid  filled   blisters  or  bullous,  erosions)  was   observed  in  all  the  patients,  and   most  prevalent  oral  site  was  buc-­‐ cal  mucosa  (92.85%),  which  is  fol-­‐ lowed  by  soft  palate  (71.42%),   lower  lip  (42.85%),  tongue   (35.71%),  labial  mucosa  (28.57%),   floor  of  mouth  (21.42%),  upper  lip   (14.28%),  gingival  (14.28%),  and   hard  palate  (7.14%).  78.57%  (11   out  of  14)    patients  had  reported   that,  oral  mucosal  manifestation   as  a  primary  site  of  disease  pro-­‐ cess.  in  the  published  reports  of   thorakkal  shamim  et  al.,  revealed   that  pemphigus  vulgaris  is  the   most  common  subtype,  with  the   primary  site  of  oral  mucosa  in   53.52%  of  cases.  the  most  com-­‐ monly  affected  site  was  buccal   mucosa,  followed  by  palate,  lips,   tongue,  floor  of  mouth  and  gingi-­‐ val.11     the  histopathological  ex-­‐ amination  of  the  pemphigus  tissue   was  characterized  by  the  presence   of  acantholytic  epithelial  cells  with   resultant  cleft  formation  within   the  oral  epithelium.  direct  immu-­‐ nofluorescence  method  demon-­‐ strated  the  presence  of  igg  and  c3   components.  all  the  patients   (100%)  showed  the  positivity  for   igg  autoantibody,  and  8  out  of  13   (61.53%)  patients  showed  c3   component  positivity.  anuradha  et   al.,  in  2011  stated  that  direct  im-­‐ munofluorescence  of  pemphgius   showed  deposition  of  igg  was  ob-­‐ served  in  80%  and  14%  of  cases   showed  c3  positivity.12     the  frequency  of  pemphi-­‐ goid  in  the  present  study  is   53.33%,  with  higher  prevalence  of   bullous  pemphigoid  87.5%  among   the  clinical  subtypes  of  pemphi-­‐ goid.  the  study  results  suggested  a   oral  mucosal  lesions  in  pa/ents  with  pemphigus  and  pemphigoid  skin  diseases:  a  cross  sec/onal  study  from  southern  india     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.59    http://dentistry3000.pitt.edu   6   female  predilection  in  pemphigoid   disease.  fadia  et  al  in  2011,  sug-­‐ gested  that  pemphigoid  is  more   common  in  females  of  fifth  or   sixth  decade  of  life.  majority  of   the  patients  (6  out  of  16,  37.5%)   fall  between  age  group  of  31-­‐40   years.13  the  median  age  of  pem-­‐ phigoid  disease  in  the  present   study  is  48  years.  oral  mucosal   manifestation  (fluid  filled  blisters   or  bullous)  in  pemphigoid  in  the   present  study  is  12.50%  (2  out  of   16  patients)  which  is  considered  to   be  relatively  low,  and  only  one  pa-­‐ tient  (6.25%)  had  reported  oral   mucosa  as  an  initial  site  of  disease   manifestation.  the  most  common-­‐ ly  affected  site  in  oral  cavity  is   hard  palate  (12.5%),  which  is  fol-­‐ lowed  by  gingiva  (6.25%),  and  soft   palate  (6.25%).    sunil  dogra  et  al  in   2003,  reported  that  palate  and   gingival  as  most  common  area  for   pemphigoid  disease.14  the  histo-­‐ pathological  examination  of  pem-­‐ phigoid  tissue  was  characterized   by  the  presence  of  sub  basilar  cleft   formation  in  the  oral  biopsied   specimens.  direct  immunofluores-­‐ cent  method  demonstrated  the   positivity  for  igg  and  c3  compo-­‐ nents  in  basement  membrane   zones  of  two  oral  tissue  speci-­‐ mens.  stephen  et  al  in  2001,  stat-­‐ ed  the  direct  immunofluorescence   of  pemphigoid  tissues  shows  a  lin-­‐ ear  deposition  of  igg  and  c3  at   basement  membrane  zone.15     conclusion   identification  of  oral  mani-­‐ festations  in  pemphigus  and  pem-­‐ phigoid  are  important  in  both  den-­‐ tal  and  dermatology  practice.  early   detection  of  oral  lesions  in  dental   practice,  and  referral  to  expert   dermatologist  may  help  in  early   intervention.  the  collaborative   approach  will  eventually  alter  the   prognosis,  and  provides  a  scope  to   reduce  morbidity  and/or  mortality   rate.     references   1. oral  lesions  in  patients   with  pemphigus  vulgaris   and  bullous  pemphigoid.   budimir  j1,  mihić  ll,  situm   m,  bulat  v,  persić  s,  tom-­‐ ljanović-­‐veselski  m.  acta   clin  croat.  2008   mar;47(1):13-­‐8.    pmid:   18714642   2. oral  manifestations  of   pemphigus  vulgaris:  clini-­‐ cal  presentation,  differen-­‐ tial  diagnosis  and  man-­‐ agement.  bascones-­‐ martinez  a,  munoz-­‐ corcuera  m,  bascones-­‐ ilundain  c,  esparza-­‐gómez   g.  j  clin  exp  dermatol  res   2010  dec;1:112.     3. clinical  and  histological   characterization  of  oral   pemphigus  lesions  in  pa-­‐ tients  with  skin  diseases:  a   cross  sectional  study  from   sudan.  nada  m  suliman,   anne  n  åstrøm,  raouf  w   ali,  hussein  salman  and   anne  c  johannessen.  bmc   oral  health.  2013  nov   21;13:66.  pmcid:   pmc3871015   4. mucous  membrane  pem-­‐ phigoid  affecting  the  oral   cavity:  short  review  on  eti-­‐ opathogenesis,  diagnosis   and  treatment.  petruzzi  m.     immunopharmacol  immu-­‐ notoxicol.  2012   june;34(3):363-­‐7.  pmid:   22564172   5. immunofluorescence  and   its  application  in  dermato-­‐ pathology  with  oral  mani-­‐ festations:  revisited.   arvind  babu  rs,  p  chan-­‐ drasekar,  k  lalith  prakash   chandra,  g  sridhar  reddy,   k  krian  kumar,  bv  ramana   reddy.  j  oro  fac  sci  2013   june;5(1):2-­‐8.   6. the  anti-­‐desmoglein  1  au-­‐ toantibodies  in  pemphigus   vulgaris  sera  are  pathogen-­‐ ic.  xiang  ding,  luis  a  diaz,   janet  a  fairley,  george  j   giudice,  zhi  liu.  j  invest   dermatol.  2005   may;124(5):939-­‐46.  pmid:   15854034   7. frequency  of  oral  condi-­‐ tions  in  dermatology  clinic.   velia  a  ramrez  amador,   lilly  esquirvel  pedraza,  ro-­‐ cio  orozco  topete.  int  j   dermatol.  2000   jul;39(7):501-­‐5.  pmid:   10940113   8. prevalence  and  socio-­‐ demographic  determinants   of  skin  disease  among  low-­‐ er  primary  school  children   in  calcicut,  kerala.  libu  gk,   thomas  bina,  lucy  rapha-­‐ el,  shyam  e  balakrishnan,   biju  george,  joan  felicita   samson.  kerala  medical   journal  2009  octo-­‐ ber;5:185-­‐90.   oral  mucosal  lesions  in  pa/ents  with  pemphigus  and  pemphigoid  skin  diseases:  a  cross  sec/onal  study  from  southern  india     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.59    http://dentistry3000.pitt.edu   7   9. incidence  of  pemphigus  in   thrissur  district,  south  in-­‐ dia.  kidangazhiyathmana   ajith  kumar.  indian  j  der-­‐ matol  venereol  leprol.   2008  jul-­‐aug;74(4):349-­‐ 51.pmid:  1879705.   10. bullous  pemphigoid  and   pemphigus  vulgaris  –  inci-­‐ dence  and  mortality  in  the   uk  population  based  co-­‐ hort  study.  langan  sm,   smeeth  l,  hubbard  r,   flemming  km,  smith  chp,   west  j.  bmj.  2008  jul   9;337:a180.  pmid:   18614511   11. pemphigus  vulgaris  in  oral   cavity:  clinical  analysis  of   71  cases.  thorakkal   shamim,  vengal  ipe  var-­‐ ghese,  pallikandi  maliyek-­‐ kal  shameena,  sivasankar   sudha.  med  oral  patol  oral   cir  bucal.  2008  oct   1;13(10):e622-­‐6.  pmid:   18830168.   12. current  concepts  of  immu-­‐ nofluorescence  in  oral  mu-­‐ cocutaneous  diseases.   anuradha  ch,  anandan,   magesh  kt,  malathi  n.  j   oral  maxillofac  pathol.   2011  sep;15(3):261-­‐6.   pmcid:  pmc3227250.   13. pemphigus  vulgaris  and   mucous  membrane  pem-­‐ phigoid:  update  on  eti-­‐ opathogenesis,  oral  mani-­‐ festations  and  manage-­‐ ment.  fadia  ata  ali,  javier   ata  ali.  j  clin  exp  dent   2011;3(3):  246-­‐50.     14. mucous  membrane  pem-­‐ phigoid.  sunil  dogra,  am-­‐ rinder  jit  kanwar.  indian  j   dermatol  2003   dec;48(4):239-­‐40.     15. immunodiagnosis  of  pem-­‐ phigus  and  mucous  mem-­‐ brane  pemphigoid.  ste-­‐ phen  j  challacombe,  jane   setter  field,  pepe  shirlaw,   karen  harman,  crispian   scully,  martin  m  black.  ac-­‐ ta  odontol  scand.  2001   aug;59(4):226-­‐34.  pmid:   11570526   vol 1, no 1 (2013) issn 2167-8677 (online) doi 10.5195/d3000.2013.14 http://dentistry3000.pitt.edu this work is licensed under a creative commons attribution 3.0 united states license. this site is published by the university library system, university of pittsburgh as part of its d-scribe digital publishing program and is cosponsored by the university of pittsburgh press. heritability of face shape in twins: a preliminary study using 3d sterophotogrammetry and geometric morphometrics seth m. weinberg 1 , trish e. parsons 1 , mary l. marazita 1 , brion s. maher 2 1center for craniofacial and dental genetics, department of oral biology, school of dental medicine, university of pittsburgh, pittsburgh, pa, usa 2department of mental health, bloomberg school of public health, johns hopkins university, baltimore, md, usa abstract introduction: previous research suggests that aspects of facial surface morphology are heritable. traditionally, heritability studies have used a limited set of linear distances to quantify facial morphology and often employ statistical methods poorly designed to deal with biological shape. in this preliminary report, we use a combination of 3d photogrammetry and landmark-based morphometrics to explore which aspects of face shape show the strongest evidence of heritability in a sample of twins. methods: 3d surface images were obtained from 21 twin pairs (10 monozygotic, 11 samesex dizygotic). thirteen 3d landmarks were collected from each facial surface and their coordinates subjected to geometric morphometric analysis. this involved superimposing the individual landmark configurations and then subjecting the resulting shape coordinates to a principal components analysis. the resulting pc scores were then used to calculate rough narrow-sense heritability estimates. results: three principal components displayed evidence of moderate to high heritability and were associated with variation in the breadth of orbital and nasal structures, upper lip height and projection, and the vertical and forward projection of the root of the nose due to variation in the position of nasion. conclusions: aspects of facial shape, primarily related to variation in length and breadth of central midfacial structures, were shown to demonstrate evidence of strong heritability. an improved understanding of which facial features are under strong genetic control is an important step in the identification of specific genes that underlie normal facial variation. citation: weinberg sm, parsons te, marazita ml, maher bs. (2013) heritability of face shape in twins: a preliminary study using 3d sterophotogrammerty and geometric morphometrics. dentistry 3000 1:a004 doi: 10.5195/d3000.2013.14 received: august 19, 2013 accepted: november 15, 2013 published: november 25, 2013 copyright: ©2013 weinberg et al. this is an openaccess article licensed under a creative commons attribution work 3.0 united states license. email: smwst46@pitt.edu introduction variation in human facial morphology is strongly influenced by genetics. this contention is supported by decades of accumulated anthropometric research on twin concordance and parent-offspring resemblance [1-16]. to summarize the major findings of the previous research: 1) estimates of heritability for individual or composite quantitative facial features generally range from moderate (0.40) to high (0.80); 2) additive genetic effects account for much of the observed phenotypic variance in facial traits; 3) measured variables in each of the principal anatomical directions (horizontal, vertical and projective) display strong evidence of heritability; 4) vertically oriented measures (e.g., facial height measures) tend to be among the most heritable facial traits. determining the genetic contribution for specific facial traits remains a relevant area of concern in a number of fields that deal with human morphology, including paleoanthropology and orthodontics. while the majority of craniofacial heritability studies are limited to measures of skeletal structure, most often derived from radiographs, a smaller number of studies have focused solely on external soft-tissue features [17-35]. these studies typically use traditional anthropometric methods (i.e., direct measurement with calipers) to quantify the head and face, reporting similar heritability estimates for these surface traits as for the underlying skull. 3d surface imaging represents an alternative, and far less invasive, method for capturing quantitative data on the facial soft tissues [36,37]. recent advances in capture speed, accuracy, and affordability have rendered traditional methods of direct facial measurement largely obsolete. moreover, by combining 3d surface imaging technology with powerful methods of landmark-based shape analysis, it is possible to quantify subtle aspects of facial morphology and variation often missed with conventional anthropometrics. nevertheless, only two published studies to date have utilized 3d surface imaging to evaluate the genetic basis of facial features [38,39]. although both studies found a very high degree of similarity among monozygotic twins compared with same-sex dizygotic pairs, with the central midfacial region exhibiting the greatest level of concordance, the quantitative analysis was limited to a small set of simple linear distances, which are often inadequate for describing complex 3d features. http://dentistry3000.pitt.edu/ http://creativecommons.org/licenses/by-nc-nd/3.0/us/ http://www.library.pitt.edu/ http://www.pitt.edu/ http://www.library.pitt.edu/articles/digpubtype/index.html http://upress.pitt.edu/ http://creativecommons.org/licenses/by-nc-nd/3.0/us/ heritability of face shape in twins: a preliminary study using 3d sterophotogrammetry and geometric morphometrics vol 1, no 1 (2013) doi 10.5195/d3000.2013.14 http://dentistry3000.pitt.edu 2 in this preliminary investigation, we utilize 3d digital stereophotogrammetry in concert with geometric morphometrics to quantify midfacial structures in a sample of monozygotic (mz) and same-sex dizygotic (dz) twins. the main objective is to identify aspects of midfacial shape that show the strongest evidence of heritability. materials and methods a set of 10 monozygotic twin pairs (20 individuals) and 11 same-sex dizygotic twin pairs (22 individuals) were ascertained at the 2005 annual twins days festival in twinsburg, ohio. the twin sample was comprised of caucasian boys and girls between the ages of 5 and 12 years (mean age = 9.3 years 2.2). following informed consent, a 3d facial photo was obtained from each child as part of a broader assessment of phenotypic traits. medical history was also collected from all participants to rule out any congenital conditions or trauma that might affect facial morphology. routine orthodontic interventions were not considered as a basis for exclusion. parents of same-sex twins completed a standard 15item questionnaire regarding the twins’ similarities and differences to determine zygosity [40]. the algorithm for determining zygosity has been described elsewhere [41]. it has been previously demonstrated that this short questionnaire has an accuracy of 94% for zygosity determination when compared with molecular genetic results [41,42]. all aspects of this study were approved by an institutional review board (irb). facial surface images were obtained via 3d digital stereophotogrammetry (facecam 250, genex technologies, kensington, md). briefly, the imaging process involves projecting a structured light pattern onto the facial surface, which is then recorded by multiple ccd cameras with overlapping fields of view. by extracting positional information from the deformation of the projected light pattern by the natural contours of the target surface, the location of points (> 300,000) distributed over the entire surface is determined. from this collection of points, an accurate 3d model of the facial surface is then generated (figure 1). color and texture information is captured simultaneously and is mapped onto the underlying surface geometry, providing lifelike rendering. the entire image acquisition process takes place in less than 0.5 seconds. the accuracy and precision of measures derived these 3d surface models has been explored previously by the authors and found to very high [43,44]. thirteen surface landmarks were collected from each 3d facial model (figure 1) and their xyz coordinate locations saved. these landmarks correspond primarily to centrally located midfacial structures and are defined as in traditional surface anthropometry [45]. there were several factors that limited the number and kinds of landmarks we were able to collect. for many subjects, the quality of the scans was compromised below the labial fissure, eliminating the opportunity to capture landmarks on the lower lips or chin. furthermore, a number of subjects failed to maintain open eyes during the capture, making the identification of exocanthion impossible. a geometric morphometric approach was utilized to perform shape analysis of the landmark coordinate data [46,47]. first, the individual landmark configurations for all 42 participants were scaled and aligned through a least-squares procrustes superimposition [48]. this procedure results in a new set of 3d coordinates (procrustes coordinates), preserving only shape information. to control for any remaining allometry effects in the data, centroid size was regressed against the procrustes shape coordinates, and the resulting residuals were saved for further analysis. principal components analysis (pca) was then applied to the allometry-adjusted procrustes coordinates in order to describe the shape variation present in the dataset. in the context of shape analysis, pca reduces the dimensionality of the procrustes coordinate data into a more manageable number of uncorrelated summary variables (components), each capturing a distinct mode of shape variation [49]. because the geometric morphometric approach preserves the intrinsic geometry present in landmark coordinate data, shape variation associated with a given principal component (pc) can be visualized intuitively as a displacement of points in 3d space [50,51]. to further enhance visualization, facial surface warps can be generated based on the pca results. figure 1. 3d facial surface showing the midfacial landmarks used in the present study. midline points: n=nasion, prn=pronasale, sn=subnasale. bilateral points: en=endocanthion, al=alare, sbal=subalare, cph=crista philtri, ch=chelion. http://dentistry3000.pitt.edu/ heritability of face shape in twins: a preliminary study using 3d sterophotogrammetry and geometric morphometrics vol 1, no 1 (2013) doi 10.5195/d3000.2013.14 http://dentistry3000.pitt.edu 3 similar to traditional pca or factor analysis, every subject in the dataset receives a score on each extracted pc. these pc scores can then be used to compute narrow-sense heritability (g) estimates with the following formula: g = 2(iccmz – iccdz) where icc is the intraclass correlation coefficient. this is a very crude method for estimating heritability, often used when ample sizes preclude the use of more refined methods, such as structural equation modeling. all geometric morphometric analyses (procrustes analysis, allometry regression, and pca) were carried out using the program morphoj [52]. facial surface warps based on the pca results were generated using the program landmark v3.6 (http://www.idav.ucdavis.edu/research/ev omorph). results a total of 17 pcs were extracted; iccs were only calculated from scores on the first 9 pcs, which accounted for approximately 90% of the total shape variance. three of the derived shape pcs displayed evidence of moderate to high heritability (pc4, pc5, and pc7); the remaining six pcs were unrevealing. the heritability estimates for pc4, pc5, and pc7 are provided in table 1; although the values reported here approach 1.0, it should be noted these are still very crude approximations. we attempted to use bootstrap resampling techniques to obtain empirical confidence intervals for these estimates. these resulting confidence intervals approached or included zero and were likely affected by the small sample size from which the permutations were based. individually, these pcs accounted for a relatively small amount of the total shape variance (pc4 = 6.8%, pc5 = 5.7%, pc7 = 4.0%). pc4 was associated with a complex suite of shape variations. in the upper face, variation in the lateral position of the left/right endocanthion points (relating to the degree of hyper/hypotelorism) was observed. variation in nasal breadth, height, and projection was due primarily to shifts in the vertical and anterior-posterior position of nasion, the lateral position of the alar cartilages (left/right alare), and the anterior-posterior position of the nasal tip (pronasale). in the orolabial region, pc4 was associated with variation in the length of the philtrum and vertical height of the upper lip; this was coupled with relative changes in the vertical position of the left and right chelion. pc5 was associated mainly with the vertical and anterior-posterior position of nasion, the anterior-posterior position of the orbits (left/right endocanthion), and the breadth of the nasal floor and philtrum. variation in shape associated with pc4 and pc5 are depicted as surface warps in figure 2. pc7 (not shown in figure 2) was related primarily to the vertical position of landmarks defining the interorbital septum (nasion and the left/right endocanthion) and variation in the nasolabial angle due to the anterior position of the upper lip relative to the nose. the first few pcs, accounting for the majority of shape variation in the sample, did not demonstrate strong evidence of heritability. instead, shape variation along these pcs was related more to sex and age/maturity related factors. the source of the variation for the remaining pcs was not immediately apparent. discussion the results of the present study revealed evidence of heritable shape variation in central midfacial structures, including the interorbital region, nose, and upper lip. principal shape components associated with the degree of horizontal separation between the eyes, the length, breadth and projection of the nose, and the height and projection of the upper lip all demonstrated high heritability. these findings are largely supported by more traditional anthropometric studies that utilize standard linear distances to quantify facial surface structures [18-20,2235]. there is also substantial overlap between the results of the present study and those of prior 3d facial surface studies on twin pairs. burke [38] employed an early form of stereophotogrammetry to assess facial similarity in a sample of 18 like-sexed twin pairs. of the 13 facial measures examined, the degree of intrapair difference was significantly greater in dz compared to mz twins for eight variables, most of which involved central midfacial structures including the nose, interorbital region, and upper table 1. intraclass correlation coefficients (icc) and g estimates for principal components showing evidence of high heritability. shape component variation explained iccmz iccdz ga pc4 6.8% 0.783* 0.013 1.0b pc5 5.7% 0.771* 0.314 0.9 pc7 4.0% 0.675* 0.284 1.0b * p < 0.05 for test of within group non-zero icc a g = 2(iccmz – iccdz) b heritability estimate capped at 1.0 figure 2. facial surface warps modeling the shape variation associated with pc4 (along the horizontal axis) and pc5 (along the vertical axis). the centrally located face represents the average or consensus facial surface for the sample. movement along the principal axes of shape variation in either direction away from the consensus face is accompanied by the 3d displacement of landmarks and modeled as a deformation of the consensus 3d surface. this deformation reflects the major modes of shape variation associated with pc4 and pc5, respectively. figure 1. 3d facial surface showing the midfacial landmarks used in the present study. midline points: n = nasion, prn = pronasale, sn = subnasale. bilateral points: en = endocanthion, al = alare, sbal = subalare, cph = crista philtri, ch = chelion http://dentistry3000.pitt.edu/ http://www.idav.ucdavis.edu/research/evomorph http://www.idav.ucdavis.edu/research/evomorph heritability of face shape in twins: a preliminary study using 3d sterophotogrammetry and geometric morphometrics vol 1, no 1 (2013) doi 10.5195/d3000.2013.14 http://dentistry3000.pitt.edu 4 lip. using a similar study design, naini and moss [39] reported comparable results in a sample of 20 like-sexed twin pairs using 3d laser scanning technology. kau et al. [53] utilized 3d laser surface scanning to evaluate facial resemblance in a single pair of monozygotic twins; within-pair differences at points spread over the facial surface were generally at the sub-millimeter level (the chin region showed the greatest disparity at 1.5-1.9mm). several prior studies report the highest heritability estimates for vertically oriented facial measures [6,9-11,24,54], although there is considerable disagreement on this point [7,14,33-35]. the current findings present a slightly more complex picture. pc4 was associated with simultaneous changes in the relative length and breadth of the midface. this pattern of correlated shifts in length and breadth contributed to an overall shift in midface shape, from narrow and long to short and broad (see figure 2). the remaining two pcs showing evidence of heritability, however, were related primarily to the vertical and anterior position of the midline landmark nasion. thus, there is only partial support for the contention that vertical aspects of the face show higher heritability relative to variation along other principal anatomical directions. determining the heritability of quantitative facial traits represents an important step in the effort to identify specific genes that underlie typical variation in face shape. to date, limited work in this area has been carried out on animal models. quantitative trait loci (qtl) have been identified in mice that relate to variation in mandibular [5557] and maxillary shape [58,59]. in particular, qtls on several chromosomes have been associated with variation in midfacial breadth and length (projection) in the mouse skull [58,59]. in dogs, polymorphisms in specific candidate genes (e.g., tcof1, fgf8, runx2) have been shown to influence the extreme variation in facial form that characterizes certain breeds [6063]. in humans, a handful of recent studies have also begun to uncover the genetic basis of facial shape [64,65]. interestingly, both of the aforementioned human studies have independently identified associations between snp variants in the pax3 gene and variation in nasal root morphology, specifically relating to the relative position of nasion and left/right endocanthion. variation in the spatial position of these same points was associated with all three relevant pcs in the present study, suggesting that our preliminary results, although quite limited, may have some external validity. it is important to keep in mind that although several derived shape pcs showed seemingly very high levels of heritability, these estimates are based on a simplistic approach to modeling heritability in a small sample and ultimately could not be substantiated using resampling methods. consequently, the heritability values for pc4, pc5, and pc7 are likely to be overestimates of the true additive genetic effects on phenotypic similarity. rather than focusing on the values in an absolute sense, a more prudent interpretation might be that pc4, pc5, and pc7 demonstrated the most compelling evidence of heritability, relative to the other components of shape variation derived here. in future studies, larger samples will hopefully permit the use of more sophisticated methods for estimating heritability, including those based on structural equation modeling. conclusion this study represents a preliminary attempt to estimate the heritability of facial shape using a combination of 3d stereophotogrammetry and landmark-based geometric morphometrics in a sample of twin pairs. our results indicate that variation in both the length and breadth of central midfacial structures showed statistical evidence of moderate to high heritability. these results are in line with previous reports, including recent studies that have identified specific genetic variants underlying facial shape in humans. nevertheless, due to the small size of the sample of twin pairs available for analysis, the results here should be viewed as preliminary and, as such, interpreted with caution. acknowledgements the authors would like to thank carla brandon and kathy bardi for their help recruiting subjects and nicole scott for helping to collect 3d images. we also thank the directors of the twins days festival for granting permission to conduct this research. this work was supported by the following grant from the national institute of dental and craniofacial research: r01-de016148. conflict of interest: there are no conflicts of interest to declare. references 1. genetic determinants of cranio-facial morphology: a twin study. nakata m, yu pl, davis b, nance we. ann hum genet 1974;37:431-43. 2. multivariate analysis of craniofacial measurements in twin and family data. nakata m, yu pl, nance we. am j phys anthropol 1974;41:423-9. 3. a family study of craniofacial dimensions in the burlington growth center sample. saunders sr, popovich f, thompson gw. am j orthod 1980;78:394-403. 4. sibling correlations for cranial measurements from serial radiographs. byard pj, lewis ab, ohtsuki f, siervogel rm, roche af. j craniofac genet dev biol 1984;4:265-9. 5. craniofacial morphology and occlusal variation in monozygotic and dizygotic twins. lobb wk. angle orthodont 1987;57:219-33. 6. comparison of some cephalometric distances and corresponding facial proportions with regard to heritability. lundstrom a, mcwilliam j. eur j orthod 1988;10:27-9. 7. heritability of craniometric and occlusal variables: a longitudinal sib analysis. harris ef, johnson mg. am j orthod dentofacial orthop 1991;99:258-68. 8. heritability of cephalometric and occlusal variables as assessed from siblings with overt malocclusions. king l, harris ef, tolley ea. am j orthod dentofacial orthop 1993;104:121-31. 9. heritability of 39 orthodontic cephalometric parameters on mz, dz twins and mn-paired singletons. manfredi c, martina r, grossi gb, giuliani m. am j orthod dentofacial orthop 1997;111:44-51. 10. a genetic study of anteroposterior and vertical facial proportions using model-fitting. savoye i, loos r, carels c, derom c, vlietinck r. angle orthodont 1998;68:467-70. 11. a quantitative genetic study of cephalometric variables in twins. carels c, van cauwenberghe n, savoye i, et al. orthod craniofacial res 2001;4:130-40. 12. heritability of craniofacial characteristics between parents and offspring from lateral cephalograms. johannsdottir b, thorarinsson f, thordarson a, magnusson te. am j orthod dentofacial orthop 2005;127:200-7. 13. maximum likelihood estimation of human craniometric heritabilities. carson ea. am j phys anthropol 2006;131:169-80. 14. heritability of facial proportions and soft-tissue profile characteristics in turkish anatolian siblings. baydaş b, erdem a, yavuz i, ceylan i. am j orthod dentofacial orthop 2007;131:504-9. 15. quantitative genetics of modern human cranial variation. sherwood rj, duren dl, demerath ew, czerwinski sa, siervogel rm, towne b. j hum evol 2008;54:909-14. 16. heritability of human cranial dimensions: comparing the evolvability of different cranial regions. martínez-abadías n, esparza m, sjøvold t, gonzález-josé r, santos m, hernández m. j anat 2009;214:19-35. 17. correlation of brothers in factor scores. howells ww. am j phys anthropol 1953;11:121-40. 18. the heritability of certain anthropometric characters as ascertained from measurements of twins. clark pj. am j hum genet 1956;8:49-54. 19. genetic basis of morphological variation. osborne rh, de george fv. cambridge: harvard university press; 1959. 20. a comparison of identical and fraternal twins on some anthropometric measures.vandenberg sg, strandskov hh. hum biol 1964;36:45-52. http://dentistry3000.pitt.edu/ heritability of face shape in twins: a preliminary study using 3d sterophotogrammetry and geometric morphometrics vol 1, no 1 (2013) doi 10.5195/d3000.2013.14 http://dentistry3000.pitt.edu 5 21. genetic and environmental influences on morphological characteristics. susanne c. ann hum biol 1975;2:279-87. 22. heritability of anthropometric characters. susanne c. hum biol 1977;49:573-80. 23. a family study of anthropometric traits in a punjabi community: ii. an investigation of familial transmission. byard pj, sharma k, russell jm, rao dc. am j phys anthropol 1984;64:97104. 24. family resemblance for components of craniofacial size and shape. byard pj, poosha dvr, satyanarayana m, rao dc. j craniofac genet dev biol 1985;5:229-38. 25. family resemblance for cranio-facial measurements in velanti brahmins from andhra pradesh, india. poosha dv, byard pj, satyanarayana m, rice jp, rao dc. am j phys anthropol 1984;65:15-22. 26. a family study of anthropometric traits in a punjabi community: i. introduction and familial correlations. sharma k, byard pj, russell jm, rao dc. am j phys anthropol 1984;63:389-95. 27. testing for the presence of genetic variance in factors of face measurements of belgian twins. hauspie rc, susanne c, defrise-gussenhoven e. ann hum biol 1985;12:429-40. 28. transmissible and nontransmissible components of anthropometric variation in the alexanderwohl mennonites: i. description and familial correlations. devor ej, mcgue m, crawford mh, lin pm. am j phys anthropol 1986;69:71-82. 29. transmissible and nontransmissible components of anthropometric variation in the alexanderwohl mennonites: ii. resolution by path analysis. devor ej, mcgue m, crawford mh, lin pm. am j phys anthropol 1986;69:83-92. 30. transmission of human craniofacial dimensions. devor ej. j craniofac genet dev biol 1987;7:95106. 31. genetics of human body size and shape: complex segregation analysis. livshits g, otremski i, kobyliansky e. ann hum biol 1995;22:13-27. 32. similarities in anthropometrical traits of children and their parents in a bulgarian population. nikolova m. ann hum genet 1996;60:51725. 33. heritability of anthropometric phenotypes in caste populations of visakhapatnam, india. arya r, duggirala r, comuzzie ag, puppala s, modem s, busi br, crawford mh. hum biol 2002;74:325-44. 34. quantitative genetic study of head size related phenotypes in ethnically homogeneous chuvasha pedigrees. ermakov s, kobyliansky e, livshits g. ann hum biol 2005;32:585-98. 35. genetic determination of head-size-related anthropometric traits in an ethnically homogeneous sample of 373 indian pedigrees of west bengal. karmakar b, ermakov s, yakovenko k, kobyliansky e. hum biol 2007;79:501-14. 36. three-dimensional surface imaging: limitations and considerations from the anthropometric perspective. weinberg sm, kolar jc. j craniofac surg 2005;16:847-51. 37. measuring adult facial morphology in three dimensions. kau ch, richmond s, savio c, mallorie c. angle orthodont 2006;76:771-6. 38. intrapair facial differences in twins. burke ph. acta genet med gemellol (roma) 1989;38:3747. 39. three-dimensional assessment of the relative contribution of genetics and environment to various facial parameters with the twin method. naini fb, moss jp. am j orthod dentofacial orthop 2004;126:655-65. 40. diagnosis of twin zygosity. nichols rc, bilbro wc. acta genet stat med 1966;16:265-75. 41. pittsburgh registry of infant multiplets (prim). strassberg m, peters k, marazita m, et al. twin res 2002;5:499-501. 42. environmental and genetics influence on dimensions of perceived parenting: a twin study. rowe dc. dev psychol 1981;17:203-8. 43. digital three-dimensional photogrammetry: evaluation of anthropometric precision and accuracy using a genex 3d camera system. weinberg sm, scott nm, neiswanger k, brandon ca, marazita ml. cleft palate craniofac j 2004;41:507-18. 44. anthropometric precision and accuracy of digital three-dimensional photogrammetry: comparing the genex and 3dmd imaging systems to one another and to direct anthropometry. weinberg sm, naidoo s, govier dp, martin ra, kane aa, marazita ml. j craniofac surg 2006;17:477-83. 45. anthropometry of the head and face. farkas lg. new york: raven press; 1994. 46. statistical shape analysis. dryden il, mardia kv. chichester: wiley; 1998. 47. in: slice de, ed. modern morphometrics in physical anthropology. slice de. modern morphometrics. new york: kluwer academic/plenum publishers; 2005:1-45. 48. extensions of the procrustes method for the optimal superimposition of landmarks. rohlf f, slice d. syst zool 1990;39:40-59. 49. geometric morphometrics for biologists: a primer. zelditch ml, swiderski dl, sheets hd, fink wl. amsterdam: elsevier academic press; 2004. 50. facial growth in cercocebus torquantus: an application of three-dimensional geometric morphometric techniques to the study of morphological variation. o'higgins p, jones n. j anat 1998;193:251-72. 51. geometric morphometric study of the regional variation of modern human craniofacial form. hennessy rj, stringer cb. am j phys anthropol 2002;117:37-48. 52. morphoj: an integrated software package for geometric morphometrics. klingenberg cp. mol ecol resour 2011;11:353-7. 53. the investigation of the changing facial appearance of identical twins employing a threedimensional laser imaging system. kau ch, zhurov a, bibb r, hunter l, richmond s. orthod craniofacial res 2005;8:85-90. 54. a study of the inheritance of craniofacial characteristics as seen in lateral cephalograms of 72 like-sexed twins. hunter ws. rep congr eur orthod soc 1965;41:59-70. 55. pleiotropic effects of individual gene loci on mandibular morphology. cheverud jm, routman ej, irschick dj. evolution 1997;51:2006-16. 56. quantitative trait loci for directional but not fluctuating asymmetry of mandibular characters in mice. leamy lj, pomp d, eisen ej, cheverud jm. genet res 2000;76:27-40. 57. genetic architecture of mandible shape in mice: effects of quantitative trait loci analyzed by geometric morphometrics. klingenberg cp, leamy lj, routman ej, cheverud jm. genetics 2001;157:785-802. 58. enu large-scale mutagenesis and quantitative trait linkage (qtl) analysis in mice: novel technologies for searching polygenetic determinants of craniofacial abnormalities. nishimura i, drake ta, lusis aj, lyons km, nadeau jh, zernik j. crit rev oral biol med 2003;14:320-30. 59. a genome segment on mouse chromosome 12 determines maxillary growth. oh j, wang cj, poole m, et al. j dent res 2007;86:1203-6. 60. the canine homeobox gene msx2: sequence, chromosome assignment and genetic analysis in dogs of different breeds. haworth k, breen m, binns m, hopkinson da, edwards yh. anim genet 2001;32:32-6. 61. canine tcof1; cloning, chromosome assignment and genetic analysis in dogs with different head types. haworth ke, islam i, breen m, et al. mamm genome 2001;12:622-9. 62. characterization of the genomic canine fgf8 locus and screen for genetic variants in 4 dogs with different face types. haworth ke, healy c, mcgonnell im, binns m, sharpe pt. dna seq 2007;18:209-19. 63. molecular origins of rapid and continuous morphological evolution. fondon jw, garner hr. proc natl acad sci usa 2004;101:18058-63. 64. genome-wide association study of threedimensional facial morphology identifies a variant in pax3 associated with nasion position. paternoster l, zhurov ai, toma am, kemp jp, st pourcain b, timpson nj, mcmahon g, mcardle w, ring sm, smith gd, richmond s, evans dm. am j hum genet 2012;90:478-85. 65. a genome-wide association study identifies five loci influencing facial morphology in europeans.liu f, van der lijn f, schurmann c, zhu g, chakravarty mm, hysi pg, wollstein a, lao o, de bruijne m, ikram ma, van der lugt a, rivadeneira f, uitterlinden ag, hofman a, niessen wj, homuth g, de zubicaray g, mcmahon kl, thompson pm, daboul a, puls r, hegenscheid k, bevan l, pausova z, medland se, montgomery gw, wright mj, wicking c, boehringer s, spector td, paus t, martin ng, biffar r, kayser m. plos genet 2012;8:e1002932. http://dentistry3000.pitt.edu/ microsoft word parsons proof update oct 28.docx vol 2, no 1 (2014) issn 2167-8677 (online) doi 10.5195/d3000.2014.21 http://dentistry3000.pitt.edu this work is licensed under a creative commonsattribution 3.0 united states license. this site is published by the university library system, university of pittsburgh as part of its d-scribe digital publishing program and is cosponsored by the university of pittsburgh press. hypoxic conditions alter developing branchial arch-derived structures in zebrafish trish e. parsons1, seth m. weinberg1, michael tsang2, alexandre r. vieira1 1department of oral biology, school of dental medicine, university of pittsburgh, pittsburgh, pa, usa 2department of developmental biology, school of medicine, university of pittsburgh, pittsburgh, pa, usa abstract background: previous epidemiological findings have implicated hypoxia as a risk factor for craniofacial defects including cleft lip, microtia and branchial arch anomalies. this study tests the hypothesis that hypoxic exposure results in craniofacial shape variation in a zebrafish model. methods: three sets of zebrafish embryos were raised in uniform conditions with the exception of dissolved oxygen level. at 24 hours past fertilization (hpf) embryos were placed in hypoxic conditions (70% or 50% dissolved oxygen tank water) and compared to unexposed control embryos. after 24 hours of exposure to hypoxia, the embryos were incubated under normoxia. larvae were collected at 5 days post fertilization (dpf) and stained for cartilage. images were taken of each specimen and subsequently landmarked to capture viscerocranial morphology. a geometric morphometric analysis was performed to compare shape variation across groups. results: the mean branchial arch shape of each exposure group was significantly different from controls (p<0.001). principal components analysis revealed a clear separation of the three groups, with controls at one end of the shape spectrum, the 50% hypoxia group at the other end, and the 70% hypoxia group spanning the variation in between. conclusions: this experiment shows that hypoxia exposure at 24hpf is capable of affecting craniofacial shape in a dose-dependent manner. these results may have implications not only for high altitude fetal health, but other environments, behaviors and genes that affect fetal oxygen delivery. citation: parsons te, weinberg sm, tsang m, vieira ar. (2014) hypoxic conditions alter developing branchial arch-derived structures in zebrafish.. dentistry 3000. 1:a001 doi:10.5195/d3000.2014.21 received: february 3, 2014 accepted: july 12, 2014 published: august 6, 2014 copyright: ©2014 parsons et al. this is an open access article licensed under a creative commons attribution work 3.0 united states license. email: smwst46@pitt.edu introduction there are multiple lines of evidence indicating that hypoxia can alter craniofacial development. most research has focused on the midface and the effects of reduced oxygen on the development of orofacial clefts. for example, maternal cigarette smoking has been shown to increase the risk of oral clefts in offspring [1], the incidence of clefting appears to be elevated in certain high-altitude populations [2], and cleft mouse models show increased incidence of clefting after hypoxia exposure [3]. there is also evidence that hypoxia can affect a broader range of structures comprising the craniofacial complex, including branchial-arch related morphology [2,4]. for example, the observed prevalence rate for microtia is five times greater for individuals living at high-altitude compared with those living at low altitude in the same general region, suggesting that development of the first and second branchial arches is susceptible to hypoxia [5]. it is critical to understand the multitude of the factors capable of altering the underlying developmental architecture ultimately resulting in craniofacial malformations. animal models are tools that help determine which factors warrant further scrutiny. zebrafish (daniorerio) provide a relatively quick and inexpensive way to screen for factors involved in phenotypes of interest [6]. while zebrafish are most often used to explore genetic questions, they also provide an opportunity to test environmental effects such as oxygen availability on phenotypic outcomes. previous experiments with zebrafish in lowoxygenated water have resulted in general growth retardation and developmental delay [7,8]. the present experiment focuses on the zebrafish viscerocranium. the structures comprising the viscerocranium are derived from branchial arches. the branchial arches consist principally of neural crest cell-derived mesenchyme and are separated by endodermal pouches. in fish, the first and second branchial arches give rise to various structures of the craniofacial complex; the first arch gives rise to meckel s cartilage and the dorsal palatoquadrate, while the second arch forms the ceratohyal, basihyal, interhyal and hyosimplectic [9]. in mammals, first and second arch derivatives include parts of the middle ear, mandibular process, maxillary process and part of the hyoid bone [10]. in this study, two hypotheses about hypoxia and craniofacial shape variation are tested with zebrafish. the first hypothesis contends that hypoxia will alter the shape of first and second branchial arch-derived structures in developing zebrafish embryos. the second hypothesis states that hypoxic stress will result in different levels of phenotypic perturbation in a dose-dependent manner. http://dentistry3000.pitt.edu hypoxic conditions alter developing branchial arch-derived structures in zebrafish vol 2, no 1 (2014) doi 10.5195/d3000.2014.21 http://dentistry3000.pitt.edu 2 figure 1: landmark key. materials and methods zebrafish maintenance adult wildtype zebrafish ab* were maintained in a zebrafish housing system (aquaneering inc.). adult fish are maintained at 25-27oc with reconstituted reverse osmosis water (ph 7.0-7.5; conductivity: 650-750 µs/cm; dissolved oxygen: 7.5mm hg) in 14 h: 10 h light: dark photoperiod. male and female adult zebrafish were set up the day before and embryos were collected in the morning and staged according to nuesslein-volhard et al [6]. embryos were transferred to petri dishes containing e3 solution (5mm nacl, 0.17 mmkcl, 0.33 mm cacl2, 0.33 mm mgso4) and incubated at 28.5oc. specimen preparation and data collection embryos from a single clutch were divided into three different conditions: negative control/normal o2 level(6.5-6.6mm hg, which was considered 100% o2), 50% o2 level, and 70% o2 level)at 24 hours post fertilization (hpf) for a period of 24 hours. oxygen levels were manipulated by bubbling nitrogen gas and measuring dissolved oxygen level with a dissolved oxygen meter (sontek/ysi modell 58, fisher scientific) in e3 media. embryos were added to the deoxygenated e3 and placed into 50ml conical tubes and sealed, thereby reducing the amount of oxygen transfer in the process. the embryos were incubated at 28.5oc and after the 24 hours the larvae under hypoxic conditions were returned to normoxic e3 media. at 120hpf, a portion of specimens from each group were sacrificed with tricaine treatment (ms-222, sigma) and fixed in 4% paraformaldehyde. the five-day time point(120 hpf) was chosen because it is within a developmental window where the visceral cartilages are well developed but just prior to the stage when feeding occurs. the mechanics of feeding could introduce mechanical influences on shape that would confound our analysis. the skin surrounding the anterior half of the embryo and the brain were removed from each specimen. at this time the eyes were also removed as they interfered with the visualization of certain craniofacial structures. after dissection the specimens were dehydrated with a series of ethanol treatments and stained for cartilage with alcian blue (0.1% alcian blue dissolved in 80% ethanol/20% glacial acetic acid) followed by the addition of sodium borate to neutralize the acid [11]. ventral images of the branchial arches were captured using a cameramounted microscope, similar to the methods used by lopez-romero and co-workers [12]. subsets of specimens from each of the three experimental rounds were included in the data collection for a total sample size of 35 (70% o2,n=19; 50% o2,n=9; normal controls, n=7). each larva was held in place with pins through their caudal end to minimize orientation error in the images. eight twodimensional landmarks were collected on first and second gill arch-derived structures by a single digitizer using tpsdig2 [13] and the corresponding x,y coordinates saved for analysis. landmark locations were based on those presented in alberston and yelick [14] which are listed and presented in figure 1.institutional ethical approval was obtained prior to any animal experimentation (university of pittsburgh iacuc protocol #0409348) statistical shape analysis a geometric morphometric (gm) approach was used to analyze the landmark coordinate data. when performing a gm analysis, the first step after landmark collection is a procrustes superimposition of the entire coordinate dataset to align all the landmarks in the same coordinate system, which makes all specimens comparable to one another [15,16]. the procrustes coordinates were then regressed on centroid size (pooled within-groups approach) to remove any variation in shape that is due to size (i.e. allometry). the residuals from this regression were then used as the shape variables for the ensuing multivariate statistical tests. all analyses were performed in morphoj 1.04a [17]. to test the first hypothesis, the branchial arch mean shape from each reduced o2 group was compared to the control group. this was accomplished by computing the procrustes distance from each group mean to the control mean; statistical significance was determined via a 5000-round permutation test. to test the second hypothesis of a directional relationship between hypoxia levels and shape variation, both a principal components analysis (pca) and a canonical variate analysis (cva) were applied to the shape data. the pca reduces the multivariate data to a set of tractable axes representing the major modes of shape variation; the first few principal components (pcs) represent the axes containing the greatest amount variation in the sample, with each subsequent pc representing a lesser amount. by using gm methods, the shape variation these axes display can be modeled with wireframes that are warped using a thin plate spline technique [18]. cva is a similar data-reduction method, but where pca reduces the data without any assumptions regarding group membership, in a cva the groups are specified a priori. in effect, the cva seeks to maximize the shape variation that most effectively discriminates among groups [16]. results results of the mean shape comparison between the hypoxia groups and the control group are listed in table 1. based on the permutation test, the mean shapes of both the 70% oxygen group and the 50% oxygen group are statistically significantly different compared to that of the control group (p<0.001). the two experimental groups were also statistically diftable 1: procrustes distances between the mean shapes of each group and their associated p-values as determined by a permutation test (5000x). comparison groups procrustes distance p-value control vs. 70% oxygen 0.162 <0.001 control vs. 50% oxygen 0.243 <0.001 70% oxygen vs. 50% oxygen 0.090 0.0072 http://dentistry3000.pitt.edu hypoxic conditions alter developing branchial arch-derived structures in zebrafish vol 2, no 1 (2014) doi 10.5195/d3000.2014.21 http://dentistry3000.pitt.edu 3 figure 2: shape analysis results. ferent from each other in terms of shape (p = 0.0072).the centroid sizes of the experimental groups were both statistically lower compared to the control group (70% versus control, p = 0.013; 50% versus control, p = 0.002). the results of the pca and cva are depicted in figure 2. the scatter plot of the first two pcs indicates that the greatest axis of variation, pc1, corresponds to hypoxia level. pc1 accounts for 57.4% of the shape variation in the dataset. the control shapes cluster at one end of the plot; the 70% oxygen specimens are largely in the middle with the 50% at the opposite end from the controls. the 70% oxygen group shows the widest range of shape variation along pc1, spanning nearly the entire axis. the variance in scores associated with pc1 did not differ statistically among the three groups (p > 0.05; non-parametric levene s test for equality of variances). the shapes corresponding to the first pc are also modeled in figure 2. the wireframe at the negative end of the axis, corresponding to the control group, shows a slightly wider first arch and a more posteriorly located anterior portion of the second arch. the anterior most point of the first arch of this shape also extends beyond that of the over-all consensus shape, which is the average shape derived from all specimens included in the analysis. the wireframe corresponding to the positive end and the 50% oxygen group appears slightly compacted relative to the consensus; it appears slightly narrower in all respects and the second arch appears anteriorly displaced, while the anterior most point of the first arch is located relatively closer to the second arch. the cva also revealed a difference in shape variation between the three groups. similar to the pca scatter plot, the first cv shows three discrete clusters with the controls at one end, 50% oxygen group at the opposite end, and the 70% oxygen group in the middle. the corresponding shapes also mimic those of the pca, largely confirming the analysis. neither 70% nor 50% hypoxia exposure at 24 hpf for 24 hours resulted in excess embryo mortality. however, mortality rates at three months were increased 50% and 22%, respectively. none of the treatments caused obvious vascular phenotypes, although this judgment was based solely on gross visual inspection. discussion the results of this hypoxia experiment show that reduced oxygen availability changes the morphology of the anterior viscerocranium in developing zebrafish. additionally, the extent of the phenotypic outcome depends on the severity of hypoxia exposure. these preliminary results support the hypothesis that hypoxia is one of many environmental factors capable of altering craniofacial development, specifically first and second arch-derived structures. while these shape differences could have been a result of developmental stage differences, every attempt was made to harvest specimens at the same time point. to further reduce error due to size, the shape data were subjected to a regression to minimize size effects in the sample. this would have also reduced shape differences due to developmental stage, as stage is highly correlated with size in zebrafish [19]. the data as presented in the pca in figure 2 reveal that the largest axis of variation in the sample separates the first and second branchial arch shapes of the three groups. the 50% oxygen group is at one extreme end of the shape spectrum and the controls at the opposite extreme. interestingly, the 70% oxygen group spans the entire area of variation between the other two, overlapping with both groups. this indicates that while there is a dosage affect and a 50% reduction in oxygen level leads to anterior viscerocranial shapes outside that of normal (control) shape variation, a 30% reduction has widely variable results. in some animals it leads to the extreme phenotype, while others are within in the realm of normal variation. the cva and permutation tests statistically confirmed the mean shape differences between the three groups. this type of result from the 70% oxygen group fits previous observations of the possible effect of hypoxia on human craniofacial anomalies; prevalence is increased, but the deleterious effects are not uniform and exhibit a range of severity [2,5]. moreover, a similar dose-dependent effect and wide range of phenotypic response has been reported in the developing craniofacial complex of chicks following acute hypoxia exposure [4]. both the pca and cva indicated similar shape deformations associated with the treatment groups. two shape differences of note between the hypoxia treatment groups and the control group were at the anterior most ends of the first and second arch. part of the first arch gives rise to the mandible, and therefore the posterior location of the anterior arches in the treatment groups relative to the control group could be interesting in several ways. conclusions would depend on their relative position to the developing upper jaw. if these arches are epigenetically integrated with the developing palate, reduced oxygen during gestation could have implications on facial outgrowth. there are indications that these structures may indeed be integrated, as the shape of the adult fish lower jaw is highly correlated with the http://dentistry3000.pitt.edu hypoxic conditions alter developing branchial arch-derived structures in zebrafish vol 2, no 1 (2014) doi 10.5195/d3000.2014.21 http://dentistry3000.pitt.edu 4 neurocranium leading some to conclude that these craniofacial elements are under the same genetic control [20]. additionally, many of the zebrafish mutants with craniofacial phenotypes exhibit both affected jaws and anterior neurocrania[11,21,22]. that these structures appear to be under similar genetic control makes it tempting to speculate that they might have similar reactions to environmental factors and therefore hypoxia could hinder outgrowth in both structures, not just the first branchial arch. as mentioned above, the shape of the second branchial arch is also affected by the hypoxia exposure. the 50% reduced oxygen second arch shape is both located anteriorly and more widely angled compared to the shape associated with the control group. altered shape of the developing arches could have implications for a relationship between hypoxia and several branchial arch syndromes. an example of one such syndrome is oculo-auriculo-vertebral syndrome (oavs; omim 144210), which encompasses a wide spectrum of phenotypic variability that includes microtia, hemifacial microsomia and other mild ear malformations such as preauricular tags, and vertebral defects either singly or in combination. the etiology of oavs is unclear, but it is thought to result from dysmorphogenesis of first and second branchial arch derivatives [23]. interestingly, when compared to low elevation populations, isolated microtia and the expansive oavs phenotype have been found at higher frequencies in infants born to women in the high altitude city of quito, ecuador [2,5]. while both hypotheses tested in this study are supported by the data, the conclusions are of limited scope. although zebrafish are phylogenetically far removed from humans, much of our biomedical knowledge has been studied through the use of animal models. indeed, the zebrafish larvae form the same skeletal elements as higher vertebrates and the zebrafish is increasingly used to study jaw formation specifically [24]. while these results do not directly apply to mammals and humans specifically, they do provide evidentiary support that further research into this interaction between hypoxia and craniofacial development is warranted. now that a clear phenotypic effect has been established, a natural next step would be to elucidate the biological mechanisms underlying it. recent experimental work by smith and colleagues [4] suggests that hypoxia-induced craniofacial malformations in chick embryos result from a combination of increased cell death and decreased cell proliferation. zebrafish provide an excellent avenue for further investigations into the possible genetic and molecular machinations of hypoxia s effects on craniofacial development. additional experiments testing hypotheses of gene-environment interactions involving hypoxia and different craniofacial phenotypes would provide useful insight into determining the etiology of complex and multifactorial human craniofacial malformations such as oavs and cleft lip/palate. acknowledgements the authors have no conflicts of interest to report. this project is supported by the university of pittsburgh central research development fund (crdf to a.r.v.). references 1. unraveling human cleft lip and palate research. vieira ar. j dent res. 2008 feb;87(2):119-25. review. pmid: 18218836. 2. altitude as a risk factor for congenital anomalies. castilla ee, lopezcamelo js, campaña h. am j med genet. 1999 sep 3;86(1):9-14. pmid: 10440822. 3. hyperoxia and hypoxia in pregnancy: simple experimental manipulation alters the incidence of cleft lip and palate in cl/fr mice. millicovsky g, johnston mc. proc natl acad sci u s a. 1981 sep;78(9):5722-3. pmid: 6946511. 4. the effect of hypoxia on facial shape variation and disease phenotypes in chicken embryos. smith f, hu d, young nm, lainoff aj, jamniczky ha, maltepe e, hallgrimsson b, marcucio rs. dis model mech. 2013 jul;6(4):915-24. doi: 10.1242/dmm.011064. epub 2013 apr 16. pmid: 23592613. 5. prevalence rates of microtia in south america. castilla ee, orioli im. int j epidemiol. 1986 sep;15(3):3648. pmid: 3771073. 6. nüsslein-volhard c, gilmour dt, dahm r (2002) zebrafish as a system to study development and organogenesis. in: nüsslein-volhard c, dahm r, editors. zebrafish; a practical approach. new york: oxford university press. pp. 1-5. 7. insulin-like growth factor-binding protein-1 (igfbp-1) mediates hypoxia-induced embryonic growth and developmental retardation. kajimura s, aida k, duan c. proc natl acad sci u s a. 2005 jan 25;102(4):1240-5. epub 2005 jan 11. pmid: 15644436. 8. understanding hypoxia-induced gene expression in early development: in vitro and in vivo analysis of hypoxia-inducible factor 1-regulated zebra fish insulin-like growth factor binding protein 1 gene expression. kajimura s, aida k, duan c. mol cell biol. 2006 feb;26(3):1142-55. pmid: 16428465. 9. molecular dissection of craniofacial development using zebrafish. yelick pc, schilling tf. crit rev oral biol med. 2002;13(4):308-22. review. pmid: 12191958. 10. sperber gh, sperber sm, guttman gd (2010) craniofacial embryogenetics and development. shelton: people's medical publishing house. 251p. 11. jaw and branchial arch mutants in zebrafish ii: anterior arches and cartilage differentiation. piotrowski t1, schilling tf, brand m, jiang yj, heisenberg cp, beuchle d, grandel h, van eeden fj, furutani-seiki m, granato m, haffter p, hammerschmidt m, kane da, kelsh rn, mullins mc, odenthal j, warga rm, nüsslein-volhard c. development. 1996 dec;123:345-56. pmid: 9007254. 12. asymmetric patterns in the cranial skeleton of zebrafish (daniorerio) exposed to sodium pentachlorophenate at different embryonic developmental stages. lópez-romero f1, zúñiga g, martínez-jerónimo f. ecotoxicol environ saf. 2012 oct;84:25-31. doi: 10.1016/j.ecoenv.2012.06.008. epub 2012 jul 19. pmid: 22818112. 13. geometric morphometrics: ten years of progress following the revolution. adams, dc, rohlf jf, slice de. ital j zool. 2002 jan;71:516. 14. albertson rc, yelick pc (2009) morphogenesis of the jaw: development beyond the embryo. in: dietrich iii hw, westerfield m, zon li, editors. essential zebrafish methods. boston: elsevier. pp. 457477. 15. rohlf fj (1990) rotational fit (procrustes) methods. in: rohlf fj, bookstein fl, editors. proceedings of the michigan morphometrics workshop. ann arbor: university of michigan museum of zoolog. pp. 227-236. http://dentistry3000.pitt.edu hypoxic conditions alter developing branchial arch-derived structures in zebrafish vol 2, no 1 (2014) doi 10.5195/d3000.2014.21 http://dentistry3000.pitt.edu 5 16. zelditch ml, swiderski dl, sheets hd, fink wl (2004) geometric morphometrics for biologists. san diego: elsevier academic press. 478 p. 17. morphoj: an integrated software package for geometric morphometrics. klingenberg cp. mol ecol resour. 2011 mar;11(2):353-7. doi: 10.1111/j.1755-0998.2010.02924.x. epub 2010 oct 5. pmid: 21429143. 18. principal warps: thin-plate splines and the decomposition of deformations. bookstein fl. ieee trans pattern anal. 1989 jun;11(6):567-85. 19. schilling tf (2002) the morphology of larval and adult zebrafish. in: nuessleid-volhand c, dalm r, editors. zebrafish. oxford: oxford university press. pp. 59-94. 20. genetic basis of adaptive shape differences in the cichlid head. albertson rc, streelman jt, kocher td. j hered. 2003 jul-aug;94(4):291-301. pmid: 12920100. 21. jaw and branchial arch mutants in zebrafish i: branchial arches. schilling tf, piotrowski t, grandel h, brand m, heisenberg cp, jiang yj, beuchle d, hammerschmidt m, kane da, mullins mc, van eeden fj, kelsh rn, furutani-seiki m, granato m, haffter p, odenthal j, warga rm, trowe t, nüsslein-volhard c. development. 1996 dec;123:329-44. pmid: 9007253. 22. specification and morphogenesis of the zebrafish larval head skeleton. kimmel cb, miller ct, moens cb. dev biol. 2001 may 15;233(2):23957. review. pmid: 11336493. 23. gorlin r (2001) branchial arch and co-acral disorders. in: gorlin r, hennekam rc, editors. syndromes of the head and neck. oxford: oxford university press. pp. 790849. 24. examination of a palatogenic gene program in zebrafish. swartz me1, sheehan-rooney k, dixon mj, eberhart jk. devdyn. 2011 sep;240(9):2204-20. doi: 10.1002/dvdy.22713. pmid: 22016187. http://dentistry3000.pitt.edu microsoft word 56 2016.docx   vol  4,  no  1  (2016)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2016.56           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     the  effect  of  polymer  burs  on  microbiological  reduction  of  carious   dentin  in  deciduous  teeth:  a  systematic  review   daniela  silva  barroso  de  oliveira1,  driely  barreiros1,  léa  assed  bezerra  da  silva1,  raquel  assed  bezerra  da  silva1,  paulo  nelson-­‐filho1,  erika  cal-­‐ vano  küchler1     1department  of  pediatric  den+stry,  school  of  den+stry  of  ribeirão  preto/university  of  são  paulo,  ribeirão  preto,  sp,  brazil abstract   background.  polymer  bur  is  a  new  technology  that  proposes  to  conserve  the  den7n  that  is   capable  of  remineraliza/on.  aim:  to  conduct  a  quan+ta+ve  systema+c  review  to  evaluate   the  effect  of  polymer  burs  on  the  reduc3on  of  streptococcus  mutans  and  lactobacillus  a"er   den$n  carious  excava$on   in  deciduous   teeth.  methods  and  material:  two  reviewers  per-­‐ formed   the   database   to   iden/fy   the   relevant   clinical   papers.   only   papers   in   english   that   compared  polymer  burs  with  other  caries  removal  techniques  with  the  microbiological  as-­‐ pects  as  an  outcome  were  included.  papers  that  evaluated  only  permanent  teeth  were  ex-­‐ cluded.  risk  of   bias  was   also   assessed.   individual   study  effect   sizes  were   calculated  using   cohen’s  d  formula  for  the  comparisons  of  microorganism  reduc5on  before  and  a&er  carious   excava&on.  results:  the  search  resulted  in  12  non-­‐duplicated  papers.  a/er  the  revision,  on-­‐ ly  2  were  selected.  the  quan3ta3ve  evalua3on  demonstrated  that  polymer  bur  reduces  mi-­‐ croorganism  levels  in  carious  den0n.  the  larger  effect  size  observed  was  for  streptococcus   mutans  in  the  polymer  bur  technique  (r=0.84;  d=3.12),  followed  by  lactobacillus  in  the  car-­‐ bide  bur   technique   (r=0.83;  d=3.03).  conclusion:  polymer  burs  promoted  a   significant   re-­‐ duc$on  of  microorganism  levels,  mainly  streptococcus  mutans  in  carious  den,n.     cita%on:  oliveira,  et  al.  (2016).  the  effect  of  pol-­‐ ymer  burs  on  microbiological  reduc1on  of  cari-­‐ ous  den(n  in  deciduous  teeth:  a  systema(c  re-­‐ view.  den$stry  3000.  1:a001   doi:10.5195/d3000.2016.56   received:  july  12,  2016   accepted:    july  23  ,  2016   published:    october  3,  2016   copyright:  ©2016  oliveira,  et  al.  this  is  an  open   access   ar!cle   licensed   under   a   crea!ve   com-­‐ mons   a"ribu"on   work   4.0   united   states   li-­‐ cense.   email:  erikacalvano@gmail.com   introduction   dentinal  caries  is  structur-­‐ ally  divided  into  infected  and  af-­‐ fected  dentin.  the  superficial  layer   is  the  infected  dentin.  it  is  irre-­‐ versible  denatured  and  extensively   demineralized.  the  deeper  layer  is   the  affected  dentin  and  is  bacte-­‐ ria-­‐free.  it  is  potentially  and  physi-­‐ ologically  remineralizable,  with   odontoblastic  process,  sound  col-­‐ lagen  fibers  and  apatite  crystal   bound  to  the  fibers  [1-­‐6].  the  min-­‐ imally  invasive  dentistry  concept   implies  that  the  infected  and  irre-­‐ versibly  denatured  dentin  should   be  removed  selectively  to  pre-­‐ serve  as  much  as  possible  sound   or  potentially  remineralizable  den-­‐ tin  [3,7].  however,  a  balance  be-­‐ tween  tissue  preservation  and  in-­‐ fected  dentin  removal  should  be   achieve  in  order  to  prevent  caries   progression  [4,8-­‐9].   conventional  methods  for   carious  dentin  removal  include   manual  excavation  with  hand  ex-­‐ cavators  and  burs  associated  with   low  or  high-­‐handpieces  [10].  both   are  dependent  of  the  operator's   optical  and  tactile  sensitivity  and   may  lead  to  an  unnecessary  re-­‐ moval  of  dental  tissues  [5,8-­‐12].   moreover,  infected  and  affected   dentin  have  some  differences  such   as  in  the  hardness,  toughness  and   resilience,  which  determine  the   relative  efficiency  of  caries  excava-­‐ tion  techniques  [2].   new  techniques  have  been   proposed  to  conserve  dental  tis-­‐ sue  that  is  able  to  remineralize  [2-­‐ 3,6,10-­‐11].  polymer  burs  were  de-­‐ veloped  (smartbur-­‐ss  white  com-­‐ pany  -­‐  lakewood,  nj,  usa)  to  be   used  with  conventional  low-­‐speed   handpieces  that  proposes  a  self-­‐ limiting  technology,  to  provide  se-­‐ lective  caries  removal.  these  burs   are  single-­‐use  instruments,  made   with  a  polymer  that  presents  value   of  hardness  knoop  (khn)  equal  to   50.  this  hardness  value  is  lower   than  healthy  dentin  (70-­‐90),  but   the  effect  of  polymer  burs  on  microbiological  reduc7on  of  carious  den7n  in  deciduous  teeth:  a  systema7c  review   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.56    http://dentistry3000.pitt.edu   2   higher  than  that  the  carious  or  in-­‐ fected  dentin  (0-­‐30)  [3-­‐5,10].  pol-­‐ ymer  burs  have  the  potential  to   selectively  remove  the  carious   dentin,  since  they  lose  their  cut-­‐ ting  capacity,  becoming  blunt,   when  touching  the  affected  and   healthy  dentin  [3-­‐5,8-­‐9,12].   although  several  in  vitro   [4-­‐5,9-­‐11,13]  and  in  vivo  [6,8,12]   papers  evaluated  the  effectiveness   of  polymer  burs  in  selectively  re-­‐ move  the  infected  carious  dentin,   their  effect  on  the  microbiological   reduction  is  still  imprecise.  thus,   the  aim  of  this  study  was  to  evalu-­‐ ate  the  effect  of  the  polymer  burs   on  the  reduction  of  streptococcus   mutans  and  lactobacillus  after   dentin  carious  excavation  in  de-­‐ ciduous  teeth,  through  a  quantita-­‐ tive  systematic  review.   materials  and  methods   protocol  and  registration   to  conduct  this  review,  the   prisma  statement  checklist  was   used  [14].  the  study  was  regis-­‐ tered  in  the  international  prospec-­‐ tive  register  of  systematic  reviews   ‘‘prospero’’   (www.crd.york.ac.uk/prospero)   (registration  number   crd42015016678).   search  strategy     the  search  strategy  was   based  on  the  followingmedical   subject  heading  terms  (mesh)  or   text  word  [tw]  in  different  combi-­‐ nation  strategy:  “polymers”   [mesh  terms]  or  “polymer”  [tw]   and  “burs”  [tw]  or  “bur”  [tw]  or   “drill”  [tw]  and  “dental  caries”   [mesh  terms]  or  “caries  removal”   [tw]  or  “caries  excavation”  [tw].   using  the  following  data-­‐ bases,  the  search  of  the  literature   was  performed:  medline  (1966–   february  2016),  web  of  science   (1900–  february  2016),  scopus   (1960–  february  2016)  and  the   cochrane  library  (1993-­‐  february   2016).   attempts  to  obtain  missing   information,  additional  manual   search  were  performed  to  find  fur-­‐ ther  papers  that  were  not  in  the   electronic  databases.  reference   lists  from  identified  papers  were   scanned  to  identify  other  poten-­‐ tially  relevant  papers.     inclusion  and  exclusion  criteria   the  inclusion  criteria  were  as  fol-­‐ lows:   1.  papers  in  english.   2.  clinical  papers.     3.  papers  that  evaluate  the  effect   of  the  polymer  burs  on  the  reduc-­‐ tion  of  microorganisms.   4.  the  outcome  was  to  evaluate   microbiological  aspects.     table&1.!!summary!of!data!reported!in!selected!articles!addressing!polymer!burs!and!caries!removal! study& number&of& participants& and&teeth& age& range& study&& design& microbiological& evaluation& microorganisms&& analyzed& authors&& conclusions& ! ! 12.!isik!et! al.,!2010! 24!patients! ! 48!teeth! ! 5a9! same!child! received!both! treatments! assigned!into!2! groups:! 1)!round!carbide! burs!! 2)!polymer!burs! growth!in!culture! media!(cfu)!and! biochemical! fermentation!test! 1)#streptococcus#mutans# 2)#lactobacillus# 3)!aerobic!microorganisms! 4)!anaerobic!microorganisms!! !!!no!statistically!significant! differences!in!microorganisms! reduction!between!round! carbide!burs!and!polymer!burs! in!all!microbiological!test!used.! ! ! 8.! zakirulla! et!al.,! 2011! 36!patients! ! 45!teeth! 6a14! division!into!3! groups:! 1)!round!carbide! burs! 2)!polymer!burs! 3)!spoon! excavator! growth!in!culture! media!(cfu)! 1)#streptococcus#mutans# 2)#lactobacillus# ! polymer!burs!showed! statistically!significant!more! reduction!for!lactobacillus,!but! not!for!s#mutans#when! compared!with!spoon! excavator.##round!carbide!burs! showed!statistically!significant! more!reduction!for!s.#mutants# and!lactobacillus#when! compared!to!polymer!burs.! note:&cfu=colony!forming!unit& ! the  effect  of  polymer  burs  on  microbiological  reduc7on  of  carious  den7n  in  deciduous  teeth:  a  systema7c  review   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.56    http://dentistry3000.pitt.edu   3   the  exclusion  criteria  included  the   following:     1.  papers  in  vitro,  ex-­‐vivo,  case  re-­‐ ports,  case  series,  studies  with  an-­‐ imal  models  and  reviews.   2.  papers  without  comparison  of   the  polymer  bur  with  other  tech-­‐ nique.   3.  papers  that  evaluated  only   permanent  teeth.   unpublished  results,  thesis,   book  chapters,  abstracts  and  re-­‐ views  were  not  included.   selection  criteria   all  potentially  relevant  pa-­‐ pers  were  identified  by  the  title   and  the  abstract.  after  that,  the   full  text  analyses  were  performed   to  include  the  paper  in  the  sys-­‐ tematic  review.     two  examiners  (dsbo  and   db)  evaluated  titles,  abstracts  and   full  text.  if  there  was  a  diverging   opinion,  disagreement  among  ex-­‐ aminers  was  reexamined  in  con-­‐ sensus  meetings.   data  extraction   one  author  (dsbo)  ex-­‐ tracted  the  relevant  data  from  the   included  papers.  from  each  in-­‐ cluded  study,  the  description  of   the  sample,  the  study  design,  mi-­‐ crobiological  evaluation  and  re-­‐ sults  were  summarized  in  the  ta-­‐ ble  1.  other  author  (eck)  checked   the  extracted  data.  disagreements   between  the  two  researchers   were  resolved  by  discussion  and   consensus.   assessment  of  risk  of  bias   the  quality  assessment  of   the  remaining  papers  was  per-­‐ formed  to  evaluate  methodologi-­‐ cal  aspects  related  to  influence   bias,  to  gain  insight  into  the  results   of  the  techniques  comparisons   and  to  guide  the  interpretation  of   qualitative  and  quantitative  find-­‐ ings.   five  criteria  were  used  to   analyze  all  included  papers:  1)   sample  size  calculation;  2)  random   allocation  of  the  participants;  3)   participants’  eligibility,  4)  inclusion   and  exclusion  criteria;  5)  standard-­‐ ization  of  the  investigator.  two  of   the  reviewers  (pnf  and  eck)  as-­‐ sessed  the  included  papers  for  risk   of  bias  and  were  blinded  to  each   other's  assessments.  disagree-­‐ ments  were  resolved  through  dis-­‐ cussions  with  a  third  review   (rabs).  domains  from  the   cochrane  collaboration  risk  of  bias   tool  were  assessed  for  each  study.   data  were  entered  into  review   manager  5.3  (revman  5.3)  for  the   graphical  representation.   calculation  of  the  effect  size   comparisons  of  microor-­‐ ganism  reduction  before  and  after   dentin  carious  excavation  were   performed  for  each  study  and   each  comparison  group.  in  order   to  pool  data,  means  and  standard   deviations  were  converted  into   the  effect  of  polymer  burs  on  microbiological  reduc7on  of  carious  den7n  in  deciduous  teeth:  a  systema7c  review   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.56    http://dentistry3000.pitt.edu   4   effect  size.  when  the  results  were   presented  as  median,  the  mean   value  and  the  standard  was  calcu-­‐ lated  using  the  formula  proposed   by  hozo  et  al.  [15].  individual   study  effect  sizes  for  the  2  includ-­‐ ed  papers,  that  presented  the  data   as  a  continuous  variable,  were  cal-­‐ culated  using  cohen’s  d  formula   [16].  positive  values  represented  a   reduction  of  microorganism  level.   the  effect-­‐size  was  classified  as   suggested  by  cohen  [16],  in  low   effect  (d≤0.2),  medium  effect   (0.21≤d≥0.79)  and  high  effect   (d≥0.8).   results   a  total  of  132  titles  and  ab-­‐ stracts  were  screened  from  the   selected  databases.  there  were   110  in  pubmed,  10  in  web  of  sci-­‐ ence,  8  in  scopus  and  4  in   cochrane.  the  duplicates  were   considered  only  once,  totaling  132   papers.   initially,  the  titles  and  ab-­‐ stracts  not  connected  with  the   topic  were  excluded  (116  papers).   afterwards,  the  papers  were  se-­‐ lected  according  to  the  inclusion   and  exclusion  criteria,  totaling  4   papers  selected  for  full  text  analy-­‐ sis.  after  reading,  2  papers  were   excluded  because  they  were  in   vitro  papers.  thus,  2  full  texts   were  included  and  analyzed  ac-­‐ cording  to  the  selection  criteria   (figure  1).   the  summary  of  the  char-­‐ acteristics  of  the  two  included  pa-­‐ pers  is  described  in  the  table  1.   the  papers  were  also  carefully   read  and  ranked  based  on  the  risk   of  bias  as  shown  in  figure  2.     the  quantitative  analysis,   that  shows  the  effect  size  of  mi-­‐ croorganisms  reduction,  are  pre-­‐ sented  in  the  table  2.  all  tech-­‐ niques  demonstrated  a  moderate   and  large  reduction  of  streptococ-­‐ cus  mutans  and  lactobacillus  lev-­‐ els.  the  larger  effect  size  observed   was  for  streptococcus  mutans  in   the  polymer  bur  technique   (r=0.84;  d=3.12),  followed  by  lac-­‐ tobacillus  in  the  carbide  bur  tech-­‐ nique  (r=0.83;  d=3.03).     discussion   the  removal  of  infected   carious  dentin  and  the  careful   maintenance  of  the  dentin  that  is   capable  of  remineralization  has   been  a  focus  of  the  conservative   dentistry  approaches  [3].  the  ac-­‐ tual  criteria  for  caries  removal   arise  from  the  concept  that     table&2.!calculation!of!effect!size!for!the!included!studies! study& caries&excavation&technique& microorganism& effect&size&score&of&the&reduction& type&of&the&effect& r& d& 12.!isik!et!al.,!2010! polymer!bur! streptococcus*mutans* 0.646! 1.695! moderate!reduction! lactobacillus* 0.458! 1.033! moderate!reduction! ! ! carbide!bur! streptococcus*mutans* 0.671! 1.811! moderate!reduction! lactobacillus* 0.573! 1.398! moderate!reduction! ! 8.!zakirulla!et!al.,!2011! polymer!bur!! streptococcus*mutans! 0.842& 3.121! high!reduction! lactobacillus! 0.703! 1.979! moderate!reduction! carbide!bur! streptococcus*mutans! 0.624! 1.600! moderate!reduction! lactobacillus! 0.835& 3.035! high!reduction! spoon!excavator! streptococcus*mutans! 0.712! 2.032! moderate!reduction! lactobacillus! 0.578! 1.418! moderate!reduction! note:&&bold&form&indicates&high&effect&size.& r:&effect!size!correlation! d:&cohen’s!d& ! the  effect  of  polymer  burs  on  microbiological  reduc7on  of  carious  den7n  in  deciduous  teeth:  a  systema7c  review   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.56    http://dentistry3000.pitt.edu   5     “sound”  tissue  or  poten-­‐ tially  remineralizable  tissue  should   not  be  unnecessarily  removed   [5,17].  nevertheless,  the  exact   endpoint  of  caries  removal  cannot   be  easily  clinically  defined.  this   may  be  explained  by  the  fact  that   dentists  have  been  trained  in  rely-­‐ ing  on  the  hardness  of  dentine  as   felt  with  a  dental  probe.  other   more  subjective  characteristics,   such  as  the  dentin  color  and  mois-­‐ ture  are  often  neglected  [17].  our   quantitative  systematic  review   aimed  to  evaluate  a  new  tech-­‐ nique  that  proposes  to  preserve   the  potentially  remineralizable   tissue  without  the  dentist´s  atten-­‐ tion  to  the  subjective  characteris-­‐ tics.   an  important  criteria  to   differentiate  infected  dentin  of   affected  dentin  is  the  number  of   viable  microorganisms  existent  in   the  remaining   dentin  [2-­‐4,6].   therefore  we   chose  this  pa-­‐ rameter  as  an   outcome  to   evaluate  the   effectiveness  of   the  polymer   burs  to  reduce   cariogenic  mi-­‐ croorganisms.  in   the  present   study,  the  effect   size  calculation   demonstrated   that  burs,  poly-­‐ mer  burs  and   spoon  excavator   were  able  to   promote  a   moderate  and  a   large  microbiological  reduction.     mutans  streptococci  are   the  main  etiologic  agent  of  dental   caries  in  humans  [18-­‐19],  which   explains  the  importance  of  its  re-­‐ duction  after  procedures  of  mini-­‐ mal  invasive  excavation.  indeed,   we  observed  that  polymer  bur   promoted  the  largest  reduction  in   streptococcus  mutans  levels.   minimal-­‐invasive  dental   preparation,  such  as  polymer  burs,   simplifies  and  standardizes  caries-­‐ removal  procedures  [17].  in  fact,   our  results  demonstrated  that   polymer  burs  may  offer  a  viable   and  efficient  means  to  achieve   minimal-­‐invasive  dental  prepara-­‐ tion.  maintain  the  healthy  dental   structures  as  previously  described   [3-­‐5].     our  results  showed  that   the  polymer  burs  were  more  ef-­‐ fective  in  streptococcus  mutans   removal,  while  conventional  burs   provided  a  further  reduction  of   lactobacillus.  as  conventional   burs  are  more  invasive  than  poly-­‐ mer  burs  and  usually  extends  into   the  affected  dentine  [3,5,13],  this   might  lead  to  a  larger  reduction  of   lactobacilli  which  are  strictly  an-­‐ aerobic,  highly  sensitive  to  ph  rais-­‐ ing  and  located  in  the  deeper  por-­‐ tions  of  the  carious  dentin  [20-­‐22].   the  included  papers  have   some  limitations  that  directly  re-­‐ flect  our  review  results.  none  of   them  attempted  to  perform  a   sample  size  calculation,  and  only   one  paper  performed  a  random   allocation  of  used  teeth  and  the   standardization  of  the  investigator   for  the  polymer  burs  use.  in  addi-­‐ tion,  it  is  important  to  emphasize   that  a  long-­‐term  follow  up  should   be  perform  in  order  to  evaluate  a   real  clinical  efficiency  regarding   the  caries  lesion  recurrence.     although  in  all  techniques   evaluated  in  the  included  papers,   there  was  reminiscent  number  of   viable  microorganisms  in  the   deeper  layers  of  dentin,  we  can   assume  that  the  subsequent  seal-­‐ ing  performed  by  the  restoration   could  solve  this  issue,  as  previous-­‐ ly  demonstrated  [23].  therefore,   the  maintenance  of  the  cavity   sealing  through  a  dentinoenamel   junction  free  from  caries  and  ad-­‐ hesive  restorations  would  inter-­‐ rupt  the  communication  of  bacte-­‐ ria  present  in  dentin  with  the  sur-­‐ face  biofilm,  with  a  consequent   reduction  in  nutrients  and  bacteri-­‐ al  viability  [24].       the  effect  of  polymer  burs  on  microbiological  reduc7on  of  carious  den7n  in  deciduous  teeth:  a  systema7c  review   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.56    http://dentistry3000.pitt.edu   6   how  does  a  polymer  bur  actually   provide  clinical  benefits?     although  this  study  aimed   to  answer  the  question  about  the   efficacy  of  the  polymer  burs  for   microorganism’s  reduction  during   carious  removal,  it  raises  ques-­‐ tions  that  should  be  considered  by   clinical  researchers.  microorgan-­‐ isms  reduction  comparison  does   not  necessary  allows  the  re-­‐ searchers  and  clinicians  to  infer   which  is  the  most  effective  meth-­‐ od  for  minimal  invasive  approach.   furthermore,  the  use  of  polymer   burs  does  not  eliminate  the  need   of  conventional  burs,  which  may   be  necessary  for  the  enamel  re-­‐ moval  to  access  the  carious  den-­‐ tin.     the  polymer  burs  proposes   for  the  clinicians  a  less  invasive   option  for  deep  decay  removal,   while  leaving  healthy  dentin  intact   (smartbur-­‐ss  white  company-­‐ lakewood,  nj,  usa).  this  study   demonstrated  that  conventional   methods  and  the  polymer  burs   reduce  levels  of  streptococcus  mu-­‐ tans  and  lactobacillus.  however,   since  the  polymer  burs  are  selec-­‐ tive  for  carious  dentin  remove,  it   should  be  an  option  for  clinicians   to  preserve  sound  dentin.  in  addi-­‐ tion,  it  has  been  proposed  that   this  technique  may  eliminate  the   use  of  local  anesthesia3.  however,   the  researchers  should  largely  in-­‐ vestigate  the  clinical  benefit  of  this   new  instrument.   suggestion  for  future  papers   based  on  this  systematic   review,  some  questions  should  be   answered  in  future  papers.  first,   although  it  is  accepted  that  the   dental  restoration  is  effective  to   deal  with  the  microorganisms  left   in  the  remaining  dentin,  a  long   term  follow  up  should  be  per-­‐ formed.  also,  since  there  were  no   remarkable  differences  in  micro-­‐ organisms  reduction  among  the   minimal  invasive  techniques,  fu-­‐ ture  clinical  papers  should  attempt   to  evaluate  the  effectiveness  of   the  polymer  bur  in  a  multilevel   design  study,  in  which  preparation   time,  peculiarities  of  this  new   technique,  operator  training,  as-­‐ sessment  of  important  clinical  cri-­‐ teria  such  as  pain,  sensitivity  to   pressure,  vibration  and  tempera-­‐ ture  changes  and  cost  (single  use   instrument)  of  the  polymer  bur   should  be  included.   finally,  definitive  clinical   conclusions  about  the  effective-­‐ ness  of  polymer  burs  were  not  de-­‐ scribed  in  this  paper  as  only  one   outcome,  the  microbiological,  was   evaluated.  other  important  pa-­‐ rameters  such  as  evaluation  of   hardness,  color  and  humidity  are   important  in  assessing  the  carious   dentin  removal.  furthermore,  pa-­‐ pers  evaluating  the  long-­‐term  re-­‐ maining  dentin  are  important  to   determine  if  the  number  of  bacte-­‐ ria  remaining  after  carious  dentin   removal  with  polymer  burs  will   not  be  viable  for  the  recurrence  of   caries.    in  conclusion,  polymer   burs  promote  the  reduction  of  mi-­‐ croorganism  levels,  mainly  strep-­‐ tococcus  mutans.     references   1. electron  microscopic  struc-­‐ ture  of  two  layers  of  cari-­‐ ous  dentin.  ogushi  k,   fusayama  t.  j  dent  res   1975;  sep-­‐oct;54(5):1019-­‐ 26.  pmid:  1058852.   2. new  device  for  selective   dentin  caries  removal.  bos-­‐ ton  dw.  quintessence  in-­‐ ternational  2003  oct;34(9):   678-­‐685.  pmid:  14982221.     3. removing  carious  dentin   using  a  polymer  instrument   without  anesthesia  versus   a  carbide  bur  with  anes-­‐ thesia.  allen  kl,  salgado  tl,   janal  mn,  thompson  vp.  j   am  dent  assoc  2005  may;   136(5):  643-­‐651.  pmid:   15966653.     4. efficiency  of  the  polymer   bur  smartprep  compared   with  conventional  tungsten   carbide  bus  bur  in  dentin   caries  excavation.   dammaschke  t,  ronden-­‐ berg  tn,  schäfer  e,  ott   khr.  operative  dentistry   2006  mar-­‐apr;  31(2):256-­‐ 260.  pmid:  16827030   5. digital  image  analysis   method  to  assess  the  per-­‐ formance  of  conventional   and  self-­‐limiting  concepts   in  dentin  caries  removal.   toledano  m,  ghinea  r,   cardona  jc,  cabello  i,   yamauti  m,  pérz  mm,  oso-­‐ rio  r.  j  den  2013;  aug;41   suppl  3:e31-­‐8.  epub  2013   mar  15.  pmid:  23507398   6. a  comparative  clinical   study  of  various  methods   of  caries  removal  in  chil-­‐ dren.  soni  hk,  sharma  a,   sood  pb.  eur  arch  paediatr   the  effect  of  polymer  burs  on  microbiological  reduc7on  of  carious  den7n  in  deciduous  teeth:  a  systema7c  review   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.56    http://dentistry3000.pitt.edu   7   dent  2015  feb;  16(1):19-­‐ 26.  pmid:  25391222.   7. minimal  intervention  den-­‐ tistry:  rationale  of  cavity   design.  mount  gj.  opera-­‐ tive  dentistry  2003  jan-­‐ fev;  28(1):  92-­‐99.  pmid:   12540125.   8. in  vivo  comparison  of  re-­‐ duction  in  bacterial  count   after  caries  excavation  with   3  different  techniques.  za-­‐ kirulla  m,  uloopi  ks,  subba   reddy  vv.  j  den  child  2011   jan-­‐apr;  78(1):  31-­‐35.   pmid:  22041006.   9. differential  resin-­‐dentin   bonds  created  after  caries   removal  with  polymer   burs.  toledano  m,  cabello   i,  yamauti  m,  osorio  r.  mi-­‐ crosc  microanal  2012  jun;   18(3):  497-­‐508.  pmid:   22640962   10. performance  of  four  den-­‐ tine  excavation  methods  in   deciduous  teeth.  celiberti   p,  francescut  p,  lussi  a.   caries  res  2006;  40(2):   117-­‐123.  pmid:  16508268.   11. comparison  of  dentin  car-­‐ ies  excavation  with  poly-­‐ mer  and  conventional   tungsten  carbide  burs.   meller  c,  welk  a,  zeli-­‐ gowski  t,  splieth  c.  quin-­‐ tessence  int  2007  jul-­‐aug;   38(7):  565-­‐569.  pmid:   17694212.   12. a  microbiological  assess-­‐ ment  of  polymer  and  con-­‐ ventional  carbide  burs  in   caries  removal.  isik  ee,   ölmez  a,  akca  g,  sultan  n.   pediatr  dent  2010  jul-­‐aug;   32(4):  316-­‐23.  pmid:   20836951.   13. evaluation  of  a  self-­‐limiting   concept  in  dentinal  caries   removal.  silva  nrfa,  carva-­‐ lho  rm,  pegoraro  lf,  tay   fr,  thompson  vp.  j  dent   res  2006  mar;  85(3):  282-­‐ 286.  pmid:  16498079.   14. preferred  reporting  items   for  systematic  reviews    and   meta-­‐analysis:  the  prisma   statement.  moher  d,  lib-­‐ erati  a,  tetzlaff  j,  altman   dg,  prisma  group.  ann  in-­‐ tern  med.  2009  aug   18;151(4):264-­‐9.  pmid:   19622511.   15. estimating  the  mean  and   variance  from  the  median,   range,  and  the  size  of  a   sample.  hozo  sp,   djulbegovic  b,  hozo  i.  bmc   med  res  mehtodol  2005   apr  20;5:13.  pmid:   15840177.   16. cohen  j  (1988)  statistical   power  analysis  for  the  be-­‐ havioral  sciences.  2nd  ed.   new  jersey:  lawrence  erl-­‐ baum  associates.   17. caries-­‐removal  effective-­‐ ness  and  minimal-­‐ invasiveness  potential  of   caries-­‐excavation  tech-­‐ niques:  a  micro-­‐ct  investi-­‐ gation.  neves  aa,  coutinho   e,  de  munck  j,  meerbeek   bv.  j  den  2011  feb;  39(2):   154-­‐162.  pmid:  21111770.     18. role  of  streptococcus  mu-­‐ tans  in  human  dental  de-­‐ cay.  loesche  wj.  microbio-­‐ logical  reviews  1986  dec;   50(4):  353-­‐380.  pmid:   3540569.   19. recovery  of  mutans  strep-­‐ tococci  on  msb,  sb-­‐20  and   sb-­‐20m  agar  media.  sara-­‐ via  me,  nelson-­‐filho  p,  sil-­‐ va  rab,  de  rossi  a,  faria  g,   silva  lab,  emilson  cg.  arch   oral  biol  2013  mar;  58(3):   311-­‐316.  pmid:  23149153.   20. assessing  the  stage  of  car-­‐ ies  lesion  activity  on  the   basis  of  clinical  and  micro-­‐ biological  examination.     nyvard  b,  fejerskov  o.   communty  dent  oral  epi-­‐ demiol  1997  feb;  25(1):69-­‐ 75.  pmid:  9088694.   21. evaluation  of  clinical  and   microbiological  features  of   deep  carious  lesions  in   primary  molars.  ayna  b,   çelenk  s,  atakul  f,  sezgin   b,  özekinci  t.  j  dent  child   2003  jan-­‐apr;  70(1):15-­‐18.   pmid:  12762602.   22. a  tissue-­‐dependent  hy-­‐ pothesis  of  dental  caries.   simón-­‐soro  á,  belda-­‐ferre   p,  cabrera-­‐rubio  r,  alcaraz   ld,  mira  a.  caries  res   2013;  47(6):591-­‐600.   pmid:  24080530.   23. partial  caries  removal  in   primary  teeth:  association   of  clinical  parameters  with   microbiological  status.  lula   eco,  almeida  jr  ljs,  alves   cmc,  monteiro-­‐neto  v,  ri-­‐ beiro  cc.  caries  res  2011;   the  effect  of  polymer  burs  on  microbiological  reduc7on  of  carious  den7n  in  deciduous  teeth:  a  systema7c  review   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.56    http://dentistry3000.pitt.edu   8   45(3):275-­‐280.  pmid:   21576960.   24. how  “clean”  must  a  cavity   be  before  restoration?   kidd  ea.  caries  res  2004   may-­‐jun;  38(3):  305-­‐313.   pmid:  15153704.     microsoft word the correlation of perceived stress and professional concerns during covid.docx the correla*on of perceived stress and professional concerns during covid-19 pandemic among iranian den*sts: the media*ng role of cogni*ve flexibility vol 8 no 1 (2020) doi 10.5195/d3000.2020.119 h#p://den*stry3000.pi#.edu the correlation of perceived stress and professional concerns during covid-19 pandemic among iranian dentists: the mediating role of cognitive flexibility afrooz afshari 1, seyedehsareh hashemikamangar 1, sedighe sadat hashemikamangar 2 * 1 department of psychology, alzahra university, tehran, iran 2 department of operative dentistry, dental school, tehran university of medical sciences, tehran, iran abstract objec&ve: to evaluate the associa.on of perceived stress and dental work-related concerns regarding to covid-19 by media.ng role of cogni.ve flexibility among den.sts during pandemic outbreak of covid-19 in iran. methods: this study was a cross-sec.onal survey conducted by means of a confiden.al on-line survey on 174 iranian den.sts. covid-19 related ques.ons were divided into three parts: den.sts’ perspec.ve about their career future, virus contagion and concerns of gelng infected covid-19 from pa.ents, and paucity of informa.on and facili.es to control the spread of the virus. perceived stress and psychological flexibility were assessed by pss (cohen et al., 1983) and cfi (dennis et al., 2010). to analyze the data path analysis method was used. results: perceived stress significantly predicted psychological flexibility (both alterna.ves and control subscales) and covid-19 factors related to dental work. in addi.on, the control subscale significantly predicted den.sts’ concerns toward their career future and fear of covid-19 contagion. hence, control subscale of cogni.ve flexibility mediated the rela.onship of perceived stress and professional concerns of den.sts during covid-19 pandemic. conclusion: given these results, elevated stress during an infec.ous disease outbreak can affect (have an impact on) perceived stress and professional concerns among den.sts. different aspects of den.sts’ lives can be affected by those concerns and stress. interven.ons to enhance cogni.ve flexibility should be applied to reduce the psychological burden of the crisis for den.sts. keywords: covid-19; den.sts; professional concerns; perceived stress; psychological flexibility. cita9on: afshari a, et al. (2020) the correla9on of perceived stress and professional concerns during covid-19 pandemic among iranian den9sts: the media9ng role of cogni9ve flexibility. den9stry 3000. 1:a001 doi:10.5195/d3000.2020.119 received: october 28, 2020 accepted: november 30, 2020 published: dezember 22, 2020 copyright: ©2020 afshari a, et al. this is an open access ar9cle licensed under a crea9ve commons asribu9on work 4.0 united states license. email: smhk58950@gmail.com introduction the first cases of coronavirus (covid-19) were reported in late december 2019 in wuhan, china, and after its rapid spread in many countries around the world, on march 11, 2020, the world health organization (who) declared it as a pandemic. the disease became a global threat quickly. it is the largest outbreak of atypical pneumonia since sars's acute respiratory syndrome in 2003. the common transmission routes of coronavirus include direct and contact transmissions such as droplet inhalation transmission, cough and sneeze, contact with oral, nasal, and eye mucous membranes [1]. this respiratory virus can be transmitted from person to person through coarse or small droplets, and can also be transmitted directly or indirectly through saliva [2]. past researches on smaller-scale epidemics and recent researches on covid-19 have reported a large amount of stress and increased psychological problems among professionals who dealt with patients suffering from corona [36]. in a review study conducted on the correla*on of perceived stress and professional concerns during covid-19 pandemic among iranian den*sts: the media*ng role of cogni*ve flexibility vol 8 no 1 (2020) doi 10.5195/d3000.2020.119 h#p://den*stry3000.pi#.edu experienced stress by health care workers during covid-19 pandemic, extensive strain due to stressful experiences, depression and anxiety symptoms were reported [7]. dentists might be one of the most stress vulnerable population during covid-19 pandemic because of high risk for transmission of infection and disease due to exposure to blood, saliva, and aerosol / droplet generated during dental treatment procedures [2]. since the virus spread can happen in the absence of clinical symptoms, [8] psychological stress of dental professionals might be more serious due to the ambiguous nature of the virus and the probability of virus contamination in their work environment. many studies mentioned the psychological problems of dental staff in work environment or in relation to patients [9, 10]. in a review study performed on key determinants of health and wellbeing among dentists working in the uk, it was revealed that workplace characteristics were the most often explored determinants by researchers and reported as being a key issue. the results also showed that the health and wellbeing of dentists are associated with a wide range of factors: personal, professional career, relationships, job specification, workplace and system [11]. in another recent study on elevated distress associated with covid-19 and psychological factors in israeli dental staff, it was proven that those who had a background illness, higher subjective overload, and fear of contracting covid-19 from patient, reported higher levels of psychological distress. being in a committed relationship and having higher scores for selfefficacy was related to lower psychological distress [12]. there are many individual differences between people when they are exposed to threats. the way individuals perceive and interpret a stressful event is considered of greater importance than the event itself. according to lazarus, perceived stress refers to the degree to which people view life events as unpredictable, uncontrollable, and stressful [13]. lazarus believes that stimuli that are evaluated as stressor call for stress responses. however, in a crisis such as the outbreak of an infectious lifethreatening virus that endangers the sense of safety and wellbeing of people, one of the crucial things which can possibly affect the mental health of individuals is cognitive flexibility. the term is described as “the ability to switch cognitive sets to adapt to the changing environmental stimuli” [14]. if a stressful situation is perceived as “beyond a person’s control”, cognitive flexibility can be reduced by the person’s appraisal. whereas, a brief social stressor can have an enhancing effect on mental flexibility [15]. therefore, one of the possible reasons for individual differences in cognitive flexibility [16] is the variety of perceptions and appraisal of stressful events. in this study, we aimed to evaluate the perceived stress and cognitive flexibility of dental staff about covid-19 factors related to work environment such as concerns about their career future, contagion control and paucity of information, and facilities to control the disease. material and methods design and sample this study is based on a path analysis designed to test a hypothetical model involving perceived stress, cognitive flexibility, and covid-19 factors related to work environment among iranian dentists during coronavirus outbreak. using convenient sampling method and the correla*on of perceived stress and professional concerns during covid-19 pandemic among iranian den*sts: the media*ng role of cogni*ve flexibility vol 8 no 1 (2020) doi 10.5195/d3000.2020.119 h#p://den*stry3000.pi#.edu online questionnaires sent to dentists, data were recruited. participants were general dentists or specialist in iran who work in either private office, clinics, or both, and aged between 25 to 65 years old from both genders. exclusion criteria were unemployment for at least one year prior to the start of the covid-19 pandemic and/or unwillingness to participate in the research. ethical considerations this is a cross-sectional study, which is approved by research ethics committee of school of dentistry in tehran university of medical science, iran (approval code: ir.tums.dentistry. rec.1399.025). before completing the questionnaires, participants signed informed consent forms which contained information about the researchers, the purpose of research, advantages of participation, issues related to confidentiality, and procedures for withdrawal from the research. measures in this study, to assess perceived stress among dentists, perceived stress scale (pss) was administered to participants. the pss, designed by cohen, kamarck, & mermelstein (1983), has different forms with 10, 4, and 14 questions. in this study, 14questions form was used. each item is rated on a 5-point scale (0= never to 4 =always). items are designed to measure the extent to which one’s life is perceived as “unpredictable, uncontrollable, and overloading” [17]. the higher a person's score on the test, the higher his/her stress level. cohen et al. (1983) reported test-retest reliability of the scale is 0.85 and 0.84 to 0.86 is its internal consistency. evaluating the psychometric properties of the perceived stress scale in an iranian sample showed good reliability and validity for pss [18]. in the present study, the reliability of this scale by means of cronbach alpha test was 0.91 to measure cognitive flexibility in dentists, the cognitive flexibility inventory (dennis & vander wal, 2010) was used (20 questions). this is a self-report inventory with 7-point likert (from strongly disagree to strongly agree) inventory with two subscales called the alternatives and control subscale. this measurement evaluates the cognitive flexibility that presents the ability to challenge irrational thoughts and replace them with efficient and balanced ones. it measures three aspects of cognitive flexibility: 1) the tendency to perceive difficult situations as controllable, 2) the ability to perceive multiple alternative explanations for life occurrences and human behavior, and 3) the ability to generate multiple alternative solutions in difficult situations. the cfi showed high internal consistency (for the total score α = 0.91, and for alternatives and control subscale respectively 0.91 and 0.86 was reported). it showed adequate test-retest reliability over 7-weeks (cfi total score: r=0.81; alternatives subscale: r=0.75; control subscale: r=0.77), and good convergent validity with other self-report measures of cognitive flexibility and negative correlations with measures of depression (14). the internal consistency of cfi in the present study was 0.90. covid-19 factors related to dental environment: fear of getting infected covid-19 from patients/dental environment and virus contagion was measured by two questions: “are you afraid to be infected with covid-19 because of your profession?” and “i am worried that because of aerosol/droplets production in the dental environment, it causes contagion and spread of the disease”. responses were coded the correla*on of perceived stress and professional concerns during covid-19 pandemic among iranian den*sts: the media*ng role of cogni*ve flexibility vol 8 no 1 (2020) doi 10.5195/d3000.2020.119 h#p://den*stry3000.pi#.edu from 0 for “not at all” to 4 for “very afraid”. receiving enough professional knowledge regarding covid-19 and ability to adhere to valid guidelines were measured by two following questions: “to maintain a safe working environment since the covid-19 outbreak, do you acquire sufficient knowledge (e.g. lectures, seminars, information leaflet, etc.)?” the second question was “i do not have enough facility to control infection by regarding guidelines and valid instructions”. responses were coded from 0 for “not at all” to 4 for “very much”. the last part was concerns of dentists about career future, which was measured by two statements; “considering the covid-19 outbreak and uncertain future of this disease, i don’t have a clear job prospect”, and “due to the covid-19 outbreak, even for emergency treatment, i am not reluctant to reopen the office and restart my clinical work”. these responses were coded from 0 for “not at all” to 4 for “very much”. demographic questions some self-report questions such as age, gender, marital status, years of work experience, work place( private office/clinic/both ) , professional status (general dentist, specialist, resident (specialty resident)). another question was about being in a risk group: “are you defined as being in a high-risk population (suffering from asthma/chronic kidney disease and who are undergoing dialysis/liver disease/serious heart conditions/immunocompromised conditions, such as cancer treatment/diabetes)?” the responses were coded as 0 for “no” and 1 for “yes”. statistical analysis this study used spss-20 and amos-20 to analyze data. the spss software was used to analyze participants’ demographic characteristics, correlation between variables, regression analysis and reliability of instruments. amos software was used to examine the feasibility of the proposed model, by considering goodness of fit indices such as adjusted goodness of fit index (agfi), the goodness of fit index (gfi), the comparative-fit index (cfi), the root mean square error of approximation (rmsea), and the normed fit index (nfi). the fit of the model was examined using χ2/degrees of freedom (d.f.) analyses which should be less than 3.00, other model fit analysis scores, such as agfi, gfi, cfi, and nfi, should be greater than 0.80. the acceptable model fit should be greater than 0.90, and rmsea should be less than 1.00. results participant characteristics the age range of the sample was 24-65 years (n=174) and the average was 38.11 (sd=9.67). 71.3% of them were female and 28.7% were male. 35.6% of the total sample were single/unmarried and 64.4% were married. 47.7% of the total sample were general dentists, 40.2% were professional dentists, and 12.1% were resident (specialty student). majority of participants (n=83, 47.7%) had more than 10 years of work experience, 17.8% less than 2 years, 14.9% of respondents 2-5 years, 19.5% had 6-10 years of work experience. more than half of the respondents did not have any child (52.3%), 25.9% had one child, 20.1% had 2 children and 1.7% had more than 2 children. 61% of whom had children were women and 39% men. additionally, the majority of the dentists were healthy (n=157, 90.2%) and 9.8% were in the highrisk group with a kind of chronic or immunocompromised condition. descriptive statistics the correla*on of perceived stress and professional concerns during covid-19 pandemic among iranian den*sts: the media*ng role of cogni*ve flexibility vol 8 no 1 (2020) doi 10.5195/d3000.2020.119 h#p://den*stry3000.pi#.edu the mean score for the variables of perceived stress was 28.08, for cognitive flexibility was 105.27, for paucity of information and facilities 3.77, for fear of covid-19 contagion in work environment 5.83, and finally for concerns of their career future 4.38. the standardized skewness and kurtosis did not exceed the critical value (±1.96) at the significance level of 0.05 [19]. the multivariate critical ratio of the whole sample was 0.688, which is less than 2.58. therefore, the conditions of normal distribution were satisfied. correlation among variables in this research, there was a significant negative correlation between perceived stress and psychological flexibility (alternatives subscale, r=-0.34, and control subscale, r=-0.59, p≤0.0001) and significant positive correlation between perceived stress and covid-19 factors related to the dental environment (fear of covid-19 contagion r= 0.21, career future r=0.24, p<0.01). additionally, there was a significant negative correlation between control subscale and fear of covid-19 contagion r= -0.16, p<0.05; and career future r=-0.28, p≤0.0001). in general, perceived stress, fear of virus contagion in dental work environment, and control subscale of cognitive flexibility explain 31.2% of the variance of professional concerns of dentists during the recent crisis. the results of comparing female and male in the variables of cognitive flexibility, concerns about virus contagion in the dental environment and getting infected covid-19 from patients, concerns about career future, facilities and dental information from reputable guidelines in preventing disease transmission showed that there was no significant difference between the two genders in any of these variables. the results of comparing the mentioned variables in dentists with less than 2 years, 2-5 years, 6-10 and more than 10 years of work experience showed that in general there is no significant difference between the groups with different work experience in terms of the mentioned variables. however, the intergroup comparison of perceived stress showed that perceived stress of dentists with less than two years of work experience (mean = 30.194 and standard deviation = 1,574) was significantly higher than that of dentists with more than 10 years of work experience (mean = 25.853 and standard deviation = 1.503) p = 048. there was no significant difference between perceived stresses in other groups. moreover, no significant difference was observed between any of the groups of assistants, general dentists and specialists in the research variables. in addition, in the variable concerns about virus contagion in the dental environment and getting infected covid-19 from patients, difference between two groups of dentists with more than 10 years of work experience (mean= 5,530 and standard deviation = 0.198) and dentists with less than two years of work experience (mean = 6.378 and standard deviation = 0.324) was significantly high p=.025. furthermore, the concern in group with less than two years of work experience was significantly higher than that of the group with more than 10 years of work experience. there was no significant difference between the other groups. also, there was no significant difference between the groups in terms of concerns about career future. additionally, no significant difference was observed between the dentists working in a private office with those working in a clinic in terms of research variables. the correla*on of perceived stress and professional concerns during covid-19 pandemic among iranian den*sts: the media*ng role of cogni*ve flexibility vol 8 no 1 (2020) doi 10.5195/d3000.2020.119 h#p://den*stry3000.pi#.edu there was no significant difference between dentists with children and dentists without children in the mentioned variables, except for the variable concerns about virus contagion in the dental environment and getting infected covid-19 from patients. this variable was significantly higher in dentists with more children (p=.018). no significant relationship was observed between the age of the dentists and the mentioned variables. mediation test of cognitive flexibility as the alternative component of cognitive flexibility did not have any correlation with covid-19 related variables in dentists, it was removed from the equation. the model included perceived stress as exogenous variable and control subscale of cognitive flexibility, fear of covid-19 contagion in the work environment, and career future concerns due to corona virus outbreak were considered as endogenous variables. a mediation effect is created when a third variable/construct intervenes between two other related constructs [19]. tests carried out by path analysis using regression methods (spss) and structural model (amos). the results can describe the final path substructure model, as shown in figure 1. by considering the control component of cognitive flexibility of dentists as the mediator variable between perceived stress and career future concerns, the results revealed that although the comparison between the full mediation model and indirect models’ goodness-of-fit indices showed that both models fit the data, the indirect model showed a significantly better fit than the full mediation model. in addition, since the indirect model showed smaller aic (akaike information correction: 18.185) than the full mediation model (akaike information correction: 20.000), and the pnfi (parsimony normed fit index) of the full mediation model was (0.0) lesser than that of the indirect model (0.166), control component was mediated between perceived stress and career future concerns in dentists. indirect model goodness of fit indices were (chi-square: 0.185, p> 0.05, nfi: .99, gfi: 0.99, agfi: 0.99, cfi: 1.00, and rmsea: 0.00). these results revealed that control subscale of cognitive flexibility is a full mediator between perceived stress and career future concerns. there were direct effects between perceived stress and fear of covid-19 contagion in the work environment, as well as perceived stress and career future (table 1). figure 1. path diagram the correla*on of perceived stress and professional concerns during covid-19 pandemic among iranian den*sts: the media*ng role of cogni*ve flexibility vol 8 no 1 (2020) doi 10.5195/d3000.2020.119 h#p://den*stry3000.pi#.edu cr: critical ratio; se: standard error moreover, the control component was mediated the relationship between perceived stress and fear of covid-19 transmission in the dental work environment. indirect model goodness of fit indices were (chi-square: 3.65, p> 0.05, nfi: .97, gfi: 0.99, agfi: 0.90, cfi: .98). discussion the first result of this study indicated that iranian dentists are under high levels of stress during the covid-19 pandemic. they reported concerns about being infected by a new virus while presenting professional services to patients. this result was similar to those previous studies conducted among dentists, nurses and medical staff population [3, 5, 6, 12]. a significant amount of stress and mental health problems among the specialists was shown in those studies. due to the unknown nature of the disease, lack of vaccine, and high probability of virus transmission in dental workplaces [2], dentists are prone to experience high levels of stress and be reluctant to provide dental services to patients. as high levels of stress can reduce the proper function of the individuals, determining the factors that lead to stress reduction is of significant importance. in this study, we investigated the effect of perceived stress of dental staff predictive variable dependent variable beta sig cr se direct effect (p value) indirect effect (p value) full mediation (p value) perceived stress control -.59 <.00001 -9.51 .06 0 -.59 (<.0001) -.59 (<.0001) perceived stress career future .24 .001 .43 .02 .14 (.03) 0 .034 (.67) control career future -.28 <.00001 -2.33 .02 0 -.20 (.002) .18 (.020) perceived stress contagion fear .21 .005 2.83 .019 .18 (.02) .21 (.005) .21 (005) control contagion fear -.16 .03 -.65 .020 0 -.16 (.03) -.06 (.52) contagion fear career future .52 <.00001 7.50 .075 .49 (<.00001) .49 (<.00001) .49 (<.00001) table 1. direct, indirect and total effects for the model (n=174) the correla*on of perceived stress and professional concerns during covid-19 pandemic among iranian den*sts: the media*ng role of cogni*ve flexibility vol 8 no 1 (2020) doi 10.5195/d3000.2020.119 h#p://den*stry3000.pi#.edu during covid-19 pandemic on their professional stress and concerns. this effect was mediated by control component of cognitive flexibility. in fact, when a dentist perceives her/his as being enough compatible to have control over changeable and challenging circumstances, he/she would experience less distress and concerns toward the profession. although the control component of cognitive flexibility could predict the professional stress in dentists, it was not a very strong predictive factor. however, fear of covid-19 contagion in the work environment was a better predictive variable for it. according to previous studies which mentioned the effective and significant role of environmental workplace of dentists [9, 11, 12] on their mental health, the authors declare that during the covid-19 pandemic, mental health of iranian dentists should be given serious attention. although by means of dentists’ reports, there was no significant correlation between lack of information and facilities with their career future concerns about the upcoming circumstances, the other related variables such as contextual factors should be investigated. perceived stress among dentists with less than two years of work experience was greater than that of dentists with a longer work experience, and the concerns about virus contagion in the dental environment and getting infected covid-19 from patients was higher among dentists with less than two years of work experience. this finding is in agreement with farrukh et al. 20. according to these findings, young people who have less clinical work experience do not seem to have the confidence to provide the condition to control disease transmission. on the other hand, more work experience may have led to false confidence in more experienced dentists. this needs further investigation. more concerns about virus contagion in the dental environment and getting infected covid-19 from have been reported in dentists with more children. it makes sense because infections would mean either risking hurting one’s children through unintentional infection or being isolated from one’s children for two weeks (which can feel longer based on how young ones children are). given the fact that most of the participants in the present study who had children were women (61%), and in iranian society women are more responsible for taking care of their children, job concerns in these people may be doubled that affect the perception of their career future. the author tried their best to follow up with these survey respondents in two years or so to see if the pandemic has had a lasting effect on how they view/treat infection, or if their professional anxieties were proven or not. conclusion given these results, elevated stress during an infectious disease outbreak can affect (have an impact on) perceived stress and career future concerns among dentists. different aspects of dentists’ lives can be affected by those concerns and stress. interventions to enhance cognitive flexibility should be applied to reduce the psychological burden of the crisis for dentists. conflicts of interest the authors declare no competing interest. references the correla*on of perceived stress and professional concerns during covid-19 pandemic among iranian den*sts: the media*ng role of cogni*ve flexibility vol 8 no 1 (2020) doi 10.5195/d3000.2020.119 h#p://den*stry3000.pi#.edu 1. 2019-ncov transmission through the ocular surface must not be ignored. lu c-w, liu x-f, jia z-f. lancet (london, england). 2020;395(10224):e39. 2. transmission routes of 2019-ncov and controls in dental pracwce. peng x, xu x, li y, cheng l, zhou x, ren b. internawonal journal of oral science. 2020;12(1):1-6. 3. covid-19 pandemic and its impact on mental health of healthcare professionals. tsamakis k, rizos e, manolis aj, chaidou s, kympouropoulos s, spartalis e, et al. [comment] experimental and therapeuwc medicine. 2020;19(6):3451-3. 4. factors associated with resilience among medical staff in radiology departments during the outbreak of 2019 novel coronavirus disease (covid-19): a cross-secwonal study. huang l, wang y, liu j, ye pf, cheng bc, xu hy, et al. medical science monitor. 2020;26. 5. covid-19 pandemic: stress experience of healthcare workers a short current review. bohlken j, schomig f, lemke mr, pumberger m, riedel-heller sg. psychiatrische praxis. 2020;47(4):190-7. 6. psychological status of surgical staff during the covid-19 outbreak. xu j, xu qh, wang cm, wang j. psychiatry research. 2020;288. 7. impact of covid-19 on the mental health of surgeons and coping strategies. balasubramanian a, paleri v, bennez r, paleri v. head and neck-journal for the sciences and specialwes of the head and neck. 8. incubawon period of 2019 novel coronavirus (2019-ncov) infecwons among travellers from wuhan, china, 20–28 january 2020. backer ja, klinkenberg d, wallinga j. eurosurveillance. 2020;25(5):2000062. 9. assessment of stress among doctors of dental medicine. jahić im, bukejlović j, alić-drina s, nakaš e. acta stomatologica croawca. 2019;53(4):354. 10. self-reported psychological problems amongst undergraduate dental students: a pilot study in seven european countries. stangvaltaite-mouhat l, pūrienė a, chałas r, hysi d, katrova l, nacaite m, et al. european journal of dental educawon. 2020;24(2):341-50. 11. key determinants of health and wellbeing of denwsts within the uk: a rapid review of over two decades of research. salazar fbc, sipiyaruk k, white s, gallagher je. briwsh dental journal. 2019;227(2):127-36. 12. covid-19 factors and psychological factors associated with elevated psychological distress among denwsts and dental hygienists in israel. shacham m, hamama-raz y, kolerman r, mijiritsky o, benezra m, mijiritsky e. internawonal journal of environmental research and public health. 2020;17(8):2900. 13. stress and emowon: a new synthesis. lazarus rs. springer publishing company; 2006. 14. the cogniwve flexibility inventory: instrument development and eswmates of reliability and validity. dennis jp, vander wal js. cogniwve therapy and research. 2010;34(3):241-53. 15. acute stressor effects on cogniwve flexibility: mediawng role of stressor appraisals and corwsol. gabrys rl, howell jw, cebulski sf, anismana h, matheson k. stress. 2019;22(2):182-9. the correla*on of perceived stress and professional concerns during covid-19 pandemic among iranian den*sts: the media*ng role of cogni*ve flexibility vol 8 no 1 (2020) doi 10.5195/d3000.2020.119 h#p://den*stry3000.pi#.edu 16. the role of stress mindset in shaping cogniwve, emowonal, and physiological responses to challenging and threatening stress. crum aj, akinola m, marwn a, fath s. anxiety, stress, & coping. 2017;30(4):379-95. 17. a global measure of perceived stress. cohen s, kamarck t, mermelstein r. journal of health and social behavior. 1983:385-96. 18. assessing stress in cancer pawents: factorial validity of the perceived stress scale in iran. journal of nursing educawon. 2014;2(1):13-22. 19. mulwvariate data analysis . uppersaddle river. hair jf, black wc, babin bj, anderson re, tatham r. nj: pearson prenwce hall; 2006. vol 1, no 1 (2013) issn 2167-8677 (online) doi 10.5195/d3000.2013.17 http://dentistry3000.pitt.edu this work is licensed under a creative commons attribution 3.0 united states license. this journal is published by the university library system, university of pittsburgh as part of its d-scribe digital publishing program and is cosponsored by the university of pittsburgh press. introducing dentistry 3000 alexandre r. vieira editor in chief, dentistry 3000 department of oral biology, university of pittsburgh, school of dental medicine, pittsburgh, pa, usa citation: vieira ar. (2013) introducing dentistry 3000. dentistry 3000 1:a006 doi: 10.5195/d3000.2013.17 published: december 12, 2013 copyright: ©2013 vieira. this is an open-access article licensed under a creative commons attribution work 3.0 united states license. email: arv11@pitt.edu a few decades ago, scientific journals were housed at university libraries, which handled the journal’s peer-review process and production. when universities faced economic hardships and library budgets were cut, one solution was to allow publishers take over journal responsibilities. most of the revenue came from readership subscriptions. more recently, subscriptions have been under attrition and the model of charging author fees for publication has emerged. parallel to these events is the surge of the world wide web and the possibility to publish scientific papers as online only. furthermore, publishers raised fees for online-access to scientific literature. publishers charge around us$35 for access to each individual paper they publish. online-only scientific journals became an attractive business, with some companies holding more than 200 journals each and charging author fees from us$300 to more than us$2,000. the quality of these journals has been questioned and evidence suggests that the quality of the published papers is lower than other more reputable journals. for dental research in particular, opportunities to publish high quality research in highly visible journals can be limited, despite the fact that more than 2,000 dental journals exist. to provide a venue for the dental community that is peer-reviewed, open access with no cost to readers, and free of charge to authors, we created dentistry 3000. the first issue includes five papers, one on the association of cleft lip and palate with interferon regulatory factor 6, one on the association of cancer with cleft lip and palate, one on the relationship of tooth and facial dimensions, one on the heritability of facial structures, and one summarizing the proceedings of the cariology symposium during the international association for dental research 2012 meeting. dentistry 3000 is published by the university library systems, university of pittsburgh, as part of its d-scribe digital publishing program, and is cosponsored by the university of pittsburgh press. http://dentistry3000.pitt.edu/ http://creativecommons.org/licenses/by-nc-nd/3.0/us/ http://www.library.pitt.edu/ http://www.pitt.edu/ http://www.library.pitt.edu/articles/digpubtype/index.html http://upress.pitt.edu/ http://creativecommons.org/licenses/by-nc-nd/3.0/us/ http://www.pitt.edu/ http://www.library.pitt.edu/dscribe/ http://www.library.pitt.edu/dscribe/ http://www.upress.pitt.edu/upressindex.aspx microsoft word 120 2021.docx incorpora(on of fluconazole and ocimum sanctum oil in so6 denture liners to treat biofilms of candida albicans associated with denture stoma((s vol 9 no 1 (2021) doi 10.5195/d3000.2021.120 h#p://den*stry3000.pi#.edu incorporation of fluconazole and ocimum sanctum oil in soft denture liners to treat biofilms of candida albicans associated with denture stomatitis aamir zahid godil 1*, diksha bhagat 2, parijat das 3, arshi ilyas kazi 1, ramandeep dugal 1, surekha satpute 2* 1department of prosthodon0cs, m. a. rangoonwala college of dental sciences and research centre, maharashtra, india 2department of microbiology, savitribai phule pune university, india 3department of biosciences and bioengineering, savitribai phule pune university, india abstract purpose: to inves(gate the in vitro ac(vity of incorporated an(fungal agents like fluconazole and ocimum sanctum oil (tulsi) in the denture so6 liners to reduce the risks associated with the biofilms of candida albicans. materials and methods: in the current study, the minimum inhibitory concentra(on (mic) of two an(fungal agents namely fluconazole and o. sanctum (tulsi oil) against c. albicans (atcc 10231) was evaluated to examine their effect in reducing the count of candida and its biofilms. analysis of physical proper(es such as surface roughness and hardness of so6 denture liners (test and control) were also performed. the effect of both an(fungal agents was also observed on cell morphology of candida cells using scanning electron microscopy (sem). results: studies confirmed mic value for fluconazole and o. sanctum oil as 600 and 400 µg/ml respec(vely. surface hardness and roughness of so6 denture material (test) remained unaltered. finally, sem studies also proved the effec(veness of incorporated an(fungal agents on the cell morphology of c. albicans at their respec(ve mic values. conclusions: this approach allows the prolonged drug release in the oral cavity which simultaneously treats the injured denture bearing (ssues and also the infec(on, biofilms of candida without compromising on their physical proper(es. these studies are significant and have tremendous medicinal and therapeu(c relevance. keywords: an(fungal agents; biofilms; candida; scanning electron microscope; so6 liners; hardness; surface roughness; minimal inhibitory concentra(on; denture stoma((s. cita8on: godil a, et al. (2021) incorpora8on of fluconazole and ocimum sanctum oil in soh denture liners to treat biofilms of candida albicans associated with denture stoma88s. den8stry 3000. 1:a001 doi:10.5195/d3000.2021.120 received: october 29, 2020 accepted: december 16, 2020 published: february 5, 2021 copyright: ©2021 godil a, et al. this is an open access ar8cle licensed under a crea8ve commons axribu8on work 4.0 united states license. email: ramandeepdugal@gmail.com introduction denture soft liners are widely used for restoring the health of abused or inflamed tissues in the oral cavity which is brought about by the hard denture materials by providing cushioning and evenly distributing the load on the oral tissues during mouth movements. however, these liners surface harbor and support the adhesion, colonization of several microbes, especially pathogens like candida albicans that result in lesion formation in denture wearers [1]. this microbial colonization on denture surface represented as a biofilm of microbial communities which are consistently encased within an extracellular polysaccharide matrix produced by the organisms themselves. candida strains are proficient in forming biofilms and this has been linked to both increased expression of virulence factors and also lesser susceptibility to antimicrobial agents [2,3]. biofilms permits the persistence as well as resistance to several antifungals agents and further affect the strategies in preventing, treating incorpora(on of fluconazole and ocimum sanctum oil in so6 denture liners to treat biofilms of candida albicans associated with denture stoma((s vol 9 no 1 (2021) doi 10.5195/d3000.2021.120 h#p://den*stry3000.pi#.edu infections instigated by candida sp [4]. biofilm related infections are efficiently tackled by incorporating powerful antimicrobial agents in denture soft liners. several investigators have advocated the usage of antibiotics, few medicinal oils in treating candida infections [5]. an ideal antimicrobial agent should have good stability, high efficiency, broad spectrum, long-lasting antimicrobial activity and biocompatibility, rendering them useful for biomedical applications [5,6]. numerous antifungal drugs, antiseptic solutions, chitosan salts, metallic oxides, surface coatings, etc. have been proposed in literature [7-10]. fluconazole, one of the most popular antifungal agents has been witnessed from the literature. fluconazole basically contains a triazole ring instead of an imidazole ring and it has a lower molecular weight (306.271 g/mol) making it more water soluble as compared to other azole compounds. it has a high bioavailability (92 %) when administered intra-orally [11,12]. the antifungal activities of medicinal herb oils like origanum oil, and numerous other essential oils have been documented [1317]. sparse literature suggest the use of tulsi – o. sanctum oil incorporated in soft liners. gupta et al demonstrated the use of o. sanctum in combination with chlorhexidine (skin – disinfectant, antiseptic) for mouthwash preparation against dental plaque and gingival inflammation [17]. shokeen et al have proposed the presence of eugenol component from o. sanctum for its medicinal potential against multi drug resistant (mdr) and susceptible strains [18,19]. even though the potential of various plant extracts or essential oils is known, it is important to highlight that the literature on the usage of such extracts in prosthodontics is inadequate. we wish to state a noteworthy observation here that in comparison with other plant extracts, o. sanctum oil has not been exploited thoroughly for prosthodontics purposes. incorporation of antifungal medicaments or additives, in any form is likely to modify or compromise the physical and mechanical properties of soft liners. it is therefore important that only the minimum inhibitory concentration (mic) of the medicament can be incorporated to ensure minimum changes in the properties of these materials. many researchers have studied the effect of incorporated antifungal medicaments on the properties of soft liners such as dimensional stability, surface roughness, hardness, tensile strength, water sorption, modulus of elasticity, and weight in soft liner materials [5,13,19-23]. among the several properties evaluated, surface roughness is the most critical one, since; rougher surfaces have a greater affinity to biofilm formation, and thus favor the emergence/maintenance of oral pathologies. release of alcohol and plasticizers from these soft lining materials to the liquid medium also result in increased roughness. the fine pores created by the rough surfaces act as an attachment point as well as breeding ground for the microbes, initiating the process of biofilm formation [24]. hardness is another such property which is regarded as a fundamental one since it represents a simple method to determine its modulus of elasticity. the greater the content of plasticizers in the material, the softer it becomes. in this context, a major disadvantage of soft lining materials is the rapid loss of plasticizer that leads the gradual hardening and even oral discomfort to the user [24]. the purpose of our present study was to evaluate in vitro antifungal activity of fluconazole and o. sanctum seed oil incorporated in a soft liner. we also investigated the effect of these inclusions on the hardness and surface roughness of the soft liner. lastly, scanning electron microscopy (sem) analysis was also carried out to demonstrate the effect of antifungal agents on the morphology of c. albicans. the null hypothesis of this study is that there is no difference in the outcomes of physical properties of denture soft liners due to the incorporation of the two test antifungal agents. material and methods incorpora(on of fluconazole and ocimum sanctum oil in so6 denture liners to treat biofilms of candida albicans associated with denture stoma((s vol 9 no 1 (2021) doi 10.5195/d3000.2021.120 h#p://den*stry3000.pi#.edu culture and growth conditions culture of candida albicans (atcc 10231) was obtained from swami ramanand teerth marathwada university srtmu, nanded, maharashtra, india fungal culture was grown in sterile yeast malt broth (ymb, himedia, india) overnight at 30˚c under shaking conditions. figure 1a represents well isolated, off-white coloured, circular, regular edged colonies of candida sp. grown on yeast malt agar plates (yma, himedia, india). morphological features of c. albicans were evaluated for freshly grown 24 hours old culture on a trinocular microscope (axiocam erc5s: zeiss, germany) under 10x magnification after staining with lactophenol cotton blue stain (himedia, india) (figure 1b). the fungal morphology was analysed on a computer screen using zen lite 2011 software (zeiss microscopy, germany) to observe oval, budding yeast like cells and confirm its active growth. determination of minimum inhibitory concentration (mic) of antifungal agents by agar well diffusion assay agar well diffusion technique was performed to evaluate the mic of the antifungal agents: fluconazole (150 mg tablet: fluka, ciplauttarakhand, india), and ocimum sanctum (p. h. gandhi chemicals, pune, maharashtra, india) against the fungal strain c. albicans (atcc 10231). the turbidity of overnight freshly grown fungal culture was adjusted to a cell count of 1×105 cells/ml and further 100 µl of the culture was uniformly spread over yma plates. wells were dug into the agar using sterile cork-borer, filled with serially increasing concentrations (100 to 1000 µg/ml) of the fluconazole, and o. sanctum oil and refrigerated for 15 minutes for pre-diffusion. after the pre-diffusion step, the plates were incubated overnight at 30˚c. fluconazole test solutions were prepared by crushing the tablet using a mortar and pestle using dimethyl sulfoxide (dmso). the lowest concentration showing a zone of inhibition (zi) > 15 mm diameter was taken as the mic for both antifungal agents. the mics of the antifungal agents were also investigated using standard microtitre plate assay. flat bottomed 96-well microtitre plates (tarsons tm, india) were filled with 100 μl of serially diluted solutions of the antifungal agents (ranging from 2000 to 200 μg/μl). to each well, 100 μl of freshly grown candida cells (1×105 cells/ml) were added. the plates were incubated at 30°c overnight and od600 was measured using a double-beam uv-vis spectrophotometer (shimadzu, japan). as the incorporation of the antifungal agents did not alter the od600 of the solution significantly, 200 μl of sterile ymb broth served as the negative control. evaluation of fungal culture viability in presence of antifungal agents fungal cultures were grown overnight in a sterile ymb medium in presence of mic values of fluconazole and o. sanctum oil. further microscopic observations were carried out up to 14 days to check the viability of fungal cells with time. the initial concentration of cells was adjusted to 1×105 cells/ml on a neubauer chamber: 1/400 mm2, depth 0.1 mm (axiva counting chamber brightline touff, delhi, india). figure 1. a) colony characteristics of c. albicans grown on yeast malt agar plates, b) microscopic image (10x) of c. albicans stained with lactophenol cotton blue incorpora(on of fluconazole and ocimum sanctum oil in so6 denture liners to treat biofilms of candida albicans associated with denture stoma((s vol 9 no 1 (2021) doi 10.5195/d3000.2021.120 h#p://den*stry3000.pi#.edu preparation of denture disk specimens soft liner denture disks of mollosil, with silicone based permanent chair side soft liner (detax gmbh & co. kg, carl-zeiss-str.4, germany) were prepared as per manufacturer’s instructions in a laminar flow (microfilt india pvt. ltd.). the base paste and catalyst paste were mixed in 1:1 w/w ratio on a sterile ceramic (figure 2). denture disc specimens of diameter = 12 mm, and thickness = 2 mm were prepared for antifungal assay. a stainless steel mould was used to prepare denture discs specimens of diameter = 20 mm, and thickness = 5 mm were also prepared for the assessment of physical properties. figure 2. prepara(on of denture so6 liner disk specimens using stainless steel mold. the antifungals to be incorporated were also pre-weighed and added according to their mic values to the pastes. the material was homogenously mixed using a stainless-steel using spatula for 30 seconds and then gently placed over the mould which was further secured between two thick glass tiles, under constant pressure to ensure the uniformity without any voids. the specimens were allowed to set for 8 10 minutes at room temperature until the mix was plastic and easy to retrieve. any specimens with voids or irregular surfaces were discarded. the excess portion was trimmed off carefully using a fine blade scissor. the specimens were then coated with lustrol gloss varnish and were allowed to dry for 5 minutes and stored further in a sterile petri dish. the procedure was repeated for both test compounds using all concentrations. for the control group, no antifungal agent was incorporated. these specimens were stored in a sterile container having 10 ml of artificial saliva. determination of physical properties (surface roughness and hardness) of the soft liner surface roughness was measured using a surface roughness tester (mitutoyo south asia pvt. ltd) calibrated with a 0.8 mm cut-off length and speed of 0.5 mm/s. the hardness was measured on one side of specimen using a shore a durometer (kori seiki, japan). a constant load of 10 n/s was applied, and the results were obtained from 0 to 100 shore a units. a mean of five readings was taken, from different sites of the sample for both the properties. both physical properties were measured at a regular interval of 24 hours (up to two weeks) [25]. demonstrating the effect of antifungal agents on c. albicans using scanning electron microscopy c. albicans was grown in ymb for an overnight to achieve a mid-log phase to reach a cell count of 1 × 105 cells. after growth; the cells of c. albicans were centrifuged (10,000 rpm for 15 minutes at 4°c) and the supernatant was discarded. further, cell pellet was washed with saline (0.85 % nacl) for one to two minutes. the pellet was made up to 1 ml with the saline and was labelled as a stock. around 1 µl from the stock was then diluted to 103 times and the cells were counted using neubauer chamber underneath a 1 mm thick coverslip under 40 x magnification. from this, the amount of stock needed to get 1×105 cells was calculated. the cells grown in mid log phase (~ 1×105 cells) were treated with their respective mics of both antifungal agents. the tubes were further incubated at 30°c for four hours at 150 rpm. the cells were collected by centrifugation at 10,000 rpm for 15 minutes at 4°c and the pellet was then washed thrice with 0.1 m phosphate buffer saline (pbs) at ph 7.4. the cell pellet was then fixed with 2.5 % glutaraldehyde at 4°c for four hours. it was then rinsed twice with pbs. after the rinsing step, the pellets were dehydrated in ethanol series ranging from 10 – 100 %, for 15 minutes per step. at the end of this stage, water content from the cell was completely replaced with ethanol and this was used further for sem studies. around 2 µl of this was spread uniformly on a incorpora(on of fluconazole and ocimum sanctum oil in so6 denture liners to treat biofilms of candida albicans associated with denture stoma((s vol 9 no 1 (2021) doi 10.5195/d3000.2021.120 h#p://den*stry3000.pi#.edu clean, grease free glass cube (1 × 1 mm) and allowed to air dry completely in a laminar air flow to observe it using sem facility. statistical analysis of data all experiments were performed in triplicates. the numerical readings for all three parameters were subjected to statistical analysis. the data obtained on continuous variables were presented as mean and standard deviation (± sd). the intergroup statistical comparison of continuous variables was done using analysis of variance with bonferroni’s correction for multiple group comparisons. the intra-group statistical comparisons were done using repeated measures analysis of variance (rmanova). the p -values < 0.05 were considered as statistically significant. the entire data were statistically analyzed using statistical package for social sciences (spss version 21.0, ibm corporation, usa) for ms windows. results in the present study, a biofilm forming strain c. albicans (atcc 10231) was selected for antifungal assays to mimic the characteristics of oral microflora. c. albicans showed atypical colony morphology on yma plate. candida cells stained with lactophenol cotton blue in absence and presence of antifungal agents are shown in figure 3a, 3b and 3c. antifungal activity performed through agar well plate assay manifested the potency of fluconazole and o. sanctum oil at mic of 600 and 400 µg/ml respectively. figure 4 represents the increased diameter of zi around the well with increased concentrations of fluconazole against c. albicans culture, proving its concentration dependent antifungal activity. a 96 well plate micro-titre assay also supported the same mic values as reflected through well plate assay for both antifungal agents. cell viability assay confirmed that the negative control, where the cell count was much higher (26 × 105/ml) as compared to the candida cells treated with both antifungal agents individually (fluconazole : 6 × 105/ml and o. sanctum oil : 4 × 105/ml). such a drastic decrease in the cell count indicates a powerful antifungal effect of both agents and proving their significance in control of candida culture. viable cell counts in the presence of antifungal agents showed significant reduction (p = 0.001) in cell count due to antifungal activity of test samples (having fluconazole and o. sanctum oil) as compared to the figure 3. a) microscopic image of c. albicans in absence (control) of antifungal agents, b) microscopic image of c. albicans in presence (test) of fluconazole (mic= 600 µg/ml), c) microscopic image of c. albicans in presence (test) of o. sanctum oil (mic = 400 µg/ml). figure 4. antifungal activity of fluconazole against c. albicans grown on yma agar plate in well diffusion assay performed with its different concentrations (100 to 1000 µg/ml). fluconazole exhibiting the zone of inhibition against c. albicans as compared to negative control (control well: centre one filled with dimethyl sulfoxide) incorpora(on of fluconazole and ocimum sanctum oil in so6 denture liners to treat biofilms of candida albicans associated with denture stoma((s vol 9 no 1 (2021) doi 10.5195/d3000.2021.120 h#p://den*stry3000.pi#.edu control (no antifungal agent) over 24 hours, 7 days and 14 days (figure 5a). these mic values were also used to prepare denture disc soft liner specimens for further studies. cell viability of the antifungal agents treated culture certainly affected the growth of c. albicans as compared to control. denture soft liner disc specimens incorporated with antifungal agents should not significantly alter their physical property which was clearly reflected in the present studies. the surface roughness (figure 5b) and hardness (figure 5c) of the denture disc specimens incorporated with antifungal agents at their respective mic over 24 hours, 7 days and 14 days shows no significant alterations. (p = 0.99) sem images of c. albicans illustrated significant changes in the morphology of candida cells in presence fluconazole and o. sanctum oil under different magnification (refer figure 6). figure 6a to 6c represents the sem images untreated or the control cells (without antifungal treatment) having its natural smooth, oval-shape and polar budding type with blastoconidia. however, treatment of candida cells with antifungal agents (at their respective mic) showed complete damaged cell surface as shown in figure 6d to 6f for fluconazole and figure 6g to 6i for o. sanctum oil. thus, sem studies also supported the outcome of the figure 5. a) graphical representa(on for cell count of c. albicans treated with (test) and without (control) an(fungal agents, b) graphical representa(on for the surface roughness of denture specimens incorporated with (test) and without (control) an(fungal agents, c) graphical representa(on for shore a hardness of denture specimens incorporated with (test) and without (control) an(fungal agents. figure 6. scanning electron microscopic images of cell surface morphology of c. albicans in the presence (test) and absence (control) of antifungal agents at their mic values under different magnification levels. a) (5000x) b) (10000x) c) (30000x): control without any antifungal agents showing normal cell morphology while, d) (5000x), e) (10000x) and f) (30000x): in the presence of fluconazole (600 µg/ml) and g) (5000x), h) (10000x) and i) (30000x): in the presence of o. sanctum oil (400 µg/ml) clearly show ruptured cells. incorpora(on of fluconazole and ocimum sanctum oil in so6 denture liners to treat biofilms of candida albicans associated with denture stoma((s vol 9 no 1 (2021) doi 10.5195/d3000.2021.120 h#p://den*stry3000.pi#.edu experiments from the agar well plate and microtitre assay proving the antifungal activity of both agents. discussion temporary soft denture liners are alike to tissue conditioners; that improves the health of inflamed, hypertrophic, hyperaemic, and traumatized denture-bearing tissues [14]. they provide cushioning effect under dentures by distributing the functional stresses, thereby can especially be beneficial in reducing the pain arising from the severely resorbed, sharp and thin residual crests [25]. during this process, plaque accumulated to the tissue surface, can cause microbial adhesion to the soft liner [14]. c. albicans is an established pathogen in denture stomatitis, especially in geriatric patients with poor oral hygiene, and in patients with physical or mental disabilities. budtz-jorgensen et al and koopmans et al showed that the percentage of yeasts was statistical significantly higher in the denture related stomatitis (drs) group [26,27]. complete denture wearers have shown higher prevalence of candida infections, due to microbial adherence on denture bases and liners; leading to varying degrees of denture stomatitis. the surface properties of denture liners, salivary composition, low ph, and diets rich in carbohydrates accelerate the colonization and adhesion of c. albicans, causing exponential growth that possibly encourage denture stomatitis. the initial adhesion of c. albicans depends on the micro porosities of denture base material and lack of oral hygiene, which encourages the formation of a sessile community and biofilm in the oral cavity [14]. in the present study, biofilm forming c. albicans was selected for fungal assays to mimic the characteristics of oral microflora. evaluation of this strain using a trinocular microscope showed actively budding yeast cells when cultured overnight in ymb. fluconazole and o. sanctum oil are known for their antifungal activity [28]. similar observations were noted in the current study. noteworthy reduction in cell viability of candida in artificial saliva proposes efficacy of antifungal against candida cells without affecting the physical properties of the denture material. this reflection agrees to bates et al and bueno et al [24,28]. the mixing of the base materials with antifungal agents may result to covalent binding of the antifungal agents that fetches their controlled discharge over prolong periods of time in the oral cavity or on site colonized by candida cell on the denture surface. the observation of the antifungal assay shows that there was a gradual reduction in the growth of the organism over the three intervals of 24 hours, 7 and 14 days. since denture stomatitis is a multi-factorial condition, the treatment is complex and host-dependent [29]. antifungal agents are considered as a potent and safe medications for both topical and systemic therapies for mucosal lesions [1,2]. although these are considered to be safe and effective topically, prolonged systemic use may cause renal toxicity or hepatotoxicity [11]. using topical antifungal agents are effective on the fungal pathogens that invade superficial tissues. however, their usage may be associated with a few undesirable effects for example, objectionable taste and necessity of frequent applications [5]. due to their shortcomings, the concept of incorporating antifungal agents in denture liners was advocated. the idea of incorporating antifungal agents into denture liners was introduced by douglas and walker, since, a plenty of research has been conducted to improvise the efficacy of tissue conditioners using a variety of antifungal medicaments [30]. thus, addition of fungicidal compounds directly to the tissue conditioners or denture liners has numerous advantages: extremely low-cost and successful technique. this method can be readily employed for those patients at high risk: patients with xerostomia and those with a history of denture stomatitis [31]. the antifungal effects of fluconazole and o. sanctum oil incorporated in denture soft liners have not been compared by in existing literature. the antimicrobial susceptibility and efficacies of any drug are analysed by determining its mic. the viability of topical antimicrobials is lowered than incorpora(on of fluconazole and ocimum sanctum oil in so6 denture liners to treat biofilms of candida albicans associated with denture stoma((s vol 9 no 1 (2021) doi 10.5195/d3000.2021.120 h#p://den*stry3000.pi#.edu its therapeutic concentration due to the cleaning action of the musculature, the diluent effect of saliva, and the microbial colonization in complex biofilms. therefore, candida cells were exposed to a limited concentration of the antifungal [32,33]. it may be assumed that the release of antimicrobial agents incorporated in denture materials after temporary relining of contaminated acrylic surfaces is uniform and steady in infected sites, maintaining an effective concentration, although it is still lower than the initial [5]. in this study, mic were determined where the diameter of zi measured for different concentrations of fluconazole and o. sanctum oil and also confirmed by using a 96 well plate micro-titre assay. the mic for fluconazole for c. albicans atcc 10231 was 600 µg/ml and that for o. sanctum was 400 µg/ml. these results are similar to that achieved by balakumar et al who assessed the activity of o. sanctum on clinically isolated dermatophytic fungi. they found the mic values of the oil in the range of 125 to 400 µg/ml [34]. surfaces with greater roughness usually exhibit higher yeast count; owing to higher irregularities and microporosities. the microscopic surface peaks and valleys protect the microbes from shear forces, even during denture cleaning, allowing the entrapped microbial cells to attach irreversibly to a surface [35]. in the current study, incorporation of the antifungal agents in the soft liner material during preparation of the soft liner discs did not result in any significant increase in the surface roughness or shore a hardness of the material (p < 0.05) even after 14 days of incubation (p < 0.05) when compared with the control. higher mean values of surface roughness for auto-polymerizing soft lining materials (2.8 to 4.2 µm) and tissue conditioners (1.3 to 7.9 µm) were previously described by kang et al and by bueno et al (5.04 to 6.00 µm) [24,36]. these variations of roughness values may be ascribed to differences among the used experimental conditions and also the chemical condition of test materials. furthermore, in in vivo conditions, the variations may occur due to thermal changes, ph variations and deformation by occlusal load [24]. there are no limitations for clinically acceptable values for the shore a hardness of temporary soft lining materials. however, a variation from 13 to 49 shore a units in 24 hours is regarded as acceptable for clinical use of these materials [37]. the results achieved in the present study are in this range for all test groups. a hardness of 20 – 25 units without any change during the life cycle of a resilient material is considered clinically suitable, as reported by bueno et al, gonzalez et al and yilmaz et al who found higher shore a hardness values (60 –78 units) for three temporary soft lining materials [24,38,39]. the scanning electron microscopy observations presented clearly confirm significant fungicidal action exerted by fluconazole and o. sanctum oil. the surface alterations are most probably due to a change in cell permeability. as for the cells treated with the fungicidal dose, some have a completely smooth surface fully comparable to those seen in the controls, though the surfaces of most cells were partly covered with vesicles. vesicular material possibly originates from the cytoplasmic components of the broken and damaged cells [40]. thus, indicating the efficacy of the two test antifungals used in the present study. although significant conclusions have been drawn from this study, it is necessary to clinically correlate the same under in vivo, conditions to promote the use of these anti-fungal medicaments in day-to-day use for management of denture stomatitis. in addition, the in vitro specimens are usually smoother and have standardized surfaces, which increases the effectiveness of the tests of antifungal agents; in vivo, denture liners lose their plasticizers, becoming hardened and rough. conclusion in conclusion antifungal agents fluconazole and o. sanctum oil tested in the current study showed comparatively greater reduction in cell count of c. albicans at the concentration of 600 and 400 μg/ml respectively. the surface roughness and hardness of soft denture liners remains more or less unaltered after incorporation of antifungal agents. incorpora(on of fluconazole and ocimum sanctum oil in so6 denture liners to treat biofilms of candida albicans associated with denture stoma((s vol 9 no 1 (2021) doi 10.5195/d3000.2021.120 h#p://den*stry3000.pi#.edu the efficacy of effect of antifungal agents demonstrated destruction of c. albicans and was evident through sem studies. work demonstrated through this approach allows the prolonged drug release in the oral cavity which simultaneously treats the injured denture bearing tissues and reduce not only candida infection as well as their colonization. these studies are significant and have promising medicinal and therapeutic perspectives. acknowledgements we express special gratitude to rashtriya uchchattar shiksha abhiyan rusa-cbs-th3.2 for financial support and prof. gajanan zore, swami ramanand teerth marathawad university, nanded for providing the necessary candia strains. conflicts of interest the authors deny any conflicts of interest in regards to the current study. references 1. comparative evaluation of two antifungal agents incorporated in auto polymerising denture base resin, heat polymerising denture base resin and permanent silicone soft liner-an in vitro study. chincholikar s, sridevi j, kalavathy n, singh s, kapoor a, saumya s. j clin diag res 2019;13. doi : 10.7860/jcdr/2019/37889.12520 2. effect of incorporation of antifungal agents on the ultimate tensile strength of temporary soft denture liners. neppelenbroek kh, lima jf, hotta j, galitesi ll, almeida al, urban vm. j prosthodont 2018;27:177-181. doi: 10.1111/jopr.12660 3. biofilm production by candida species and inadequate antifungal therapy as predictors of mortality for patients with candidemia. tumbarello m, posteraro b, trecarichi em, fiori b, rossi m, porta r, de gaetano donati k, la sorda m, spanu t, fadda g, cauda r. j clin microbiol 2007;45:1843-1850. doi: 10.1128/jcm.00131-07 4. predisposing factors, prevention, diagnosis and alternative treatment. martins n, ferreira icfr, barros l, silva s, henriques m. candidiasis: mycopathologia 2014;177:223-240. doi: 10.1007/s11046-014-9749-1 5. role of antifungal medicaments added to tissue conditioners: a systematic review. iqbal z, zafar ms. j prosthodont res 2016;60:231-239. doi: 10.1016/j.jpor.2016.03.006 6. denture plaque and adherence of candida albicans to denture-base materials in vivo and in vitro. radford d, challacombe sj, walter jd. crit rev oral biol med 1999;10:99-116. doi: 10.1177/10454411990100010501 7. a comparative efficacy of nystatin and fluconazole incorporated into tissue conditioner as drug delivery method for denture stomatitis. kumar n, kumari a, priyadarshi v, kumar a, prasad rs, kumar b. j adv med dent scie res 2020;8(1):159-62. doi: 10.4103/ccd.ccd_542_17 8. evaluation of the physical and antifungal effects of chlorhexidine diacetate incorporated into polymethyl methacrylate. maluf cv, peroni lv, menezes lr, coutinho w, lourenço ej, telles dd. j appl oral sci 2020;28. doi: 10.1590/1678-7757-2019-0039 9. structural insights into conformation of amphiphilic quaternary ammonium chitosans to control fungicidal and anti-biofilm functions. jung j, bae y, cho yk, ren x, sun y. carbohydr polym 2020;228:115391. doi: 10.1016/j.carbpol.2019.115391 10. effect of incorporation of silver nanoparticles on the tensile bond strength of a long term soft denture liner. habibzadeh s, omidvaran a, eskandarion s, shamshiri ar. eur j dent 2020;14(2):268. doi: 10.1055/s-0040-1709923 11. clinical pharmacology of fluconazole. dudley mn. pharmacotherapy: j human pharmacology and drug therapy 1990;10:141-145 https://doi.org/10.1002/j.18759114.1990.tb02599.x 12. fluorinated vs. zwitterionicpolymer grafted surfaces for adhesion prevention of the fungal pathogen candida albicans. masotti e, poma n, guazzelli e, fiaschi i, glisenti a, vivaldi f, bonini a, francesco fd, tavanti a, galli g, martinelli e. polymers 2020;12(2):398. doi: 10.3390/polym12020398 13. evaluation of the properties of a tissue conditioner containing origanum oil as an antifungal additive. srivatstava a, ginjupalli k, perampalli nu, bhat n, ballal m, j prosthet dent 2013;110:313-319. incorpora(on of fluconazole and ocimum sanctum oil in so6 denture liners to treat biofilms of candida albicans associated with denture stoma((s vol 9 no 1 (2021) doi 10.5195/d3000.2021.120 h#p://den*stry3000.pi#.edu doi: 10.1016/s0022-3913(13)60381-9 14. comparative evaluation of antifungal activity of melaleuca oil and fluconazole when incorporated in tissue conditioner: an in vitro study. sharma s, hegde v. j prosthodont 2014;23:367-373. doi: 10.1111/jopr.12117 15. effect of incorporating seed oils on the antifungal property, surface roughness, wettability, weight change, and glucose sorption of a soft liner. muttagi s, subramanya jk. j prosthet dent 2017;117:178-185. doi: 10.1016/j.prosdent.2016.05.010 16. nature curesan alternative herbal formulation as a denture cleanser. sushma r, sathe tt, farias a, sanyal pk, kiran s. ann afr med 2017;16:6¬12. doi: 10.4103/aam.aam_43_16 17. randomized controlled clinical trial of ocimum sanctum and chlorhexidine mouthwash on dental plaque and gingival inflammation. gupta d, bhaskar dj, gupta rk, karim b, jain a, singh r, karim wa. j ayurveda integr med 2014;5:109116. doi: 10.4103/0975-9476.131727 18. in vitro activity of eugenol, an active component from ocimum sanctum, against multiresistant and susceptible strains of neisseria gonorrhoeae. shokeen p, bala m, singh m, tandon v. int j antimicrob agents 2008;32:174-179. doi: 10.1016/j.ijantimicag.2008.03.018 19. an in vitro analysis of a sustained release system for the treatment of denture stomatitis. schneid tr. special care in dentistry 1992;12(6):245-50. doi: 10.1111/j.17544505.1992.tb00458.x 20. effect of 3 types of antifungal agents on hardness of 2 different commercially available tissue conditioners: an in vitro study. jadhav v, shetty mm, kalavathy n, kumar r. srm j res in dental sci 2013;1;4(4):150. doi: 10.4103/0976-433x.125590 21. effect of the addition of antimicrobial agents on shore a hardness and roughness of soft lining materials. urban vm, lima tf, bueno mg, giannini m, arioli filho jn, de almeida al, neppelenbroek kh. j prosthodont 2015;24(3):207-214. doi: 10.1111/jopr.12205 22. effect of the association of nystatin with a tissue conditioner on its ultimate tensile strength. urban vm, de souza rf, galvao arrais ca, borsato kt, vaz lg. j prosthodont 2006;15:295-299. doi: 10.1111/j.1532849x.2006.00130.x 23. superficial distribution and identification of antifungal/antimicrobial agents on a modified tissue conditioner by semeds microanalysis: a preliminary study. urban vm, seó rs, giannini m, arrais ca. j prosthodont: implant, esthet reconst dent 2009;18(7):603610. doi: 10.1111/j.1532849x.2009.00479.x 24. surface properties of temporary soft liners modified by minimum inhibitory concentrations of antifungals. bueno mg, sousa ej, hotta j, porto vc, urban vm, neppelenbroek kh. brazilian dental j 2017;28:158-164. doi: 10.1590/0103-6440201701266 25. candida albicans adherence and proliferation on the surface of denture base materials. koch c, bürgers r, hahnel s. gerodontology 2013;30:309-313. doi: 10.1111/ger.12056 26. an epidemiologic study of yeasts in elderly denture wearers. budtzjorgensen e, stenderup a, grabowski m. community dent oral epidemiol 1975;3:115-119. doi: 10.1111/j.16000528.1975.tb00291.x 27. bacterial involvement in dentureinduced stomatitis. koopmans a, kippuw n, graaff j. j dent res 1988;67:1246-1250. doi: 10.1177/00220345880670091901 28. diminished antimicrobial peptide and antifungal antibiotic activities against candida albicans in denture adhesive. bates am, garaicoa jl, fischer cl, brogden ka. antibiot 2017;6:6. doi: 10.3390/antibiotics6010006 29. candida-associated denture stomatitis. aetiology and management: a review. part 3. treatment of oral candidosis. webb bc, thomas cj, willcox md, harty dw, knox kw. australian dental journal. 1998;43:244-249. doi: 10.1111/j.18347819.1998.tb00152.x 30. nystatin in denture linersan alternative treatment of denture stomatitis. douglas cj, walker dm. brit dent j 1973;135:55-58. doi: 10.1038/sj.bdj.4803045 incorpora(on of fluconazole and ocimum sanctum oil in so6 denture liners to treat biofilms of candida albicans associated with denture stoma((s vol 9 no 1 (2021) doi 10.5195/d3000.2021.120 h#p://den*stry3000.pi#.edu 31. effect of triazine derivative added to denture materials on a microcosm biofilm model. de moraes ap, barwaldt ck, nunes tz, sarkis-onofre r, ogliari fa, boscato n, pereira-cenci t. j biomed mater res b appl biomater 2012;100:1328-1333. doi: 10.1002/jbm.b.32699 32. microwave disinfection of dentures for the treatment of oral candidiasis. banting dw, hill sa. special care in dentist 2001;21:4-8. doi: 10.1111/j.17544505.2001.tb00216.x 33. adhesion of oral candida albicans isolates to denture acrylic following limited exposure to antifungal agents. ellepola an, samaranayake lp. arch oral biol 1998;43:999-1007. doi: 10.1016/s0003-9969(98)00075-2 34. antifungal activity of ocimum sanctum linn. (lamiaceae) on clinically isolated dermatophytic fungi. balakumar s, rajan s, thirunalasundari t, jeeva s. asian pacific j tropical med 2011;4:654657. doi: 10.1016/s1995-7645(11)60166-1 35. development of candidaassociated denture stomatitis: new insights. pereira-cenci t, del bel cury aa, crielaard w, ten cate jm. j appl oral science 2008;6:86-94. doi: 10.1590/s167877572008000200002 36. influence of surface characteristics on the adhesion of candida albicans to various denture lining materials. kang sh, lee hj, hong sh, kim kh, kwon ty. acta odontologica scandinavica. 2013;1;71(1):241-248. doi: 10.3109/00016357.2012.671360 37. prosthetics applications of polymers. craig rg. 10th ed. st. louis: mosby 1997;551. 38. use of tissue conditioners and resilient liners. gonzalez jb. dent clin north am 1977;21:249-259. 39. effects of different disinfectants on physical properties of four temporary soft denture-liner materials. yilmaz h, aydin c, turhan bal b, ocak f. quintessence int 2004;35:826-834. 40. the effects of miconazole on the ultastructure of candida albicans. sonja de nollin, borgersand m, vanbelle h. roc roy soc med 1977;supplement 4. microsoft word rea 40 april 30.docx   vol  3,  no  1  (2015)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2015.40           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     haitian  orphan  population  and  protective  factors  against  caries   madelyn  rea1     1  university  of  pi.sburgh  school  of  dental  medicine,  pi.sburgh  pa,  usa abstract   objec&ve  in  hai',  families  were  torn  apart  and  children  were  le6  orphans  a6er  the  2010  earthquake.   in  the  a(ermath  of  this  natural  disaster  many  children  were  relocated  to  orphanages  across  the  coun-­‐ try  and  adopted  interna,onally.  years  later  these  children  find  themselves  catching  up  in  growth  physi-­‐ cally,  mentally  and  emo-onally  a.er  an  extremely  trauma-c  event  during  a  crucial  -me  in  their  health   development.  another  important  marker  of  development  is  the  primary  den55on  and  the  presence  of   caries.    we  report  es(mates  of  early  childhood  caries  (ecc)  frequency,  risk  factors  and  quality  of  health   among  hai)an  children.  methods  medical  and  dental  professionals  conducted  a  descrip1ve  cross  sec-­‐ !onal   study   through   the   pi2sburgh   kids   founda!on   and   their   partnership   with   idadee   children’s   home,  ebac  orphanage  and  new  vision  children’s  home.  vital  signs  were  taken  and  recorded  to  create   a   health/growth   history   for   each   child.   brief   dental   screenings   were   conducted   and   topical   fluoride   treatments  were  administered.  risk   factors  and  quality  of  health   informa6on  was  obtained  from  dis-­‐ cussions  with   the   caregivers  present.   the   children  and  caregivers  were  given  oral  hygiene  educa6on   and  supplies  (i.e.  toothbrushes,  toothpaste,  floss).    results  physical  exams  and  dental  screenings  were   conducted  on  the  40  children  ages  3-­‐10  years  of  age  living  in  the  idadee  children’s  home.  two  children   had  cavitated  teeth.  eight  children  had  teeth  that  were  stained.  four  children  had  evidence  of  dental   trauma.  26  out  of  the  40  children  had  otherwise  healthy  den//on.  conclusion  the  idadee  children’s   home   and   new   vision   children’s   home   have   hopes   to   expand   their   capacity  with   new   construc9on   scheduled   to   be   finished   in   2016.   as  more   children   enter   these   homes   ac8on   is   needed   to   educate   caregivers  on  ways  to  iden0fy  high-­‐risk  children  to  prevent  ecc  and  ways  they  can  be  treated  before  ir-­‐ reversible  damage  is  done  to  the  developing  permanent  den22on.     cita%on:  rea  m.  (2016)  hai$an  orphan  popula-­‐ !on  and  protec!ve  factors  against  caries.  den-­‐ !stry  3000.  1:a001  doi:10.5195/d3000.2015.40   received:  april  2,  2016   accepted:    april  8,  2016   published:    may  10,  2016   copyright:  ©2016  rea,  m.  this  is  an  open  access   ar!cle   licensed   under   a   crea-ve   commons   a!ribu!on  work  4.0  united  states  license.   email:  mar262@pi).edu   introduction   in  cap-­‐haïtien,  a  city  in  northern   haiti,  multiple  orphanages  are  the   home  to  children  who  found   themselves  orphans  after  the  2010   earthquake  that  hit  the  southern   capital  city  port  au  prince.  as  in   any  natural  disaster  aid,  clean  up   and  rebuilding  can  be  a  long  pain   staking  process  and  many  children   when  found  were  fighting  to  hang   on  for  their  lives.  the  original  mis-­‐ sion  of  the  idadee  childrens’   home  was  in  fact  not  to  be  a  home   at  all,  but  rather  a  place  for  mere   infants  and  toddlers  to  respectful-­‐ ly  die.  many  of  these  children  ini-­‐ tially  exhibited  failure  to  thrive,  [1]   and  some  continue  to  show  these   symptoms,  however  the  vast  ma-­‐ jority  have  experienced  catch  up   growth  and  are  developing  within   normal  limits  for  their  sex  and  age   [2].  this  can  be  in  large  attributed   to  the  organizational  set  up  of  the   idadee  children’s  home.  the  chil-­‐ dren  of  idadee  find  themselves  in   a  very  unique  situation  in  which   they  are  divided  and  assigned  to  1   of  8  different  house  “mothers”   who  permanently  live  at  idadee   cooking,  cleaning  and  caring  for   the  basic  needs  of  the  children   under  their  care,  just  as  any  par-­‐ ent  does.  the  house  “mothers”   are  also  the  ones  brushing  the   children’s  teeth  or  supervising   them  brushing  their  own  teeth.      hai$an  orphan  popula$on  and  protec$ve  factors  against  caries   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.40    http://dentistry3000.pitt.edu   2   the  idadee  children’s  home  is  no-­‐ tably  located  in  a  more  rural  por-­‐ tion  of  cap  haitien  known  as   mourne  rouge,  however  it  is   unique  in  that  the  home  is  located   on  a  rather  large  compound  that   includes  the  idadee  school  and   the  joshua  house  retreat  center.   the  joshua  house  retreat  center   is  open  all  year  long,  accommodat-­‐ ing  church,  business  and  medical   missions.  the  idadee  house   “mothers”  are  very  aware  of  the   presence  of  medical  personnel   that  frequent  the  center  and  they   use  that  as  their  main  source  of   medical  attention  for  the  children.   when  our  specific  medical  group   was  there,  the  “mothers”  were   ready  and  prepared  to  express   their  concerns  both  medically  and   dentally  for  many  of  the  children   [3].  in  this  report,  we  describe  the   preliminary  findings  related  to  oral   health  of  children  that  survived   the  haitian  2010  earthquake.   subjects  and  methods   physical  exams  were  conducted  on   the  idadee  compound.  idadee   staff  was  given  notification  in  the   weeks  prior  to  the  visit  and  con-­‐ sent  was  given  and  arrangements   were  made  for  the  physicals.  vital   signs  were  measured  and  record-­‐ ed  for  height,  weight,  occipital   frontal  circumference  (ofc),  blood   pressure,  and  heart  rate.  these   records  are  then  filed  in  the  per-­‐ sonal  records  being  kept  for  each   child  to  track  growth  patterns.   additionally  the  children  under-­‐ went  a  limited  oral  exam  screen-­‐ ing  for  decay,  trauma,  staining  and   oral  hygiene.  they  were  then  giv-­‐ en  brief  oral  hygiene  education   and  had  their  teeth  brushed  by   the  dental  and  medical  volunteers   (figures  1  and  2).  once  superficial   plaque  removal  was  achieved   prevident  varnish  (5%  naf)  was   applied  and  the  caregivers  and  the   children  were  given  instruction  to   not  brush  their  teeth  until  the  next   day.  questions  about  diet,  oral   hygiene,  previous  medical  and   dental  history  answered  by  care-­‐ givers  via  and  interpreter.   results   table  1  summarizes  the  oral   health  outcomes  found.  table  2   describes  the  contents  of  the  chil-­‐ dren  weekly  diet  and  oral  hygiene   habits  as  reported  by  the  chil-­‐ dren’s  caregivers.   discussion    according  to  the  aapd  (american   association  of  pediatric  dentis-­‐ try)  caries-­‐risk  assessment  this   population  of  children  would   all  be  classified  as  high  risk  for   early  childhood  caries  (ecc)   due  to  the  factors  of  having  a   low  ses  status,  non-­‐ fluoridated  drinking  water,  lit-­‐ tle  availability  of  oral  hygiene   products  (i.e.  toothbrushes   and  fluoridated  tooth  paste,   minimal  access  to  or  regular   dental  care  [4].  the  results  of   our  oral  exam  findings  howev-­‐ er  only  report  5%  of  these  children   presenting  with  tooth  decay.  this   is  a  relatively  large  difference  in   comparison  to  the  42%  of  children   affected  with  tooth  decay  in  the   united  states.  the  children  in  our   sample  that  presented  with  such   extensive  decay  that  their  dmf   score  (decayed  missing  filled)  for   their  age  further  classified  as  hav-­‐ ing  severe  early  childhood  carried   (s-­‐ecc).  s-­‐ecc  is  defined  as  having   a  dmf  score  of  greater  than  or   equal  to  4  surfaces  at  age  3,   greater  than  or  equal  to  5  surfaces   at  age  4  or  greater  than  equal  to  6   surfaces  at  age  5  [4].  among  the   children  with  staining,  no  further   classifications  of  ecc  or  s-­‐ecc   were  made  because  the  staining   appeared  to  be  mainly  external  or   confined  to  the  pits  and  fissures  of   the  teeth.  it  was  suspected  that   much  of  this  external  staining   would  have  been  able  to  be  re-­‐ moved  with  a  proper  dental   prophylaxis.  additionally  it  was   suspected  that  the  pit  and  fissure   staining  was  the  sign  of  an  arrest-­‐ ed  caries  process  and  that  appro-­‐ priate  treatment  for  this  would   have  been  the  placement  of  seal-­‐ ants  or  a  preventive  resin  restora-­‐ tion  (prr)  confined  to  these  af-­‐ fected  pits  and  fissures  [5].     the  results  of  the  diet  his-­‐ tory  given  by  the  caregivers  nota-­‐ bly  report  a  diet  that  would  be   considered  relatively  low  caries   risk.  the  children  have  essentially   no  processed  sugar  intake  and   table  1.  frequency  of  dental  operations.   finding   n   percent   (%)   staining   8   20   cavitations   2   5   trauma   4   10   normal  dentition   26   65     40   100      hai$an  orphan  popula$on  and  protec$ve  factors  against  caries   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.40    http://dentistry3000.pitt.edu   3   their  consumption  of  fruits  is   mainly  in  the  form  of  whole  fruits.   in  the  case  of  whole  fruits  there  is   little  evidence  to  show  causality  of   caries.  fiber  rich  fruits  such  as   mangos  and  bananas  require  more   chewing,  therefore  stimulate  more   saliva  flow  potentially  acting  as  a   protective  mechanism  against  car-­‐ ies  [6].    children  that  these  organi-­‐ zations  are  seeking  to  bring  into   their  programs  are  those  that  are   physically,  mentally  and  emotion-­‐ ally  delayed.  action  is  needed  to   educate  these  caregivers  regard-­‐ ing  the  specialized  care  required   by  these  children  to  prevent  as   many  permanent  complications  as   possible.  the  organizational  set  up   of  dividing  the  children  among  in-­‐ dividual  “  house”  mothers  has  in-­‐ creased  individual  attention  that   children  receive  and  has  arguably   allowed  these  children  to  over-­‐ come  developmental  obstacles   early  on  in  life.  additionally  it  can   be  argued  that  oral  hygiene  pro-­‐ vided  under  the  supervision  of   these  “mothers”  has  resulted  in   prevention  of  early  childhood  car-­‐ ies  for  the  vast  majority  of  these   children.    these  women  are  the   common  denominator  in  control-­‐ ling  crucial  environmental  factors   such  as  hygiene,  nutrition  and  ac-­‐ cess  to  healthcare.     these  women  are  also  a   common  denominator  as  a  reser-­‐ voir  for  streptococcus  mutans,  the   prime  organism  involved  in  caries   initiation  and  progression.  in  bio-­‐ logic  mothers  and  children  the   levels  of  streptococcus  mutans  are   highly  associated  due  to  their  con-­‐ tact  normally  being  most  intimate,   of  highest  frequency  and  duration.   additionally,  immunological  fac-­‐ tors  are  shared  between  mother   and  child,  a  phenomenon  known   as  vertical  transmission.  although,   the  children  of  idadee  no  longer   are  in  contact  with  their  biologic   mothers  they  still  have  their  im-­‐ munological  factors  that  were  ac-­‐ quired  in  utero  or  potentially  via   breastfeeding.  in  the  case  of  these   women,  while  their  contact  with   these  children  can  also  be  de-­‐ scribed  as  intimate,  in  high  fre-­‐ quency  and  duration,  their  immu-­‐ nological  factors  are  not  being   conferred  to  these  children.  this   type  of  transmission  is  known  as   horizontal  transmission.  other   children  in  idadee  also  serve  as  a   reservoir  of  streptococcus  mutans   and  contact  with  each  other  is  an-­‐ other  form  of  horizontal  transmis-­‐ sion  [7].     with  so  many  of  these  envi-­‐ ronmental  factors  kept  constant   from  child  to  child  in  idadee,  the   major  difference  in  these  children   is  their  genetics  and  their  past  envi-­‐ ronmental  exposures.     caries  is  defined  as  a  multi-­‐ factorial  infectious  disease  caused   by  an  imbalance  between  protec-­‐ tive  and  risk  factors  [8].  keeping  in   mind  the  multifactorial  nature  of   this  disease  it  can  be  proposed   that  the  genetic  mode  of  inher-­‐ itance  would  best  be  described  as   being  polygenic.  genes  of  interest   when  investigating  cariology  in-­‐ clude  dentition,  salivary,  immuno-­‐ logical,  and  gustation  genes  [9].  in   some  children  the  protective  ef-­‐ fect  of  some  of  these  genes  is   great  enough  to  outweigh  many  of   the  established  environmental  fac-­‐ tors  such  as  diet,  oral  hygiene,  and   ses  that  place  children  at  high  risk.   in  the  case  of  the  children  at   idadee  it  can  be  argued  that  the   75%  of  children  with  healthy  den-­‐ tition  and  history  of  dental  trauma   have  genes  that  in  fact  do  provide   that  protection  against  the  caries   process.  in  the  5%  of  children  with   ecc  we  can  argue  that  their  genes   are  not  providing  adequate  pro-­‐ tection  against  the  caries  process   and  are  being  heavily  outweighed   by  known  risk  factors.  in  the  20%   of  children  with  staining,  the  exact   nature  of  the  staining  would  be   further  characterized  as  either  ex-­‐ ternal  or  pit  and  fissure.  in  the   case  of  pit  and  fissure  staining  the   staining  could  be  the  result  of  an   arrested  caries  process.  if  this  is  in   fact  the  case,  an  argument  can  be   made  that  while  the  balance  at   some  point  was  in  favor  of  risk  fac-­‐ tors  and  the  progression  of  the   caries  process,  it  has  now  been   tipped  to  the  other  direction  and   protective  genetic  factors  could  be   predominating.        hai$an  orphan  popula$on  and  protec$ve  factors  against  caries   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.40    http://dentistry3000.pitt.edu   4   follow-­‐up  is  definitely  indicated  in   this  sample,  with  potential  re-­‐ search  expanding  to  the  other  as-­‐ sociated  orphanages  that  were  in   place  before  the  earthquake.   while,  the  resilient  nature  of  the   idadee  children  is  more  than  evi-­‐ dent  the  synergistic  effect  of  their   resilience  and  the  diligence  of  the   “house”  mothers  to  provide  ex-­‐ ceptional  care  to  an  otherwise   marginalized  population  has  re-­‐ sulted  in  better  than  expected  oral   health.  it  would  be  very  valuable   future  research  to  collect  saliva   samples  and  identify  the  exact   markers  expressed  in  the  children   with  s-­‐ecc.  additionally,  this   would  be  a  very  interesting  sam-­‐ ple  population  to  study  longitudi-­‐ nally  caries  and  other  oral  health   outcomes.  overall,  this  is  an  ex-­‐ traordinarily  unique  group  of  chil-­‐ children  that  can  teach  us  the   power  of  environment  on  genet-­‐ ics.     acknowledgements   the  pittsburgh  kids  foundation   provided  funding  and  support.     references   1.  mclean  hs,  price  dt.  failure  to   thrive.  in:  kliegman  rm,  stanton   bf,  st.  geme  jw  iii,  et  al.,  eds.   nelson  textbook  of  pediatrics.   19th  ed.  philadelphia,  pa:  elsevier   saunders;  2011:chap  38.   2.  growth  charts  -­‐  homepage.   cdcgov.  2016.  available  at:   http://www.cdc.gov/growthchart s.  accessed  march  14,  2016.   3.  pittsburgh  kids  foundation  -­‐   haiti.  pittsburghkidsfounda-­‐ tionorg.  2016.  available  at:   http://www.pittsburghkidsfounda tion.org/haiti/.  accessed  march   14,  2016.   4.  nowak  aj,  casamassimo  ps.   the  handbook  of  pediatric  den-­‐ tistry.  american  academy  of  pe-­‐ diatric  dentistry,  2011   5.  dean  j,  mcdonald  r,  avery  d.   mcdonald  and  avery's  dentistry   for  the  child  and  adolescent.  st.   louis,  missouri:  elsevier;  2016.   6.  dental  caries  in  pre-­‐school  chil-­‐ dren:  associations  with  social   class,  toothbrushing  habit  and   consumption  of  sugars  and  sugar-­‐ table  2.  interveiw  responses  obtained  from  caregivers  in  regards  to  diet  and   oral  health.   diet     protein   chicken,  fish,  beans,  peanut  butter   carbohydrates   rice,  white  bread,  oatmeal   vegetables   plantains   fruits   mango,  oranges,  pineapple,  bananas   water   bagged     juice   occasionally  tampico  (orange  and  other  fruit  juices)   soda   rarely   sugar/sweets   rarely;  have  somewhat  of  an  aversion   dental  health  behav-­‐ iors     brushing  frequency   2x  a  day;  by  caregiver  some  of  the  older  children  can   brush  on  their  own   fluoride  sources   toothpaste  when  available   varnish  ~1-­‐2x  a  year   water  non-­‐fluoridated   history  of  pain     2  children  with  severely  cavitated  teeth,  cry  a  lot  at   night,  won’t  open  their  mouths      hai$an  orphan  popula$on  and  protec$ve  factors  against  caries   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.40    http://dentistry3000.pitt.edu   5   containing  foods.  further  analysis   of  data  from  the  national  diet  and   nutrition  survey  of  children  aged   1.5-­‐4.5  years.  gibson  s,  william  s.   caries  res.  1999;33(2):101-­‐13.   pmid:  9892777   7.  an  investigation  into  the  use  of   restriction  endonuclease  analysis   for  the  study  of  transmission  of   mutans  streptococci.  kulkarni  gv,   chan  kh,  sandham  hj.  j  dent  res.   1989;68(7):1155–61.  pmid:   2632600   8.  early  childhood  caries  is  associ-­‐ ated  with  genetic  variants  in   enamel  formation  and  immune   response  genes.  abbasoğlu  z,   tanboğa  i̇,  küchler  ec,  deeley  k,   weber  m,  kaspar  c,  korachi  m,   vieira  ar.  caries  res.   2015;49(1):70-­‐7.  pmid:  25531160   9.  vieira,  alexandre  r  (january   2016)  genetic  influences  on  den-­‐ tal  caries.  in:  els.  wiley  &  sons,   ltd:  chichester.   doi:10.1002/9780470015802.a00 24258   durability of different types of mouthwashes on the salivary buffering system in orthodontic patients vol 9 no 1 (2021) doi 10.5195/d3000.2021.161 http://dentistry3000.pitt.edu durability of different types of mouthwashes on the salivary buffering system in orthodontic patients israa r. alkasso1, sarmad s. salih al qassar2, ghada a. taqa1 1 basic science department, college of dentistry, university of mosul, mosul, iraq 2 orthodontics and preventive dentistry department, college of dentistry, university of mosul, mosul, iraq abstract the fast elevation of the salivary ph in orthodontic patients has acritical role to safe the teeth and encourage the orthodontic treatment after acidic challenger. as their baseline salivary ph is dropped during fixed orthodontic treatments. aims: this study was aimed to evaluate the durability of three types of mouthwashes on the salivary buffering capacity in orthodontic patients at 0, 5, 10, 15 mints after acidic challenger using direct ph meter. material and method: 80 orthodontic patients; age range (13-21years) were distributed into four groups; 20/ each, the salivary ph were recorded using compact ph meter (laquatwin, horiba's, kyoto, japan) before and after administration of carbonated beverage (100 mm coca cola) and directly after gargling with 5 ml of the tested mouthwashes (herbal, green tea and chlorohexidine mouthwash) beside tap water as placebo control. the ph was documented again at 0, 5, 10 and 15 mins after each mouthwash gargling. one way anova analysis and duncan multiple were used for salivary ph analysis, p≤0.05. results: non-significant differences were found at 0, 5, 10, 15 mints in comparison with baseline ph after herbal mouthwash gargling, the same result was shown with chlorohexidine mouth wash but with less defined border. salivary ph after green tea gargling showed clear oscillatory results after the same tested periods. conclusion: herbal mouthwash is the best type to improve salivary ph for longer duration after acidic challenger. while chlorohexidine mouthwash shows beneficial effects in elevation salivary ph with less duration. green tea mouthwash fail to control salivary ph for long duration. keywords: salivary ph; orthodontic appliance; herbal mouthwash; green tea mouthwash; chlorhexidine mouthwash. citation: alkasso i, et al. (2021) durability of different types of mouthwashes on the salivary buffering system in orthodontic patients. dentistry 3000. 1:a001 doi:10.5195/d3000.2021.161 received: march 12, 2021 accepted: april 14, 2021 published: september 16, 2021 copyright: ©2021 alkasso i, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: sarmadsobhi@yahoo.com | sarmadsobhi@uomosul.edu.iq introduction dental caries is the highest routine infectious disease worldwide. the interaction of shielding and deleterious factors in saliva and plaque considered as the elements that associate with caries incidence [1]. this incidence have a significant impact on patients wearing fixed orthodontic appliance, as the fixed appliance increase the occurrence of dental caries. saliva is entirely unique oral fluid secreted from major and minor salivary glands. the usage of saliva in recognizing caries threat status for patients is well believed, which can be performed by evaluation the salivary flow rate, ph stage and lastly, its buffering capability. [1] saliva ph ranges from (6.2 -7.6) with 6.7 practicality considered as the common ph. saliva ph of non caries teeth shouldn’t fall below 6.3. saliva, in turn, takes its role to control the ph of the oral cavity, which is maintained near neutrality (6.7 to 7.3). the saliva mechanism to preserve the ph consist of the draught of saliva eliminates carbohydrates that would be metabolized by the bacteria and removes acids that created by microorganism. also, drinks and foods acidity alongside bacterial waste products, is canceled out by the saliva http://dentistry3000.pitt.edu/ mailto:sarmadsobhi@yahoo.com mailto:sarmadsobhi@uomosul.edu.iq durability of different types of mouthwashes on the salivary buffering system in orthodontic patients vol 9 no 1 (2021) doi 10.5195/d3000.2021.161 http://dentistry3000.pitt.edu buffering action [2]. the ammonia release from urea metabolism by plague, this ammonia act to buffer the acidity of the saliva. however, the critical ph was reported to be 5.5 or less, in dental studies, thus any further drops in salivary ph would leads to teeth decay as a result of alteration of calcium and phosphate. as this ph is risky on progression of dental enamel [3]. mouthwashes are antibacterial liquids held in the mouth temporarily and swished by the perioral muscles to eliminate the oral infections. as a liquid, these mouthwashes reach all mouth areas that are hard to reach using dental brush. according to their content, mouthwashes are generally classified into cosmetic and therapeutic products [4]. chlorhexidine is the golden option for treatment and controlling dental plaque and gingival inflammations. it is well known act against gram-positive and gramnegative bacteria both aerobes and anaerobes, as well as yeasts, fungi and virus’s lipid envelope [5, 6,7]. up to date, several synthetic mouthwashes are offered in the supermarkets. however, their side effects such as toxicity, hypersensitivity, and tooth staining limited it’s used. the alternative option is medicines that are industrialized from medicinal plants. these plants are usually containing natural phytochemicals and henceforth, can replace synthetic medications [6]. green tee also, has a multiple advantage for oral health. the previous studies focused on treatment aspects of mouthwashes regardless their effects on the saliva. the public queries that commonly arise is which one is well in improving saliva buffering capacity (chlorhexidine and herbal mouth). hence, this study was designed to ask this question. also, this study used to evaluate the effects of herbal, chlorohexidine as well as green tea as mouthwash to test the durability of buffering capacity of the saliva over 0, 5, 10, 15 mints using direct ph meter. material and methods study design this study was designed as a double blinded randomized controlled trail. the protocol of this study was revised and accepted by research ethics committee of the collage of dentistry, university of mosul (no. 26139 date 22\9\2019). according to the pilot study results, sample size was calculate utilizing g power software as follow: power of study was 85%, α= 0.05, constant proportion was 0.5, the result was total sample size was 18 per group. we recruited 20 patients per group for more precise the results. eighty orthodontic patients seeking dental treatments in dental hospital at the dental school/ university of mosul were asked to participate in this study. each patient signed special constant form, which was previously prepared for this purpose. the patients’ age range was (13-21 years). the inclusion criteria include healthy orthodontic patients wearing fixed orthodontic appliance (roth “22 stainless steel brackets, ultratrim, dentaurum, ispringen, germany) within the last two months that were neither complain from any systemic disease nor under daily drug course that could excreted in the saliva and hinder the results. the recruited patients were divided into four groups (20 for each) according to the type of the mouthwashes under investigation as follow: herbal group (hg), chlorohexidine group (cg), green tea group (fg) and tap water group (tg) which act as placebo negative. the patients and the observer were blinded to the type of the mouth wash used. measurement procedure each patient was instructed to stop eating and drinking at all for an hour before the morning appointment after well brushing of his teeth one night before. the patient set at morning in upright http://dentistry3000.pitt.edu/ durability of different types of mouthwashes on the salivary buffering system in orthodontic patients vol 9 no 1 (2021) doi 10.5195/d3000.2021.161 http://dentistry3000.pitt.edu position in the dental chair and initial salivary ph was measure by spitting a sufficient amount directly on the dish part of the ph meter (horiba's laquatwin, horbia instruments incorporated, kyoto, japan). then the subject was instructed to gargle with a (100 ml) of coca cola® (carbonated beverage) for 10 seconds before swelling it. patient saliva ph was re-measured and recorded immediately after coca cola swallowing and 5, 10, 15 mints after respectively, using the same above procedures and device. for hg, the above procedure was repeated one day after, as the patient was instructed to gargle with constant amount (5 ml) of herbal mouthwash (vitex maximum orthodontic care mouthwash (cjsc “vitex” in republic of belarus) for 5 10 sec [according to british national formulary 77] before coca cola swallowing. the salivary ph was measured immediately after that and 5, 10, 15 mints subsequently. the same above procedure was repeated using the chlorohexidine mouth wash (biofresh, scitra co. in united arab emirates), tap water and green tea (ahmad tea, london, united kingdom) according to their group. green tea mouth wash was prepared by dipping five tea bags in 100ml of boiling water, then let it cool at room temperature for about one hour before give it to patients. all the mouthwashes were packed in totally black bottles to a chive a double blind of the observer and participates. the ph meter was cleaned and washed with plenty of distal water after each measurement and re calibrated using special wellknown ph liquids that was provided with it. statistical analysis the data that had been collected throughout the study have been processed and analysed by using spss 23 (statistical package for the social sciences) (spss inc., chicago, usa). it was used to calculate mean and stander deviation for each salivary ph measurement for all groups. one way anova analysis and duncan multiple range test was used to find differences in salivary ph measurements for mouthwashes in compare with baseline salivary ph and to find differences in salivary ph measurement between all locally active tested products (p≤0.05). results table 1. shows the mean age for all patients participated in this study classified according to type of mouth wash used, which was (15.97 sd 4.01) years, with no significant difference between age groups, female represent (80%) for whole patients that participate in our study compare with (20%) for male with no significant difference between the two genders at (p≤0.05) p value was 0.763. salivary ph measurement after herbal mouthwash gargling herbal mouthwash ph was (7.2). the results of measurement salivary ph for patients after herbal mouthwash were shown in table 2, that shows the means and stander deviation of each salivary ph measurements, and the result of comparison of salivary ph for patients at different intervals from gargling with herbal mouthwash using one way analysis of variance (anova) that reveal a significant difference in salivary ph after coca cola in compare with baseline, 0, 5, 10 and 15 min salivary ph after gargling with herbal mouthwash (p≤0.05), while there were no significant differences between the salivary ph at 0, 5, 10 and 15 and baseline ph at (p ≤0.05). salivary ph measurement after chlorohexidine gargling chlorohexidine mouthwash ph was 6.3. the results of measurement salivary ph for patients after chlorohexidine mouthwash were shown in table 3 that shows the means and stander http://dentistry3000.pitt.edu/ durability of different types of mouthwashes on the salivary buffering system in orthodontic patients vol 9 no 1 (2021) doi 10.5195/d3000.2021.161 http://dentistry3000.pitt.edu deviation of each salivary ph measurement and the result of comparison for salivary ph measurement at different time from chlorohexidine gargling by one way analysis of variance (anova) that reveal a significant difference in salivary ph after coca cola in compare with baseline 0, 5, 10 and 15min salivary ph after gargling with chlorohexidine mouthwash (p≤0.05), there were also a significant differences between 0 min salivary ph after gargling with 15 min salivary ph measurement (p≤0.05), while there were no significant differences between salivary ph measurement at 5, 10 and baseline ph in compare with 0 and 15 min salivary ph measurement after chlorohexidine mouthwash. tap water mouthwash and salivary ph measurements tap water ph was (7.4) the results of salivary ph measurement for patients after tap water mouthwash were shown in table 4 at which we reveal the means and stander deviation of each salivary ph measurement and the result of comparison for these salivary ph measurement by one way analysis of variance (anova) which reveal a significant difference in coca cola salivary ph in compare with baseline, 0, 5, 10 and 15 min salivary ph after tap water mouthwash (p≤0.05), there were also a significant difference in 15 min salivary ph from tap water mouthwash in compare with baseline and 0 min salivary ph from tap water mouthwash at (p≤0.05), while there were no significant differences between baseline ph in compare with 0, 5, and 10 min salivary ph measurement after tap water gargling at (p≤0.05). green tea mouthwash and salivary ph measurements the results of measurement salivary ph for patients after green tea mouthwash (mouthwash solution ph 5.7) were shown in table 5 at which we reveal the means and stander deviation of each salivary ph measurement and the result of comparison for salivary ph measurement after green tea mouthwash by one way analysis of variance (anova) that reveal a significant difference in baseline salivary ph in compare with coca cola salivary ph 0, 5, 10 and 15 min salivary ph after gargling with green tea mouthwash (p≤0.05), there were a significant difference at 0 min salivary ph after green tea mouthwash in compare with coca cola salivary ph 5, 10 and 15 min salivary ph after green tea mouthwash gargling (p≤0.05) and there were a significant difference between coca cola salivary ph in compare with salivary ph after 15min from green tea mouthwash (p≤0.05), while there were no significant differences between 0min and baseline, coca cola in compare with 5 and 10 min. also no significant differences between 15 min in compare with 0, 5 and 15 min salivary ph measurement at (p≤0.05). comparison between different mouthwashed used in this study the results of comparison salivary ph measurement between orthodontic patients’ groups after gargling with different mouthwash (herbal, chlorohexidine, green tea and tap water) were shown in table 6 at which we reveal the means and stander deviation of salivary ph measurement for each group. the result of comparison by one way analysis of variance (anova) which reveal a significant difference between herbal group and green tea group at 0 min from gargling, a significant difference between green tea groups at 5 min with herbal, chlorohexidine and tap water groups at 5 min, significant difference between green tea groups at 10 min with herbal, chlorohexidine and tap water groups at 10min, also a significant difference between tap water groups at 10min with herbal and green tea groups and a significant difference between herbal at 15min with tap water and green tea groups. http://dentistry3000.pitt.edu/ durability of different types of mouthwashes on the salivary buffering system in orthodontic patients vol 9 no 1 (2021) doi 10.5195/d3000.2021.161 http://dentistry3000.pitt.edu groups age (mean (years) ± sd) f p-value herbal mouthwash hg (17.1 ± 4.35) 0.025 0.994 chlorohexidine mouthwash cg (15.3 ± 3.20) tap water mouthwash tg (15.7 ± 2.53) green tea mouthwash fg (15.8 ± 4.960) groups ph (mean ± sd) f p-value baseline (6.65 ± 0.303) a 22.254 0.000 after coca cola (5.47 ± 0.689) b 0 min from gargling (6.79 ± 0.110) a 5 min from gargling (6.7 ± 0.115) a 10 min from gargling (6.58 ± 0.139) a 15 min from gargling (6.53 ± 0.188) a table 1. the mean age differences between the groups. table 2. one way anova for salivary ph after herbal mouthwash a: homogenous group, b and c: significant differences at p ≤ 0.05. p ≤ 0.05. http://dentistry3000.pitt.edu/ durability of different types of mouthwashes on the salivary buffering system in orthodontic patients vol 9 no 1 (2021) doi 10.5195/d3000.2021.161 http://dentistry3000.pitt.edu groups ph (mean ± sd) f p-value baseline ph (6.54 ± 0.195) ab 18.494 0.000 after coca cola (5.65 ± 0.636) b 0 min from gargling (6.73 ± 0.231) a 5 min from gargling (6.59 ± 0.159) ab 10 min from gargling (6.51 ± 0.166) ab 15 min from gargling (6.37 ± 0.194) b groups ph (mean ± sd) f p-value baseline ph (6.61 ± 0.281) a 23.184 0.000 after coca cola (5.54 ± 0.532) b 0 min from gargling (6.62 ± 0.155) a 5 min from gargling (6.49 ± 0.129) ac 10 min from gargling (6.42 ± 0.114) ac 15 min from gargling (6.31 ± 0.099) c table 3. one way anova of salivary ph after chlorohexidine mouthwash a: homogenous group, b significant differences at p ≤ 0.05. table 4. one way anova of salivary ph after tap water mouthwash a: homogenous group, b and c: significant differences at p ≤ 0.05. http://dentistry3000.pitt.edu/ durability of different types of mouthwashes on the salivary buffering system in orthodontic patients vol 9 no 1 (2021) doi 10.5195/d3000.2021.161 http://dentistry3000.pitt.edu there were no significant differences at 0 min salivary ph between all groups as they elevated salivary ph directly after gargling. herbal mouthwash has the highest ph (7.2), chlorohexidine was in the second place by (6.73), tap water at the third place by (6.62) and lastly home-made green tea mouthwash (6.54). after five minutes from gargling with mouthwashes there were a significant difference between green tea (6.02) in compare with herbal (6.7), chlorohexidine (6.59) and tap water (6.49) that is also lesser than baseline salivary ph, these mean mouths become more acidic after 5 minutes from green tea mouthwash. after 10 minutes from mouthwashes gargling there were a significant difference between green tea ph (6.18) with herbal (6.58), chlorohexidine (6.51) and tap water (6.42) mouthwashes, also there were significant differences between tap water (6.42) with herbal (6.58) and green tea (6.51) mouthwashes. after 15 minutes from gargling with mouthwashes there were a significant difference between green tea (6.31) salivary ph with herbal mouthwash (6.53) also, significant differences between tap water (6.32) mouthwashes with herbal mouthwash (6.53), while there were no significant differences between chlorohexidine (6.37) and other mouthwashes after 15 minutes from gargling. the durability of each mouth washes used in this study after acid challenger are shown in figure 1 for different intervals. groups ph (mean ± sd) f p-value baseline ph (6.69 ± 0.202) a 6.496 0.000 after coca cola (5.91 ± 0.574) b 0 min from gargling (6.54 ± 0.408) ac 5 min from gargling (6.02 ± 0.426) bc 10 min from gargling (6.18 ± 0.234) bc 15 min from gargling (6.32 ± 0.244) c table 5. one way anova of salivary ph after green tea mouthwash a: homogenous group, b and c: significant differences at p ≤ 0.05. http://dentistry3000.pitt.edu/ durability of different types of mouthwashes on the salivary buffering system in orthodontic patients vol 9 no 1 (2021) doi 10.5195/d3000.2021.161 http://dentistry3000.pitt.edu p v a lu e 0 .1 4 0 0 .0 0 0 0 .0 0 0 0 .0 3 3 ta p w at e r m o u th w as h (m e an ± s d ) (6 .6 2 ±0 .1 5 5 ) ab (6 .4 9 ±0 .1 2 9 ) a (6 .4 2 ±0 .1 2 9 ) c (6 .3 1 ±0 .0 9 9 ) b g re e n t e a m o u th w as h (m e an ± s d ) (6 .5 4 ±0 .4 0 8 ) b (6 .0 2 ± 0 .4 2 6 ) b (6 .1 8 ± 0 .2 3 4 ) b (6 .3 2 ± 0 .2 4 4 ) b c h lo ro h e xi d in e m o u th w as h ( m e an ± s d ) (6 .7 3 ± 0 .2 3 1 ) a (6 .5 9 ± 0 .1 5 9 ) a (6 .5 1 ± 0 .1 6 6 ) ac (6 .3 7 ± 0 .1 9 4 ) ab h e rb al m o u th w as h (m e an ± s d ) (6 .7 9 ± 0 .1 1 0 ) a (6 .7 ± 0 .1 5 5 ) a (6 .5 8 ± 0 .1 3 9 ) a (6 .5 3 ± 0 .1 8 8 ) a g ro u p s 0 m in 5 m in 1 0 m in 1 5 m in table 6. one way anova of salivary ph after green tea mouthwash a: homogenous group, b and c: significant differences at p ≤ 0.05. http://dentistry3000.pitt.edu/ durability of different types of mouthwashes on the salivary buffering system in orthodontic patients vol 9 no 1 (2021) doi 10.5195/d3000.2021.161 http://dentistry3000.pitt.edu discussion saliva acidity consider as critical factor in dental erosion, which defined as the chemical process that lead to loss of tooth hard tissue without cooperation of bacteria, dental erosion incidence has been expanded in current years and it is presently taken into consideration to be a worldwide oral sickness, for that salivary acidity is the important threshold for this chemical process that cause dental tissue dissolution [7]. in orthodontic patients, the control of the ph of the saliva has a primary importance, as it was reported that the ph of saliva in orthodontic patients dropped significantly after only one month from starting the fixed orthodontic therapy [8]. additionally, toodehzaeim and khanpayeh, [8], clarified that acidic saliva has an adverse effect on brackets bonding, and archwire surface roughness that could be delay orthodontic treatment. the checking out of the erosive capability on human teeth makes feel with substances having ph values under 5.5. saliva had the ability to compensate these dropping in ph by its dynamic system withen 15-60 min, but this not consider a fast process as dental erosion progression start only after 5 min from exposure to acidic challenger [9]. in this study, a new ph meter was used to measure the ph of the saliva of the volunteers, which is more realable and accurate than ph strip, as these strips has no exact clear measurements of the salivary ph. three types of mouthwashes were evaluated in this study which are the most popular used, tap water also was used as placebo control, which is most accessible one. different intervals were selected (0, 5, 10, 15 mints) to find out the faster mouthwash that could elevated the salivary ph for longer duration. coca cola and salivary ph our results showed that the consumption of the coca cola dropping the ph of the saliva to reach the critical ph which was reported to be the main causative factor of teeth demineralization and subsequent teeth decays [2]. this could be due to the high percentage of carbonate induced in the coca cola. this fallen ph was re-elevated gradually with time to reach 83% from baseline ph after only 5 mints, and to reach 88.95% after 15 mints of beverage intake. however, there are a significant difference between these time intervals. normally the saliva has an ability to control and balance the acidic challenger within 15-60 mints [2, 10, 11]. herbal mouthwash as herbal mouthwash has alkaline ph (7.2), saliva ph up surged directly to higher ph than baseline ph after gargling with it, that persist for 5 min then it retains to 5 5.5 6 6.5 7 7.5 8 baseline coca cola 0 t 5mins 10mins 15mins p h time herbal chlorohe xdine green tea tap water figure 1 durability of salivary ph for different mouthwashes used in this study. http://dentistry3000.pitt.edu/ durability of different types of mouthwashes on the salivary buffering system in orthodontic patients vol 9 no 1 (2021) doi 10.5195/d3000.2021.161 http://dentistry3000.pitt.edu approach baseline ph and dropped after 10, 15 mints respectively with good durability, with no significant between these intervals and base line ph. herbal mouth washes has a refreshing feeling with no bitter test or loss of sensation which seen with other chemical mouthwash, with less staining and pleasant taste induced in the synthetic mouthwashes and also, they're better in cost [6, 12]. recently, the trend to used natural products become more popular as they have high antimicrobial efficiency, safe and reasonably-priced as well. triclosan content of the herbal mouthwashes has an ability to increase the salivary ph immediately [6, 12]. sandhya, in 2017 [13] reported that the traditional mouthwash comprises of the high alcoholic content which can cause harm to the teeth, gums and the oral mucosa. while the natural mouthwash doesn’t contain any alcoholic residue and is gentler on the mouth. beside all of above, the use of herbal mouthwash enhances the saliva buffering capacity immediately. one of its limitation is that it’s difficult to be taking every time as it is bulk to be transported or taking everywhere. chlorohexidine mouthwash our result showed that taken chlorohexidine mouthwash, which have alkaline ph, directly after carbonated beverage elevated salivary ph directly higher than baseline ph, that persist for 10 min insignificantly, then drop back to have ph lower than baseline ph after 15mins from chlorohexidine mouthwash however with no significant difference between them. thus, the immediate one time used of chlorohexidine mouthwash has a significant, but a short-lasting effect on salivary ph. this conclusion is agree with kalyani & leelavathi [14] as they found that chlorohexidine mouthwash have a positive effect on the salivary ph, which is also prolonged in nature. on the other hand, this study is in disagreement with bescos et al., [15] how found that salivary ph and salivary buffering capacity are highly diminished after using chlorohexidine mouthwash, however, their study conducted twice daily for one week, as chlorohexidine use is associated with high increase in saliva lactate, glucose and nitrate concentration. green tea mouthwash as saliva ph drop immediately after drinking carbonated beverage green tea mouthwash succeed to increase ph directly to reach ph higher than critical salivary acidic ph but lower than baseline ph, then it dropped again to almost reach ph after carbonated beverage (after 5 mints), then retain to elevation after 15mins from green tea mouthwash. the green tea mouthwash has a significant, but a short-lasting effect on salivary ph (less durability). talreja et al., [16] and lee et al., [17] uncovered that the catechins (green tea extract) were held for 60 min in saliva and protected plaque ph toward neutrality and also it have antiplaque and antibacterial properties by collaboration with boundary work of microorganism and consumption of this bunch, in this way contributing to caries prevention. this results agreed with singh, [18] kamalaksharappa et al., [19] srinidhi et al., [20] rathod, [21] and sangameshwar, [22] there were a significant increase in salivary ph after rinsing with green tea mouthwash. however, our results shows short improvements effects. tap water mouthwash tap water mouthwash (ph 6.8), after carbonated beverage, rise the salivary ph directly to reach to ph higher than baseline ph, that persist for few minutes then drop http://dentistry3000.pitt.edu/ durability of different types of mouthwashes on the salivary buffering system in orthodontic patients vol 9 no 1 (2021) doi 10.5195/d3000.2021.161 http://dentistry3000.pitt.edu back to have ph lower than baseline ph after 15mins, but higher than ph after carbonated beverage. here we demonstrate that tap water mouthwash has a significant, but a short-lasting effect of salivary ph. this type of mouthwash considers the simplest and most accessible type of mouthwashes, with safe and cheap considerations with insignificant durability from baseline ph after 10 mint. we agreed with kalyani and leelavathi, [14], as they claimed that quick increment within salivary ph after 5 min. from water gargling. also, we agree with mosallam & mohamed, [24, 23] who found that water gargling after dropping in salivary ph leads to neutralization of salivary ph, thus could be consider as a simple preventive measure of caries process in young adults. in 2014 lim [23] discover that water mouth rinsing after suppers plays a basic part in maintaining mouth health. as already depicted, water with ph at 7.0 demonstrates to be cost‑effective preventive degree to prevent dental caries. saliva’s fundamental constituent is water. in addition to that, there are few chemicals and microscopic organisms which help in oral health status support. it is a well‑known fact that the low salivary ph would result in higher rate of caries. the higher ph will alter the ordinary microflora, which in turn will change the mouth balance influencing the periodontium antagonistically. singh et al., [18] studied reinforce rinsing with plain water may be a good preventive step to be consolidated in preventive, promotive programs pointed at decreasing dental caries and gingivitis. panchal and gurunathan, [25] found that water flushing increments salivary ph after the diminishment with 50% sucrose solution. this in a roundabout way improves the caries anticipation and a straight forward mouth cleanliness hone which can be effectively used. in tolentino et al, [26] study the volunteers flushed with refined water, and they found there was an increment in salivary ph amid the estimations. it can be clarified since of the increment within the saliva’s bicarbonate concentration when salivary stream is increased, a condition that's common over time: salivary ph increments with the increment of stream. panchal and gurunathan, [25] expressed that the clearance rate and the amount of salivary secretion alter the ph. also, in their study, the protective impact of tap water flush can be ascribed to increments in the clearance rate, treatment changing the ph having a more protective effect toward caries anticipation. hence, modifying salivary buffering capacity by changing salivary ph can help as a preventive degree in dental caries. mouth flushing with water having a more alkaline ph leads to extend in salivary ph, which in this manner aids as a halt within the handle of demineralization by acidic corrosive generation. comparison between mouthwashes as these mouthwashes have different ph and different in persisting time in the mouth, herbal mouthwash enhances salivary buffering capacity and show the highest salivary ph in compare with baseline salivary ph and other tested groups as it has highest ph (7.2) and persisted for longer period in the mouth (long durability), chlorohexidine in second place showed result approach to herbal mouthwash, homemade green tea mouthwash increases salivary ph after gargling but not reach baseline ph then ph decrease almost become acidic then increase gradually, green tea mouthwash show no enhancement for saliva buffering capacity, on the other hand tap water mouthwash increase salivary ph approach to baseline ph as it has an alkaline ph, that persist for short period as it http://dentistry3000.pitt.edu/ durability of different types of mouthwashes on the salivary buffering system in orthodontic patients vol 9 no 1 (2021) doi 10.5195/d3000.2021.161 http://dentistry3000.pitt.edu readily wash from the mouth, that mean enhance saliva buffering capacity for short period. kalyani and leelavathi, 2019 [14] concluded that chlorohexidine mouthwash had alkaline ph, which is also drawn out in nature when compared to plain water, but we disagreed that chlorohexidine has higher and prolonger effect on salivary ph than herbal mouthwash as we use different herbal mouthwash. however, they have been measured salivary ph at 5 and 60 mints. the kukreja and dodwad, 2012 [27] declared that if people can utilize and advance such cost-effective measures (herbal mouthwashes) of keeping up the oral health which are moreover destitute of any untoward side impacts, it may offer assistance in overcoming some common dental issues. bagchi et al., [6] owing to the side effects reported due to utilize of chlorhexidine mouth rinse and biocompatibility and well acknowledgment of herbal mouthwash by the consumer, it can be viably utilized as a replacing to chlorhexidine mouth rinse. one of the limitations of the herbal mouthwashes that was reported by abdullah et al., [1] study, as they found that herbal mouthwashes are corrosive to orthodontic appliance as the number of ions released from orthodontic appliance in herbal mouthwashes was higher than chlorohexidine mouthwash but didn’t exceed the daily recommended to be release from orthodontic appliance according to who. so prolonged use of these mouthwashes is not recommended. dehghan et al., [28] found that mouth washing with chlorohexidine after an acidic challenge expanded salivary ph. the chlorohexidine mouthwashes raised ph higher than water. mouthwashes with a neutralizing impact can possibly decrease tooth disintegration from corrosive presentation. flötra et al., [29] instructed utilizing of chlorhexidine mouth washes for brief time only, due to their staining effects on the teeth. vilela et al., [30] study supports the effectiveness of green tea mouthwash as an antibacterial option and as substitutes to chlorohexidine mouthwashes. however our results showed it failed to improve the salivary ph after extended period of time. kaur et al., [31] study out comes showed that green tea catechin mouthwash contains a comparable antiplaque viability to chlorhexidine when utilized in a period of 7 days. moreover, green tea catechin mouthwash due to its superior taste and no known sideeffects can be utilized on an everyday premise as an elective for chlorhexidine gluconate as an anti-plaque operator. it ought to be investigated as a long-term antiplaque flush with prophylactic benefits. conclusion herbal mouthwash is the best one to improve salivary ph and for longer duration after acidic challenger. chlorohexidine mouthwash shows beneficial effects in elevation salivary ph higher than baseline ph and fast return to higher ph of saliva after acidic challenge. while green tea mouthwash showed limited effects in elevation salivary ph after acidic challenge for long duration. conflicts of interest the authors declare no competing interest. acknowledgments we thank the collage of dentistry university of mosul, iraq on their support in conducting this research. references 1. in-vitro evaluating the effect of different mouthwashes on the ions released from orthodontic bondable molar tubes. in-vitro abdullah, n. a., hassan, f. a., and nahidh, m. 2020; 25(02), 645654. http://dentistry3000.pitt.edu/ durability of different types of mouthwashes on the salivary buffering system in orthodontic patients vol 9 no 1 (2021) doi 10.5195/d3000.2021.161 http://dentistry3000.pitt.edu 2. the salivary factors and dental erosion romila, l., sachelarie, l., burlui, a., vasiliu, m., and farcas, d. m. international journal of medical dentistry.2020; 24(1), 23-27. 3. ) effect of various sugary beverages on salivary ph, flow rate, and oral clearance rate amongst adults. hans r, thomas s, garla b, dagli r j. and hans m k. hindawi publishing corporation, scientifica 2006; 10(2), 6. 4. evaluation of antibacterial effect of propolis and its application in mouthwash production nazeri, r., ghaiour, m. and abbasi, s. frontiers in dentistry. 2019; 16(1), 1. 5. effects of chlorhexidine mouthwash on the oral microbiome. bescos, r., ashworth, a., cutler, c., brookes, z. l., belfield, l., rodiles, a. and white, d. scientific reports.2020; 10(1), 18. 6. evaluation of efficacy of a commercially available* herbal mouthwash on dental plaque and gingivitis: a double‑blinded parallel randomized controlled trial. bagchi s, saha s, jagannath g v, reddy v k, sinha p. journal indian association public health dentistry. 2015; 13(3), 222-227. 7. correlation between parotid saliva composition and dental caries using 31p-nmr and icdas score. rovera, a., rovera, g., alzahrani, a., hector, m., and anderson, p. archives of oral biology. 2020; 111(1), 104651. 8. effect of saliva ph on shear bond strength of orthodontic brackets. toodehzaeim, m. h. and khanpayeh, e. journal of dentistry (tehran, iran). 2015; 12(4), 257. 9. comparative proteomic analysis on acquired enamel pellicle at two time points in cariessusceptible and caries-free subjects. luo, j., wang, y., wang, k., jiang, w., li, x., and zhang, l. journal of dentistry, 2020; 94(10), 3301. 10. dental erosion in mice with impaired salivary gland function.tulek, a., mulic, a., refsholt stenhagen, k., galtung, h. k., saeed, m., utheim, t. p., and sehic, a.). acta odonatological scandinavica. 2020; 78(50), 1-11. 11. are vitamin beverages good for dental health?. kang, a. r., park, s. h., woo, j. w., hong, d. j., kim, k. r., sung, c. y, and jung, e. hjournal of dental hygiene science . 2020; 20(1), 9-15. 12. comparative evaluation of antimicrobial properties of pomegranate peel extract against streptococcus mutans and lactobacillus-an in vitro study. kunte, s., kadam, n., patel, a., shah, p., lodaya, r., and lakde, l. international dental and medical journal of advanced research 2018; 4(1), 1-6. 13. herbal product as mouthwash-a review. sandhya, r. international journal of sciences and researches. 2017; 6(7), 1334. 14. comparison between the effect of plain water, herbal mouthwash, and chlorhexidine mouthwash on salivary ph. kalyani, p., and leelavathi, l. drug invention today. 2019; 11(5), 1184-1187. 15. effects of chlorhexidine mouthwash on the oral microbiome. bescos, r., ashworth, a., cutler, c., brookes, z. l., belfield, l., rodiles, a., and white, d. scientific reports. 2020; 10(1), 1-8. 16. an in vivo comparison of plaque ph changes in children aged 8-12 years after consumption of milk and green tea with sugar.talreja, n., devendrappa, s. n., singla, s. s., agrawal, n., and mali, s. journal of international oral health. 2018; 10(1), 10. 17. delivery of tea polyphenols to the oral cavity by green tea leaves and black tea extract. lee, m. j., lambert, j. d., prabhu, s., meng, x., lu, h., maliakal, p. and yang, c. s. cancer epidemiology and prevention biomarkers. 2004; 13(1), 132-137. 18. effectiveness of green tea mouth rinse over combination mouth rinse in restoring salivary ph post sugar exposure in children. singh, n. journal of scientific research. (2020); 64(1), 20. 19. efficacy of probiotic and green tea mouth rinse on salivary ph. kamalaksharappa, s.k., rai, r., babaji, p., and pradeep, m.c. journal of indian society of pedodontics and preventive dentistry. 2018; 36(2), 279-82. http://dentistry3000.pitt.edu/ durability of different types of mouthwashes on the salivary buffering system in orthodontic patients vol 9 no 1 (2021) doi 10.5195/d3000.2021.161 http://dentistry3000.pitt.edu 20. effect of two different commercially available tea products on salivary ph:a randomized double blinded concurrent parallel study. srinidhi, p.b., basha, s., naveen kumar, p., prashant, g.m., sushanth, v.h., and imranulla, m. dentistry and medical research. 2014; 2(39), 42. 21. green tea effects on salivary ph and streptococcus mutans count. rathod, v. c., pundir, s., dixit, s., chandraker, n. k., and desai, v. international journal of innovation researches in dental scinces. 2017; 2(6), 4. 22. effect of green tea on salivary ph and streptococcus mutans count in healthy individuals. sangameshwar, m., vanishree, m., surekha, r., santosh, h., anila, k., and vardendra, m. international journal of oral and maxillofacial pathology. 2014; 5(1), 13-16. 23. the effects of mouthwash on lactoperoxidase and ph in human saliva: helpful or harmful. lim, o. journal of future science leaders. 2014; 9(7), 6. 24. comparison of salivary ph changes of young adults with mineral and alkaline water rinse after acidic challenge. a clinical study. mosallam, r. s., & mohamed, m. s. egyptian dental journal. 2019; 65(3-july (fixed prosthodontics, dental materials, conservative dentistry & endodontics)), 2511-2516. 25. comparison of salivary ph changes with tap water and mineral water rinse after 50% sucrose solution rinse: a crossover trial. panchal, v., and gurunathan, d. journal clinical diagnosis researches (2017); 6(140), 2376-0311. 26. saliva and tongue coating ph before and after use of mouthwashes and relationship with parameters of halitosis. tolentino, e. d. s., chinellato, l. e. m., & tarzia, o. journal of applied oral science. 2011; 19(2), 90-94. 27. herbal mouthwashes-a gift of nature. kukreja, b. j., and dodwad, v. international journal of pharmaceutical and biological sciences; 2012; 3(2), 46-52. 28. neutralizing salivary ph by mouthwashes after an acidic challenge. dehghan, m., tantbirojn, d., kymer-davis, e., stewart, c. w., zhang, y. h., versluis, a., and garcia-godoy, f. journal of investigative and clinical dentistry. 2017; 8(2) e12198. 29. side effects of chlorhexidine mouth washes. flötra, l., gjermo, p. e. r., rölla, g., and waerhaug, j. european journal of oral sciences. 1971; 79(2), 119-125. 30. efficacy of green tea and its extract, epigallocatechin-3-gallate, in the reduction of cariogenic microbiota in children: a randomized clinical trial. vilela, m. m., de souza salvador, s. l., teixeira, i. g. l., del arco, m. c. g., and de rossi, a. archives of oral biology. 2020; 114(1), 104727. 31. comparative evaluation of the antiplaque effectiveness of green tea catechin mouthwash with chlorhexidine gluconate. kaur, h., jain, s., and kaur, ajournal of indian society of periodontology. 2014; 18(2), 178. http://dentistry3000.pitt.edu/ microsoft word 78 2017.docx   vol  5,  no  1  (2017)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2017.78         http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     fluctuating  dental  asymmetry  in  rabbits  with  familial  nonsyndromic   coronal  suture  synostosis   mae  a.  hyre1,  seth  m.  weinberg2,  gregory  m.  cooper3,  james  gilbert3,  michael  i.  siegel2,  mark  p.  mooney2      1oro-­‐maxillo-­‐facial  surgeon,  private  prac+ce,  charleston  wv   2university  of  pi.sburgh,  school  of  dental  medicine   3university  of  pi.sburgh,  department  of  plas0c  surgery abstract   fluctua'ng  dental   asymmetry  has  been   linked   to   condi'ons  of  unstable  pre-­‐  and  peri-­‐natal   development.   familial,   nonsyndromic  craniosynostosis  disrupts  early  craniofacial  development  through  localized  excessive  calvarial  ossifica-­‐ !on  leading  to  the  premature  fusion  of  the  calvarial  sutures.  such  abnormal  gene  expression  may  also  produce  sys-­‐ temic   stress   resul+ng   in  developmental   instability,   thereby  affec+ng  normal   trait   symmetry.  the  present   study  was   designed  to  test  this  hypothesis  by  examining  fluctua4ng  dental  asymmetry  in  an  inbred  strain  of  rabbits  with  familial,   nonsyndromic  coronal  craniosynostosis.    the  mesiodistal  (md)  and  buccolingual  (bl)  dimensions  of  the  right  and  le:   maxillary   first  molars  were  measured   in   four   groups   of   new   zealand  white   rabbits   (n=176;   n=40  with   early-­‐onset   synostosis,   n=65  with   delayed-­‐onset   synostosis,   n=46   in-­‐colony,   phenotypically   normal   rabbits,   and  n=25  wild-­‐type   normal  controls).  for  each  variable,  raw  signed  asymmetry  was  calculated  (le:-­‐right)  and  tested  for  assump2ons  of   fluctua&ng  asymmetry   (i.e.,  normality  and  non-­‐direc&onality).  any  group   that  did  not  meet   these  assump.ons  was   excluded  from  further  analysis.  using  a  standard  size-­‐adjusted,  fluctua,ng  asymmetry  index,  mean  fluctua,ng  asym-­‐ metry  was  calculated  and  compared  across  groups  with  non-­‐parametric  sta+s+cs.    for  the  md  dimension,  no  signifi-­‐ cant   (p  >  0.05)  group  differences   in  mean  fluctua8ng  asymmetry  were  observed  among  groups.   in  contrast,   rabbits   with  early-­‐onset  synostosis  had  significantly  (p  <  0.05)  more  fluctua;ng  asymmetry  in  the  bl  dimension  compared  to   wild-­‐type  controls.    results  demonstrate  increased  fluctua1ng  dental  asymmetry  in  rabbits  with  nonsyndromic,  early-­‐ onset  coronal  suture  synostosis  and  suggest  that  the  molecular  events  producing  suture  synostosis  locally  may  have   also  have  systemic  effects.  knowledge  of  these  systemic  interac/ons  may  contribute  to  a  fuller  understanding  of  the   phenotypic  spectrum  observed  in  individuals  with  nonsyndromic  craniosynostosis.   cita%on:  hyre  m,  et  al.  (2017)  fluctua'ng  dental   asymmetry  in  rabbits  with  familial  nonsyndromic   coronal  suture  synostosis.  den$stry  3000.   1:a001  doi:10.5195/d3000.2017.78   received:    july  11,  2017   accepted:    august  18,  2017   published:    october  10,  2017   copyright:  ©2017  hyre  m,  et  al.  this  is  an  open   access   ar!cle   licensed   under   a   crea!ve   com-­‐ mons   a"ribu"on   work   4.0   united   states   li-­‐ cense.   email:  mpm4@pi&.edu   introduction   nonsyndromic,  simple  cranio-­‐ synostosis  is  a  relatively  rare  condi-­‐ tion  (0.06  –  1.9/1000)  that  occurs   when  one  or  more  of  the  calvarial   sutures  fuses  prematurely  during  the   period  of  active  brain  growth,  result-­‐ ing  in  aberrant  patterns  of  growth  in   both  the  neurocranium  and  viscer-­‐ ocranium  [1].  in  recent  years,  cranio-­‐ facial  biologists  have  begun  to  unrav-­‐ el  the  biological  pathways  underlying   abnormal  suture  morphogenesis.  the   emerging  picture  is  one  of  complexi-­‐ ty,  involving  multiple  osteogenic   growth  factors  and  genes  (fgfs,   tgfβs,  bmps,  msxs,  etc.)  operating  at   various  levels  of  regulation  [2,3].   while  the  genetic  mutations  respon-­‐ sible  for  syndromic  cases  of  cranio-­‐ synostosis  have  been  known  for  some   time  [4,5],  only  recently  have  specific   mutations  in  a  number  of  genes   (fgfr3  and  msx2  for  e.g.)  been  iden-­‐ tified  in  individuals  affected  with  non-­‐ syndromic  coronal  suture  synostosis   [5-­‐8].  the  number  of  familial  cases,   however,  remains  relatively  small  at   around  14%  for  coronal  cases  and  6%   for  sagittal  cases  [1,  9,10],  suggesting   that  sporadic  mutations  typically  are   involved.     while  significant  progress  has   been  made  in  elucidating  the  molecu-­‐ lar  causes  of  premature  suture  fusion,   our  understanding  of  the  disease  at   the  phenotypic  level  still  remains  in-­‐ complete.  in  particular,  it  is  unclear   how  alterations  at  the  genotypic  level   are  translated  into  the  range  of  varia-­‐ ble  phenotypes  that  present  under   the  heading  nonsyndromic  cranio-­‐ synostosis.  the  presence  of  certain   environmental  exposures  (e.g.  ciga-­‐ rette  smoke),  systemic  factors  (e.g.   circulating  levels  of  thyroxin  or  an-­‐ drogens)  and/or  the  additive  effects   of  modifier  genes  may  all  act  to  mod-­‐ ulate  phenotypic  severity  [1].  there-­‐ fore,  given  a  genetic  predisposition   for  nonsyndromic  craniosynostosis,   the  specific  phenotypic  outcome  may   depend  in  part  on  the  presence  of   additional  deleterious  factors,  both   intrinsic  and  extrinsic  to  the  organ-­‐ ism.  the  presence  of  such  factors  dur-­‐ ing  ontogeny  may  create  a  develop-­‐ mental  environment  characterized  by   amplified  stress,  leading  to  both  in-­‐ creased  synostotic  severity  and,  pos-­‐ sibly,  other  phenotypic  manifesta-­‐ fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.78    http://dentistry3000.pitt.edu   tions.  unfortunately,  little  is  known   about  the  phenotypic  correlates  of   nonsyndromic  craniosynostosis  apart   from  the  well-­‐documented  secondary   growth  effects  on  the  skull.  however,   because  many  of  the  growth  factors   involved  in  the  regulation  of  suture   morphogenesis  have  expression  pat-­‐ terns  in  a  variety  of  other  tissues   throughout  the  developing  organism   [1,2-­‐5,11,12]  it  is  unlikely  that  the   extent  of  the  disruption  in  nonsyn-­‐ dromic  craniosynostosis  is  limited   entirely  to  the  suture  site.   increased  stress  during  de-­‐ velopment,  either  of  genomic  or  envi-­‐ ronmental  origin,  has  been  linked  to   specific  phenotypic  patterns  in  organ-­‐ isms.  one  of  the  most  commonly   used  phenotypic  indicators  of  devel-­‐ opmental  stress  is  the  degree  of  ran-­‐ dom  asymmetry  present  in  normally   symmetrical,  paired  traits,  commonly   referred  to  as  fluctuating  asymmetry   [13].  mather  [14]  reasoned  that  be-­‐ cause  bilateral  characteristics  are   programmed  by  the  same  genes,  ran-­‐ dom  deviations  from  symmetry  will   represent  a  phenotypic  record  of  the   level  of  instability  present  during  de-­‐ velopment.  waddington  [15,16]  used   the  term  canalization  to  refer  to  the   degree  to  which  genetically  pro-­‐ grammed  developmental  processes   are  able  to  resist  perturbations  during   ontogeny.  developmental  instability   can  be  understood  as  the  degree  to   which  developmental  processes  are   unable  to  buffer  against  such  pertur-­‐ bations,  the  consequences  of  which   are  manifested  in  the  phenotype   [17,18].   interest  in  developmental  in-­‐ stability  as  a  mechanism  for  elucidat-­‐ ing  the  complex  relationship  between   genotype  and  phenotype  has  in-­‐ creased  dramatically  over  the  last   decade  [18-­‐20].  importantly,  elevated   levels  of  fluctuating  asymmetry  have   been  associated  with  a  variety  of   stressors  during  development,  includ-­‐ ing  a  number  of  genetic  diseases  [21].   for  example,  there  is  evidence  of  ele-­‐ vated  fluctuating  asymmetry  in  hu-­‐ mans  affected  with  orofacial  clefts   [22-­‐25],  down’s  syndrome  [26,27],   fragile-­‐x  syndrome  [28],  premature   birth  [29,30]  attention  deficit-­‐ hyperactivity  disorder  [31],  general-­‐ ized  mental  retardation  [32],  spinal   malformation  [33]  and  a  wide  range   of  psychiatric  diseases  including   schizophrenia,  major  depression  and   bipolar  disorder  [21,34].  because   nonsyndromic  craniosynostosis  is  a   disease  that  disrupts  craniofacial  de-­‐ velopment  through  abnormal  calvari-­‐ al  bone  homeostasis  resulting  in  ex-­‐ cessive  ossification  and  premature   suture  fusion  [2,3,35-­‐37],  affected   organisms  likely  experience  increased   systemic  stress  during  ontogeny.  in   principle,  such  stress  should  manifest   itself  in  the  phenotype  as  elevated   levels  of  fluctuating  asymmetry.   however,  de  leon  and  richtsmieir   [38]  reported  little  fluctuating  asym-­‐ metry  in  the  cranial  vault  bones  of   children  with  nonsyndromic  sagittal   suture  synostosis  and  deserves  fur-­‐ ther  study.  given  the  overlap  in  mo-­‐ lecular  expression  between  calvarial   suture  and  dental  morphogenesis   [39],  the  association  between  dental   abnormalities  and  syndromic  forms  of   craniosynostosis  [40],  and  the  fact   that  both  syndromic  and  nonsyn-­‐ dromic  cases  may  share  a  common   genetic  basis  [5-­‐8],  it  is  reasonable  to   suspect  that  the  teeth  of  nonsyn-­‐ dromic  craniosynostotic  organisms   develop  within  a  stressed  physiologi-­‐ cal  environment  and  may  be  a  prime   candidate  for  manifesting  elevated   levels  of  fluctuating  asymmetry.  in   order  to  test  the  hypotheses  that  ge-­‐ nomic  stress  is  elevated  in  the  pres-­‐ ence  of  nonsyndromic  craniosynosto-­‐ sis,  and  that  such  stress  increases   with  the  severity  of  the  cranio-­‐ synostotic  phenotype,  we  propose  to   investigate  patterns  of  fluctuating   dental  asymmetry  in  a  rabbit  model   with  variably  expressed,  familial,  non-­‐ syndromic,  coronal  suture  synostosis   under  controlled  laboratory  condi-­‐ tions.   figure  1.  superior  view  of  skulls  from  a  42  day  wild-­‐type  normal  control  rabbits  and  rabbits   with  late-­‐onset  (postnatal)  and  early-­‐onset  (prenatal)  coronal  suture  synostosis.  note  the   shorter,  wider  cranial  vaults  in  the  rabbits  with  coronal  suture  synostosis  compared  to  the   wild-­‐type  rabbit  skull.   fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.78    http://dentistry3000.pitt.edu   materials  and  methods   study  sample   our  study  sample  consisted  of  crani-­‐ odental  skeletal  material  from  176   new  zealand  white  rabbits   (oryctolagus  cuniculus)  affected  with   varying  degrees  of  familial,  nonsyn-­‐ dromic,  primary  coronal  suture   synostosis.  the  rabbits  were  part  of  a   unique  breeding  colony,  established   in  1993,  at  the  university  of  pitts-­‐ burgh  [41-­‐46].  it  has  served  as  a  use-­‐ ful  model  for  the  study  of  familial,   nonsyndromic  craniosynostosis  in   human  infants  [35,  41-­‐47].  all  crania   were  obtained  from  rabbits  enrolled   in  ongoing  research  protocols.  all   protocols  were  reviewed  and  ap-­‐ proved  by  the  university  of  pitts-­‐ burgh,  institutional  animal  care  and   use  committee  (iacuc).   rabbits  from  this  colony  ex-­‐ hibit  a  wide  range  of  phenotypes,  in-­‐ cluding:  phenotypically  normal  carri-­‐ ers;  rabbits  with  delayed-­‐onset   synostosis  (presenting  at  about  42   days  post-­‐natal),  and;  rabbits  with   early-­‐onset  complete  unilateral  or   bilateral  synostosis  (presenting  at   about  21  days  of  gestation)  [37,  41-­‐ 45]  (figures  1  and  2).  it  has  been   shown  recently  that  rabbits  with  ear-­‐ ly-­‐onset  coronal  suture  synostosis   (i.e.,  the  most  severe  phenotype)   were  the  most  inbred  and  exhibited   the  highest  coefficients  of  inbreeding   [46].  for  the  present  study,  dentition   from  the  skulls  of  40  rabbits  with  ear-­‐ ly-­‐onset  synostosis,  65  rabbits  with   delayed-­‐onset  synostosis,  and  46  in-­‐ colony,  phenotypically  normal  rabbits   were  analyzed.  further,  25  wild-­‐type   rabbits  of  the  same  breed  were  in-­‐ cluded  as  a  separate  control  group.   table  1  shows  basic  descriptive  statis-­‐ tics  of  the  sample  by  group.  no  dif-­‐ ference  in  the  distribution  of  males   and  females  across  groups  was  ob-­‐ served.    thus,  rabbits  of  both   sexes  were  pooled  during   analysis.    further,  due  to  sam-­‐ ple  size  limitations,  bilateral   and  unilateral  affected  animals   were  combined  for  all  anal-­‐ yses.  all  rabbits  were  housed   and  fed  under  similar  labora-­‐ tory  conditions.     measurement  procedure     the  mesiodistal  (md)   and  buccolingual  (bl)  dimen-­‐ sions  of  the  right  and  left  first   maxillary  molars  were  meas-­‐ ured  with  a  set  of  mitotoyo   digital  calipers  (japan)  (figure   3).  the  first  maxillary  molar   was  chosen  because  evidence   suggests  it  is  a  very  stable   tooth  developmentally  [48].   this  conservative  approach   should  bias  our  results  against   finding  fluctuating  asymmetry,   thus  minimizing  the  chances  of   reporting  a  false-­‐positive.  for  the   md  measurement,  the  arms  of   the  calipers  were  positioned  at   the  widest  portion  of  the  tooth   at  the  occlusal  surface  (figure  3).   for  the  bl  measurement,  one   arm  of  the  calipers  was  positioned   flush  against  the  buccal  edge  of  the   tooth.  with  the  one  arm  in  place,  the   measurement  was  then  taken  across   the  tooth  at  the  level  just  below  the   occlusal  plane  on  the  lingual  edge   (figure  3).  the  bl  measurement  was   not  taken  directly  at  the  occlusal  sur-­‐ face  in  order  to  avoid  measuring  the   effects  of  functional  wear.  all  meas-­‐ urements  were  taken  by  one  individ-­‐ ual  (mah).   statistical  analysis     testing  assumptions  of  fluc-­‐ tuating  asymmetry:     following  standardized   methodological  guidelines  [49,50],   the  statistical  nature  of  the  asym-­‐ metry  was  explored  prior  to  further   analysis.  for  fluctuating  asymmetry,   two  conditions  must  be  met.  the  raw,   signed  left-­‐right  difference  scores   must  have  both:  1)  a  normal  distribu-­‐ tion  and  2)  a  mean  equal  to  zero.  the   first  assumption  was  tested  with  the   kolmogorov-­‐smirnov  test  for  normali-­‐ ty,  while  a  one  sample  t-­‐test  was  per-­‐ formed  to  determine  whether  the   mean  of  the  signed  asymmetry  scores   is  significantly  different  from  zero.  a   mean  significantly  different  from  zero   indicates  directional  asymmetry,   which  may  have  different  biological   figure  2.  superior  view  of  the  coronal  suture  from  a   42  day  wild-­‐type  normal  control  rabbits  and  rabbits   with  late-­‐onset  (postnatal)  and  early-­‐onset  (prena-­‐ tal)  coronal  suture  synostosis.  note  the  obliterated   coronal  suture  in  the  early-­‐onset  synostosis  rabbit   skull  (bottom  picture),  the  dysmorphic  and  incom-­‐ plete  coronal  suture  in  the  late-­‐onset  synostosis   rabbit  skull  (middle  picture),  compared  to  a  normal   coronal  suture  in  the  wild-­‐type  rabbit  skull  (top   picture).   fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.78    http://dentistry3000.pitt.edu   implications  than  fluctuating  asym-­‐ metry.  tests  of  fluctuating  asymmetry   assumptions  were  carried  out  for   each  variable  within  each  group  sepa-­‐ rately.     for  both  md  and  bl  varia-­‐ bles,  the  phenotypically  normal  rab-­‐ bits  were  the  only  group  that  failed   the  test  of  normality  (p  <  0.05).  the   phenotypically  normal  (in-­‐colony)   control  group  was  therefore  dropped   from  subsequent  analyses.  for  the  bl   variable,  mean  raw  asymmetry  in  the   delayed-­‐onset  group  was  significantly   different  from  zero  (t  =  3.869;  p  <   0.001),  indicating  the  presence  of  di-­‐ rectional  asymmetry.  as  a  result,  the   delayed-­‐onset  group  was  dropped   from  further  analysis  of  the  bl  varia-­‐ ble.   because  random  measure-­‐ ment  error  is  statistically  identical  to   fluctuating  asymmetry,  the  degree  of   error  present  in  the  data  must  also  be   assessed.  repeat  measurements   were  obtained  on  all  176  animals  by   the  same  observer  (mah).  for   each  variable,  measurement   error  was  quantified  by  compu-­‐ ting  the  mean  absolute  differ-­‐ ence  (mad)  from  the  replicate   measures  [51]:   mean  absolute  difference  =  ∑   |time  1  –  time  2|  /  n   this  value  was  then   compared  directly  against  the   standard  unsigned  mean  fluc-­‐ tuating  asymmetry  index:   fluctuating  asymmetry  =  ∑   |left  –  right|  /  n   if  the  magnitude  of  the   mean  absolute  difference  is   determined  to  be  less  than  that   of  mean  fluctuating  asymmetry   for  a  given  variable,  this  indi-­‐ cates  that  residual  fluctuating   asymmetry  exists  even  after  meas-­‐ urement  error  is  taken  into  account.   conversely,  if  the  mean  absolute  dif-­‐ ference  is  greater  than  mean  fluctuat-­‐ ing  asymmetry,  then  we  cannot  be   certain  that  any  measurable  fluctuat-­‐ ing  asymmetry  exists.  for  both  varia-­‐ bles  in  the  present  study,  mean  abso-­‐ lute  difference  was  less  than  mean   fluctuating  asymmetry  (md:  0.05  vs.   0.07;  bl:  0.04  vs.  0.09).  this  pattern   held  true  both  overall  and  within   each  group  separately,  indicating  that   fluctuating  asymmetry  was  not   swamped  by  random  measurement   error.   for  some  biological  traits,  in-­‐ creased  trait  size  is  related  to  elevat-­‐ ed  levels  of  fluctuating  asymmetry.   thus,  the  effects  of  size  on  fluctuating   asymmetry  magnitude  must  be  con-­‐ sidered.  for  each  variable,  pearson   correlation  coefficients  were  calcu-­‐ lated  between  original  trait  values   and  unsigned  fa  values  (|left  –   right|).  because  a  weak  but  signifi-­‐ cant  correlation  (r  =  0.20;  p  <  0.01)   was  observed  for  the  md  variable,  an   alternative  size-­‐adjusted  fa  index  was   used  for  subsequent  group  compari-­‐ sons:   fluctuating  asymmetry  =  (|left  –   right|  /  0.5  left  +  right)     group  fluctuating  asymmetry   comparisons:   using  the  standard  size-­‐ adjusted  index,  mean  fluctuating   asymmetry  across  groups  was  com-­‐ pared  using  nonparametric  statistics,   followed  by  post-­‐hoc  comparisons   where  necessary.  the  nonparametric   approach  was  employed  because  the   fluctuating  asymmetry  index  chosen   for  group  comparisons  converts  left-­‐ right  differences  into  absolute  values,   which  by  nature  are  not  distributed   normally.  because  certain  groups   failed  to  meet  the  assumptions  of   fluctuating  asymmetry,  certain  group   comparisons  could  not  be  carried  out.   for  the  md  variable,  only  three   fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.78    http://dentistry3000.pitt.edu   groups  (early-­‐onset,  delayed-­‐onset,   and  wild-­‐type  controls)  were  com-­‐ pared  with  a  kruskal-­‐wallis  anova.   for  the  bl  variable,  only  two  groups   (early-­‐onset  and  wild-­‐type  controls)   were  compared,  thus  a  mann-­‐ whitney  u  t-­‐test  was  used.  statistical   analyses  were  performed  with  the   program  spss  11.5.  all  differences   were  considered  significant  if  p  <   0.05.   results     for  the  md  dimension,  rab-­‐ bits  with  early-­‐onset  synostosis  had   more  fluctuating  asymmetry  than   rabbits  with  delayed-­‐onset  synostosis   while  wild-­‐type  control  rabbits  had  a   greater  magnitude  of  fluctuating   asymmetry  than  either  affected   group.  however,  no  significant  (p  >   0.05)  group  differences  in  mean  fluc-­‐ tuating  asymmetry  were  observed  for   the  md  dimension  (table  2).     in   contrast  for  the  bl  dimension,  rabbits   with  complete  early-­‐onset  synostosis   had  a  significantly  (p  <  0.05)  greater   magnitude  of  fluctuating  asymmetry   than  wild-­‐type  control  rabbits  (table   2).  although  mean  age  differed  con-­‐ siderably  across  groups,  no  significant   correlation  (p>  0.05)  between  fa  and   age  was  observed  for  either  tooth   dimension.       discussion     familial,  nonsyndromic  crani-­‐ osynostosis  is  a  disease  characterized   by  abnormal  calvarial  bone  regulation   early  in  prenatal  life  [2,3,35-­‐37].  it   was  hypothesized  that  this  disruption   in  homeostasis  would  lead  to  in-­‐ creased  systemic  physiological  stress   during  the  critical  period  of  den-­‐ togenesis,  resulting  in  increased  fluc-­‐ tuating  dental  asymmetry.  results  of   the  present  study  confirm  this  hy-­‐ pothesis;  rabbits  with  early-­‐onset   synostosis  had  a  significantly  greater   magnitude  of  fluctuating  dental   asymmetry  for  the  bl  dimension  of   the  first  maxillary  molars  compared   to  unaffected  controls.  this  pattern   was  not  observed,  however,  for  the   md  dimension.  these  results  suggest   that  the  teeth  of  rabbits  with  congen-­‐ ital  nonsyndromic  craniosynostosis   develop  within  a  stressed  physiologi-­‐ cal  environment.  unfortunately,  be-­‐ cause  certain  groups  did  not  satisfy   the  statistical  requirements  of  fluctu-­‐ ating  asymmetry,  all  group  compari-­‐ sons  were  not  possible.  it  could  not   be  determined,  therefore,  whether   fluctuating  asymmetry  was  related  to   craniosynostosis  in  a  severity-­‐ dependent  manner.  these  results,   although  limited  in  scope,  suggest   that  a  broader  array  of  primary  de-­‐ fects  may  be  present  in  individuals   with  nonsyndromic  craniosynostosis,   and  that  these  may  not  be  limited   entirely  to  the  suture  site.  in  princi-­‐ ple,  a  fuller  understanding  of  the   phenotypic  correlates  present  in  non-­‐ syndromic  craniosynostosis  may  shed   light  on  disease  etiology.  for  in-­‐ stance,  predictions  can  be  generated   about  patterns  of  phenotypic  varia-­‐ tion  from  the  known  expression  pat-­‐ terns  of  specific  candidate  genes.     prior  to  the  present  study,   dental  fluctuating  asymmetry  had  not   been  examined  in  individuals  affected   with  craniosynostosis.  previous  re-­‐ search  into  dental  development  with-­‐ in  the  craniosynostoses  has  been  fo-­‐ cused  almost  entirely  on  syndromic   forms  of  the  disease.  in  apert  syn-­‐ drome,  for  instance,  a  wide  variety  of   dental  anomalies  have  been  report-­‐ ed;  these  include  delayed  develop-­‐ ment,  ectopic  teeth,  hypodontia  and   supernumerary  teeth  [52,53].  similar-­‐ ly,  in  cases  of  crouzon  syndrome,  ec-­‐ topic  eruption,  delayed  development   and  hypodontia  have  all  been  ob-­‐ served  [54,55,12].  pfeiffer  syndrome   may  be  characterized  by  natal  teeth   [56],  suggesting  precocious  dental   development.  this  is  further  support-­‐ ed  by  reports  of  supernumerary  and   ectopic  teeth  [57].  given  the  fact  that   fgfs  play  a  role  in  both  dental  devel-­‐ opment  and  suture  morphogenesis,  it   is  perhaps  not  surprising  that  the   teeth  are  affected  in  cases  of  syn-­‐ fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.78    http://dentistry3000.pitt.edu   dromic  craniosynostosis  [39,11,58].  in   light  of  this  overlap  in  molecular  ex-­‐ pression,  these  conspicuous  dental   defects  are  most  likely  the  result  of   aberrant  fgf  signaling  within  the  de-­‐ veloping  dentition  and  its  surround-­‐ ing  tissues  [40].     it  remains  unclear,  however,   whether  the  observed  pattern  of  den-­‐ tal  fluctuating  asymmetry  results   from  such  pleiotropic  effects.  be-­‐ cause  fluctuating  asymmetry  is  a  ra-­‐ ther  generic  response  to  stress  during   development,  we  can  only  speculate   as  to  whether  the  underlying  cause  of   the  fluctuating  asymmetry  is  the   same  as  that  of  the  craniosynostosis.   dental  dimensions  have  been  shown   to  be  highly  heritable  and  are  thought   to  be  under  relatively  tight  genetic   control  [59-­‐61].  that  being  said,  it  is   also  clear  that  environmental  influ-­‐ ences  such  as  local  and  systemic   physiological  factors,  teratogens,  and   extra-­‐organismic  variables  play  a  crit-­‐ ical  role  in  determining  the  final  form   and  size  of  teeth,  especially  during   the  early  stages  of  dental  develop-­‐ ment  [62,63].  increased  fluctuating   dental  asymmetry  has  been  reported   in  a  number  of  naturally  and  experi-­‐ mentally  stressed  human  and  non-­‐ human  populations,  although  the   source  of  the  stress  is  sometimes  not   well  defined  [24,  27,  28,  64-­‐70].     a  number  of  possible  expla-­‐ nations  for  the  present  findings  may   be  offered.    one  possibility  is  that  the   same  genetic  mutation  that  causes   premature  suture  fusion  also  results   in  increased  dental  fluctuating  asym-­‐ metry.  the  genetic  mutation  in  these   rabbits  has  been  identified  recently  as   belonging  to  the  fgfr  family  of  genes   working  in  tandem  with  a  modifying   gene  (manuscript    in   preparation)  so  this   may  be  a  possibility;   however,  no  obvious   dental  anomalies   have  been  observed   in  this  model,  sug-­‐ gesting  that  overall   dentogenesis  is   probably  not  affect-­‐ ed  in  these  rabbits.     accumulating  evi-­‐ dence  also  suggests   that  altered  calvarial   bone  regulation,   resulting  in  hyperos-­‐ tosis  and  subsequent   premature  suture   fusion  in  cases  of   nonsyndromic  crani-­‐ osynostosis,  stems  from  localized,   abnormal  expression  of  various  bone   regulating  growth  factors  (e.g.,  bmps,   fgfs,  and/or  tgfβs)  [1-­‐3,  36].  such   abnormal  growth  factor  expression   may  also  primarily  affect  other  devel-­‐ oping  antimeric  osseous  structures   resulting  in  fluctuating  asymmetry.   however,  deleon  and  richtsmieir   [38]  reported  no  significant  fluctuat-­‐ ing  asymmetry  in  the  crania  of  22   children  with  nonsyndromic  sagittal   synostosis,  although  the  effects  could   have  been  ameliorated  with  age  and   remodeling.  in  addition,  no  significant   changes  in  postcranial  skeletal  (e.g.,   first  metacarpal)  growth  have  been   observed  in  synostotic  rabbits  [41-­‐ 45],  although  fluctuating  skeletal   asymmetry  has  not  been  examined   yet  and  this  mechanism  remains  a   possibility.     another  possibility  is  that  in-­‐ creased  intracranial  pressure  result-­‐ ing  from  premature  suture  fusion   could  invoke  a  generalized  stress  re-­‐ sponse  by  activating  the  hypothalam-­‐ ic-­‐pituitary-­‐adrenal  axis,  which  may   increase  levels  of  fluctuating  calcium   during  dentogenesis  [65,71],  and  sec-­‐ ondarily  result  in  increased  dental   fluctuating  asymmetry  in  rabbits  with   early-­‐onset  synostosis.  increased  fluc-­‐ tuating  asymmetry  has  also  been   linked  to  external  environmental   stressors  such  as  heat,  cold  and  noise   [64,65,67].  however,  these  factors   are  unlikely  to  play  a  major  role  in   this  study  because  all  the  rabbits  in   the  present  sample  were  raised  under   similar  controlled,  environmental   conditions.  it  is  also  unlikely  that   functional  wear  effects  account  for   these  results  since  such  effects  are   typically  associated  with  directional   asymmetries  and  should  increase   with  age  (no  association  between  age   and  fluctuating  asymmetry  was  ob-­‐ served).  in  addition,  rabbit  teeth  are   continually  erupting  and  the  lack  of   age  related  differences  in  dental  fluc-­‐ tuating  asymmetry  suggest  that  the   stress  associated  with  craniosynosto-­‐ sis  may  be  chronic  in  rabbits  with  ear-­‐ ly-­‐onset  synostosis.    finally,  canaliza-­‐ tion  of  dental  development  may  be   under  separate  genetic  control.  un-­‐ der  this  premise,  less  canalized  organ-­‐ isms  would  be  predisposed  to  mani-­‐ festing  elevated  dental  fluctuating   asymmetry  in  the  presence  of  delete-­‐ rious  factors,  such  as  aberrant  growth   factor  signaling.  we  recently  reported   figure  3.    inferior  view  of  the  rabbit  skull  showing  the  post-­‐ incisor  maxillary  dentition  (p1-­‐m3)  (first  three  premolars  and   molars).    the  arrows  on  m1  (first  molar)  represent  the  mesi-­‐ odistal  and  buccolingual  dimensions  that  were  measured  in  the   present  study.   fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.78    http://dentistry3000.pitt.edu   a  significant,  negative  relationship   between  coronal  suture  growth  and   the  coefficient  of  inbreeding  [46]  in   rabbits  from  this  colony.  rabbits  with   early  onset  synostosis  showed  the   least  growth  and  greatest  amount  of   inbreeding.  it  is  possible  that  addi-­‐ tional  modifier  genes  accumulated   from  inbreeding  were  responsible  for   increased  systemic  stress  and  the  in-­‐ creased  levels  of  fluctuating  dental   asymmetry  in  these  rabbits.  hueze  et   al.  [72]  also  showed  that  human  cra-­‐ nia  exhibiting  a  phenotypic  continu-­‐ um  with  skulls  with  bicoronal  sutures   synostosis  displaying  more  intense   asymmetry  that  skulls  with  unicoro-­‐ nal  shape  which  supports  this  hy-­‐ pothesis.   unfortunately,  the  present   study  was  not  designed  to  test  these   competing  hypotheses.  fluctuating   asymmetry  is  a  subtle  marker  of  de-­‐ velopmental  disturbance  and  its  per-­‐ vasiveness  both  in  the  craniodental   complex  and  other  anatomical  re-­‐ gions  of  craniosynostotic  organisms   will  need  to  be  explored  further  in   order  to  begin  to  unravel  its  biological   basis.    given  the  numerous  reports  of   various  dental  defects  associated   with  syndromic  craniosynostosis,  it   may  be  worthwhile  to  investigate   other  aspects  of  the  dentition  in  non-­‐ syndromic  synostotic  populations  as   well.   acknowledgements   the  authors  would  like  to   thank  kate  quinlin  for  her  assistance   in  the  lab.  this  work  was  supported  in   part  by  a  grant  from  nih/nidcr  (p60   de13078).   references   1. cohen  mm  (2000)  history,   terminology,  and  classifica-­‐ tion  of  craniosynostosis.   in:  cohen  mm,  maclean   re,  editors.    cranio-­‐ synostosis:  diagnosis,  eval-­‐ uation,  and  management.   new  york:  oxford  universi-­‐ ty  press.  pp.  1-­‐20.   2. opperman  la,  ogle  rc   (2002)  molecular  studies  of   craniosynostosis:  factors   affecting  cranial  suture   morphogenesis  and  paten-­‐ cy.  in:  mooney  mp,  siegel   mi,  editors.  understanding   craniofacial  anomalies:  the   etiopathogenesis  of  crani-­‐ osynostoses  and  facial   clefting.  new  york:  wiley-­‐ liss,  inc.  497-­‐517  p.   3. mooney  mp,  cray  jj  jr,   losee  je,  moursi  am,   siegel  mi,  cooper  gm   (2012)  the    development   of  molecular  therapies  for   craniosynostosis.  in:   schachner  p,  editor.  pro-­‐ ceedings  of  the  9th  euro-­‐ pean  craniofacial  con-­‐ gress.  bologna  italy:  mon-­‐ duzzi  editore.  pp.  9-­‐18.   4. genetics  of  craniosynosto-­‐ sis:  genes,  syndromes,  mu-­‐ tations  and  genotype-­‐ phenotype  correlations.   passos-­‐bueno  mr,  serti   eacute  aeal,  jehee  fs,   fanganiello  r,  yeh  e.  front   oral  biol.  2008;12:107–43.   pmid:  18391498.   5. prevalence  and  complica-­‐ tions  of  single-­‐gene  and   chromosomal  disorders  in   craniosynostosis.  wilkie   aom,  byren  jc,  hurst  ja,   jayamohan  j,  johnson  d,   knight  sjl,  lester  t,  rich-­‐ ards  pg,  twigg  srf,  wall   sa.  pediatr.    2010;  126:   e391–400.  pmid:   20643727.   6. prevalence  of  pro250arg   mutation  of  fibroblast   growth  factor  receptor  3  in   coronal  synostosis.  molo-­‐ ney  dm,  wall  sa,  ashworth   gj,  oldridge  m,  glass  ia,   francomano  ca,  muenke   m,  wilkie  aom.  lancet.   1997;349:1059-­‐62.  pmid:   9107244.   7. craniosynostosis  associat-­‐ ed  with  fgfr3  pro250arg   mutation  results  in  a  range   of  clinical  presentations  in-­‐ cluding  unisutural  sporadic   craniosynostosis.  reardon   w,  wilkes  d,  rutland  p,   pulleyn  lj,  malcolm  s,   dean  jcs,  evans  rd,  jones   bm,  hayward  r,  hall  cm.  j   med  genet.  1997;34:632-­‐ 6.  pmid:  9279753.   8. identification  of  the  first   genetic  cause  for  isolated   unilateral  coronal  synosto-­‐ sis:  a  unique  mutation  in   the  fibroblast  growth  fac-­‐ tor  receptor  3  (fgfr3).   gripp  kw,  mcdonald-­‐ mcginn  dm,  gaudenz  k,   whitaker  la,  bartlett  sp,   glat  pm,  cassileth  lb,  may-­‐ ro  r,  zackai  eh,  muenke  m.   j  pediatr.  1998;132:714-­‐6.   pmid:  9580776.   9. genetic  study  of  nonsyn-­‐ dromic  coronal  cranio-­‐ fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.78    http://dentistry3000.pitt.edu   synostosis.  lajeunie  e,  le   merrer  m,  bonaiti-­‐pellie  c,   marchac  d,  renier  d.  am  j   med  genet.  1995;55:500-­‐ 4.  pmid:  7762595.   10. genetic  study  of  scapho-­‐ cephaly.  lajeunie  e,  le   merrer  m,  bonaiti-­‐pellie  c,   marchac  d,  renier  am  j   med  genet.  1996;62:282-­‐ 5.  pmid:  8882788.   11. from  genotype  to  pheno-­‐ type:  the  differential  ex-­‐ pression  of  fgf,  fgfr,  and   tgfβ  genes  characterizes   human  cranioskeletal  de-­‐ velopment  and  reflects   clinical  presentation  in   fgfr  syndromes.  britto  ja,   evans  rd,  hayward  rd,   jones  bm.  plast  reconstr   surg.  2001;108:2026-­‐39.   pmid:  11743396.   12. crouzonodermoskeletal   syndrome.  jeftha  a,  ste-­‐ phen  l,  morkel  ja,  beigh-­‐ ton  p.  j  clin  pediatr  dent.   2004;28:173-­‐6.  pmid:   14969379.   13. a  study  of  fluctuating   asymmetry.  van  valen  l.   evol.  1962;16:125-­‐142.   14. genetical  control  of  stabil-­‐ ity  in  development.  mather   k.  heredity.  1953;7:297-­‐ 336.   15. canalisation  of  develop-­‐ ment  and  the  inheritance   of  acquired  characters.   waddington  ch.  nature.   1942;150:563-­‐5.   16. waddington  ch.  (1957)the   strategy  of  the  genes.   london:  allen  and  unwin.   115  p.   17. fluctuating  asymmetry:  a   biological  monitor  of  envi-­‐ ronmental  and  genomic   stress.  parsons  pa.  heredi-­‐ ty.  1992;68:361-­‐364.  pmid:   1563968.   18. møller  ap,  swaddle  jp   (1990)  asymmetry,  devel-­‐ opmental  instability,  and   evolution.  oxford:  oxford   university  press.  56  p.   19. markow  ta  (1994)  devel-­‐ opmental  instability:  its  or-­‐ igins  and  evolutionary  im-­‐ plications.  dordrecht:   kluwer  academic  publish-­‐ ers.  102  p.     20. polak  m  (2003)  develop-­‐ mental  instability:  causes   and  consequences.  oxford:   oxford  university  press.  87   p.   21. developmental  stability,   disease  and  medicine.   thornhill  r,  møller  ap.  biol   rev  camb  philos  soc.   1997;72:497-­‐548.  pmid:   9375532.   22. congenital  cleft  lip  and   fluctuating  dermatoglyphic   asymmetry.  woolf  cm,   gianas  ad.  am  j  hum   genet.  1976;28:400-­‐3.   pmid:  941907.   23. a  study  of  fluctuating   asymmetry  in  the  sibs  and   parents  of  cleft  lip  proposi-­‐ ti.  woolf  cm,  gianas  ad.   am  j  hum  genet.   1977;29:503-­‐7.  pmid:   900124.   24. human  tooth-­‐size  asym-­‐ metry  in  cleft  lip  with  or   without  cleft  palate.  sofaer   ja.  arch  oral  biol.   1979;24:141-­‐6.  pmid:   299139.   25. cleft  lip  with  or  without   cleft  palate  and  dermato-­‐ glyphic  asymmetry:  evalua-­‐ tion  of  a  chinese  popula-­‐ tion.  neiswanger  k,  cooper   me,  weinberg  sm,  flod-­‐ man  p,  keglovitz  ab,  liu  y,   hu  dn,  melnick  m,  spence   ma,  marazita  ml  orthod   craniofacial  res.   2002;5:140-­‐6.  pmid:   12194662   26. down  syndrome—a  disrup-­‐ tion  of  homeostasis.   shapiro  b.  am  j  med   genet.  1983;14:241–269.   pmid:  6220605.   27. fluctuating  dental  asym-­‐ metry:  a  measure  of  devel-­‐ opmental  instability  in   down  syndrome.  barden   hs.  am  j  phys  anthropol.   1980;52:169-­‐173.  pmid:   6445164.   28. crown  size  asymmetry  in   males  with  fra  (x)  or  mar-­‐ tin-­‐bell  syndrome.  peretz   b,  ever-­‐hadani  p,   casamassimo  p,  eidelman   e,  shellhart  c,  hagerman  r.   am  j  med  genet.   1988;30:185-­‐190.  pmid:   2972204.   fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.78    http://dentistry3000.pitt.edu   29. decreased  developmental   stability  as  assessed  by   fluctuating  asymmetry  of   morphometric  traits  in  pre-­‐ term  infants.  livshits  g,   davidi  l,  kobyliansky  e,   ben-­‐amitai  d,  levi  y,  mer-­‐ lob  p.  am  j  med  genet.   1988;29:793–805.  pmid:   3400724.   30. left-­‐right  asymmetries  in   embryonic  development:  a   comprehensive  review.   levin  m.  mech  dev.  2005;   122:3–25.  pmid:   15582774.   31. attention-­‐deficit  hyperac-­‐ tivity  disorder  (ad/hd)  and   fluctuating  asymmetry  in  a   college  sample:  an  explora-­‐ tory  study.  burton  c,  ste-­‐ venson  jc,  williams  dc,   everson  er,  trimble  je.  am   j  hum  biol.  2003;15:601-­‐ 19.  pmid:  12953172.   32. anthropometric  asym-­‐ metry  in  normal  and  men-­‐ tally  retarded  males.  ma-­‐ lina  rm,  buschang  ph.  ann   hum  biol.  1984;11:515-­‐31.   pmid:  6524867.   33. fluctuating  asymmetry  and   vertebral  malformation:  a   study  of  palmar  dermato-­‐ glyphics  in  congenital  spi-­‐ nal  deformities  goldberg   cj,  fogarty  ee,  moore  dp,   dowling  fe.  spine.   1997;22:775-­‐9.  pmid:   9106319.   34. psychological  perspective   on  human  developmental   stability  and  fluctuating   asymmetry:  sources,  appli-­‐ cations  and  implications.   kowner  r.  brit  j  psychol.   2001;92:447-­‐69.  pmid:   11802884.   35. cohen  mm.  sutural  pa-­‐ thology  (2000)  in:  cohen   mm,  maclean  re,  editors.   craniosynostosis:  diagno-­‐ sis,  evaluation,  and  man-­‐ agement.  new  york:  ox-­‐ ford  university  press.  pp.   51-­‐68.   36. transforming  growth  fac-­‐ tor-­‐β  isoform  expression  in   the  perisutural  tissues  of   craniosynostotic  rabbits.   poisson  e,  sciote  jj,   koepsel  r,  cooper  gm,   opperman  la,  mooney   mp.  cleft  palate  craniofac   j.  2004;41:392-­‐402.  pmid:   15222795.   37. coronal  suture  pathology   and  synostotic  progression   in  rabbits  with  congenital   craniostenosis.  mooney   mp,  smith  td,  langdon  hl,   burrows  am,  stone  ce,   losken  hw,  siegel  mi.  cleft   palate-­‐craniofac  j.  1996;   33:  369-­‐78.  pmid:   8891367.   38. fluctuating  asymmetry  and   developmental  instability   in  sagittal  craniosynostosis.   de  leon  vb,  richtsmeier   jt.  cleft  palate-­‐craniofac  j.   2009;  46:  187–96.  pmid:   19254065.   39. genotype,  phenotype,  and   developmental  biology  of   molar  tooth  characters.   jernvall  j,  jung  hs.  am  j   phys  anthropol.   2000;31(suppl):171-­‐190.   pmid:  11123840.   40. harris  ef  (2002)  dental  de-­‐ velopment  and  anomalies   in  craniosynostoses  and  fa-­‐ cial  clefting.  in:  mooney   mp,  siegel  mi,  editors.  un-­‐ derstanding  craniofacial   anomalies:  the  etiopatho-­‐ genesis  of  craniosynosto-­‐ ses  and  facial  clefting.   new  york:  wiley-­‐liss,  inc.   pp.  425-­‐467.   41. craniosynostosis  with  au-­‐ tosomal  dominant  trans-­‐ mission  in  new  zealand   white  rabbits.  mooney   mp,  aston  ce,  siegel  mi,   losken  hw,  smith  td,  bur-­‐ rows  am,  wenger  sl,  ca-­‐ ruso  k,  siegel  b,  ferrell  re.   j  craniofac  genet  dev  biol.   1996;16:52-­‐63.  pmid:   8675615.   42. development  of  a  strain  of   rabbits  with  congenital   simple,  nonsyndromic  cor-­‐ onal  suture  synostosis.  part   ii:  somatic  and  craniofacial   growth  patterns.  mooney   mp,  losken  hw,  siegel  mi,   lalikos  jf,  losken  a,  bur-­‐ rows  a,  smith  td.  cleft   palate  craniofac  j.   1994;31:8-­‐16.  pmid:   8130248.   43. development  of  a  strain  of   rabbits  with  congenital   simple,  nonsyndromic  cor-­‐ onal  suture  synostosis.  part   i:  breeding  demographics,   fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.78    http://dentistry3000.pitt.edu   inheritance  pattern,  and   craniofacial  anomalies.   mooney  mp,  losken  hw,   siegel  mi,  lalikos  jf,   losken  a,  smith  td,  bur-­‐ rows  a.  cleft  palate  crani-­‐ ofac  j.  1994;31:1-­‐7.  pmid:   8130237.   44. a  rabbit  model  of  human   familial,  nonsyndromic   unicoronal  suture  synosto-­‐ sis.  part  i:  synostotic  onset,   pathology,  and  sutural   growth  patterns.  mooney   mp,  siegel  mi,  burrows   am,  smith  td,  losken  hw,   dechant  j,  cooper  g,   kapucu  mr.  childs  nerv   syst.  1998;14:236-­‐246.   pmid:  9694335.   45. mooney  mp,  siegel  mi,   opperman  la  (2002)  ani-­‐ mal  models  of  cranio-­‐ synostosis:  experimental,   congenital,  and  transgenic.   in:  mooney  mp,  siegel  mi,   editors.  understanding   craniofacial  anomalies:  the   etiopathogenesis  of  crani-­‐ osynostoses  and  facial   clefting.  new  york:  wiley-­‐ liss,  inc.  pp.251-­‐272.   46. genetic  homozygosity  and   phenotypic  variability  in   craniosynostotic  rabbits.     gilbert  j,  cray  jj  jr,   kriethen  a,  marazita  ml,   cooper  gm,  losee  je,   siegel  mi,  mooney  mp.   cleft  palate  craniofac  j.   2017  jan;54(1):94-­‐9.  pmid:   26882022.   47. craniosynostosis:  novel  in-­‐ sights  into  pathogenesis   and  treatment.  wilkie   aom,  wall  sa  curr  opin   neurol.  1996;9:146-­‐52.   pmid:  8782984.   48. kieser  ja  (1990)  human   adult  odontometrics.   cambridge:  cambridge   university  press.  237  p.     49. palmer  ar(1994)  fluctuat-­‐ ing  asymmetry  analyses:  a   primer.  in:  markow  ta,  ed-­‐ itor.  developmental  insta-­‐ bility:  its  origins  and  evolu-­‐ tionary  implications.  dor-­‐ drecht:  kluwer  academic   publishers.  pp.  335-­‐64.   50. fluctuating  asymmetry:   measurement,  analysis,   patterns.  palmer  ar,  stro-­‐ beck  c.  annu  rev  ecol  syst.   1986;17:391-­‐421.   51. interobserver  error  in  a   large  scale  anthropometric   survey.  gordon  cc,   bradtmiller  b.  am  j  hum   biol.  1992;4:253-­‐63.  pmid:   28524349.   52. the  oral  manifestations  of   apert  syndrome.  kreiborg   s,  cohen  mm.  j  craniofac   genet  dev  biol.   1992;12:41-­‐48.  pmid:   1572940.   53. dental  development  in   apert  syndrome.  kaloust  s,   kazuhiro  i,  vargervik  k.   cleft  palate  -­‐  craniofac  j.   1997;34:117-­‐21.  pmid:   9138505.   54. oral  manifestations  of   crouzon's  disease.  kelln   ec,  chaudhry  ap,  gorlin   fj.oral  surg  oral  med  oral   pathol.  1960;13:1245-­‐8.   pmid:  13752352.   55. partial  anodontia  of  both   the  deciduous  and  perma-­‐ nent  dentitions  in  a  case  of   crouzon's  disease.  stein   gm,  wahl  h.  oral  surg  oral   med  oral  pathol.   1969;28:808-­‐12.  pmid:   5260651.   56. natal  molars  in  pfeiffer   syndrome  type  3:  a  case   report.  alvarez  mp,  crespi   pv,  shanske  al.  j  clin  pedi-­‐ atr  dent.  1993;18:21-­‐4.   pmid:  8110608.   57. pfeiffer  syndrome:  report   of  a  family  and  review  of   the  literature.  naveh  y,   friedman  a.  j  med  genet.   1976;13:277-­‐80.  pmid:   957376.   58. evolution  and  develop-­‐ ment  of  teeth.  mccollum   ma,  sharpe  pt.  j  anat.   2001;199:153-­‐9.  pmid:   11385628.   59. genetic  covariance  struc-­‐ ture  of  incisor  crown  size  in   twins.  dempsey  pj,  town-­‐ send  gc,  martin  ng,  neale   mc.  j  dent  res.   1995;74:1389-­‐98.  pmid:   7560390.   60. genetic  analysis  of  decidu-­‐ ous  tooth  size  in  australian   twins.  hughes  t,  dempsey   p,  richards  l,  townsend  g.   fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis     vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.78    http://dentistry3000.pitt.edu   arch  oral  biol.   2000;45:997-­‐1004.  pmid:   11000386.   61. genetic  and  environmental   contributions  to  variation   in  human  tooth  size.   dempsey  p,  townsend  gc.   heredity.  2001;86:685-­‐93.   pmid:  11595049.   62. the  effect  of  environmen-­‐ tal  factors  on  tooth  devel-­‐ opment:  an  experimental   investigation.  riesenfeld  a.   acta  anat  (basel).   1970;77:188-­‐215.  pmid:   5508460.   63. normal  development  of   the  jaws  and  teeth  in  pigs,   and  the  delay  and  maloc-­‐ clusion  produced  by  calorie   deficiencies.  tonge  ch,   mccance  ra.  j  anat.   1973;115:1-­‐22.  pmid:   4199500.   64. fluctuating  dental  asym-­‐ metry  and  audiogenic   stress.  siegel  mi,  smookler   hh.  growth.  1973;37:35-­‐9.   pmid:  4700428.   65. heat  stress,  fluctuating   asymmetry  and  prenatal   selection  in  the  laboratory   rat.  siegel  mi,  doyle  wj,   kelley  c.  am  j  phys  an-­‐ thropol.  1977;46:121-­‐6.   pmid:  556888.   66. on  the  meaning  of  in-­‐ creased  fluctuating  dental   asymmetry:  a  cross  popu-­‐ lational  study.  doyle  wj,   johnston  o.  am  j  phys  an-­‐ thropol.  1977;46:127-­‐34.   pmid:  319679.   67. the  effects  of  cold  stress   on  fluctuating  asymmetry   in  the  dentition  of  the   mouse.  siegel  mi,  doyle   wj.  j  exp  zool.   1975;193:385-­‐9.  pmid:   1176912.   68. fluctuating  asymmetry  in   molar  dimensions  and  dis-­‐ crete  morphological  traits   in  pima  indians.  noss  jf,   scott  gr,  potter  rhy,   dahlberg  aa.  am  j  phys   anthropol.  1983;61:437-­‐ 45.  pmid:  6624887.   69. fluctuating  dental  asym-­‐ metry  as  a  measure  of   odontologic  canalization  in   man.  kieser  ja,  groeneveld   ht,  preston  cs.  am  j  phys   anthropol.  1986;71:437-­‐ 44.  pmid:  3812659.   70. comparison  of  fluctuating   asymmetry  level  between   normal  and  pathological   specimens  from  modern   thai  skeletal  group.  jung  h,   woo  ej,  von  cramon-­‐ taubadel  n.  amer  j  phys   anthropol.  2017;  162   (s64):  136.   71. prenatal  stress  and  in-­‐ creased  fluctuating  asym-­‐ metry  in  the  parietal  bones   of  neonatal  rats.  mooney   mp,  siegel  mi,  gest  tr.  am   j  phys  anthropol.   1985;68:131-­‐4.  pmid:   4061598.   72. unilateral  and    bilateral   expression  of  a  quantita-­‐ tive  trait:  asymmetry  and   symmetry  in  coronal  crani-­‐ osynostosis.  heuzé  y,  mar-­‐ tínez-­‐abadías  n,  stella  jm.,   senders  cw,  boyadjiev  sa,   lo  l-­‐j,  richtsmeier  jt.  j  exp   zool  b  mol  dev  evol.  2012;   318:  109–122.  pmid:   22532473.   evaluation of the most common dental procedural errors leading to lawsuits and the relevant reasons in three dental clinics in tehran vol 9 no 1 (2021) doi 10.5195/d3000.2021.139 http://dentistry3000.pitt.edu evaluation of the most common dental procedural errors leading to lawsuits and the relevant reasons in three dental clinics in tehran mohsen yazdanian1, esmail rafiei11,2, aghil rahmani3, elahe tahmasebi1*, abbas hajiesfandiari4 1 research center for prevention of oral and dental diseases, baqiyatallah university of medical sciences, tehran, iran 2 school of dentistry, baqiyatallah university of medical sciences, tehran, iran 3 dental materials research center, health research institute, babol university of medical sciences, babol, iran 4 faculty of management, azad university, tehran central branch, tehran, iran abstract one of the most important occupational stresses that dentists encounter in their profession is patients’ lawsuits. the present study aimed to evaluate common procedural errors that lead to lawsuits in dentistry. in the present cross-sectional study, all the patient files of three large dental clinics in tehran were evaluated from 2014 to 2019, in which a dentist’s negligence had been confirmed by the specialty dental committee. files with incomplete clinical data, without the dentist’s name, and the type of the lawsuit were excluded. after extraction of the data from the existing files and completing the relevant checklist, data were analyzed with spss 23. most lawsuits were of the therapeutic type (82%), with 31.9%, 24.2%, and 17.6% related to prosthodontics, endodontics, and surgery, respectively. most plaintiffs were 30–49 years of age (52%), and most were women (65%). over half of the lawsuits had been filed against dentists with a job experience of <10 years and against general dental practitioners (76%). one of the most important ways to prevent errors leading to lawsuits is to increase dentists’ knowledge about previous lawsuits. knowledge about the most common lawsuits and the provision of organizational guidelines will help manage and decrease such lawsuits. keywords: dentistry; error; patient complaints. citation: yazdanian m, et al. (2021) evaluation of the most common dental procedural errors leading to lawsuits and the relevant reasons in three dental clinics in tehran dentistry 3000. 1:a001 10.5195/d3000.2021.139 received: january 17, 2021 accepted: february 09, 2021 published: august 18, 2021 copyright: ©2021 yazdanian m, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: elahe.tahmasebi.delfan@gmail.com introduction human errors are one of the most important current issues in the medical and dental profession; therefore, it is of utmost importance to identify and decrease these errors [1]. human errors are defined as the deviation of human performance from the regulations and the defined responsibilities that go beyond the system’s accepted norm, negatively affecting the system’s efficacy [2]. occupational responsibilities and their complexity might, in some cases, affect human performance and behavior in some occupations, resulting in procedural errors and unsafe procedures. these errors and their unfavorable consequences will negatively affect the operator’s and other’s health [3]. based on a report by the american institute of medicine in 2008, on average, one million people are hurt all over the world annually due to medical errors, with an annual cost of $17,000,000[4, 5]. based on the who report, of every 10 people, one is affected by medical errors [6]. recent studies on medical errors have shown that the reasons for 88% of the procedural errors are unsafe procedures, 10% due to unsafe http://dentistry3000.pitt.edu/ evaluation of the most common dental procedural errors leading to lawsuits and the relevant reasons in three dental clinics in tehran vol 9 no 1 (2021) doi 10.5195/d3000.2021.139 http://dentistry3000.pitt.edu conditions and 2% due to unknown factors [7]. various surveys worldwide have shown that human errors in the medical profession have inflicted high financial and human costs in different communities. procedural errors by the medical staff and dentists lead to a lack of confidence among the community members in this personnel, increases stress in patients, and increases lawsuits against the medical and dental personnel [4, 5]. errors in different occupations do not have a single reason and usually occur due to several reasons. they are usually the result of errors in the system and latent errors [8]. kiani et al (2009) investigated dental patients’ lawsuits in tehran. their retrospective study evaluated dental patients’ lawsuits in tehran from 2002 to 2006 according to the decision of the specialty committee on medical negligence of tehran medical council. the results showed that during the 5-year period, 412 lawsuits had been filed against dentists, the majority of which were related to fixed prosthodontics and oral surgery treatments. besides, the majority of complaints were related to therapeutic activities in the private sector. most lawsuits were against general dental practitioners, and in 56.7% of the clinical files and 40% of the nonclinical files, the dentists had been convicted [9]. pereail et al (2014) determined the characteristics of dental procedural errors and classified them in terms of origin and type. they evaluated 4159 lawsuit filed due to dental errors from 2000 to 2910 in spain. the results showed that implant treatments, root canal therapy, and oral surgeries comprised the most common dental procedural errs (25.5%, 20.7%, and 20.4%, respectively). besides, 44.3% of the errors were due to predictable errors and could be prevented [10]. prevention of errors increases patient and personnel satisfaction, decreases surgery costs, and increases the validity, and decreases stress in healthcare providers. in most organizations, the prevention costs are significantly less than the costs of procedural accidents [11]. some errors during dental procedures are due to human errors, and some are due to other factors, including the environmental conditions, dental tools, and equipment, and the system prevailing in dentistry, finally resulting in patient injuries [12]. despite the limited studies on the most common errors leading to lawsuits in dentistry, no study has evaluated the details and the exact nature of these errors and the relationship between different types of errors and the patients’ and clinicians’ demographic factors in each dental specialty. therefore, the present study aimed to evaluate errors leading to lawsuits against dental practitioners and their etiologic factors so that methods can be suggested to prevent these errors and lawsuits. material and methods study design in the present cross-sectional study, all the lawsuit files (n=462) in imam khomeini, shahid chamran, and shahid shokri dental clinics in tehran were collected from 2014 to 2019. a total of 120,000 patients refer to these three clinics annually. the inclusion criteria consisted of dentists’ negligence cases confirmed by the special committee of dental specialists, consisting of 280 files. the exclusion criteria consisted of files with incomplete clinical data, a lack of the dentist’s name and his/her demographic data, and a lack of the plaintiff’s demographic data. after the application of http://dentistry3000.pitt.edu/ evaluation of the most common dental procedural errors leading to lawsuits and the relevant reasons in three dental clinics in tehran vol 9 no 1 (2021) doi 10.5195/d3000.2021.139 http://dentistry3000.pitt.edu inclusion and exclusion criteria, 107 files were finally evaluated. data collection tool a researcher-made checklist was used to collect data and research variables. content validity was used since the aim was to design a special checklist to extract data and the demographic variables of the clinicians and patients (as plaintiffs). first, previous studies on the most common errors in the dental clinics and hospitals were reviewed to prepare a list of required parameters. then, some of the files with confirmed dental errors were thoroughly reviewed to complete the checklist, consisting of plaintiffs’ demographic data (age, gender, education, insurance), the clinicians’ academic degree/level, clinicians’ gender, clinicians’ experience, the error location, error type, the steps taken to compensate for the error and the shift of the error (morning/afternoon). three specialists evaluated the checklist, including a healthcare specialist (kb), an oral disease specialist (er), and a healthcare management specialist (er), and the necessary amendments were made and confirmed to validate the tool. the researchers were then granted permission by the three clinics mentioned above to gain access to the relevant files, and each file was evaluated in the presence of the deputy dean for treatment of each clinic. the study was carried out for 14 months, and the data were collected in two months. after extracting the required data from the available files and completing the checklists, the data were analyzed with spss 23. ethics the ethics committee of the baqiatollah university of medical sciences approved the protocol of the study. for the sake of confidentiality, the files were evaluated in the presence of the deputy dean for treatment of each clinic, and by only completing the designed checklist. results of 107 lawsuit files confirmed by the special committee of dental specialists from 2014 to 2019 in the three large dental clinics in tehran, 88 files were related to treatment errors (82.2%), and 19 files were related to diagnostic errors (17.7%). the most frequent reason for filing lawsuits was the root perforation during post space preparation (13 cases, 12%), extracting the wrong tooth (9 cases, 8.4%), implant fixture loosening (9 cases, 8.4%), and root canal perforation, resulting in abscess formation and tooth loss (8 cases, 7.4%). of all the lawsuits related to treatment, the most frequent one was related to the root perforation during the preparation of post spaces (13 cases, 12%). of all the complaints related to diagnosis, the most frequent complaint was related to unjustified and unnecessary crown lengthening procedures (interdisciplinary error) (6 cases, 5.6%) (table 1). evaluation of the frequencies of the evaluated variables showed that 67 (62.6%) of the plaintiffs were female, and 40 (37.2%) were male. more than half of the plaintiffs were in the 30–49 age range, and only 19.7% of them were <30 years of age. the youngest plaintiff was 21, and the oldest was 81; 10% of the plaintiffs had some high school education, 66% were high school graduates and had associate’s and bachelor's degrees, and 24% had masters and doctorate degrees. 67% of the plaintiffs had insurance coverage, and 33% had no insurance coverage. of all the dental practitioners evaluated, 45% were female, and 55% were male. besides, 76% were general dental practitioners, http://dentistry3000.pitt.edu/ evaluation of the most common dental procedural errors leading to lawsuits and the relevant reasons in three dental clinics in tehran vol 9 no 1 (2021) doi 10.5195/d3000.2021.139 http://dentistry3000.pitt.edu and 24% were specialists. the majority of the dentists in the lawsuit cases had <10 years of experience (51%). the minimum number of lawsuits had been filed against dentists with >20 years of experience (20%). the results showed that 40% of the dentists had committed the errors in the morning shift, and 33 dentists had committed the errors in the afternoon shift. the most frequent dental fields involved were prosthodontics (31.9%), endodontics (24.2%), surgery (17.6%), restorative dentistry (14.3%), periodontics (8.8%), and periodontics (3.3%) (figure 1). the measured adopted to compensate for the errors consisted of paying the implant cost to the patient (56 cases, 52%), returning the cost of the treatment to the patient (28 cases, 26%), and compensating the error with re-treatment (23 cases, 21%). the measured adopted to compensate for the errors consisted of paying the implant cost to the patient (56 cases, 52%), returning the cost of the treatment to the patient (28 cases, 26%), and compensating the error with re-treatment (23 cases, 21%). figure 1. the frequency percentages of the lawsuits separately for each dental field 0 5 10 15 20 25 30 35 t h e p er ce n ta g e http://dentistry3000.pitt.edu/ evaluation of the most common dental procedural errors leading to lawsuits and the relevant reasons in three dental clinics in tehran vol 9 no 1 (2021) doi 10.5195/d3000.2021.139 http://dentistry3000.pitt.edu percentage number (frequency) error types (complaints) 5.6% 6 unnecessary surgery (interdisciplinary error) diagnostic error 5.6% 6 unnecessary endodontic treatment (interdisciplinary error) 1.8% 2 wrong diagnosis (leading to unnecessary endodontic treatment) 1.8% 2 unnecessary endodontic treatment 0.9% 1 inattention to c/r ratio (leading to the fracture of cantilever bridge abutment) 0.9% 1 wrong diagnosis (restoration instead of endodontic treatment) 0.9% 1 wrong diagnosis (restoration instead of placing a crown) 12% 13 root perforation during preparation of the root canal for a post therapeutic error 8.4% 9 extraction of the wrong tooth 8.4% 9 implant failure (fixture loosening) 7.4% 8 perforation of the root canal (leading to abscess formation, making the tooth hopeless) 5.6% 6 trauma to the adjacent tooth during tooth extraction 4.6% 5 restoration of the wrong tooth (wrong tooth) 4.6% 5 vertical root fracture (vrf) due to post placement 3.7% 4 crown under-occlusion 2.8% 3 file separation within the root canal 1.8% 2 an open contact of the crown with the adjacent tooth 1.8% 2 mild burning due to acid etching 1.8% 2 non-tooth-colored restoration without informing the patient 1.8% 2 cementation of the bridge with short margins (leading to caries and loss of the abutment teeth) 1.8% 2 not reducing the cavity walls (leading to cavity wall fracture, making the tooth hopeless) 1.8% 2 rendering treatment without informing the patient 1.8% 2 complete evacuation of gutta-percha during root canal preparation for a post-and-core treatment (leading to abscess formation, making the tooth hopeless) 1.8% 2 missed root canal(s) 1.8% 2 over-contoured implant-supported crowns 0.9% 1 dissatisfaction with crown color match 0.9% 1 trauma to the patient’s lip due to anesthetic syringe needle stick 0.9% 1 fracture of the abutment tooth crown during removal of the bridge 0.9% 1 trauma to the adjacent tooth during surgery 0.9% 1 removing the bridge without informing the patient 0.9% 1 restoring the tooth without informing the parents 0.9% 1 extrusion of gutta-percha from the root end 0.9% 1 under-obturation (leading to abscess formation, making the tooth hopeless) 100% 107 total table 1. direct, indirect and total effects for the model (n=174) http://dentistry3000.pitt.edu/ evaluation of the most common dental procedural errors leading to lawsuits and the relevant reasons in three dental clinics in tehran vol 9 no 1 (2021) doi 10.5195/d3000.2021.139 http://dentistry3000.pitt.edu discussion similar to other healthcare providers, dentists aim to promote patients’ health. despite all the efforts, unfavorable outcomes might be achieved. the present study is the first one to evaluate procedural errors leading to lawsuits in dentistry by evaluating the dentists' and plaintiffs’ demographic data. the study was carried out in three large dental clinics in tehran. the patients’ complaints are not properly recorded and classified; therefore, only a limited number of studies are available to compare them with the present study. the present study showed that the most frequent complaints were therapeutic, consistent with a study by kiani et al [9]. the present study results showed that the most frequent dental fields involved in lawsuits were prosthodontics, endodontics, and surgery, consistent with a study in spain [13, 14]. besides, these findings are consistent with two studies in tehran, in which the most frequent treatments leading to lawsuits were prosthodontic procedures [15]. in addition, in two previous studies in sweden and the united states, too, the most frequent complaints were related to prosthodontics [16, 17], which might be because prosthetic treatments are expensive, complex, and varied, and the dentist should cooperate with dental laboratories, too, increasing the odds of errors and patient complaints compared to other dental fields. besides, patients' high expectations from the treatment results and psychological factors in some patients give rise to increased frequency of complaints in this field. the frequencies of complaints in the fields of restorative dentistry and periodontics were low in the present study, consistent with a study by kiani et al. this might be attributed to a low rate of patients’ awareness and knowledge about these fields. the lowest frequency of complaints was related to the field of pedodontics, which might be attributed to the short duration of the presence of deciduous teeth in children's oral cavities and the low expectation of parents from the treatment outcomes. in the present study, most plaintiffs were in the 30–49 age range. in this context, in a study by pakk et al, too, in sweden, most plaintiffs were 40–59 years old, and in the study by pinchi et al in italy, they were mostly 30–40 years of age, consistent with the present study. this might be attributed to a higher rate of dental caries in this age range and a higher need for dental treatments with a higher referral rate of the patients for dental procedures [13, 18]. in the present study, most plaintiffs were female, consistent with studies by moles et al, shahsavari et al, and pukk et al [1, 3-5]. women’s predominance of in filing lawsuits compared to men might be due to the greater attention and higher sensitivity of women to their health and esthetic appearance than men. however, in a study by kiani et al in tehran, most plaintiffs were male [9]. the plaintiffs in the present study were mostly high school graduates and associate and bachelor’s degree holders. unfortunately, no comprehensive study is available to have evaluated the demographic data of patients filing lawsuits against dental practitioners. even in similar studies, too, the plaintiffs’ demographic data have not been evaluated with such detail, which might be because the community members’ educational levels are mostly in this rage. studies in tehran, riyadh, and the united kingdom have shown that the lawsuits have been filed against general dental http://dentistry3000.pitt.edu/ evaluation of the most common dental procedural errors leading to lawsuits and the relevant reasons in three dental clinics in tehran vol 9 no 1 (2021) doi 10.5195/d3000.2021.139 http://dentistry3000.pitt.edu practitioners in most cases, and most convicts have been in this group, with the majority of specialists being acquitted. this is because dental specialists usually select patients according to their academic and practical abilities due to higher knowledge and skill levels, decreasing the frequency of complaints against them [15, 19]. in the present study, more than half of the complaints were against dental practitioners with <10 years of experience, indicating that dentists' procedural errors decrease over time with an increase in their clinical experience, resulting in a decrease in lawsuits against them. this finding is consistent with a study by jafarian et al, who concluded that an increase in the dental practitioners’ experience to >15– 20 years decreased the frequency of lawsuits against them [20]. this might be attributed to dentists’ greater skill and experience in rendering the treatment and interacting with patients more effectively. in the present study, an attempt was made to evaluate the plaintiffs’ demographic data details and treatment processes. the error type that has been evaluated in a limited number of studies was evaluated in the present study. the present study showed that dentists should be very careful in proper patient selection (proportional to their academic ad practical skills). besides, it is necessary to increase dentists’ theoretical knowledge and practical skills in the diagnosis and treatment planning and in establishing a rapport with the patients to decrease errors and patient dissatisfaction and lawsuits against them. lawsuits against dentists are increasing in iran, which might be due to an increase in dentists’ knowledge and dental treatments, patients’ interest in preserving their teeth, and an increase in patients’ dental visits. besides, patients’ knowledge of their legal rights has increased in recent years. increasing dentists’ awareness and knowledge about dental lawsuits might increase their awareness, resulting in increased attention to details during treatment. unfortunately, a large number of dental clinics and centers lack a system to record lawsuits. besides, some clinics were not interested in cooperating and allowing access to the lawsuit cases. conclusion one of the most important ways to avoid errors that lead to lawsuits is to increase dental practitioners’ awareness and knowledge about previous lawsuits. knowledge about the most frequent causes of lawsuits and the provision of institutional guidelines will help decrease such cases. conflicts of interest the authors declare no competing interest. references 1. engineering of human error. haji hoseini a. tehran: fanavaran. 2010. 2. ramiro js, aisa pb, risk analysis and reduction in the chemical process industry. 2012: springer science & business media. 3. survey the cognitive abilities of professional drivers and its role in driving error [phd thesis]. allahyari t. health school of tehran university of medical science. 2007. 4. ‘global trigger tool’shows that adverse events in hospitals may be ten times greater than previously measured. classen dc, resar r, griffin f, federico f, frankel t, kimmel n, whittington jc, frankel a, seger a, james bc. health affairs. 2011; 30(4):581589. http://dentistry3000.pitt.edu/ evaluation of the most common dental procedural errors leading to lawsuits and the relevant reasons in three dental clinics in tehran vol 9 no 1 (2021) doi 10.5195/d3000.2021.139 http://dentistry3000.pitt.edu 5. the $17.1 billion problem: the annual cost of measurable medical errors. van den bos j, rustagi k, gray t, halford m, ziemkiewicz e, shreve j. health affairs. 2011; 30(4):596-603. 6. in-patients’ satisfaction with food served in imo state university teaching hospital, orlu. vincent cc. contemp econ policy. 14(112-24. 7. brauer rl, safety and health for engineers. 2016: john wiley & sons. 8. evaluating your quality management program. katz j, green e. st. louis: mosby. 1992. 9. a five-year survey for dental malpractice claims in tehran, iran. kiani m, sheikhazadi a. journal of forensic and legal medicine. 2009; 16(2):76-82. 10. analysis of 415 adverse events in dental practice in spain from 2000 to 2010. perea-pérez b, labajo-gonzález e, santiago-sáez a, albarrán-juan e, villa-vigil a. medicina oral, patologia oral y cirugia bucal. 2014; 19(5):e500. 11. are bad outcomes from questionable clinical decisions preventable medical errors? a case of cascade iatrogenesis. hofer tp, hayward ra. annals of internal medicine. 2002; 137(5_part_1):327-333. 12. patient safety in dentistry: dental care risk management plan. perea pérez b, santiago sáez a, garcía marín f, labajo gonzález e, villa vigil a. med oral patol oral cir bucal. 2011; 1(16 (6)):805-809. 13. trends in endodontic claims in i taly. pinchi v, pradella f, gasparetto l, norelli ga. international dental journal. 2013; 63(1):43-48. 14. the role of a professional dental organization in the resolution of malpractice claims the professional dentist college in the region of murcia (spain). lopez-nicolas m, falcon m, perezcarceles md, osuna e, luna a. med. & l. 2011; 30(55. 15. assessment of frequency of complaints against dentists in tehran during 2001-2008. shahsavari f, sadri d, mohammadzade m, sedighi a. the iranian journal of medical law. 2010; 4(13):121-32. 16. malpractice claims during 1988-1992: a national survey of dentists. milgrom p, fiset l, whitney c, conrad d, cullen t, o'hara d. journal of the american dental association (1939). 1994; 125(4):462-469. 17. malpractice reports in prosthodontics in sweden. rene n, owall b. swedish dental journal. 1991; 15(5):205. 18. do women simply complain more? national patient injury claims data show gender and age differences. pukk k, lundberg j, penaloza-pesantes rv, brommels m, gaffney fa. quality management in healthcare. 2003; 12(4):225-231. 19. dental negligence: a study of cases assessed at one specialised advisory practice. moles d, simper r, bedi r. british dental journal. 1998; 184(3):130133. 20. a survey of the complaints entering the medical council organization of tehran in three time periods: the years ending on 20 march 1992, 20 march 1997 and 20 march 2002. jafarian a, parsapour a, haj-tarkhani a, asghari f, razavi she, yalda a. journal of medical ethics and history of medicine. 2009; 2. http://dentistry3000.pitt.edu/ in-vitro study to assess effect of a glide path on canal transportation and centering ratio in double curved canals. vol 9 no 1 (2021) doi 10.5195/d3000.2021.131 http://dentistry3000.pitt.edu in-vitro study to assess effect of a glide path on canal transportation and centering ratio in double curved canals. raghad alhalabi1, mohammad salem rekab1, khaldaon hossein alhroob2, maher al-assaf3 1 department of operative dentistry, faculty of dentistry, damascus university, damascus, syria 2 department of periodontology, faculty of dentistry, damascus university, damascus, syria 3 department of oral histology and pathology, faculty of dentistry, damascus university, damascus, syria abstract objective: in endodontic treatment, creating a glide path became one of the recommended essential principles to obtain a safe preparation as possible; so the aim of the research is to evaluate the efficiency of the r-pilot system in glide path preparation within the double curved canals. study design: a laboratory study to assess the efficiency r-pilot mechanical patency system on the shape marinating of the canal. those criteria were used to evaluate the change in the original shape of the canal, canal transportation and centering ratio. the data were analyzed using spss v.13 program and the correlation between the variables were defined using descriptive statistics and inductive statistical tests (chi-square, t student for independent samples, anova). results: no significant difference between the two glide path creation systems was found in terms of the deviation amount from the canal centric. whereas, between the r-pilot system and the manual preparation, significant difference was found in the preserving rate of the canal centric at both the coronal area and the entire canal. conclusion: glide path creation in the previously mentioned methods with confirmation on the importance of the mechanical patency in terms of speed, reducing the effort and burden imposed on the practitioner compared to the use of manual files. xp endo shaper had lower deviation values and more ability to save the shape and centric of the canal. keywords: endodontic treatment; canal centric; r-pilot system; deviation amount. citation: alhalabi r, et al. (2021) in-vitro study to assess effect of a glide path on canal transportation and centering ratio in double curved canals. dentistry 3000. 1:a001 doi: 10.5195/d3000.2021.131 received: january 01, 2021 accepted: february 02, 2021 published: july 19, 2021 copyright: ©2021 alhalabi r, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: dr.maher.assaf1990@gmail.com introduction the primary goal of endodontic treatment is to eliminate or reduce bacteria in the root canal system as much as possible while preserving the original shape and path of the canal [1]. where preparing the root canals is considered one of the most important steps of endodontic treatment, including the removal of infected and affected tissues within the root canal walls and obtaining smooth walls that facilitate irrigation and filling, by following the utmost ways to make the preparation as conservative as possible, enough to protect the tooth from the risk of refraction in the long term [2]. root canals with anatomical curves are considered a challenge to the practicing physician, due to the risk of complications incidence such as instruments refraction and deviation occurrence [3], stainless steel files tend to restore their original straight shape during the preparation of these curves, unlike nickel titanium files, which has a high elasticity, so it reduces the efforts and lateral forces applied on the canal walls as much as possible in intense curving canals [3]. the insertion of nickel titanium alloys in endodontic has allowed manufacturers to produce many instruments with high flexibility http://dentistry3000.pitt.edu/ in-vitro study to assess effect of a glide path on canal transportation and centering ratio in double curved canals. vol 9 no 1 (2021) doi 10.5195/d3000.2021.131 http://dentistry3000.pitt.edu and resistance. today, nickel titanium files are considered part of the daily uses of endodontists and even general practitioners with their variety of shapes, tapering, cross-sections, and many of the advantages and developments which happened to the original alloy, made it correspond with different cases [4]. because of the sensitivity of dealing with curved canals, and because of the multiple complications that face the practitioner when preparing such canals, such as loss of working length, change of canal path, obturations, perforations, instruments refraction, ledge formation and zipping occurrence due to the difficulty of reaching the entire working length[5] and the accompanying difficulties in completing the remaining therapeutic procedures [6], the use of nickel titanium files, with their varied tapers and high flexibility, gave the practitioner a great ability in maintaining the original curvature of the root canal system [7], especially if the preparation was preceded by creating a glide path for the canal using a glide path-creation nickel titanium files [8,9]. securing the glide path in endodontic therapy became one of the recommended basic principles for obtaining as safe preparation as possible [10], wither the glide path was secured using manual or mechanical files [11] and is defined as a smoothwalled path extending from the canal orifice and ending with the physiological apical foramen. this is achieved when the file by which the glide path was created enters the entire canal length smoothly and without any impedances [12], and that will ease the canal preparation procedures as it provides a path that allows larger files to easily enter the canal [13,14]. hence, the idea of the research emerged in evaluating the ability of mechanical patency -to create the glide pathin maintaining the original shape and centric of the canal during canal preparation using three single mechanical preparation systems, that is in one of the most difficult cases in endodontic treatment, which is the double curved canals. so, the aim of the research is to evaluate the effectiveness of the r-pilot system in creating the glide path within the simulated double curved canals in terms of its ability to maintain -the centric and the shape of the canal compared with manual patency. material and methods study sample: 60 double curved canals were used in this study, each canal made of transparent resin and had two coronal and apical curvature with diameter and taper corresponding to the iso measurement, the apex measurement was 0.15, a taper of 2%, and a working length of 16 mm for each canal. depending on the used patency technique the sample split into two groups a and b, manually and mechanically respectively, each of them had 30 canals (n = 30) . each subgroup was split to three basic groups and each of them was named after the used preparation system type ((o, x, h)), each preparation system group contains 10 canals, the blocks were also numbered in each group from 1 to 10. study design: a laboratory study to evaluate the effectiveness of three single-file preparation systems in preparing artificial double curved canals made of transparent resin, after performing mechanical or manual patency and studying the changes and the complications that occur when preparing such canals to determine the best patency http://dentistry3000.pitt.edu/ in-vitro study to assess effect of a glide path on canal transportation and centering ratio in double curved canals. vol 9 no 1 (2021) doi 10.5195/d3000.2021.131 http://dentistry3000.pitt.edu pattern and the best preparation system used. used materials: three different preparation systems were used, one curve, hyflex edm, xp endo shaper, rpilot mechanical patency system and manual stainless-steel k-files were used in accordance with specifications specified earlier in the sample group (figure 1-a), a privet hand-made device was used to fix the resin blocks, a singledimensional fixed-angle camera was used for all samples, during which time a unified fixed angle camera was used. (figure 1-b). the practical steps of the research: these resin blocks, which contain the double curved canals, were fixed within a specially designed base for this study and solar images were taken under the same conditions in terms of dimension, angle and lighting using a digital camera, then the study was conducted on 10 levels that were determined perpendicular to the longitudinal axis of the canal where these points were determined based on the initial image, and each canal was divided into three areas (the straight coronal area, the first curvature, and the apical curvature). by identifying these levels and areas, we can study what will happen along the length of the canal, depending on the following criteria, where the change in the natural shape of the canal is evaluated according to the following: amount of deviation (expressing the centric of the canal): the maximum value of the removed resin width from two sides. centering ratio (rate of the canal centric). results the research sample consisted of 600 different sections determined in 60 simulated double curved canals. the sample was divided into two groups according to the glide path creation technique (rpilot, and manual files), and each group was divided into three subgroups (one curve preparation system, xp endo shaper preparation system, hyflex edm preparation system). in addition, 10 different sections were made in each simulated canal; the sections were divided according to the studied area to the apical curvature area; the first curvature area, and the straight coronal area. studying the deviation from the canal centric: table no. 1 shows the various between the two means of the deviation value from the canal centric after creating the glide path using the r-pilot system or by using manual files. a student test was performed for the independent samples and there were no significant differences in all the studied areas, as the significance level value was greater than (0.05)) for all groups. an anova test was performed to determine the differences between the used preparation systems (one curve, xp endo shaper, and hyflex edm) in each of the studied areas in terms of figure 1: (a) the stimulated resin canals, (b) photo fixing device. http://dentistry3000.pitt.edu/ in-vitro study to assess effect of a glide path on canal transportation and centering ratio in double curved canals. vol 9 no 1 (2021) doi 10.5195/d3000.2021.131 http://dentistry3000.pitt.edu sig: significance. the amount of deviation from the canal centric after preparation. when using the r -pilot system to create the glide path, a statistically significant difference was found in the first curvature area (0.001), the coronal area (0.043) and the entire canal (0.007). whereas, with the use of manual files, there was no significantly important difference except in the coronal area (0.044) (table no. 2). the bonferroni test was used to compare the deviation amount variable in the canal centric after preparation between two different preparation systems separately, according to different studied areas and different glide path creation techniques. (table no. 3) shows the results of the values of this test. studying the rate of preserving the canal centric: table no. 4 shows the mean and the standard deviation of the rate of preserving the canal centric after creating the glide path, the independent sample t-test was also used to study the existence of a significant difference between the r-pilot system and the manual files, as it found a significant difference between the two preparation systems at each of the coronal area and the entire canal (0.000, 0.002), respectively, while there was no significant difference in both the apical and the first curvature area. figure no.2 demonstrated the canal centric preserving rate value in the research sample, the values were higher in all groups, which used the r-pilot system. table 1: studying the deviation from the canal centric method n mean sd min max means difference t-value p-value sig apical curvature r-pilot 120 -0.01 0.84 -2.25 2.77 0.07 0.59 0.556 hand files 120 -0.07 0.87 -2 2.39 first curvature r-pilot 120 0.46 0.73 -1.22 2.39 0.04 0.395 0.693 hand files 120 0.43 0.73 -1.71 2.71 coronal curvature r-pilot 60 -0.2 1.04 -2.78 3.62 0.12 0.706 0.482 hand files 60 -0.32 0.75 -2.74 1.35 full canal r-pilot 300 0.14 0.88 -2.78 3.62 0.06 0.911 0.363 hand files 300 0.08 0.85 -2.74 2.71 http://dentistry3000.pitt.edu/ in-vitro study to assess effect of a glide path on canal transportation and centering ratio in double curved canals. vol 9 no 1 (2021) doi 10.5195/d3000.2021.131 http://dentistry3000.pitt.edu table 2: studying the deviation from the canal centric anova test. method preparation system n sd se min max f-value p-value sig r-pilot apical curvature one curve 40 0.2 1.35 -3.22 3.39 0.314 0.731 xp endo shaper 40 -0.1 1.55 -3.41 2.84 hyflex edm 37 0.05 2.11 -3.73 3.36 first curvature one curve 40 1.27 1.12 -0.77 3.64 7.391 0.001 * xp endo shaper 40 0.48 1.28 -2.31 2.65 hyflex edm 40 1.45 1.2 -1.53 3.87 coronal curvature one curve 20 -0.37 0.92 -1.79 1.92 3.323 0.043 * xp endo shaper 20 -0.83 1.02 -3.05 1.18 hyflex edm 20 0.07 1.34 -1.53 4.44 full canal one curve 100 0.52 1.34 -3.22 3.64 5.103 0.007 * xp endo shaper 100 -0.01 1.43 -3.41 2.84 hyflex edm 97 0.63 1.75 -3.73 4.44 hand files apical curvature one curve 40 0.45 1.75 -2.86 5.28 0.435 0.648 xp endo shaper 40 0.03 2.2 -3.95 4.57 hyflex edm 40 0.42 2.64 -4.44 9.54 first curvature one curve 40 1.65 0.92 -0.61 3.19 2.723 0.07 xp endo shaper 40 1.06 1.21 -1.76 3.16 hyflex edm 40 1.51 1.41 -2.49 3.85 coronal curvature one curve 20 0.28 0.78 -1.28 1.27 3.297 0.044 * xp endo shaper 20 -0.23 1.08 -2.16 1.45 hyflex edm 20 -0.4 0.72 -2.11 0.59 full canal one curve 100 0.9 1.43 -2.86 5.28 2.123 0.121 xp endo shaper 100 0.39 1.74 -3.95 4.57 hyflex edm 100 0.69 2.04 -4.44 9.54 http://dentistry3000.pitt.edu/ in-vitro study to assess effect of a glide path on canal transportation and centering ratio in double curved canals. vol 9 no 1 (2021) doi 10.5195/d3000.2021.131 http://dentistry3000.pitt.edu table 2: sig: significance. *: statistically significant. *: statistically significant table 3: studying the deviation from the canal centric after preparation, bonferroni test variables preparation method ( i-j ) se p-value significance r-pilot first curvature one curve xp endo shaper 0.79 0.27 0.011 * hyflex edm -0.18 0.27 1 xp endo shaper hyflex edm -0.97 0.27 0.001 * coronal area one curve xp endo shaper 0.46 0.35 0.577 hyflex edm -0.44 0.35 0.64 xp endo shaper hyflex edm -0.9 0.35 0.038 * full canal one curve xp endo shaper 0.53 0.21 0.042 * hyflex edm -0.12 0.22 1 xp endo shaper hyflex edm -0.64 0.22 0.009 * manual coronal area one curve xp endo shaper 0.51 0.28 0.203 hyflex edm 0.68 0.28 0.051 xp endo shaper hyflex edm 0.17 0.28 1 http://dentistry3000.pitt.edu/ in-vitro study to assess effect of a glide path on canal transportation and centering ratio in double curved canals. vol 9 no 1 (2021) doi 10.5195/d3000.2021.131 http://dentistry3000.pitt.edu *: statistically significant discussion the importance of mechanical preparation of root canals comes not only in terms of removing all live and dead tissues inside the root canal, but also from the importance of creating a sufficient place to allow the entry of both irrigating fluids, intra-canalicular dressing, and filling materials in order to complete their role in disinfection the root canal system and seal it tightly [15] while preserving the original shape of the canal [16]. where canals curvature and canal anatomical anomalies are one of the most important challenges faced by the practitioner during endodontic treatment, due to the many potential complications during treatment such as zipping occurrence, deviation of the canal, opening or changing in the shape or location of the apex, which causes subsequent failure of the endodontic treatment [17]. despite the advantages and the massive flexibility of nickeltitanium mechanical files, securing an initial glide path on the canal before using the mechanical preparation instruments is considered among the recommended matters, whether manually or by using glide path creation mechanical files [18]. in this study, double curved resin blocks were used instead of using natural teeth, due to the ineffectiveness of those last for comparison between preparation systems, as differences, even minor ones, between those teeth may make clear variances in results more than these made by different preparation systems [19], thus giving the resin blocks the possibility of unification the required standards in the study sample in terms of length, hardness, manufacture's material, curvature radius and angle [20,21], and, although it may not significantly reflect the clinical aspect due to the difference between the hardness of the dentin and the resin [22], though, it is a good and valid measure for studying different preparation systems [23,24]. the results of our study showed that when comparing the two methods of creating a glide path table 4: the rate of preserving the canal centric after glide path variables n mean sd t-value p-value significance apical curvature r-pilot 120 0.493 0.285 0.754 0.451 manual 120 0.466 0.268 first couverture r-pilot 120 0.53 0.258 1.653 0.1 manual 120 0.474 0.265 coronal area r-pilot 60 0.713 0.215 4.158 0.000 * manual 60 0.529 0.268 full canal r-pilot 300 0.552 0.274 3.173 0.002 * manual 300 0.482 0.267 http://dentistry3000.pitt.edu/ in-vitro study to assess effect of a glide path on canal transportation and centering ratio in double curved canals. vol 9 no 1 (2021) doi 10.5195/d3000.2021.131 http://dentistry3000.pitt.edu 'with an r-pilot mechanical file and using manual k-files up to the size of # 20' in studying the amount of deviation of the canal centric, by subtracting the output of the removed resin in the right side, -in each level of the study levelsfrom the removed resin in the left side, in two stages after creating the glide path and after preparation, our study did not show a significant difference in the deviation amount from the canal centric after creating the glide path, regardless the followed method. whereas the study of the rate of preserving the canal centric after the glide path creating step, there were no clear differences between the two methods. thus, the two methods were equal in preserving the canal centric in the apical curvature area. however, in the first curvature area, the coronal area and in the entire canal, our study had proved that the glide path creation using r-pilot mechanical file is better than creating it using manual files. our study showed that the deviation amount values in the centric of the canal after preparing the apical area did not differ according to the different preparation systems applied and the used method to create the glide path. as for in the first curvature area, coronal area and the entire canal, the xp endo shaper preparation group outperformed which made her the smallest among the preparation groups compared to the one curve preparation system group and the hyflex edm preparation system group. this can be explained by the difference in tapering between the singlefiles used in the preparation as the xp endo shaper has the smallest taper between them. our study agreed with the results of a previous study [25], which compared different types of glide path creation using single-file mechanical files (r-pilot wave one gold glider proglide) in terms of the ability of r-pilot file to maintain the shape and centric of the canal. compared to other glide path creation systems. we also agreed with another study [26], which compared five different types of mechanical preparation systems on a 120 sshaped manufactured canal sample with or without glide path creation and showed that the files with smaller taper were more preserver on the shape and the centric of the canal, where (schilder h 1974) [27] showed that the elasticity and hardness of the file varies according to the size, diameter and taper of the used instrument. therefore, instruments with smaller tapering led to better results in terms of maintaining centric. also, when comparing this study with that of (alfadley et al, 2020) we show the superiority of the xpendo shaper over wave one gold in terms of maintaining the centrality and the shape of the figure 2: average value for canal centric after creating the glide path using different methods. http://dentistry3000.pitt.edu/ in-vitro study to assess effect of a glide path on canal transportation and centering ratio in double curved canals. vol 9 no 1 (2021) doi 10.5195/d3000.2021.131 http://dentistry3000.pitt.edu canal in severely curved canals; due to the mixed-made nature of xp-endo shaper alloy, where the elasticity of the alloy may have the largest influence on the mechanical performance and behavior of the file upon preparation [16]. while when comparing the hyflex edm preparation system in the study [28] with protaper next, the first protagonist outperformed the others and had the best results in terms of conserving the centeralization of the canals; and this may be due to the difference in the study design and the systems used to compare each other. as for the one curve preparation system, it is considered a recent file. until now, there are only two studies that discussed susceptibility formation when studying it. our study agreed with previous study [29], which compared four preparation systems in an 80 curved mesial canal sample, so the used preparation systems had close results. in terms of preserving the shape and the centric of the canal, the one curve preparation system showed similar results to the other systems, which can be explained by its high flexibility due to the heat-treated c-wire alloy. the same thing with the r-pilot mechanical file, which is considered very recent, and despite the presence of many studies that discussed its ability to resist efforts in various forms and degrees of curvature of the root canals, until now there was only one study dealt with the ability of maintaining the shape and the centric of the canal. it is important to mention that in this research, we studied the ability to maintain the shape and the centric of the canal only. the absence of differences between the glide path creation systems does not mean neglecting the importance of mechanical patency, including speed, reducing the effort imposed on the practitioner, especially in curved canal and safety in preparation. conclusion creating a glide path in one of the described methods, emphasizing the importance of mechanical patency in terms of speed and reducing the effort and burden imposed on the practitioner compared to the use of manual files. emphasis on the one-time use of mechanical preparation systems whenever possible, especially in canals with anatomical anomalies, to avoid complications resulting from preparation. consider depending on high flexibility and smaller tapering preparation systems for preparing curved root canals with complex anatomical anomalies. xp endo shaper had lower deviation values and more ability to save the canal's shape and centric. conflicts of interest the authors declare no competing interest. references 1. comparative evaluation of root canal transportation by three niti single-file systems in curved canals: a cone beam computed tomography study. saberi e, farhad-mollashahi n, bijari s, daryaeian m. int j dent. 2018 may;14:4151692. doi: 10.1155/2018/4151692. pmid: 29861728. 2. shaping ability of protaper universal, waveone and protaper next in simulated l-shaped and sshaped root canals. wu h, peng c, bai y, hu x, wang l, li c. bmc oral health. 2015 mar;15(1):27. doi: 10.1186/s12903-015-0012-z. pmid: 25886847. 3. analysis of ni-ti versus stainless steel instrumentation in resin simulated canals. coleman cl, svec ta. j endod. 1997 apr;23(4):232-235. doi: http://dentistry3000.pitt.edu/ in-vitro study to assess effect of a glide path on canal transportation and centering ratio in double curved canals. vol 9 no 1 (2021) doi 10.5195/d3000.2021.131 http://dentistry3000.pitt.edu 10.1016/s0099-2399(97)80053-2. pmid: 9594772. 4. bending resistance and cyclic fatigue life of reciproc blue, waveone gold, and genius files in a double (s-shaped) curved canal. özyürek t, gündoğar m, yılmaz k, uslu g. j dent res dent clin dent prospects. 2017 fall;11(4):241246. doi: 10.15171/joddd.2017.042. pmid: 29354251. 5. safe root canal preparation using reciprocating nickel-titanium instruments. ha jh. restor dent endod. 2015 aug;40(3):253-254. doi: 10.5395/rde.2015.40.3.253. pmid: 26295031. 6. the effect of preparation procedures on original canal shape and on apical foramen shape. weine fs, kelly rf, lio pj. j endod. 1975 aug;1(8):255-262. doi: 10.1016/s0099-2399(75)80037-9. pmid: 10697472. 7. the shaping effects of three nickel-titanium rotary instruments in simulated s-shaped canals. yoshimine y, ono m, akamine a. j endod. 2005 may;31(5):373-275. doi: 10.1097/01.don.0000140568.4046 2.43. pmid: 15851932. 8. shaping ability of protaper next, protaper universal and irace files in simulated s-shaped canals. hiran-us s, pimkhaokham s, sawasdichai j, ebihara a, suda h. aust endod j. 2016 apr;42(1):3236. doi: 10.1111/aej.12117. pmid: 26420685. 9. apical root canal transportation and remaining dentin thickness associated with protaper universal with and without pathfile. zanette f, grazziotin-soares r, flores me, camargo fontanella vr, gavini g, barletta fb. j endod. 2014 may;40(5):688-693. doi: 10.1016/j.joen.2013.09.001. pmid: 24767565. 10. comparison of glide paths created with k-files, pathfiles, and the proglider file, and their effects on subsequent waveone preparation in curved canals. zheng l, ji x, li c, zuo l, wei x. bmc oral health. 2018 aug 29;18(1):152. doi: 10.1186/s12903-018-0614-3. pmid: 30157822. 11. comparison among manual instruments and pathfile and mtwo rotary instruments to create a glide path in the root canal preparation of curved canals. alves vde o, bueno ce, cunha rs, pinheiro sl, fontana ce, de martin as. j endod. 2012 jan;38(1):117120. doi: 10.1016/j.joen.2011.10.001. pmid: 22152634. 12. endodontic update 2006. west j. j esthet restor dent. 2006;18(5):280-300. doi: 10.1111/j.17088240.2006.00039.x. pmid: 16987326. 13. a dozen ways to prevent nickel-titanium rotary instrument fracture. di fiore pm. j am dent assoc. 2007 feb;138(2):196-201; quiz 249. doi: 10.14219/jada.archive.2007.0136. pmid: 17272374. 14. peters o a, koka r s (2009) preparation of coronal and radicular spaces. 15. comparison of shaping ability of different single-file systems using microcomputed tomography. elashiry mm, saber se, elashry sh. eur j dent. 2020 feb;14(1):70-76. doi: 10.1055/s0040-1701393. pmid: 32005040. 16. shaping ability of xp endo shaper file in curved root canal models. alfadley a, alrajhi a, alissa h, alzeghaibi f, hamadah l, alfouzan k, jamleh a. int j dent. 2020 feb 17;2020:4687045. doi: 10.1155/2020/4687045. pmid: 32148503. 17. canal transportation and centering ratio after preparation in severely curved canals: analysis by micro-computed tomography and double-digital radiography. poly a, almalki f, marques f, karabucak b. clin oral investig. 2019 dec;23(12):4255-4262. doi: 10.1007/s00784-019-02870-8. pmid: 30834991. 18. higher speed and no glide path: a new protocol to increase the efficiency of xp shaper in curved canals-an in vitro study. webber m, piasecki l, jussiani ei, andrello ac, dos reis pj, azim ka, azim aa. j endod. 2020 jan;46(1):103-109. doi: 10.1016/j.joen.2019.10.014. pmid: 31761330. http://dentistry3000.pitt.edu/ in-vitro study to assess effect of a glide path on canal transportation and centering ratio in double curved canals. vol 9 no 1 (2021) doi 10.5195/d3000.2021.131 http://dentistry3000.pitt.edu 19. hanges in root canal geometry after preparation assessed by high-resolution computed tomography. peters oa, laib a, göhring tn, barbakow f. c. j endod. 2001 jan;27(1):1-6. doi: 10.1097/00004770-20010100000001. pmid: 11487156. 20. a microcomputed tomography evaluation of the shaping ability of three thermallytreated nickel-titanium rotary file systems in curved canals. huang z, quan j, liu j, zhang w, zhang x, hu x. j int med res. 2019 jan;47(1):325-334. doi: 10.1177/0300060518801451. pmid: 30282507. 21. transportation assessment in artificial curved canals after instrumentation with reciproc, reciproc blue, and xp-endo shaper systems. pacheco-yanes j, gazzaneo i, pérez ar, armada l, neves mas. j investig clin dent. 2019 aug;10(3):e12417. doi: 10.1111/jicd.12417. pmid: 30955238. 22. comparative analysis of canal transportation and centring ability of three ni-ti rotary endodontic systems: protaper®, mtwo® and revo-s™, assessed by micro-computed tomography. vallaeys k, chevalier v, arbabchirani r. odontology. 2016 jan;104(1):83-88. doi: 10.1007/s10266-014-0176-z. pmid: 25248755. 23. shaping ability of 4 different single-file systems in simulated s-shaped canals. saleh am, vakili gilani p, tavanafar s, schäfer e. j endod. 2015 apr;41(4):548-552. doi: 10.1016/j.joen.2014.11.019. pmid: 25576206. 24. a comparison of the shaping ability of three nickeltitanium rotary instruments: a micro-computed tomography study via a contrast radiopaque technique in vitro. wei z, cui z, yan p, jiang h. bmc oral health. 2017 jan 9;17(1):39. doi: 10.1186/s12903-016-0326-5. pmid: 28068971. 25. microcomputed assessment of transportation, centering ratio, canal area, and volume increase after single-file rotary and reciprocating glide path instrumentation in curved root canals: a laboratory study. aydın zu, keskin nb, özyürek t, geneci f, ocak m, çelik hh. j endod. 2019 jun;45(6):791-796. doi: 10.1016/j.joen.2019.02.012. pmid: 30935620. 26. shaping ability of different nickel-titanium systems in simulated s-shaped canals with and without glide path. bürklein s, poschmann t, schäfer e. j endod. 2014 aug;40(8):1231-1234. doi: 10.1016/j.joen.2014.01.043. pmid: 25069939. 27. cleaning and shaping the root canal. schilder h. dent clin north am. 1974 apr;18(2):269296. pmid: 4522570. 28. a micro-computed tomography evaluation of the shaping ability of two nickeltitanium instruments, hyflex edm and protaper next. venino pm, citterio cl, pellegatta a, ciccarelli m, maddalone m. j endod. 2017 apr;43(4):628-632. doi: 10.1016/j.joen.2016.11.022. pmid: 28215346. 29. micro-computed tomographic evaluation of canal transportation and centering ability of 4 heat-treated nickeltitanium systems. razcha c, zacharopoulos a, anestis d, mikrogeorgis g, zacharakis g, lyroudia k. j endod. 2020 may;46(5):675-681. doi: 10.1016/j.joen.2020.01.020. pmid: 32171564. http://dentistry3000.pitt.edu/ microsoft word 55 2016.docx   vol  4,  no  1  (2016)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2016.55           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     prevalence  of  oral  tori  among  medical  and  dental  students  at  the   university  of  the  west  indies   arvind  babu  rajendra  santosh1,  thaon  jones1,  hima  venugopal1,  keisha  smith1,  j  suzanne  turpin  mair1,  mark  edwards1,  errol  williams1     1den$stry  programme,  faculty  of  medical  sciences,  the  university  of  the  west  indies,  mona,  kingston,  jamaica,  west  indies.   abstract   objec&ve:    to  determine   the  prevalence  and  gender   varia2on  of   torus  pala2nus   (tp)   and   torus  mandibularis  (tm)  amongst  medical  and  dental  students  at  the  mona  campus  of  the   university  of  the  west  indies.  subjects  and  methods:  the  study  observed  335  medical  and   dental  students  from  the  mona  campus  of  the  university  of  the  west  indies.  rou:ne  dental   examina'ons  were  conducted  to  check  the  presence/absence  of  tp  and  tm  by   inspec6on   and  palpa'on.  results:  the   overall   prevalence   of   tp   and   tm  was   27.76%.   the   oral   torus   prevalence  was  as  follows,  torus  pala1nus  was  44.08%,  torus  mandibularis  was  36.55%  and   individuals   with   both   torus   pala*nus   and  mandibularis   was   19.35%.   the   study   observed   that   the   prevalence   of   tp   and   tm   in   females   was   16.11%   (54/335),   and   males   11.64%   (39/335).   it  was  also  found  that  bilateral  torus  mandibularis  (40.38%)  was  more  prevalent   than   unilateral   right   torus   mandibularis   (36.53%)   and   unilateral   le7   torus   mandibularis   (23.07%)  conclusions:  the  prevalence  of  tp  and  tm  are  compara2vely  higher   than  neigh-­‐ boring   caribbean   na+ons   and  west   african   countries.   our   observa+ons   also   highlighted   that  tp  is  highly  prevalent  among  various  types  of  oral  tori.  the  study  also  recorded  higher   prevalence   of   oral   tori   among   females.   due   to   higher   prevalence   of   oral   tori   among   the   study  group,  jamaican  den3sts  need  be  knowledgeable  about  the  high  prevalence  and  clini-­‐ cal  significance  of  tp  and  tm  for  dental  treatment.       cita%on:   santosh,   et   al.   (2016).   prevalence   of   oral   tori   among  medical  and  dental   students  at   the   university   of   the   west   indies.   den$stry   3000.  1:a001  doi:10.5195/d3000.2016.55   received:  july  12,  2016   accepted:    july  23  ,  2016   published:    october  3,  2016   copyright:  ©2016  santosh,  et  al.  this  is  an  open   access   ar!cle   licensed   under   a   crea!ve   com-­‐ mons   a"ribu"on   work   4.0   united   states   li-­‐ cense.   email:  arvindbabu2001@gmail.com     introduction   torus  palatinus  [tp]  and   torus  mandibularis  [tm]  are  nor-­‐ mal  anatomical  variants,  which  are   categorized  under  developmental   alterations  of  the  jaw  bone.  tp   and  tm  are  slow  growing  osseous   outgrowths  (exophytic)  in  the  mid-­‐ line  of  the  hard  palate  (torus   palatinus)  and  in  the  lingual  corti-­‐ cal  bones  of  mandible  (torus  man-­‐ dibularis).  it  is  a  non-­‐pathological   condition  and  in  most  cases  they   remain  asymptomatic  [1].  tp  and   tm  are  clinically  identified  as  a   hard  palpable  bony  mass  localized   in  the  mid-­‐palatal  region  specifi-­‐ cally  posterior  hard  palate  and/or   lingual  cortical  bone  of  mandibular   jaw  [2].  tm  can  be  either  unilat-­‐ eral  or  bilateral,  where  as  tp  are   solitary  and  usually  occurs  over   the  posterior  hard  palate  in  the   mid-­‐palatal  suture  region  [3].   morphologically  tp  and  tm  pre-­‐ sent  either  a  smooth  bony  projec-­‐ tion  or  multi-­‐nodular/lobular  bony   projections.    the  shapes  of  tp  and   tm  are  categorized  as  flat,  spindle,   nodular,  or  lobular.  tp  and  tm  are   considered  to  be  a  variant  of  exos-­‐ toses.  exostoses  are  benign  bony   projections  arising  from  the  cortex   of  the  maxillary  or  mandibular  al-­‐ veolar  region  [4].  tp  and  tm   shows  female  predilection,  and   the  size  may  vary  between  less   than  1cm  to  greater  than  4  cms  in   the  greatest  diameter  [5].     prevalence  of  oral  tori  among  medical  and  dental  students  at  the  university  of  the  west  indies   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.55    http://dentistry3000.pitt.edu   2   etiology  of  tp  and  tm  are   not  clear,  but  the  majority  of  in-­‐ vestigators  believed  that  it  was   either  genetic  or  environmental   making  the  etiology  multi-­‐ factorial.  studies  stated  the  etiol-­‐ ogy  of  tp  and  tm  were  related  to   genetics,  superficial  injuries,  envi-­‐ ronmental  factors,  masticatory   forces,  marine  diets,  drugs,  para-­‐ functional  habits  and  number  of   existing  teeth  or  abraded  teeth   due  to  occlusion  [4].    the  most   widely  accepted  etiological  associ-­‐ ation  of  tp  and  tm  was  of  a  genet-­‐ ic  cause.  autosomal  dominant  in-­‐ heritance  pattern  was  proposed   for  tp  and  tm,  due  to  the  higher   vertical  transmission  rate  and   penetrance  [6].    another  theory   hypothesized  that  tp  and  tm  was   a  functional  response  in  individu-­‐ als  with  well  developed  masticato-­‐ ry  muscles.  this  theory  suggested   that  masticatory  forces  transmit-­‐ ted  to  the  mid-­‐palatine  sutural   region  leads  to  increased  bony   growth,[7]    and  probably  proximity   of  ossification  center  in  maxillary   bone  may  contribute  in  initiation   of  osseous  growth.  marine  dietary   habits  such  as  consumption  of  fish   or  salted  fish  promote  a  higher   prevalence  of  tp  and  tm  due  to   the  nutritional  substances  (poly-­‐ unsaturated  fatty  acid  and  vitamin   d)  that  can  enhance  osseous   growth  [8].  drugs  such  as  phenyto-­‐ in  were  linked  with  occurrence  of   tp  and  tm  because  of  the  influ-­‐ ence  of  the  drug  with  calcium  ho-­‐ meostasis  and  initiating  osteogen-­‐ esis  [9].  one  study  mentioned  that   ratio  of  un-­‐erupted  mandibular   canines  were  higher  among  indi-­‐ viduals  with  tm  [10].  theories   have  mentioned  that  para-­‐ functional  habits  such  as  thumb   sucking  or  tongue  thrusting  influ-­‐ ences  occurrences  of  tp  due  to   the  increased  pressure  over  the   mid-­‐palatine  suture  which  triggers   osteogenesis  [11].    etiological  fac-­‐ tors  of  tp  and  tm  are  summa-­‐ rized  in  table  1.     the  importance  and  sig-­‐ nificance  of  tp  and  tm  are  ex-­‐ tensively  discussed  in  dental  lit-­‐ erature.  tp  and  tm  are  consid-­‐ ered  to  be  an  obstruction  while   fabricating  removable  (partial  or   complete)  dentures  for  dental   rehabilitation  procedures  [12].   presence  of  tp  and/or  tm  pro-­‐ vides  a  significance  in  forensic   investigations  contributing  to   personal  identification  proce-­‐ dures  [13].  tp  and  tm  osseous   tissue  are  useful  as  autogenous   or  osseous  coagulum  during  os-­‐ seous  replacement  in  periodon-­‐ tal  procedures  or  alveolar  ridge   defect  correction  surgeries   [14,15].  tm  can  also  be  used  to   indicate  the  risk  of  appearance  of   temporomandibular  disorders   [16].  a  significant  relationship  be-­‐ tween  the  duration  of  renal  dialy-­‐ sis  and  the  size  of  tp  has  been  re-­‐ ported  [17].    one  published  report   revealed  a  relationship  between   autosomal  dominant  osteosclero-­‐ sis  with  tp  and  tm  [18].  recently   tp  has  been  associated  in  a  new   anatomical  correlation  with  bone   density  in  postmenopausal  wom-­‐ en  [19].  clinical  significance  of  to-­‐ rus  palatinus  and  torus  mandibu-­‐ laris  are  summarized  in  table  2.     epidemiological  studies  of   tp  and  tm  from  caribbean  and   west  africian  countries  revealed   higher  prevalence  in  their  geo-­‐ graphic  location.  therefore  the   purpose  of  the  present  study  is  to   determine  the  prevalence  of  torus   palatinus  and  torus  mandibularis   and  gender  variation  in  their  dis-­‐ tribution  among  medical  and  den-­‐ table&1.&etiology&of&torus&palatinus&and&torus&mandibularis& & a. widely&accepted&etiological&factor&of&tp&and&tm& 1. genetic)influence& & & b. moderately&accepted&etiological&factors&of&tp&and&tm& 1. environmental)factors) 2. marine)dietary)habits) 3. masticatory)forces) 4. para=functional)habits) ) & c. less&accepted&etiological&factors&of&tp&and&tm& 1. drugs)(phenytoin)) 2. number)of)existing)teeth) 3. abraded)teeth)due)to)occlusion) 4. superficial)injuries.)) ) !1! prevalence  of  oral  tori  among  medical  and  dental  students  at  the  university  of  the  west  indies   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.55    http://dentistry3000.pitt.edu   3   tal  students  at  the  mona  campus   of  the  university  of  the  west  in-­‐ dies,  in  kingston,  jamaica.     materials  and  methods:   the  study  was  approved  by   ethics  committee  of  the  university   of  the  west  indies,  mona  campus.   the  study  obtained  written  con-­‐ sent  from  all  participants  involved   in  the  present  study;  also  ethics   committee  of  the  university  of  the   west  indies  approved  the  consent   form  that  was  used  in  the  present   study.  the  present  study  has  been   conducted  in  full  accordance  with   the  world  medical  association   declaration  of  helsinki.   dental  examination  was   conducted  in  the  dental  teaching   laboratory  at  the  faculty  of  medi-­‐ cal  sciences  teaching  and  re-­‐ search  complex  in  the  mona  cam-­‐ pus  of  the  university  of  the  west   indies,  jamaica.  during  this  study  a   routine  dental  and  oral  examina-­‐ tion  were  done  on  335  medical   and  dental  students  by  the  dental   surgeons  and  the  findings  were   systematically  recorded.  for  the   diagnosis  of  tp  and  tm,  tp  was   defined  as  a  bony  outgrowth  situ-­‐ ated  on  mid  palatal  region  and  tm   was  defined  as  a  bony  outgrowth   situated  on  lingual  cortical  bone  at   premolar-­‐molar  region  of  mandib-­‐ ular  jaw  bone.  the  size  and  shape   of  the  tp  and  tm  were  not  con-­‐ sidered  and  any  questionable   bony  outgrowths  on  mid-­‐ palate/lingual  cortical  bone  of   mandibular  jaw  were  excluded.   study  participants  were  aged  be-­‐ tween  19-­‐30  years  and  the  study   was  conducted  during  the  period   of  april-­‐may,  2015.  all  the  data   were  transferred  to  microsoft  ex-­‐ cel  data  sheet  and  subjected  for   descriptive  analysis.       results   a  total  of   335  participants   were  identified  in   the  present  study,   230  (68.65%)  were   female  and  105   (31.34%)  were   male.  the  preva-­‐ lence  of  all  tori  was   27.76%  (93  out  of   335  participants).   higher  prevalence   of  tori  was  ob-­‐ served  among  fe-­‐ males  16.11%  (54   out  of  335  partici-­‐ pants),  than  the   males  11.64%  (39   out  of  335  partici-­‐ pants)  (table  1)   descriptive  analysis  of  the   study  population  according  to  to-­‐ rus  type  showed  that  tp  was  ob-­‐ served  in  12.23%  (41  out  of  335   participants),  tm  10.14%  (34  out   of  335  participants),  participants   with  both  tp  and  tm  was  5.37%   (18  out  of  335  participants).  the   distribution  of  the  study  popula-­‐ tion  according  to  gender  showed   that  tp  in  males  was  18.09%  (19   out  of  105  male  participants),  and   tp  in  females  was  9.56%  (22  out  of   230  female  participants);  tm  in   males  was  13.33%  (14  out  of  105   male  participants),  and  tm  in  fe-­‐ males  was  8.69%  (20  out  of  230   female  participants);  males  with   both  tp  and  tm  was  5.71%  (6  out   of  105),  and  females  with  both  tp   and  tm  was  5.21%(12  out  of  230   female  participants).  (table  3)   !1! table&2.&clinical&significance&of&torus&palatinus&and&torus&mandibularis& 1. #interrupts#in#the#fabrication#of#removable(#partial/complete)#denture.## 2. #contributes#in#personal#identification#procedures#in#forensic#investigation.## 3. #osseous#tissue#from#tp#and#tm#can#be#used#as#autogenous#or#osseous# coagulum#during#osseous#replacement#in#periodontal#or#minor#oral#surgical# procedures# 4. #indicates#the#risk#of#appearance#of#temporomandibular#disorders# 5. #provides#significant#relationship#between#duration#of#renal#dialysis#and#size#of# tp.# 6. #new#anatomical#correlation#with#bone#density#in#postmenopausal#women.## 7. #overlying#oral#mucosa#is#thinner#and#linked#with#traumatic#oral#ulcerations.## 8. #phonetic#disturbance#specifically#palatal#sounds#while#pronouncing#the#words# by#the#articulation#of#the#body#of#the#tongue#towards#the#hard#palate## 9. #extremely#large#tp/tm#may#cause#limitation#in#mastication#process.## 10. #compromised#esthetic#appearance.## 11. #related#sleep#obstructive#apnea# prevalence  of  oral  tori  among  medical  and  dental  students  at  the  university  of  the  west  indies   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.55    http://dentistry3000.pitt.edu   4   the  prevalence  of  torus   type  and  gender  variation  analysis   showed  that  tp  was  observed  in   44.08%  (41  out  of  93  participants),   tm  36.55  %  (34  out  of  93  partici-­‐ pants),  participants  with  both  tp   and  tm  was  19.35%  (18  out  of  93   partici-­‐ pants).  the   distribution   of  the  study   population   according  to   gender   showed  that   tp  in  males   was  48.71%   (19  out  of  39   male  partic-­‐ ipants),  and   tp  in  fe-­‐ males  was   40.74%  (22  out  of  54  female  par-­‐ ticipants);  tm  in  males  was   35.89%  (14  out  of  39  male  partici-­‐ pants),  and  tm  in  females  was   37.03%  (20  out  of  54  female  par-­‐ ticipants);  males  with  both  tp  and   tm  was  15.38%  (6  out  of  39),  and   females  with   both  tp  and   tm  was   22.22%(12  out   of  54  female   participants).   (table  3)   analysis   of  the  tm  oc-­‐ currence  type   and  gender   distribution   showed  that   unilateral  right   tm  was  ob-­‐ served  in   36.53%  (19  out   of  52  partici-­‐ pants);  unilat-­‐ eral  left  tm   was  observed   in  23.07%  (12   out  of  52  par-­‐ ticipants);  bi-­‐ lateral  tm  was   observed  in  40.38%  (21  out  of  52).   tm  distribution  according  to  gen-­‐ der  showed  that  unilateral  right   tm  in  males  was  30%  (6  out  of  20   male  participants),  and  unilateral   right  tm  in  females  was  40.62%   (13  out  of  32  female  participants);   table&3.&distribution&of&study&population&according&to&torus&type&and&gender&variation.& & torus&type& study&population& male&population&in&the& study& female&population&in&the& study&& total& population& in&the& study&& (n&=335)& total& population& with&oral& tori& (n&=93)& 27.76%& male& (total& population& n=105)& male& population& with&oral& tori&(n=39)& 41.93%& female& population& in&the&study& (n=230)& female& population& with&oral& tori&& (n=54)& 58.06%& individuals& with&torus& palatinus&only& 41# (12.23%)# 41# (44.08%)# 19# (18.09%)# 19#(48.71%)# 22#(9.56%)# 22# (40.74%)# individuals& with&torus& mandibularis& only&& 34# (10.14%)# 34# (36.55%)# 14# (13.33%)# 14#(35.89%)# 20#(8.69%)# 20# (37.03%)# individuals& with&both& torus& palatinus&and& torus& mandibularis& 18#(5.37%)# 18# (19.35%)# 6#(5.71%)# 6#(15.38%)# 12#(5.21%)# 12# (22.22%)# total& 93#(27.76%)# 39#(11.64%)# 54#(16.11%)# !1! table&4.&distribution&of&torus&mandibularis&in&study&population&according&to&occurrence&type&and& gender.& torus&mandibularis& distribution& individuals&with&torus& mandibularis&(n=34)& and&individuals&with& both&torus&palatinus& and&mandibularis& (n=18)& (n=34+18=52)& males& (n=20)& females& (n=32)& unilateral&right&torus& mandibularis& 19#(36.53%)# 6#(30%)# 13#(40.62%)# unilateral&left&torus& mandibularis& 12#(23.07%)# 5#(25%)# 7#(21.87%)# bilateral&torus& mandibularis& 21#(40.38%)# 9#(45%)# 12#(37.5%)# !1! prevalence  of  oral  tori  among  medical  and  dental  students  at  the  university  of  the  west  indies   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.55    http://dentistry3000.pitt.edu   5   unilateral  left  tm  in  males  was   25%  (5  out  of  20  male  partici-­‐ pants),  and  unilateral  left  tm  in   females  was  21.87%  (7  out  of  32   female  participants);  and  bilateral   tm  in  males  was  40.38%  (21  out  of   52  male  participants),  and  bilateral   tm  in  females  was  37.5%  (12  out   of  32  female  participants)  (table   4)     discussion   our  observations  recorded   that  overall  prevalence  of  tp  and   tm  was  27.76%  (93  out  of  335   study  participants)  and  the  most   common  type  of  oral   tori  is  tp  44.08%,  and   females  (58.06%)  were   more  commonly  ob-­‐ served  with  oral  tori   than  males  (41.93%.)    a   higher  prevalence  of   oral  tori  was  recorded   in  the  present  study   while  comparing  previ-­‐ ous  reports  on  jamai-­‐ can  blacks,  trinidad   and  tobago  or  west   african  population   studies.(table  5)     ogunsalu  reported  a   prevalence  of  oral  tori   as  6.6  percent  among   jamaican  blacks  [20].   the  prevalence  of  oral   tori  is  12.3%  among   trinidad  and  tobago   population  [7].  the   overall  prevalence  rate   of  west  african  popula-­‐ tion  from  the  ghanaian   community  was  14.6%   [21].    the  variation  in   the  prevalence  could   be  related  to  the  di-­‐ verse  ethnic  group  in   the  present  study.  the  prevalence   rate  of  the  present  study  results   may  not  be  generalized  to  the  ja-­‐ maican  population  as  our  findings   demonstrate  university  students   come  from  areas  other  than  ja-­‐ maica,  which  include  most  of  car-­‐ ibbean  countries,  latin  or  central   american  countries  and  the  north   american  mainland.    further,  the   present  study  did  not  attempt  to   distinguish  between  ethnic  varia-­‐ tions  as  the  previous  study  from   trinidad  and  tobago  did  which,   mentioned  that  oral  tori  did  not   show  any  ethnic  difference.    how-­‐ ever,  higher  prevalence  of  oral  tori   could  be  possibly  due  to  the  rich   salt  fish  in  the  diet  of  jamaican   and  other  caribbean  countries  and   well  developed  masticatory  mus-­‐ cles  among  the  jamaican  popula-­‐ tion.  trinidad  and  tobago  study   also  mentioned  that  higher  preva-­‐ lence  of  oral  tori  could  be  possibly   due  to  the  marine  diet  [7].     in  this  study  oral  tori  was   more  prevalent  in  females   (58.06%),  and  sub  group  analysis   of  torus  type  also  showed  higher   female  prevalence  of  all  torus   table&5.&comparative&table&showing&the&prevalence&rate&of&oral&tori&in&various&geographic&areas.& study& population& sample& prevalence& gender& predilection& reference& woo#jk.#1950# eskimos# 0# 66%# 0# [23]# kolas#et#al.#1953# united#states# 2478# 20.9%# 0# [24]# vidic#b.#1966# yugoslavian# 0# 49.7%# 0# [25]# bernaba#jm.#1977# brazilian#indian# 200# 10%# 0# [26]# chew#cl#et#al.#1984# singapore# 0# 48%# equal#distribution# [27]# axelsson#g#et#al.#1985# icelandic,#south#–# thingeyjarsysla# 763# 33.3%# 0# [28]# axelsson#g#et#al.#1985# icelandic,#north#–# thingeyjarsysla# 213# 14.6%# 0# [29]# bouquot#et#al.#1986# white#americans# 23616# 2.7%# 0# [30]# salem#g#et#al.#1987# saudi#arabia# 1932# 1.4%# 0# [31]# reichart##et#al.#1988# german# 1317# 13.5%# 0# [32]# reichart#et#al.#1988# thailand# 947# 32.3%# male#predilection# [32]# eggen#et#al.#1989# norway# 326# 31.3%# 0# [33]# haugen#lk#et#al.#1992# norway,#oslo#area# 5000# 9.2%# female#predilection# [34]# shah#ds#et#al.#1992# india# 1000# 10.9%# female#predilection# [35]# eggen#et#al.#1994# norway,#lofoten# 1181# 38.2%# female#predilection# [36]# ogunsalu#cu.#1994# jamaica# 958# 6.6%# 0# [21]# eggen#et#al.#1994# norway,#gudbrandsdalen# 829# 32.7%# female#predilection# [36]# nair#et#al.#1996# vietnam# 550# 1.44%# female#predilection# [37]# gorsky#m#et#al.#1996# israel# 1002# 21.0%# female#predilection# [38]# gorsky#m#et#al.#1998# israel# 168# 38.7%# female#predilection# [39]# kerdpon#et#al.#1999# southern#thailand# 609# 61.7%# female#predilection# [22]# sonnier#et#al.1999# united#states#(african# american)# 328# 47.20%# female#predilection# [40]# sirirungrojying#et#al.1999# thailand# 412# 27.8%# 0# [22]# gozil#r#et#al.#1999# turkish# 86# 45.4%# 0# [41]# jainkittivong#et#al.#2000# thailand# 960# 26.9%# 0# [42]# al0bayaty#et#al#in#2001# trinidad#and#tobago# 667# 12.3%# 0# [7]# aphinhasmit#w#et#al.# 2002# thailand# 1200# 58.1%# female#predilection# [43]# virgina#g#et#al.#2003# spanish# 278# 20.3%# female#predilection# [44]# bruce#i#et#al.#2004# ghana# 926# 14.6%# female#predilection# [20]# cagirankaya#lb#et#al.# 2004# turkish# 253# 20.9%# female#predilection# [45]# yildiz#e#et#al.#2005# turkish# 1943# 30.9%# female#predilection# [46]# al#quran#fa#et#al.#2006# jordan# 338# 29.8%# female#predilection# [47]# jainkittivong#a#et#al.#2007# thailand# 1520# 60.5%# female#predilection# [48]# sisman#et#al.#2007# turkish# 2660# 4.1%# female#predilection# [49]# hiremath#vk#et#al.#2011# malay# 65# 55.4%# female#predilection# [50]# santosh#patil#et#al.#2014# india# 3087# 6.9%# male#predilection# [51]# present#study# jamaica# 335# 27.76%# female#predilection# present# study#!1! prevalence  of  oral  tori  among  medical  and  dental  students  at  the  university  of  the  west  indies   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.55    http://dentistry3000.pitt.edu   6   types  tp  (40.74%),  tm  (37.03%),   both  tp  and  tm  (22.22%)).  inter-­‐ estingly  similar  female  predilec-­‐ tion  was  also  observed  in  the  stud-­‐ ies  from  jamaica,  trinidad  and  to-­‐ bago,  ghana  and  thailand   [7,20,21,22].    certain  prevalence   with  respect  to  age  has  also  been   observed.  tori  tend  to  appear   more  frequent  during  middle  age   of  life  [4].  in  the  present  study,  we   could  not  conclude  on  age  fre-­‐ quency  of  tori,  due  to  population   involved  in  the  present  study  were   medical  and  dental  students  who   fall  in  the  age  range  of  19-­‐30   years.  the  onset  of  tp  appears  to   be  earlier  than  tm  and  the  cases   have  been  described  from  birth   and  the  first  decade  of  life.  in  con-­‐ trast,  appearance  of  tm  is  rare   before  the  first  decade  of  life   [4,42].  it  is  worth  noting  that  pre-­‐ sent  study  is  documenting  preva-­‐ lence  of  tm  as  37.03%  with  the   age  range  of  19-­‐30  years.  most   published  reports  suggested  that   tp  appears  to  have  female  predi-­‐ lection  and  it  is  believed  that  there   may  be  dominant  type  linked  to   the  x  chromosome.  although  most   studies  reported  female  predilec-­‐ tion  of  tp,  the  difference  was  not   statistically  significant.  on  other   hand  tm  was  also  reported  to   have  higher  predilection  in  males,   the  difference  had  no  statistical   significance.   consistently,  epidemiologi-­‐ cal  studies  are  reported  from  1950   to  current  date  regarding  oral  tori   from  various  geographical  areas.   comparing  prevalence  rates   among  our  literature  search  from   the  countries  alaska,  southern   thailand,  malaysia,  yugoslavia,   singapore,  united  states,  turkey,   israel,  norway,  jordan,  jamaica,   spain,  iceland,  ghana,  german,   trinidad  and  tobago,  india,  vi-­‐ etnam  and  saudi  arabia  revealed  a   higher  prevalence  rate  of  oral  tori   in  eskimos  and  the  least  in  saudi   arabians.                    (table  5)  a  true   conclusion  about  a  country  having   a  high  or  low  prevalence  rate  of   oral  tori,  could  not  be  made  from   the  comparison  table  because  the   sample  size  between  each  study   shows  great  variation.  interesting-­‐ ly,  most  countries  reported  a   higher  prevalence  of  oral  tori   among  females.  hence,  it  can  be   concluded  that  oral  tori  is  a  com-­‐ mon  normal  anatomical  variant   that  can  be  seen  in  most  of  geo-­‐ graphical  areas  and  female  predi-­‐ lection  is  strongly  supported.  fur-­‐ ther,  from  caribbean  and  west   african  studies,  the  present  study   from  jamaica  seems  to  have  high   prevalence  of  oral  tori.       clinical  diagnosis  of  tori  is   usually  straight  forward  and  inves-­‐ tigations  are  generally  not  re-­‐ quired.  dental  clinicians  should   note  that  tori  can  have  slow  and   continuous  growth  in  later  age.   however,  although  growth  is  slow   and  continuous,  it  may  stop  spon-­‐ taneously.  it  is  also  worth  to  note   that  differential  diagnosis  of  bony   and  unilateral  growing  lesion  in-­‐ cludes  tori,  peripheral  ossifying   firboma,  osteoma,  osteochon-­‐ droma,  osteoid  osteoma,  osteo-­‐ blastoma  and  osteosarcoma.  the   presence  of  pain  or  paresthesia  in   bony  unilateral  growing  should   prompt  further  investigation  [52].       conclusion   the  prevalence  of  tp  and   tm  are  higher  in  our  observation.   our  results  showed  a  female  pre-­‐ dilection  among  gender  and  simi-­‐ lar  interpretation  was  observed   from  previous  studies  from  vari-­‐ ous  geographic  locations.  the  pre-­‐ sent  study  supports  that  etiology   of  tp  and  tm  being  multifactorial,   specifically  genetic,  marine  diet,   and  well-­‐developed  masticatory   muscles.    due  to  the  higher  preva-­‐ lence  of  tp  and  tm  in  the  present   study,  jamaican  dentists  need  to   be  knowledgeable  about  the  high   prevalence  and  clinical  significance   of  tp  and  tm  on  dental  treatment.     references   1. clinical,  tomographic  as-­‐ pects  and  relevance  of  to-­‐ rus  palatinus:  case  report   of  two  sisters.  alexandre   simo˜es  nogueira,  eduardo   sanches  gonc¸ales,  paulo   sergio  da  silva  santos,  jo-­‐ se´  humberto  damante,   phillipe  nogueira  barbosa   alencar,  fernanda  arau´jo   sampaio  et  al.  surg  radiol   anat  2013  nov;35:867-­‐871.   pmid:  24170193   2. prevalence  of  torus  palati-­‐ nus  in  cappadocia  region   population  of  turkey.  yild-­‐ iray  sisman,  elif  tarim  er-­‐ tas,  cumali  gokce  and  fa-­‐ ruk  akgunlu.  european   journal  of  dentistry  2008   oct;2:269-­‐75.  pmid:   19212533   3. prevalence  of  torus  palati-­‐ nus  and  torus  mandibularis   in  jordanian  population.   prevalence  of  oral  tori  among  medical  and  dental  students  at  the  university  of  the  west  indies   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.55    http://dentistry3000.pitt.edu   7   derar  al-­‐sebaie,  mashoor   al  wrikat.  pakistan  oral  and   dental  journal  2011   may;31(1):  214-­‐16.  pmid:   16685302   4. current  status  of  the  torus   palatinus  and  torus  man-­‐ dibularis.  andres  s  garcia-­‐ garcia,  jose-­‐maria  mar-­‐ tinez-­‐gonzalez,  rafael   gomez-­‐font,  angeles  soto-­‐ rivadeneira,  lucia  ovideo-­‐ roldan.  med  oral  patol   oral  cir  buccal  2010  mar;   15(2):e353-­‐60.  pmid:   19767716   5. prevalence  of  torus  man-­‐ dibularis  among  various   ethnic  groups  in  karachi   city:  a  cross  sectional   study.  daud  mizra,   nadeem  hafeez  khokhar,   shah  jahan  katpar,  yawar   khan,  saman  hakeem,  mo-­‐ hammad  ayub  musani.  pa-­‐ kistan  oral  and  dental   journal  2013;33(2):277-­‐ 280.   6. genetic  influence  on  the   prevalence  of  torus  palati-­‐ nus.  gorsky  m,  bukai  a,   shohat  m.  am  j  med   genet.  1998  jan;75(2):138-­‐ 40.  pmid:  9450873   7. an  epidemiological  study   of  tori  among  667  dental   outpatients  in  trinidad  and   tobago,  west  indies.  al-­‐ bayaty  hf,  murti  pr,  mat-­‐ thews  r.  gupta.  interna-­‐ tional  dental  journal   2001aug;  51:300-­‐04.   pmid:  11570546   8. the  effect  of  culture   change  upon  the  eskimo   dentition.  mayhal  j  t.  arc-­‐ tic  antbrapol  1970;51:  117-­‐ 121.   9. pronounced  palatal  and   mandibular  tori  observed   in  a  patient  with  chronic   phenytoin  therapy:  a  case   report.  sasaki  h,  ilkeo  d,   kataoka  m,  kido  j,  kitamu-­‐ ra  s,  nagata  t.  j  periodontl   1999  apr;70:445-­‐8.  pmid:   10328658   10. relationship  between  to-­‐ rus  mandibularis  and  num-­‐ ber  of  present  teeth.  eggen   s,  natvig  b.  scand  j  dent   res  1986  jun;94:233-­‐40.   pmid:  3461543   11. torus  palatinus  and  torus   mandibularis  in  edentulous   patients.  al  quran  f,  al-­‐ dwairi  z.  j  contemp  dent   pract  2006  may;7(2):  112-­‐ 119.  pmid:  16685302   12. palatal  tubercles,  palatal   tori,  and  mandibular  tori:   prevalence  and  anatomic   features  in  a  u.s.  popula-­‐ tion.  sonnier  ke,  horning   gm,  cohen  me.  j  periodon-­‐ tol  1999  mar;70:329-­‐36.   pmid:  10225550   13. palatal  rugae  patterns  as   bioindicators  of  identifica-­‐ tion  in  forensic  dentistry.   ismar  eduardo  martins,  sil-­‐ via  helena  de  carvalho   sales-­‐peres,  arsenio  sales-­‐ peres,  suzana  papile   maciel  carvalho.  rfo  2009;   14(3):  227-­‐233.   14. torus  mandibularis  bone   chips  combined  with  plate-­‐ let  rich  plasma  gel  for   treatment  of  intrabony  os-­‐ seous  defects:  clinical  and   radiographic  evaluation.   hassan  ks,  alagl  as,  abdel-­‐ hady  a.  int  j  oral  maxillo-­‐ fac  surg.  2012   dec;41(12):1519-­‐26.  pmid:   22483445   15. surgical  removal  of  man-­‐ dibular  tori  and  its  use  as   an  autogenous  graft.   rastogi  k,  verma  sk,  bhu-­‐ shan  r.  bmj  case  reports.   2013  apr;   2013:bcr2012008297.   doi:10.1136/bcr-­‐2012-­‐ 008297.  pmid:  23605821   16. relationship  between  oral   tori  and  temporomandibu-­‐ lar  disorders.  sirirungrojy-­‐ ing  s,  kerdpon  d.  int  dent   j.  1999  apr;49(2):101-­‐4.   pmid:  10858740   17. oral  tori  in  chronic  hemo-­‐ dialysis  patients.  pei-­‐jung   chao,  huang-­‐yu  yang,   wen-­‐hung  huang,  et  al.,   biomed  research  interna-­‐ tional  2015  mar,  article  id   897674,  7  pages.   doi:10.1155/2015/897674   pmid:  25918724   18. autosomal  dominant  oste-­‐ osclerosis:  report  of  kin-­‐ dred.  curran  ae,  pfeffle  rc,   miller  e.  oral  surg  oral   med  oral  pathol  oral  radi-­‐ ol  endod  1999   may;87:600-­‐4.  pmid:   10348520   prevalence  of  oral  tori  among  medical  and  dental  students  at  the  university  of  the  west  indies   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.55    http://dentistry3000.pitt.edu   8   19. torus  palatinus:  a  new  an-­‐ atomical  correlation  with   bone  density  in  postmeno-­‐ pausal  women.  joseph  l   belsky,  josephine  s  hamer,   janet  e  hubert,  karl  in-­‐ sogna.  the  journal  of  clini-­‐ cal  endocrinology  and  me-­‐ tabolism  2003   may;88(5):2081-­‐86.  pmid:   12727958   20. oral  tori  in  jamaicans  of   african  origin:  a  clinical   study.  ogunsalu  c  u.  west   indian  dent  j  1994  1:  5-­‐7.     21. epidemiological  aspects  of   oral  tori  in  a  ghanaian   community.  bruce  i,  ndanu   ta,  addo  me  accra,  ghana.   international  dental  jour-­‐ nal  2004  apr;54:78-­‐82.   pmid:  15119797   22. a  clinical  study  of  oral  tori   in  southern  thailand:  prev-­‐ alence  and  the  relation  to   parafunctional  activity.   kerdpon  d,  sirirungrojying   s.  eur  j  oral  sci  1999   feb;107:9-­‐13.  pmid:   10102745   23. torus  palatinus.  woo  jk.   am  j  phys  antopol  1950   mar;8:81-­‐111.  pmid:   15410865   24. the  occurrence  of  torus   palatine  and  torus  man-­‐ dibularis  in  2478  dental  pa-­‐ tients.  kolas  s,  halperin  v,   jefferis  k,  huddleston  s,   rbinson  hb.  oral  sur  oral   med  oral  pathol  1953;   6:1134-­‐1141.   25. incidence  of  torus  palati-­‐ nus  in  yugoslav  skulls.  vidic   b.  j  dent  res  1966  oct;   45:1511-­‐1515.  pmid:   5225326   26. morphology  and  incidence   of  torus  palatinus  and   mandibularis  in  brazillian   indians.  bernaba  jm.  j   dent  res  1977may;56:499-­‐ 501.  pmid:  267104   27. torus  palatinus.  a  clinical   study.  chew  cl,  tan  ph.   aust  dent  j  1984  aug;   29:245-­‐48.  pmid:  6596933   28.  torus  palatinus  and  torus   mandibularis  in  edentulous   patients.  al-­‐quran  fa,  al-­‐ diwari  zn.  j  contemp  dent   pract  2006  may;7:112-­‐19.   pmid:  16685302   29. torus  palatinus  in  icelandic   school  children.  axelsson   g,  hedegaard  b.  am  j  phys   anthropol  1985;67:105-­‐ 112.   30. oral  exophytic  lesions  in   23,616  white  americans   over  35  years  of  age.   bouquot  je,  gundlach  kk.   oral  surg  oral  med  oral   pathol  1986  sep;62:284-­‐ 91.  pmid:  3462634   31. developmental  oral  anom-­‐ alies  among  school  chil-­‐ dren  in  gizan  region,  saudi   arabia.  salem  g,  holm  sa,   fattah  r,  basset  s,  nasser   c.  community  dent  oral   epidemiol  1987;15:150-­‐51.     32. prevalence  of  torus  palati-­‐ nus  and  torus  mandibularis   in  germans  and  thai.   reichart  pa,  neuhas  f,   sookasem  m.  community   dent  oral  epidemiol  1988   feb;16:61-­‐4.  pmid:   3422622   33. torus  mandibularis:  an  es-­‐ timation  of  the  degree  of   genetic  determination.  eg-­‐ gen  s.  acta  odontol  scand   1989  dec;47:409-­‐15.  pmid:   2609949   34. palatine  and  mandibular   tori.  a  morphologic  study   in  the  current  norwegian   population.  haugen  lk.  ac-­‐ ta  odontol  scand.  1992   apr;50:65–77.  pmid:   1604967   35. prevalence  of  torus  palati-­‐ nus  and  torus  mandibularis   in  1000  patients.  shah  ds,   sanghavi  sj,  chawda  jd,   shah  rm.  indian  j  dent   res.  1992  oct;3(4):107-­‐10.   pmid:  1344979   36. variation  in  torus  palatinus   prevalence  in  norway.  eg-­‐ gen  s,  natvig  b,  gasemyr  j.   scand  j  dent  res.  1994   feb;102:54–59.  pmid:   8153581   37. prevalence  of  oral  lesions   in  a  selected  vietnamese   population.  nair  rg,    sa-­‐ marnayake  lp,  philipsen   hp,  graham  rg,  itthagarun   a.  int  dent  j  1996   feb;46:48-­‐51.  pmid:   8744917   38. prevalence  of  torus  palati-­‐ nus  in  a  population  of   young  and  adult  israelis.   prevalence  of  oral  tori  among  medical  and  dental  students  at  the  university  of  the  west  indies   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.55    http://dentistry3000.pitt.edu   9   gorsky  m,  raviv  m,  kfir  e,   moskona  d.  arch  oral  biol.   1996  jun;41:623–625.   pmid:  8937655   39. genetic  influence  on  the   prevalence  of  torus  palati-­‐ nus.  gorsky  m,  bukai  a,   shohat  m.  am  j  med   genet.1998;75:138–140.   40. palatal  tubercles,  palatal   tori,  and  mandibular  tori:   prevalence  and  anatomical   features  in  a  u.s.  popula-­‐ tion.  sonnier  ke,  horning   gm,  cohen  me.    j  perio-­‐ dontol  1999  mar;70:329-­‐ 36.  pmid:  10225550   41. is  torus  palatinus  a  feature   of  a  well-­‐developed  maxil-­‐ la.  cagirankaya  lb,  kansu   o,  hatipoglu  mg.  clin  anat.   2004  nov;17:623–625.   pmid:  15494968   42. buccal  and  palatal  exosto-­‐ ses:  prevalence  and  con-­‐ currence  with  tori.  jain-­‐ kittivong  a,  langlais  rp.   oral  surg    oral  med  oral   pathol  oral  radiol  endod.   2000  jul;  90:48-­‐53.  pmid:   10884635   43. torus  palatinus  and  torus   mandibularis  in  a  thai   population.  apinhasmit  w,   jainkittivong  a,  swasdison   s.  sci  asia.  2002;28:105– 111.   44. oral  tori  in  a  sample  of  the   spanish  university  stu-­‐ dents:  prevalence  and   morphology.  virginia  ga-­‐ lera,  josefa  maría  moreno,   esperanza  gutiérrez.  an-­‐ tropologia  portuguesa   2003;20:281-­‐305.   45. is  torus  palatinus  a  feature   of  a  well-­‐developed  maxil-­‐ la.  cagirankaya  lb,  kansu   o,  hatipoglu  mg.  clin  an-­‐ at.  2004;17:623–625   46. prevalence  of  torus  palati-­‐ nus  in  turkish  school  chil-­‐ dren.  yildiz  e,  deniz  m,   ceyhan  o.  surg  radiol  an-­‐ at.  2005  dec;27:368–371.   pmid:  16075159   47. torus  palatinus  and  torus   mandibularis  in  edentulous   patients.  al  quran  fa,  al-­‐ dwairi  zn.  j  contemp  dent   pract.  2006;7:112–119.   48. prevalence  and  clinical   characteristics  of  oral  tori   in  1,520  chulalongkorn   university  dental  school   patients.  jainkittivong  a,   apinhasmit  w,  swasdison   s.  surg  radiol  anat.  2007   mar;29:125–131.  pmid:   17340055   49. prevalence  of  torus  palati-­‐ nus  in  cappadocia  region   population  of  turkey.  sis-­‐ man  y,  ertas  et,  gokce  c,   akgunlu  f.  european  jour-­‐ nal  of  dentistry.  2008   oct;2:269-­‐275.  pmid:   19212533   50. prevalence  of  torus  palati-­‐ nus  and  torus  mandibularis   among  malay  population.   hiremath  vk,  husein  a,   mishra  n.  journal  of  inter-­‐ national  society  of  preven-­‐ tive  and  community  den-­‐ tistry.  2011jul;1(2):60-­‐64.   pmid:  24478956   doi:10.4103/2231-­‐ 0762.97704.   51. prevalence  of  torus  palati-­‐ nus  and  torus  mandibularis   in  an  indian  population.   patil  s,  maheshwari  s,   khandelwal  sk.  saudi  j  oral   sci  2014;1:94-­‐7.       52. peripheral  osteoma  of  the   oral  and  maxillofacial  re-­‐ gion:  a  study  of  35  new   cases.  sayan  nb,  ucok  c,   karasu  ha.  j  oral  maxillo-­‐ fac  surg  2002   nov;60:1299-­‐301.  pmid:   12420263     microsoft word 74 2017.docx   vol  5,  no  1  (2017)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2017.74           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     oral  candidiasis:  relation  to  systemic  diseases  and  medications   xiaozhu  chu1      1university  of  pi.sburgh,  school  of  dental  medicine abstract   background:  oral  candidiasis  is  by  far  the  most  common  oral  fungal  infec!on  in  humans.  it  is  caused  by  the  fungal  or-­‐ ganism  candida  albicans.  when  the  host  is  debilitated  by  other  diseases  and  condi!ons,  c.  albicans,  which  is  usually  a   part  of  the  normal  oral  flora,  can  turn  pathogenic  and  invade  the  host  2ssue  to  cause  the  infec2on.  the  purpose  of   this   paper   is   to   inves-gate   the   role   of   systemic   diseases   and  medica-ons   in   the   development   of   oral   candidiasis.   methods:  a  total  of  12  cases  with  code   indica2ng  oral  candidiasis  were  collected   from  the  university  of  pi.sburgh   school  of  dental  medicine  dental  registry  and  dna  repository.  the  systemic  diseases  and  medica*ons  were  descrip-­‐ !vely  analyzed.  results:  50%  of  the  subjects  had  more  than  two  systemic  diseases.  the  most  prevalent  diseases  were   mental   illnesses   (50%),   cardiovascular   diseases   (41.7%),   and   respiratory   system   diseases   (33.3%).   50%   of   subjects   were  on  polypharmacy  therapies  and  75%  of  subjects  were  taking  medica-ons  that  may  contribute  to  oral  candidiasis.   among   the  medica!ons,   an!depressants   and   inhala#onal   cor#costeroids  may   have   strong   poten#als   to   cause   oral   candidiasis.  conclusion:  oral  candidiasis  is  associated  with  having  systemic  diseases  and  intake  of  medica.on,  espe-­‐ cially  with  those  medica!ons  can  cause  xerostomia.  as  the  number  of  systemic  diseases  and  medica!ons  increases,   the  risk  of  developing  oral  candidiasis  may  increase  too.   cita%on:  chu,  x.  (2017)  oral  candidiasis:  rela,on   to   systemic  diseases  and  medica-ons.  den$stry   3000.  1:a001  doi:10.5195/d3000.2017.74   received:    may  5,  2017   accepted:    may  26,  2017   published:    june  19,  2017   copyright:  ©2017  chu,  x.  this  is  an  open  access   ar!cle   licensed   under   a   crea!ve   commons   a!ribu%on  work  4.0  united  states  license.   email:  xic81@pi(.edu   introduction   candidiasis  is  by  far  the   most  common  oral  fungal  infec-­‐ tion  in  humans  and  has  a  variety   of  clinical  manifestations.  it  is  the   infection  caused  by  the  yeast-­‐like   fungal  organism  candida  albicans.   as  many  other  pathogenic  fungi,   c.  albicans  can  exist  in  two   forms—the  yeast  form  and  the   hyphal  form.  the  hyphal  form  is   believed  to  be  able  to  invade  the   host  tissue  and  cause  the  infec-­‐ tion.  [1]  c.  albicans  is  considered   as  part  of  the  normal  oral  flora   and  the  prevalence  of  oral  can-­‐ dida  carriage  in  the  healthy  popu-­‐ lation  has  been  estimated  to  range   from  23%  to  68%.  [2]  candidiasis   used  to  be  considered  as  only  an   opportunistic  infection  but  it  has   been  recognized  that  people  who   are  otherwise  healthy  can  develop   oral  candidiasis  too.  however,  as  a   result  of  complex  host  and  organ-­‐ ism  interaction,  a  large  percentage   of  patients  with  candidal  infection   are  still  individuals  who  are  debili-­‐ tated  by  another  diseases  and   conditions.  many  risk  factors  can   modify  an  individual’s  susceptibil-­‐ ity  to  oral  candidiasis,  including   compromised  autoimmunity,  to-­‐ bacco  consumption,  hyposaliva-­‐ tion,  denture  wearing,  systemic   disease,  and  medication.   compromised  autoimmunity     t-­‐cell  immunity  has  a  criti-­‐ cal  role  in  host  defense  against   candidal  infection.  chronic  muco-­‐ cutaneous  candidiasis  (including   oral  candidiasis)  is  an  infectious   phenotype  in  patients  with  inher-­‐ ited  or  acquired  t-­‐cell  deficiency.   studies  have  shown  that  th17  cell,   as  well  as  other  cell  expressing  ret-­‐ inoic  acid-­‐related  orphan  receptor   γt  (rorγt),  produce  interleukin   (il)-­‐17,  which  has  an  essential  role   in  defensing  against  candidal  in-­‐ fections  in  humans.  four  genetic   etiologies,  ar  il-­‐17  receptor  a,  il-­‐ 17  receptor  c  and  act1  deficien-­‐ cies,  and  ad  il-­‐17f  deficiency,  are   identified  as  underlying  risk  fac-­‐ tors  for  chronic  mucocutaneous   candidiasis.  each  of  these  gene   defects  has  a  direct  negative  effect   on  il-­‐17  signaling.  patients  with   these  gene  defects  may  have  se-­‐ verely  reduced  frequencies  of  cir-­‐ culating  il-­‐17-­‐producing  t  cells.  or   they  may  produce  neutralizing  au-­‐ toantibodies  against  il-­‐17.  as  a   result,  the  ability  of  neutrophil   cells  to  kill  c.  albicans  is  impaired   and  chronic  mucocutaneous  can-­‐ didiasis  may  develop.  [3]     tobacco  consumption   e!ology  and  familial  inheritance  of  pleomorphic  adenomas   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.73    http://dentistry3000.pitt.edu    tobacco  smoking  has  been   considered  as  one  of  the  many   factors  that  can  predispose  indi-­‐ viduals  to  oral  candidiasis.  litera-­‐ tures  revealed  that  the  rate  of  oral   candidal  carriage  in  tobacco   smokers  is  significantly  higher   than  in  non-­‐smokers.  besides  c.   albicans,  the  oral  carriage  of  other   species  such  as  c.  glabrata,  c.   dubliniensis  and  c.  tropicalis  are   also  associated  with  smoking.  the   observation  is  even  more  striking   in  hiv-­‐infected  individuals.  it  has   been  reported  that  hiv-­‐positive   smokers  are  50  times  more  likely   to  be  oral  candidal  infection  posi-­‐ tive  compared  to  hiv-­‐infected   non-­‐smokers.  it  has  not  been   cleared  yet  that  why  tobacco  con-­‐ sumption  can  increase  candidal   carriage.  however,  there  are  stud-­‐ ies  suggesting  that  smoking  may   lead  to  localized  epithelial  altera-­‐ tions,  which  facilitate  candidal   colonization.  another  hypothesis   is  that  cigarette  smoke  may  con-­‐ tain  nutritional  factors  for  c.  albi-­‐ cans.  [4]     hyposalivation   adequate  salivation  is  es-­‐ sential  for  oral  health,  since  it  pro-­‐ vides  support  for  the  microbial   population  of  the  mouth,  while  at   the  same  time  containing  antimi-­‐ crobial  products  that  control  these   microbial  populations.  histatin  5   (hst  5)  is  an  important  salivary  an-­‐ timicrobial  protein.  it  has  been   shown  that  hst  5  has  potent  and   selective  antifungal  activity.  after   coupling  with  the  carrier  molecule   spermidine,  hst  5  significantly  en-­‐ hances  the  killing  of  c.  albicans.   [5]  reduced  salivary  secretion,  as   occurred  in  patients  with  sjögren's   syndrome,  can  re-­‐ sult  in  a  significant   increase  in  oral   candidal  carriage.   the  prevalence  of   oral  candidal  car-­‐ riage  among   sjögren's  syndrome   patients  is  estimat-­‐ ed  to  range  from   68%  to  100%,  com-­‐ pared  to  the  range   from  23%  to  68%  in   normal  population   [2].     denture  wearing    denture  stomatitis  is  con-­‐ sidered  to  be  a  form  of  erythema-­‐ tous  candidiasis  and  it’s  a  common   inflammatory  lesion  of  the  oral   mucosa  covered  by  denture.  epi-­‐ demiological  studies  show  a   prevalence  of  denture  stomatitis   among  denture  wearers  from  15%   to  over  70%.  etiological  factors   include  poor  denture  hygiene,   continual  and  nighttime  wearing   of  removable  dentures,  accumula-­‐ tion  of  denture  plaque,  and  bacte-­‐ rial  and  yeast  contamination  of   denture  surface.  all  together,  the-­‐ se  factors  may  increase  the  ability   of  c.  albicans  to  colonize  both   denture  and  oral  mucosal  surfac-­‐ es,  exerting  its  pathogenic  effort.   [6]     systemic  diseases  many  systemic   diseases  have  been  associated   with  oral  candidiasis.  the  primary   cause  is  attributed  to  the  de-­‐ creased  salivary  secretion,  leading   to  the  reduced  concentration  of   immunoglobulin  in  the  saliva  and   less  efficient  humoral-­‐mediated   host  defense  against  c.  albicans.   e!ology  and  familial  inheritance  of  pleomorphic  adenomas   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.73    http://dentistry3000.pitt.edu   for  patients  with  diabetes  melli-­‐ tus,  besides  the  reduced  salivary   flow,  the  high  level  of  blood  glu-­‐ cose  also  plays  a  significant  role.  it   is  associated  with  reduced  salivary   ph  and  facilitates  oral  candidal   overgrowth  and  colonization.  [7]   as  an  opportunistic  infection,  oral   candidiasis  is  also  associated  with   a  wide  spectrum  of  systemic  dis-­‐ eases  that  suppress  the  host  auto-­‐ immunity  [8].     medication   several  drugs  may  cause   the  development  of  oral  candidia-­‐ sis  by  many  mechanisms.  the   pharmacological  action  of  the   broad-­‐spectrum  group  of  antibiot-­‐ ics  may  break  the  balance  within   the  normal  oral  flora,  resulting  in   the  overgrowth  of  c.  albicans.   drugs  such  as  corticosteroids  may   suppress  either  the  nonspecific   inflammatory  response  or  the  t-­‐ cell-­‐mediated  immunity,  which   can  in  turn  predispose  individuals   to  oral  candidiasis.  drugs  that   have  xerogenic  effects  can  cause   oral  candidiasis  by  directly  reduc-­‐ ing  the  salivary  flow.  [8]     it  appears  that  the  associa-­‐ tion  between  oral  candidiasis  and   systemic  diseases,  intake  of  medi-­‐ cations  has  been  well  established.   the  purpose  of  this  study  is  to   analyze  the  12  cases  collected   from  the  university  of  pittsburgh   school  of  dental  medicine  dental   registry  and  dna  repository,  and   further  investigate  which  systemic   diseases  and  medications  may  be   potent  and  contribute  to  the  de-­‐ velopment  of  oral  candidiasis.   methods   by  the  time  of  the  study  started,   5,869  subjects  from  the  university   of  pittsburgh  school  of  dental   medicine  dental  registry  and  dna   repository  were  available  for   analysis.  since  the  september  of   2006,  the  registry  invited  all  pa-­‐ tients  who  seek  treatment  at  the   university  of  pittsburgh  school  of   dental  medicine  to  participate  and   give  written  consent  to  authorize   their  clinical  information  to  be   used  for  academic  research  pur-­‐ pose.  12  cases  with  codes  indicat-­‐ ing  oral  candidiasis  were  collected   from  the  5,869  subjects.  com-­‐ pared  to  the  estimated  prevalence   of  oral  candidal  carriage,  which   ranges  from  23%  to  68%  in  the   normal  population  [2],  the  number   12  in  5,869  subjects  (0.2%)  seems   to  be  unreasonably  small.    this  can   be  a  result  of  the  inconsistent  re-­‐ port  of  the  oral  lesions  by  clinical   faculty  members  and  student  den-­‐ tists.  for  the  12  cases  with  oral   candidiasis,  the  data  on  current   (i.e.  time  of  examination)  systemic   diseases/medical  conditions,  daily   intake  of  prescribed  or  otc  medi-­‐ cations  were  obtained  from  the   registry.  the  total  number  of  sys-­‐ temic  diseases  and  medical  condi-­‐ tions  were  counted.  all  medica-­‐ tions  were  categorized  by  using   the  internationally  approved  ana-­‐ tomical  therapeutic  chemical   (atc)  classification  system.  and   their  impacts  on  oral  hygiene  and   dental  treatment  were  obtained   from  the  website  lexicomp  online   for  dentistry   (http://www.wolterskluwercdi.co m/online-­‐for-­‐dentistry/).  all  data   then  were  descriptively  analyzed.     results   characteristics  of  the  12  study   samples  are  presented  in  table  1.   among  the  12  cases,  only  one  par-­‐ ticipant    (8.3%)  did  not  report  any   systemic  disease  or  medical  condi-­‐ tion.  for  the  other  11  cases,  the   number  of  systemic  diseas-­‐ es/medical  conditions  reported  by   the  participants  varied  from  1  to  8   (33.3%  reporting  more  than  four   diseases).  the  highest  scores  by   e!ology  and  familial  inheritance  of  pleomorphic  adenomas   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.73    http://dentistry3000.pitt.edu   number  of  diseased  individuals   were  found  for  neurological  sys-­‐ tem  disease  (58.3%,  n  =  7)  includ-­‐ ing  depression,  epilepsy,  and  bipo-­‐ lar  disorder,  cardiovascular  system   diseases  (41.7%,  n  =  5)  including   mainly  hypertension,  irregular   heartbeat,  and  heart  failure,  and   respiratory  system  disease  (33.3%,   n  =  4)  including  asthma  (figure  1).   less  frequent  diagnosis  included   gastrointestinal  tract  and  metabo-­‐ lite  diseases  (liver  diseases  and   diabetes,  25%,  n  =  3),  neoplasms   (neuroblastoma  and  unspecified   throat  cancer,  16.7%,  n  =  2),  geni-­‐ tourinary  system  diseases  (kidney   disease,  8.3%,  n  =  1),  and  muscu-­‐ loskeletal  system  diseases  (pros-­‐ thetic  joint,  8.3%,  n  =1).     all  the  study  subjects  had   intake  of  prescribed  or  otc  (over   the  counter)  medications.  the   number  of  medications  taken  on   daily  basis  varied  from  1  to  14.   among  the  12  cases,  three  indi-­‐ viduals  took  only  one  medication   (25%),  three  took  2-­‐4  medications   (minor  polypharmacy,  25%),  and   six  took  more  than  4  medications   (major  polypharmacy,  50%).  the   distribution  of  prescribed  medica-­‐ tion  (figure  2)  shows  that  antihy-­‐ pertensive  agents,  antidepressant,   systemic  hormones,  analgesics,   and  agents  for  obstructive  airway   diseases  predominated.  this  med-­‐ ication  pattern  reflected  the  dis-­‐ ease  burden  among  the  study  sub-­‐ jects.     since  intake  of  medication   is  an  important  risk  factor  to  the   development  of  oral  candidiasis,   all  the  medications  taken  by  study   subjects  were  analyzed  by  their   impacts  on  oral  cavity  environ-­‐ ment  and  dental  treatment.  based   on  the  information  obtained  from   the  website  lexicomp  online  for   dentistry,  all  medications  that  can   cause  xerostomia,  change  in  sali-­‐ vation,  candidal  infection  were   labeled  as  “may  contribute  to  oral   candidiasis”.  the  63  medications   were  fitted  into  22  categories   based  on  the  anatomical  thera-­‐ peutic  chemical  (atc)  classifica-­‐ tion  system.  among  the  predomi-­‐ nated  categories,  antidepressant   contained  10  medications,  all  of   which  may  contribute  to  the  de-­‐ velopment  of  oral  candidiasis.  it   had  been  followed  by  agents  for   obstructive  airway  diseases  and   analgesics,  which  had  5  out  of  9   (55.5%)  and  2  out  of  7  (28.6%)   medications,  respectively,  fell  into   the  category  “may  contribute  to   oral  candidiasis”.  the  other  cate-­‐ gories,  which  contained  medica-­‐ tions  that  may  contribute  to  oral   candidiasis,  included  anticonvul-­‐ sant,  antimanic,  antiulcer,  hypnot-­‐ ic  and  immunosuppressant  agents.   (figure  3)  among  all  12  subjects,  9   (75%)  were  taking  medications   that  fitting  into  the  “  may  contrib-­‐ ute  to  oral  candidiasis”  category.   discussion  and  conclusion   in  my  study,  50%  of  the   study  samples  were  having  three   or  more  than  three  systemic  dis-­‐ eases,  which  is  in  agreement  with   previous  observations  suggesting   that  having  multiple  systemic  dis-­‐ eases  and  medical  conditions  is   associated  with  oral  candidiasis.   the  most  frequent  diseases  involv-­‐ ing  in  this  study  were  mental  dis-­‐ eases  including  depression  and   bipolar  disorder,  and  obstructive   airway  diseases  including  asthma,   chronic  obstructive  pulmonary   disease,  and  an  unspecified  lung   disease.  those  are  not  diseases   that  directly  cause  change  in  sali-­‐ vary  secretion  and  salivary  glands   or  those  suppress  the  host  auto-­‐ immunity.  their  primary  associa-­‐ tion  with  the  development  of  oral   candidiasis  is  more  attributed  to   the  medications  that  were  taken   by  subjects  to  treat  the  systemic   diseases.  however,  it  may  not  be   true  for  one  subject  who  was  di-­‐ agnosed  as  insulin-­‐dependent  dia-­‐ betes.  on  the  morning  the  dental   examination  was  performed,  the   subject  reported  that  her  blood   glucose  level  was  235mg/dl.  com-­‐ pared  with  the  preferable  goal  of  a   good  diabetic  management,  which   is  close  to  the  70  to  120mg/dl   e!ology  and  familial  inheritance  of  pleomorphic  adenomas   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.73    http://dentistry3000.pitt.edu   range,  it’s  safe  to  assume  the  dia-­‐ betes  was  not  well  controlled.  be-­‐ sides  all  other  drugs  she  took,  the   level  of  blood  glucose  may  play  an   important  role  in  the  development   of  her  oral  candidiasis.  it  has  been   reported  that  highest  rate  of  c.   albicans  colonization  occurs  in  di-­‐ abetic  patients  with  poor  glycemic   control.  in  addition,  subjective  oral   dryness  is  a  frequent  complaint   among  diabetic  patients.  [7]     the  five  most  commonly   reported  medications  included   systemic  hormones,  antidepres-­‐ sants,  antihypertensive  agents,   agents  for  obstructive  airway  dis-­‐ eases,  and  analgesics.  among  the   five  categories,  the  association   between  antidepressants  and  oral   candidiasis  has  been  well  estab-­‐ lished.  all  salivary  secretion  is  me-­‐ diated  by  neurotransmitters  and   relies  on  stimulations  from  the   autonomic  nervous  system.  stimu-­‐ lation  of  the  cholinergic  system   leads  to  secretion  of  “watery”  sali-­‐ va  thus  anticholinergic  drugs  re-­‐ duce  the  secretion.  many  antide-­‐ pressants  have  an  anticholinergic   effect  that  blocks  the  peripheral   cholinergic  innervation,  resulting   in  hyposalivation  and  xerostomia.   [9]  among  other  agents  used  to   manage  obstructive  airway  dis-­‐ eases,  corticosteroids  are  widely   used  as  oral  inhalers  due  to  its  an-­‐ ti-­‐inflammatory  and  immunosup-­‐ pressive  properties.  however,  the   repeated  use  of  corticosteroids   oral  inhalers  also  suppress  an  indi-­‐ vidual’s  resistance  to  candidal  in-­‐ fection  by  suppressing  either  the   nonspecific  inflammatory  re-­‐ sponse  or  the  t-­‐cell-­‐mediated  im-­‐ munity,  which  predispose  the  oral   inhaler  users  to  oral  candidiasis.   [10]  in  this  study,  75%  of  the  study   subjects  was  on  polypharmacy   therapies  and  took  medications   that  had  xerogenic  or  immuno-­‐ suppressive  properties,  including   10  antidepressants  and  2  inhala-­‐ tional  corticosteroids.  the  finding   is  in  line  with  previous  studies  in-­‐ dicating  that  intake  of  medications   is  an  important  risk  factor  for  the   development  of  oral  candidiasis.     when  treating  patients   with  systemic  diseases  and  taking   multiple  medications,  the  dental   professionals  must  be  able  to  rec-­‐ ognize  the  early  signs  and  symp-­‐ toms  of  oral  candidiasis  and  adjust   the  treatment  plans  to  meet  the   special  needs  of  those  patients   and  prevent  the  development  of   oral  candidiasis.     in  conclusion,  based  on  the   descriptive  analysis  of  12  cases   with  oral  candidiasis,  the  findings   support  the  concept  that  the  de-­‐ velopment  of  oral  candidiasis  is   associated  with  having  multiple   systemic  diseases  and  intake  of   medications.   acknowledgements   data  from  the  study  was  obtained   from  the  university  of  pittsburgh   school  of  dental  medicine  dental   registry  and  dna  repository,   which  is  supported  by  the  univer-­‐ sity  of  pittsburgh  school  of  dental   medicine.   references   1. neville  b,  damm  d,  allen  c,   bouquot  j.  (2009)  oral  and   maxillofacial  pathology.   3rd  ed.  st.  louis:  saunders,   an  imprint  of  elsevier.  213-­‐ 224  p.     2. elucidating   the  role  of  hyposalivation  a nd  autoimmunity  in  oral   candidiasis.  billings  m,  dye   ba,  iafolla  t,  grisius  m,  al-­‐ evizos  i.  oral  dis.  2017   apr;23(3):387-­‐394.  doi:   10.1111/odi.12626.  epub   2017  feb  8.  pmid:   27998016   3. chronic  mucocutaneous   candidia-­‐ sis  disease  associated  with   inborn  errors  of  il-­‐17  im-­‐ munity.  okada  s,  puel  a,   casanova  jl,  kobayashi  m.   clin  transl  immunology.   2016  dec  2;5(12):e114.   doi:  10.1038/cti.2016.71.   ecollection  2016  dec.  re-­‐ view.  pmid:  28090315   4. the  impact  of  ciga-­‐ rette/tobacco  smok-­‐ ing  on  oral  candidosis:  an   overview.  soysa   ns,  ellepola  an.  oral  dis.   2005  sep;11(5):268-­‐73.   review.  pmid:  16120112   5. saliva-­‐ microbe  interactions  and   salivary  gland  dysfunction.   baker  oj,  edgerton  m,   kramer  jm,  ruhl  s.  adv   dent  res.  2014   may;26(1):7-­‐14.  doi:   10.1177/00220345145262 39.  review.  pmid:   24736699   6. epidemiology   and  etiology  of  denture   e!ology  and  familial  inheritance  of  pleomorphic  adenomas   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.73    http://dentistry3000.pitt.edu   stomatitis.  gendreau   l,  loewy  zg.  j  prosthodont.   2011  jun;20(4):251-­‐60.  doi:   10.1111/j.1532-­‐ 849x.2011.00698.x.  epub   2011  apr  4.  review.  pmid:   21463383   7. can-­‐ didal  overgrowth  in  diabeti c  patients:  potential  pre-­‐ disposing  factors.  belazi  m,   velegraki  a,  fleva  a,  gida-­‐ rakou  i,  papanaum  l,  baka   d,  daniilidou  n,  karamitsos   d.  mycoses.  2005   may;48(3):192-­‐6.  pmid:   15842336   8. fungal  infections  of   the  oral  mucosa.  krish-­‐ nan  pa.  indian  j  dent  res.   2012  sep-­‐oct;23(5):650-­‐9.   doi:  10.4103/0970-­‐ 9290.107384.  review.   pmid:  23422613   9. risks  for  oral  health  with   the  use  of  antidepressants.   peeters  fp,  devries  mw,   vissink  a.  gen  hosp  psy-­‐ chiatry.  1998   may;20(3):150-­‐4.  review.   pmid:  9650032   10. inhalational   and  topical  steroids,   and  oral  candidosis:  a  mini   review.  ellepola  an,  sama-­‐ ranayake  lp.   oral  dis.  2001  jul;7(4):211-­‐ 6.  review.  pmid:  11575870     multifactorial contributions to central incisor tooth morphology and the presence of black triangles: a case study vol 9 no 1 (2021) doi 10.5195/d3000.2021.101 http://dentistry3000.pitt.edu multifactorial contributions to central incisor tooth morphology and the presence of black triangles: a case study steven e. kohane, katelyn c. means university of pittsburgh, school of dental medicine abstract abstract: dentists are often faced with patient concerns of smile esthetics in addition to oral health and should be equipped to help patients manage both. black triangle disease, or a lack of interdental papilla, is esthetically displeasing particularly in the central incisor region and has a hoard of etiologies ranging from gum disease to inherited tooth morphologic features. addressing patients’ esthetic concerns must first be met with understanding the causation of the black triangles before treatment can be rendered. genetic contributions are multifactorial, and this case report provides evidence for a case of a central incisor black triangles without other affected family members. background: black triangles are a prominent feature for those experiencing a lack of interdental papilla between central incisors and may dramatically affect the presentation of a person’s smile. the aim of this case report is to convey the importance of managing patients’ esthetic chief complaints while addressing the etiology of the esthetic concern. case description: a 24-year-old man presented with concern of his black triangle spaces. the patient does not have gingival recession in the area of interest, and his biological mother and brother do not have black triangle spaces. pathology is not an issue, but instead inadequate length of the central incisor mesial contact area prevents the interdental papilla from reaching the contact. esthetics are a concern, but the patient is not interested in irreversible treatment for his healthy tissue. practical implications: dentists should be aware of the multifactorial contributions to patients’ esthetic concerns. dentists should also be aware that meeting a patient’s expectations may require a more complex treatment plan than the patient expected. keywords: black triangles; multifactorial inheritance; cosmetic dentistry; esthetics; gingival embrasures; interdental papilla; contact point; tooth morphology; odontometry. citation: kohane se, et al. (2021) multifactorial contributions to central incisor tooth morphology and the presence of black triangles: a case study. dentistry 3000. 1:a001 doi:10.5195/d3000.2021.101 received: april 30, 2020 accepted: february 4, 2021 published: march 10, 2021 copyright: ©2021 kohane se, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: skohane@pitt.edu introduction though dentistry is primarily focused on establishing or restoring patients’ oral health, hygiene, and maintenance, dentistry’s breadth also covers a wide number of patient esthetic concerns. the smile is a hotspot for quick decision making on appearance, and research shows that esthetic perception—whether by dentists, specialists, or laypeople—of a person’s facial type should not be evaluated separately from the smile as it significantly impacts facial type perception [1]. specifically, the maxillary incisors seem to be the most prominent feature of a smile that affects facial perception, if for no other reason than they are the most anterior and obviously seen in a person’s smile. it is no wonder that among patient esthetic smile concerns are straightness, color, and proportional size of teeth, as well as incisal lip coverage. however, beyond tooth morphology contributing to esthetic concerns of straightness and size, a more overlooked contributor to smile esthetics is the presence or absence of interdental papilla in the mesial http://dentistry3000.pitt.edu/ mailto:skohane@pitt.edu multifactorial contributions to central incisor tooth morphology and the presence of black triangles: a case study vol 9 no 1 (2021) doi 10.5195/d3000.2021.101 http://dentistry3000.pitt.edu gingival embrasure of central incisors. scientifically, an absence of interdental papilla in this highly esthetic region has been named a “black triangle.” a black triangle is technically defined as a cosmetic deformity due to the loss of interdental papilla [2]. however, a black triangle appearance is not simply a matter of gingival recession and may be caused by many other factors, including bone level, tooth morphology, and contact points [2]. there are great esthetic implications of a “black triangle” being included in such a prominent contributor to facial type perception. though the presence of black triangles may be a result of underlying gingival recession or decreased bone height, this is not always the case [2]. lip length and position also contribute to the visibility of a black triangle – with a high smile line or a thin upper lip, the loss of interdental papilla is more noticeable and contributory to an esthetic judgement [3]. additionally, it has been noted that black triangles become more prominent with age [4]. another contributing factor to black triangles is tooth size and shape. tooth sizes vary between individuals most noticeably in mesiodistal width and incisocervical length. the ratio of these two measurements have been shown to predict the likelihood of black triangle, and these measurements also contribute to tooth shape or “form” [5]. three main tooth shapes – square, triangular, and ovoid – are determined by the greatest mesiodistal width of the tooth and whether or not the width is continuous [2, 5]. the pronounced cervical scallop and incisally located, short contact area of triangular teeth are predisposing to a black triangle appearance [2, 5, 6]. on average and as noted in the case report, the interproximal contact in patients with black triangles is either shorter or located 1mm more incisally than patients with adequate interdental papilla [5]. emergence profile variation – specifically divergent crown form – also contributes to open gingival embrasures, and incisal symmetry should be considered during restorative treatment to maintain esthetics [7, 8, 9]. root angulation and divergence, which can be adjusted with orthodontics, also determine crown alignment and presence of interdental bone and papilla. however, prior orthodontic treatment has been associated with longer distances from contact point to crest of the alveolar bone, though shorter distances lead to smaller embrasure areas and consequently a higher chance of sufficient interdental papilla [6, 10, 11]. relevant to the scope of this report is the discussion of genetic influence on tooth morphology. any process which may affect the developing permanent tooth bud can influence the morphology of the incisor, thereby affecting symmetry, contact regions, and interdental papilla spaces. although patient height and tooth size appear not to correlate [12], tooth organ growth may be modified by nutrition or genetic variation in growth genes. the developing central incisor tooth bud structure is also susceptible to trauma by primary incisor intrusion [13], while the erupted tooth is susceptible to trauma, erosion, attrition, and abrasion. of note in this report is abrasion, as the interproximal area can be susceptible to abrasion due to improper floss technique. the objective of this article is to discuss the causation and implication of a less frequently studied dental concern relating to patient aesthetics. though dentists’ primary role in the healthcare field is oral health, as experts of the teeth and mouth it is appropriate to address our patients’ chief complaints, even if aesthetic. in order to best address issues of black triangle disease, it is pivotal that we understand the multiple routes of causation before implementing any treatments. case report a 24-year-old man presented with concern of a dark space between his maxillary central incisors in february 2020. the patient’s chief complaint was stated as “i’ve got a hole between my teeth and it bothers me. i am of the understanding that it can’t be fixed due to my tooth shape, but i also don’t think my gingiva is http://dentistry3000.pitt.edu/ multifactorial contributions to central incisor tooth morphology and the presence of black triangles: a case study vol 9 no 1 (2021) doi 10.5195/d3000.2021.101 http://dentistry3000.pitt.edu recessed. i am often told by close friends that ‘there is something between my teeth,’ but closer friends know that the dark spot is just my anatomy. i’m sure strangers find it off-putting.” the patient was well briefed on the factors which may influence his black triangle appearance, as he had done some research before presentation. however, the patient stated that he wanted to be sure that there was no oral health concern to potentially unveil the cause of his black triangle, and to weigh the pros and cons of any permanent esthetic treatment. the patient’s medical history was significant for adhd medication for the last 14 years, which he reports causing xerostomia. he reported normal vitals and a bmi of 24. his dental history was significant for two traditional orthodontic treatments and two invisalign treatments. he reported brushing twice daily with fluoride and flossing daily, but with improper technique moving floss straight up and down “without much rubbing of the side of each tooth.” upon clinical examination, the patient’s hard tissue charting was unremarkable with third molar extractions and no caries or restorations. approximately 2mm of gingival recession was noted on the facial of mandibular anterior teeth. the patient admitted to infrequent dental visits, estimating once every two years. the patient presented with a triangular tooth form (figure 1a) with tall incisocervical length and short contact length. maxillary gingival recession was not noted, but an exaggerated scalloped appearance was, which is consistent with triangular tooth form [2]. the patient’s black triangle measured 2.1mm, but both his mother and brother lacked black triangles (figures 1a, b). the patient’s brother’s tooth form was triangular and extremely similar to that of the patient, however the brother’s contact length was 2.7mm longer than that of the patient a probable cause of the brother’s gingival embrasure closure (table 1). the patient’s mother, however, exhibited a square tooth form. as is typical, this square tooth form was accompanied by a longer contact length and a closed gingival embrasure. the patient’s father’s information was not available as he was a denture wearer. to standardize the measurements, images were superimposed on each other using quarter size as a reference. quarter diameter is 24.26mm, and figure measurements in mm (table 1) were calculated using an on-screen pixel ruler calibrated to the quarter’s pixel length. this method of measurement was used to maintain consistent results across locations the three subjects lived in different regions of the united states, and not all have access to the same calibrated periodontal probe or caliper. ultimately, the patient’s interdental papilla length was long upon visual inspection. however, it was not long enough to reach the beginning of the contact area, so the patient presented with an unaesthetic black triangle. the patient chose not to pursue treatment for his black triangle because he did not want to subject his healthy tissue to figure 1. photos of the patient and family. a) the patient, with open gingival embrasure, b) the patient’s biological mother, c) the patient’s biological brother http://dentistry3000.pitt.edu/ multifactorial contributions to central incisor tooth morphology and the presence of black triangles: a case study vol 9 no 1 (2021) doi 10.5195/d3000.2021.101 http://dentistry3000.pitt.edu irreversible treatment like composite bonding or veneers, which could lead to secondary decay down the road. discussion the category where the patient shows greatest divergence from the other subjects is the incisocervical length of the contact point. adding the length of the black triangle would bring the measurement of incisal edge to tip of interproximal papilla to within 1mm of the patient’s relatives who have closed gingival embrasures. indeed, it had been noted that shorter interproximal contacts are associated with open gingival embrasures [5]. additionally, the patient’s crown form was more triangular when compared to those of his relatives and a triangular crown form is associated with black triangles [5, 6]. although some might classify gingival recession as a simple calculation of distance between tip of the papilla to the contact area [10], a pathological process of recession was not thought to be involved, based on the healthy and pointed appearance of the patient’s interdental papilla as well as the fact that the papilla extended further incisally than other maxillary anterior papilla. additionally, if an occlusal radiograph were to be taken of the patient, it would be reasonable to predict adequate bone levels. the patient also did not seem to have a thin gingival biotype, which again indicates that the primary etiology of the patient’s black triangle is contact area length. in respect to treatment, a periodontal surgery would not be advised as the gingival tissue is healthy and there was no clinical recession in the maxillary arch beyond the definition of a measurable distance between the papillary tip to the contact area. additionally, in the esthetic circumstances of this particular case the teeth being already quite wide and generally large filling the open gingival embrasure with composite may not have been esthetic either. other options, including no treatment, must be considered. as in any esthetic case, veneers are an option in restoring the anterior appearance of the smile. however, the patient’s esthetics were otherwise ideal, and he opted for no treatment in the case of conservation of healthy tooth structure. as previously stated, no clear association was found with regards to genetic contribution to body height and tooth size. however, it is possible that the tooth organ’s growth may be modified by genetic mechanisms affecting general body growth. some have identified numerous loci that influence height via genome-wide association analysis [14]. others have discussed the hormones, paracrine factors, extracellular matrix molecules, and intracellular proteins that affect the growth plates of long bones [15]. with the abundance of genes known to contribute to body growth, it is postulated by the authors that further research will discover a more definitive association to tooth growth and size. the patient is a few inches taller than his relatives, which suggests an explanation to the differences in tooth morphology. measurements (mm) patient patient’s mother patient’s brother contact length, incisocervical 2.5 5.3 5.2 widest mesiodistal (md) dimension 8.8 9.1 8.7 incisal edge to widest md point 3.9 1.8 1.4 tip of papilla to widest md point 2.3 4.6 3.5 black triangle height 2.1 table 1. dimensions of points of interest for black triangles in millimeters. only the patient exhibits a black triangle, while his biological mother and brother do not. the most notable measurement is the patient’s contact length, which is drastically smaller. http://dentistry3000.pitt.edu/ multifactorial contributions to central incisor tooth morphology and the presence of black triangles: a case study vol 9 no 1 (2021) doi 10.5195/d3000.2021.101 http://dentistry3000.pitt.edu conclusions patients often present to the dentist with esthetic concerns including black triangles. periodontal surgery and grafting are options to restore interdental papilla length in the case of true gingival recession. however, if the main factor contributing to the presence of a black triangle is tooth morphology, then composite bonding and veneers may be an option. different “black triangle” cases have specific characteristics which fit more neatly into one category than another for treatment and causation. references 1. the influence of the smile on the perceived facial type esthetics. batwa w. biomed res int. 2018;2018:3562916. ecollection 2018. pmid: 30112381. 2. black triangle dilemma and its management in esthetic dentistry. singh vp, uppoor as, nayak dg, shah d. dent res j (isfahan). 2013 may;10(3):296-301. review. pubmed pmid: 24019795. 3. maxillary anterior papilla display during smiling: a clinical study of the interdental smile line. hochman mn, chu sj, tarnow dp. int j periodontics restorative dent. 2012 aug;32(4):375-83. pubmed pmid: 22577642. 4. clinical studies on the appearance of natural anterior teeth in young and old adults. hartmann r, müller f. gerodontology. 2004 mar;21(1):10-6. pmid: 15074535. 5. open gingival embrasures after orthodontic treatment in adults: prevalence and etiology. kurth jr, kokich vg. am j orthod dentofacial orthop. 2001 aug;120(1):116-23. pmid: 11500652. 6. an analysis on the factors responsible for relative position of interproximal papilla in healthy subjects. kim jh, cho yj, lee jy, kim sj, choi ji. journal of periodontal & implant science. 2013 aug 31; 43(4): 160-167. pmid: 24040568. 7. esthetics: the orthodonticperiodontic restorative connection. kokich vg. semin orthod. 1996 mar;2(1):21-30. review. pubmed pmid: 9161280.selct 8. incidence and size of pretreatment overlap and posttreatment gingival embrasure space between maxillary central incisors. burke s, burch jg, tetz ja. am j orthod dentofacial orthop. 1994 may;105(5):506-11. pubmed pmid: 8166102. 9. significance of crown shape in the replacement of a central incisor with a single implant-supported crown. gobbato l, paniz g, mazzocco f, chierico a, tsukiyama t, levi pa jr, weisgold as. quintessence int. 2013 may;44(5):407-13. pmid: 23479574. 10. risk factors associated with open gingival embrasures after orthodontic treatment. an ss, choi yj, kim jy, chung cj, kim kh. angle orthod. 2018 may;88(3):267-274. epub 2018 jan 16. pmid: 29337634. 11. factors influencing the presence of interproximal dental papillae between maxillary anterior teeth. chen mc, liao yf, chan cp, ku yc, pan wl, tu yk. j periodontol. 2010 feb;81(1):318-24. pmid: 20151812. 12. age-related changes in crown and root length in sri lankan sinhalese. jayawardena ck, abesundara ap, nanayakkara dc, chandrasekara ms. j oral sci. 2009 dec;51(4):587-92. pubmed pmid: 20032612. 13. intrusive luxation in primary teeth review of literature and report of a case. gupta m. saudi dent j. 2011 oct;23(4):167-76. epub 2011 sep 16. pubmed pmid: 23960512. 14. genome-wide association analysis identifies 20 loci that influence adult height. weedon mn, lango h, lindgren cm, wallace c, evans dm, mangino m, freathy rm, perry jr, stevens s, hall as, samani nj, shields b, prokopenko i, farrall m, dominiczak a, johnson t, bergmann s, beckmann js, vollenweider p, waterworth dm, mooser v, palmer cn, morris ad, ouwehand wh, zhao jh, li s, loos rj, barroso i, deloukas p, sandhu ms, wheeler e, soranzo n, inouye m, wareham nj, caulfield m, munroe pb, hattersley at, mccarthy mi, frayling tm. nat genet. 2008 may;40(5):575-83. epub 2008 apr 6. pubmed pmid: 18391952. 15. short and tall stature: a new paradigm emerges. baron j, sävendahl l, de luca f, dauber a, phillip m, wit jm, nilsson o. nat rev endocrinol. 2015 dec;11(12):735-46. epub 2015 oct 6. review. pubmed pmid: 26437621. http://dentistry3000.pitt.edu/ https://www.ncbi.nlm.nih.gov/pubmed/30112381/ microsoft word 70 2017.docx   vol  5,  no  1  (2017)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2017.70           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     etiology  and  familial  inheritance  of  pleomorphic  adenomas   krysten  clark1      1university  of  pi.sburgh,  school  of  dental  medicine abstract   background:  a  pleomorphic  adenoma  is  the  most  common  salivary  gland  neoplasm  in  both  children  and  adults.  ple-­‐ omorphic  adenomas  are  derived  from  ductal  and  myoepithelial  cells  and  are  most  commonly  found  in  the  superficial   lobe  of  the  paro,d  gland.  the  purpose  of  this  ar,cle  is  to  discuss  the  genes  involved  in  pleomorphic  adenomas  and   the  possible  autosomal  dominant  mode  of  inheritance.  case  descrip+on:  the  first  pa+ent  was  a  white  male  who  was   diagnosed  with  carcinoma  ex  pleomorphic  adenoma,  a  highly  aggressive  tumor,  at  the  age  of  57.  he  had  an  undiag-­‐ nosed  pleomorphic  adenoma  for  approximately  15  years  prior.  the  tumor  was  excised  and  the  pa9ent  underwent  ra-­‐ dia$on   in   the   loca$on  of  his  paro!d  gland   for  4  years  un!l  he  deceased.  the  second  pa!ent   is  a  white   female,  his   daughter,  who  was  diagnosed  with  a  benign  pleomorphic  adenoma  at  the  age  of  46.  her  salivary  gland  tumor  was  ex-­‐ cised  and  normal   follow  up  appointments  occurred.  prac%cal   implica&ons:  pleomorphic  adenomas  most  commonly   affect  the  paro+d  gland,  the  largest  of  the  three  major  salivary  gland  tumors.  occurrence  and  excision  of  this  salivary   gland  tumor  will  cause  a  decrease  in  the  secre2on  of  saliva,  leading  to  a  dry  mouth  and  an  increased  risk  of  caries.     cita%on:   clark,   k.   (2017)   e"ology   and   familial   inheritance  of  pleomorphic  adenomas.  den$stry   3000.  1:a001  doi:10.5195/d3000.2017.70   received:    may  9,  2017   accepted:    may  10,  2017   published:    june  16,  2017   copyright:   ©2017   clark,   k.   this   is   an   open   ac-­‐ cess  ar!cle   licensed  under  a  crea!ve  commons   a"ribu%on  work  4.0  united  states  license.   email:  kdc40@pi).edu   introduction   pleomorphic  adenomas  rep-­‐ resent  about  65%  of  all  salivary  gland   neoplasms.  they  are  the  most  com-­‐ mon  salivary  gland  neoplasm  in  both   children  and  adults.    the  majority  oc-­‐ cur  in  the  parotid  gland,  although  it  is   possible  for  them  to  arise  in  minor   salivary  glands  or  the  submandibular   and  sublingual  glands.  it  is  a  painless,   slow  growing  mass  that  can  be  pal-­‐ pated  just  behind  the  ear  once  large   enough.  they  are  defined  as  mixed   tumors  because  they  contain  both   epithelial,  mesenchymal,  and  myoepi-­‐ thelial  cells.  histologically,  pleo-­‐ morphic  adenomas  are  arranged  in   various  patterns  between  these  three   types  of  cells  [1].   pleomorphic  adenomas  most   commonly  arise  in  4th  decade  of  life   and  have  a  slight  predilection  towards   females.  the  prognosis  of  a  pleo-­‐ morphic  adenoma  is  typically  very   good,  and  the  chance  of  malignancy  is   less  than  5%  if  it  is  diagnosed  within  a   few  years.  this  chance  increases  up   to  9.5%  after  15  years  [1].  the  rate  of   recurrence  is  under  5%  as  long  as   complete  excision  occurs.  however,  if   the  tumor  is  poorly  encapsulated,  the   rate  of  recurrence  can  be  quite  high.   the  treatment  for  a  pleomorphic  ad-­‐ enoma  consists  of  removal  of  the  tu-­‐ mor  and  surrounding  salivary  gland   tissue,  leading  to  a  decreased  salivary   flow.     carcinoma  ex  pleomorphic   adenoma  is  a  rare  and  aggressive  ma-­‐ lignant  tumor  that  can  arise  from  a   pleomorphic  adenoma.  it  represents   only  about  12%  of  malignant  salivary   gland  tumors  [1].  normally,  the  risk  of   malignancy  is  very  low  unless  the  ini-­‐ tial  neoplasm  is  untreated  for  a  pro-­‐ longed  period  of  time  or  recurrence   of  the  primary  tumor  occurs.  the   mass  is  usually  painless  but  can  cause   facial  nerve  paralysis.  treatment  in-­‐ cludes  complete  excision  of  the  tu-­‐ mor,  most  likely  followed  by  radia-­‐ tion.  during  the  time  of  radiation,  it  is   extremely  important  for  the  patient   to  visit  a  dentist.  the  five-­‐year  surviv-­‐ al  rate  of  carcinoma  ex  pleomorphic   adenoma  is  only  about  30%.  the  pur-­‐ pose  of  this  report  is  to  to  provide   evidence  for  an  autosomal  dominant   mode  of  inheritance  of  pleomorphic   adenomas.   subjects   a  46-­‐year-­‐old  female  patient   was  diagnosed  with  a  pleomorphic   adenoma  of  the  parotid  gland  by  an   ent  after  her  dentist  palpated  a  lump   in  the  area  during  a  routine  head  and   neck  exam.  the  tumor  was  excised,   defined  as  benign,  and  after  follow  up   appointments  for  5  years,  no  further   treatment  was  necessary.  the  likeli-­‐ hood  of  recurrence  in  her  case  is  un-­‐ der  5%  [2]..  this  patient’s  father  was   diagnosed  with  carcinoma  ex  pleo-­‐ morphic  adenoma  at  the  age  of  57,   and  deceased  4  years  later  after  met-­‐ astatic  spread  to  his  lungs.  this  pa-­‐ tient  also  had  the  tumor  excised  and   received  radiation.  upon  diagnosis  of   e!ology  and  familial  inheritance  of  pleomorphic  adenomas   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.70    http://dentistry3000.pitt.edu   his  malignant  tumor,  he  admitted  to   feeling  a  lump  in  the  same  location   for  at  least  15  years.  figure  1  suggests   a  family  segregation  compatible  with     autosomal  dominant  mode  of  inher-­‐ itance.   discussion   the  exact  cause  of  pleo-­‐ morphic  adenomas  is  unknown,  alt-­‐ hough  70%  of  cases  show  some  form   of  chromosomal  abnormality.  there   are  both  genetic  and  environmental   risk  factors.  one  well  known  envi-­‐ ronmental  risk  factor  is  exposure  to   therapeutic  radiation.  according  to   research,  patients  treated  with  radia-­‐ tion  for  childhood  malignancies  had   an  increased  risk  of  developing  a  sali-­‐ vary  gland  neoplasm.  survivors  of  the   atomic  bomb  drop  have  also  shown   an  increased  risk.  however,  many  of   these  developing  tumors  were  malig-­‐ nant  mucoepidermoid  carcinomas   instead  of  pleomorphic  adenomas  [2].   also,  according  to  research  studies,   smoking  surprisingly  does  not  seem   to  have  a  strong  association  with  the   development  of  pleomorphic  adeno-­‐ mas,  although  it  plays  a  significant   role  in  the  develop-­‐ ment  of  warthin’s  tu-­‐ mor,  a  different  type  of   benign  salivary  gland   tumor  that  also  com-­‐ monly  affects  the  pa-­‐ rotid  gland  [3].  the  on-­‐ set  and  progression  of   pleomorphic  adenomas   has  also  potentially   thought  to  be  linked  to   the  use  of  mobile   phones,  although  more   research  needs  to  be   done.  one  last  envi-­‐ ronmental  risk  factor  to   the  formation  of  ple-­‐ omorphic  adenomas   has  viral  origin.  alt-­‐ hough  ebv  and  cyto-­‐ megalovirus  do  not   seem  to  be  correlated,  infection  with   the  simian  virus,  a  highly  oncogenic   tumor  virus,  seems  to  play  a  role  in   the  development  of  pleomorphic  ad-­‐ enomas  [4].     the  simian  virus  (sv40)  was   originally  in  monkeys,  but  accidental-­‐ ly  transferred  to  many  humans  who   received  an  anti-­‐polio  vaccine.  how-­‐ ever,  based  on  research,  it  seems  that   even  individuals  who  did  not  receive   the  polio  vaccine  have  been  infected   with  sv40[4].  the  virus  has  recently   been  found  to  be  involved  with  dif-­‐ ferent  types  of  human  tumors,  includ-­‐ ing  brain  and  bone  tumors,  mesothe-­‐ liomas,  thyroid  carcinomas,  and  pitui-­‐ tary  adenomas.  through  the  use  of   pcr,  it  was  discovered  that  the  sv40   tag  sequence  was  often  found  in  ple-­‐ omorphic  adenomas  but  not  in  nor-­‐ mal  salivary  gland  tissue  [4].  these   results  indicate  that  the  simian  virus   likely  plays  a  role  in  the  onset  or  pro-­‐ gression  of  pleomorphic  adenomas.       three  genes  have  been  corre-­‐ lated  with  the  development  of  pleo-­‐ morphic  adenomas.  the  first  is  pleo-­‐ morphic  adenoma  gene1  (plag1),   which  is  found  on  chromosome  8  and   present  in  about  39%  of  diagnosed   patients  [5].  the  second  gene  is  the   high  mobility  group  at-­‐hook  2  gene   (hmga2),  located  on  chromosome  12   and  found  in  8%  of  patients.  the   above  two  mutations  are  both  trans-­‐ locations.  lastly,  the  mucin  1  gene   (muc1)  has  been  correlated  to  the   recurrence  and  malignant  transfor-­‐ mation  of  pleomorphic  adenomas.     plag1  and  hmga2  mutations   are  both  proto-­‐oncogenes,  also   known  as  gain  of  function  mutations.   proto-­‐oncogenes  are  normal  genes  in   the  body  that  are  converted  to  onco-­‐ genes  by  three  different  mechanisms.   the  first  mechanism  is  a  point  muta-­‐ tion  that  results  in  a  continuously  ac-­‐ tivated  protein.  the  second  mecha-­‐ nism  is  localized  duplication  of  a  dna   segment,  leading  to  gene  amplifica-­‐ tion  and  overexpression  of  that  pro-­‐ tein.  the  third  mechanism  is  chromo-­‐ somal  translocation  that  causes  a  dif-­‐ ferent  promoter  to  promote  inappro-­‐ priate  expression  of  a  protein  that  is   normally  under  gene  control  [6].   hmga2  and  most  commonly  plag1   are  both  transformed  into  oncogenes   by  translocation.  however,  trisomy  8   is  a  possible  mechanism  for  the  over-­‐ expression  of  plag1.     plag1,  found  on  chromo-­‐ some  8,  has  been  consistently  rear-­‐ ranged  and  over-­‐expressed  in  indi-­‐ viduals  with  pleomorphic  adenomas.   the  most  common  plag1  transloca-­‐ tion  is  found  at  8q12,  leading  plag1   to  be  ubiquitously  expressed.  the  two   most  common  translocation  genes   are  ctnnb1  found  on  chromosome  3   and  lifr  found  on  chromosome  5  [5].   also,  mosaic  trisomy  8  is  the  most   common  chromosomal  deviation  as-­‐ sociated  with  salivary  gland  tumors   [7].  based  on  recent  research  it  has   been  noted  that  plag1  acts  as  a  pro-­‐ figure  1:  family  pedigree.  patient  1  is  in  generation  1  and  had  carci-­‐ noma  ex  pleomorphic  adenoma  after  approx.  15  years  without  removal   of  primary  tumor.  patient  2  is  in  generation  2  and  had  a  pleomorphic   adenoma.  offspring  in  generation  3  are  still  in  their  20’s  and  younger   than  the  average  age  range  for  diagnosis.   e!ology  and  familial  inheritance  of  pleomorphic  adenomas   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.70    http://dentistry3000.pitt.edu   to-­‐oncogene,  and  the  overexpression   of  this  gene  is  crucial  to  the  develop-­‐ ment  of  pleomorphic  adenomas.  it   has  also  been  noted  that  the  role  of   plag1  is  confined  to  pleomorphic   adenomas  and  carcinoma  ex  pleo-­‐ morphic  adenoma  and  does  not  play   a  role  in  other  types  of  salivary  gland   neoplasms.  plag1  encodes  a  zinc  fin-­‐ ger  protein  and  activates  dna  tran-­‐ scription  by  binding  to  dna  in  a  se-­‐ quence-­‐specific  manner.  one  poten-­‐ tial  binding  site  of  plag1  was  found   on  the  promoter  region  of  human   insulin-­‐like  growth  factor  ii  (igf-­‐ii),   leading  to  its  overexpression  [7].  the   activation  of  igfii  causes  growth   promoting  activity,  which  could  lead   to  the  formation  of  a  tumor.       mutations  in  hmga2  seem  to   be  more  common  in  carcinoma  ex   pleomorphic  adenoma,  and  are  only   found  in  around  8%  of  benign  pleo-­‐ morphic  adenomas.  hmga2,  a  small   non-­‐histone  associated  protein  locat-­‐ ed  on  chromosome  12,  can  modify   transcription  by  altering  the  chroma-­‐ tin  structure.  this  gene  also  mutates   through  translocation,  leading  to  the   expression  of  hmga2,  loss  of  regula-­‐ tion,  and  promotion  of  cell  growth   [8].  the  amplification  of  this  gene  has   been  suggested  to  play  a  role  in  the   malignant  transformation  of  pleo-­‐ morphic  adenomas.  since  both  plag1   and  hmga2  mutations  are  consistent   with  pleomorphic  adenoma,  the  de-­‐ tection  of  these  mutations  can  aid  in   the  diagnosis.       although  the  recurrence  rate   of  pleomorphic  adenomas  is  low  in   many  cases  where  surgical  excision  is   properly  performed,  if  recurrence   does  occur,  the  chance  of  re-­‐ recurrence  is  high.  these  tumors  are   often  multi-­‐nodular  and  poorly  en-­‐ capsulated.  mucin-­‐1  gene  is  one  of   the  glycoproteins  that  makes  up  mu-­‐ cus.  there  is  no  expression  of  muc1   in  normal  salivary  glands.  however,  in   a  study  by  hamada  et  al,  the   muc1/df3  mutation  was  found  in  4   of  9  patients  with  recurrent  pleo-­‐ morphic  adenomas,  but  only  in  3  of   40  patients  with  non-­‐recurrent  pleo-­‐ morphic  adenomas  [9].  according  to   literature,  the  expression  of   muc1/df3  could  be  used  as  a  marker   to  detect  the  possible  recurrence  and   malignant  transformation  of  pleo-­‐ morphic  adenomas.     the  evidence  shows  that  ple-­‐ omorphic  adenomas  do  tend  to  have   a  familial  component  and  the  two   modes  of  inheritance  for  pleomorphic   adenomas  are  autosomal  dominant   inheritance  and  a  somatic  mutation   [7].  if  pleomorphic  adenomas  are   passed  in  an  autosomal  fashion,  a   patient’s  offspring  would  have  a  50%   chance  of  passing  the  disease  down   to  offspring.  based  on  the  presented   case,  the  first  male  patient  likely  had   a  mutation  and  passed  the  mutated   gene  to  his  daughter  in  an  autosomal   dominant  type  fashion.  however,  fur-­‐ ther  research  needs  to  be  done  to   potentially  link  familial  inheritance  to   the  plag1  mutation,  the  mutated   gene  most  commonly  found  in  pleo-­‐ morphic  adenomas.     pleomorphic  adenomas  can   cause  an  increase  in  dental  caries.   the  parotid  gland  is  entirely  serous   and  drains  into  the  oral  cavity   through  stenson’s  duct.  it  produces   approximately  20%  of  the  salivary   content  in  the  oral  cavity.  the  sub-­‐ mandibular  gland,  produces  around   65%  of  saliva  and  enters  the  oral  cavi-­‐ ty  through  wharton’s  duct.  although   pleomorphic  adenomas  most  typically   arise  in  the  parotid  gland,  it  can  arise   in  either  of  the  major  salivary  glands.   complete  removal  of  these  tumors   causes  a  decrease  in  the  production   of  one  of  the  major  salivary  glands.   this  will  almost  certainly  lead  to  a   decrease  in  the  production  of  saliva.   saliva  plays  an  essential  role  in  the   prevention  of  caries,  and  a  decreased   salivary  flow  in  the  oral  cavity  makes   an  individual  more  prone  to  the  de-­‐ velopment  of  caries.  if  a  pleomorphic   adenoma  does  develop  into  carcino-­‐ ma  ex  pleomorphic  adenoma  and   radiation  therapy  is  required  after   surgical  removal,  the  likelihood  of   caries  increases  exponentially.  radia-­‐ tion  therapy  causes  an  individual  to   lose  almost  all  salivary  flow,  and  fre-­‐ quent  dental  visits  are  required  dur-­‐ ing  this  time.       genetic  testing  for  pleo-­‐ morphic  adenomas  could  cause  po-­‐ tentially  lead  to  ethical  implications   related  to  insurance  companies.  also,   there  may  be  a  fine  line  between   normal  and  abnormal  palpation  dur-­‐ ing  a  head  and  neck  exam.  it  is  very   important  that  a  dentist  refers  a  pa-­‐ tient  to  a  specialist  if  they  feel  any-­‐ thing  abnormal,  and  missing  a  mass  in   a  major  or  minor  salivary  gland  could   be  an  ethical  implication.  overall,   pleomorphic  adenomas  are  very   treatable  when  caught  early  and  do   not  recur.  there  does  seem  to  be  a   genetic  link  between  pleomorphic   adenomas  and  a  few  genes,  like  the   plag1  gene.  more  research  needs  to   be  completed  on  families  with  history   of  pleomorphic  adenoma  to  discover   a  possible  autosomal  mode  of  inher-­‐ itance.   references   1.  plag1  gene  alterations  in  sali-­‐ vary  gland  pleomorphic  adenoma   and  carcinoma  ex-­‐pleomorphic   adenoma:  a  combined  study  using   chromosome  banding,  in  situhy-­‐ bridization  and  immunocytochem-­‐ istry.  martins  c,  fonseca  i,  roque   l,  pereira  t,  ribeira  c,  bullerdiek  j,   e!ology  and  familial  inheritance  of  pleomorphic  adenomas   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.70    http://dentistry3000.pitt.edu   soares  j.  modern  pathology.  2005;   18:1048–1055.  6  may  2006.  pmid:   15920557.   2.  salivary  gland  tumors  in  survi-­‐ vors  of  childhood  cancer.  whatley   ws,  thompson  jw,  rao  b.  2006   march;  124  (3):385-­‐388.  pmid:   16500432   3.  smoking  and  risk  of  parotid   gland  tumors:  a  nationwide  case-­‐ control  study.  sadetzki  s,  ober-­‐ man  b,  mandelzweig  l,  chetrit  a,   ben-­‐tal  t,  jarus-­‐hakak  a,   duvdevani  s,  cardis  e,  wolf  m.   2008  may  1;  112(9):  1974-­‐82.   pmid:  18361448   4.  simian  virus  40  sequences  and   expression  of  the  viral  large  t  an-­‐ tigen  oncoprotein  in  human  ple-­‐ omorphic  adenomas  of  parotid   glands.  martinelli,  m,  martini,  f,   rinaldi,  e,  caramanico,  l,  magri,  e,   grandi,  e,  carinci  f,  pastore  a,   tognon,  m.  2002  oct;  161(4):   1127–1133.  pmid:  pmc1867276   5.  conserved  mechanism  of  plag1   activation  in  salivary  gland  tumors   with  and  without  chromosome   8q12  abnormalities:  identification   of  sii  as  a  new  fusion  partner   gene.  astrom  ak,  voz  ml,  kas  k,   roijer  e,  wedell  b,  mandahl  n,   van  de  ven  w,  mark  j,  stenman  g.   1999  feb  15;  59(4):  918-­‐923.  pmid   10029085   6.  lodish  h,  berk  a,  zipursky  sl,   matsudaira  p,  baltimore  d,  darnell   j  (2000)  molecular  cell  biology.   new  york:  w.  h.  freeman.     7.plag1  gene  alterations  in  salivar y  gland  pleomorphic  adenoma  and  carcinoma  ex-­‐ pleomorphicadenoma:   a  combined  study  using  chromoso me  banding,   in  situ  hybridization  and  immunoc ytochemistry.  martins  c,  fonseca   i,  roque  l,  pereira  t,  ribeiro  c,   bullerdiek  j,  soares  j.  2005  aug;   18(8):  1048-­‐1055.  pmid:   15920557   8.  aberrant  plag1  expression  in   pleomorphic  adenomas  of  the  sal-­‐ ivary  gland:  a  molecular  genetic   and  immunohistochemical  study.   matsuyama  a,  hisaoka  m,  nagao   y,  hashimoto  h.  2011  may;  458(5):   583-­‐92.  pmid  21394649   9.  mucin  expression  in  pleo-­‐ morphic  adenoma  of  salivary   gland:  a  potential  role  for  muc1   as  a  marker  to  predict  recur-­‐ rence.  hamada  t,  matsukita  s,  go-­‐ to  m,  kitajima  s,  batra  s  k,  irimura   t,  sueyoshi  k,  sugihara  k,  yoneza-­‐ wa  s.  2004;  57(8),  813–821.  pmid   pmc1770389     microsoft word gorski 7-1.docx   vol  3,  no  1  (2015)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2015.34           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.   familial  aggregation  of  anxiety  associated  with  bruxism   david  gorski1       1  university  of  pittsburgh  school  of  dental  medicine,  pittsburgh  pa,  usa   abstract   background:   this   paper   reports   the   co-­‐occurrence   of   heritable   anxiety-­‐related   disorders   and  awake  bruxism  in  a  family  and  discusses  the  heritability  of  anxiety  and  implica7ons  of   awake  bruxism  as  comorbidity  to  anxiety  disorders.   subjects:  ten  out  of  14  members  of  an  extended  family  reported  having  a  professionally  di-­‐ agnosed   anxiety-­‐related   disorder.   all   individuals   with   anxiety   disorders   also   showed   in-­‐ traoral  signs  of  wear  from  bruxism  and  reported  being  aware  of  grinding  their  teeth  while   awake.  addi*onally,  three  out  of  the  four  family  members  without  an  anxiety-­‐related  dis-­‐ order  did  not  report  grinding  their  teeth  and  showed  no  occlusal  wear  from  bruxism.  all  of   the   individuals   that   were   examined   were   educated   about   the   short-­‐term   and   long-­‐term   complica(ons  associated  with  bruxism,  but  all  of  them  elected  to  not  have  treatment  per-­‐ formed.   prac%cal  implica%ons:  anxiety  disorders  are  highly  related  to  suicidal  behaviors,  par7cular-­‐ ly  in  children  and  adolescents.  addi-onally,  awake  bruxism  can  o-en  serve  as  an  indicator   of  anxiety  or  stress.  by  recognizing  bruxism  as  a  possible  manifesta+on  of  psychological  dis-­‐ tress,  the  dental  prac..oner  may  be  able  to  direct  pa.ents  to  life-­‐saving  services  like  psy-­‐ chologists  and  crisis  hotlines  when  appropriate.     cita%on:  gorski  d.  (2015)  familial  aggrega*on  of   anxiety  associated  with  bruxism.  den$stry  3000.   1:a001  doi:10.5195/d3000.2015.34   received:  june  2,  2015   accepted:    june  26,  2015   published:    july  20,  2015   copyright:  ©2015  gorski  d.   this   is   an  open  ac-­‐ cess  ar!cle   licensed  under  a  crea!ve  commons   a"ribu%on  work  4.0  united  states  license.   email:  deg32@pi$.edu   introduction   bruxism  is  defined  as  “a  repetitive   jaw-­‐muscle  activity  characterized   by  clenching  or  grinding  of  the  teeth   and/or  by  bracing  or  thrusting  of   the  mandible”  that  can  occur  while   asleep  (sleep  bruxism)  or  while   awake  (awake  bruxism)  [1].  awake   bruxism  is  reported  in  20%  of  the   adult  population  in  the  us  [2],  but   researchers  have  only  recently   linked  its  occurrence  to  anxiety  dis-­‐ orders  [3].   the  heritability  estimates  for  anxie-­‐ ty  disorders  range  from  20%  to   65%  [4].  from  family  and  twin   studies,  the  heritability  of  general-­‐ ized  anxiety  disorder  (gad)  has   been  measured  at  32%,  indicating   significant  heritability  for  this  anxi-­‐ ety-­‐based  condition  [5].     awake  bruxism  and  anxiety  are  of-­‐ ten  only  considered  in  their  respec-­‐ tive  fields  of  dentistry  and  psychol-­‐ ogy.  this  report  describes  the  co-­‐ occurrence  of  anxiety  disorders  and   their  manifestation  as   awake  bruxism  in  family,   and  it  discusses  the  clin-­‐ ical  implications  of  these   findings.   subjects   a  28  year-­‐old  male  was   complaining  of  “jaw   pain”  and  “headaches”   that  had  been  worsening  in  the  past   two  years.  the  patient  had  a  rela-­‐ tively  unremarkable  medical  histo-­‐ ry.  other  than  minor  seasonal  aller-­‐ gies  for  which  he  was  taking  no   medications,  his  only  notable  condi-­‐ tions  were  generalized  anxiety  dis-­‐ order  and  major  depressive  disor-­‐ der.  for  these  conditions,  he  was      familial  aggrega*on  of  anxiety  associated  with  bruxism   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.34    http://dentistry3000.pitt.edu   2   taking  pristiq,  a  selective  serotonin   and  norepinephrine  reuptake  inhib-­‐ itor,  and  lithium  carbonate,  a   pharmaceutical  typically  used  to   treat  bipolar  disorder.  a  thorough   head  and  neck  exam  with  inspection   of  hard  and  soft  tissues  showed  bi-­‐ lateral  sensitivity  of  the  masseter   and  medial  pterygoid  muscles,  with   well-­‐defined  wear  facets  on  the  in-­‐ cisal  and  lingual  surfaces  of  his   maxillary  canines  (figure  1).  addi-­‐ tionally,  trauma  from  occlusal  wear   was  noted  on  his  single  mandibular   incisor  (figure  2).  when  ques-­‐ tioned,  the  patient  stated  that  he   was  aware  that  he  had  been  grind-­‐ ing  his  teeth  during  the  day.  he  re-­‐ called  that  his  sister  had  recently   been  having  similar  symptoms  and   had  received  a  “night  guard”  that   had  provided  some   relief  from  the  head-­‐ aches  and  jaw  pain.       figures  3  and  4   show  the  phenotypi-­‐ cal  distribution  in   the  family  after  oral   examinations  and   medical  history  in-­‐ terviewing  of  the   proband’s  family.   out  of  13  individuals  examined,  ten   had  professional  diagnoses  of  anxie-­‐ ty-­‐related  conditions  and  also   showed  intraoral  signs  of  bruxism   (figures  5,  6,  and   7).  additionally,   out  of  the  four   family  members   who  did  not  have   an  anxiety-­‐related   condition,  only  in-­‐ dividual  ii.1   showed  any  in-­‐ traoral  signs  of   bruxism  or  admit-­‐ ted  to  “grinding  or   clenching  their   teeth”  (figure  8).     none  of  the  family  members  that   were  inspected  wished  to  have   treatment  performed.   individuals  iii.1  and   ii.4  already  had  occlu-­‐ sal  splints  that  they   wore  at  night.  they   both  rated  their   symptom  relief  from   their  occlusal  splints   as  “moderate.”  indi-­‐ vidual  ii.4  said  that   his  occlusal  splint  was   “uncomfortable  and   awkward”  to  wear  but  did  not  wish   to  have  it  remade.  all  members  of   the  family,  regardless  of  whether   they  presented  with  signs  of  brux-­‐ ism,  were  educated  on  bruxism  and   the  short-­‐term  and  long-­‐term  com-­‐ plications  associated  with  the  con-­‐ dition.   discussion   recent  studies  on  twins  have  found   a  significant  degree  of  heritability   for  anxiety  disorders.  but,  these   studies  have  been  unable  to  pin-­‐ point  any  single  genes  that  are  re-­‐ sponsible  for  the  occurrence  of  anx-­‐ iety  [6].  instead,  it  has  been  sug-­‐ gested  that  anxiety  has  a  highly   multifactorial  pattern  of  inher-­‐ itance,  with  the  environment  play-­‐ ing  a  role  and  a  large  number  of   genes  contributing  a  small  individ-­‐ ual  effect  [7].  that  being  said,  based   on  the  pedigrees  for  anxiety  (figure   3)  and  associated  bruxism  (figure   4),  it  would  appear  that  at  least   some  of  the  genes  that  contribute  to   anxiety  in  the  proband’s  family  are   inherited  in  an  autosomal  dominant   fashion,  with  all  three  noted  genera   tions  affected  by  the  condition.      familial  aggrega*on  of  anxiety  associated  with  bruxism   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.34    http://dentistry3000.pitt.edu   3   awake  bruxism  has  been  noted  to   have  a  number  of  varied  associa-­‐ tions—smoking,  caffeine  intake,  and   alcohol  consumption  [8].  however,   in  a  study  of  86  children  by  monaco   et  al,  72%  percent  of  children  that   bruxed  showed  significant  anxiety   scores,  while  only  12%  of  non-­‐ bruxers  had  similar  psychological   findings  [9].  this  would  indicate   that  the  act  of  bruxing  is  highly  cor-­‐ related  with  anxiety  disorders.  the   results  from  this  study  agree  with   the  findings  from  the  family  report-­‐ ed  here.  indeed,  in  this  family,  brux-­‐ ism  and  multifactorially  inherited   anxiety  disorders  appear  to  be  in-­‐ tertwined.  each  person  with  a  diag-­‐ nosed  anxiety-­‐related  disorder  also   had  signs  of  occlusal  wear  associat-­‐ ed  with  bruxism  and  were  aware   that  they  bruxed  while  awake.  simi-­‐ larly,  three  out  of  four  of  the  indi-­‐ viduals  (i.1,   iii.4,  and  iii.6)   who  did  not   have  an  anxie-­‐ ty  disorder   also  did   not  brux.   only  one   family   member   (individual  ii.1)  showed  signs  of   occlusal  trauma  from  bruxism,   but  was  not  diagnosed  with   anxiety.  this  family  member   attributed  the  wear  patterns  to   “occasional  grinding”  due  to  stress   in  the  workplace.   this  report  highlights  the  im-­‐ portance  for  dental  health  profes-­‐ sionals  to  understand  the  signifi-­‐ cance  of  dental  manifestations  due   to  other  conditions.  many  dental   professionals  are  aware  of   the  evidence  linking  perio-­‐ dontal  disease  to  heart  dis-­‐ ease  [10]  and  diabetes  [11].   as  a  result,  many  dentists   are  able  to  act  as  “early   warning  indicators”  and   counsel  their  patients  about   possible  systemic  comor-­‐ bidities.  simi-­‐ larly,  the  dental   field  should   become  aware   of  bruxism  as  a  possible   predictor  of  heritable   anxiety-­‐related  disor-­‐ ders.  anxiety  disorders   are  highly  related  to   suicidal  behaviors,  par-­‐ ticularly  in  children  and   adolescents  [12].  if   dentists  are  able  to  rec-­‐ ognize  the  oral  mani-­‐ festations  of  anxiety  and  stress  in   their  patients,  they  may  be  able  to   counsel  these  patients  and  ultimate-­‐ ly  direct  them  to  life-­‐saving  services   such  as  therapists  and  crisis  cen-­‐ ters.      familial  aggrega*on  of  anxiety  associated  with  bruxism   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.34    http://dentistry3000.pitt.edu   4   references   1. bruxism  de+ined  and  grad-­‐ ed:  an  international  consen-­‐ sus.  lobbezoo  f,  ahlberg  j,   glaros  ag,  kato  t,  koyano  k,   lavigne  gj,  de  leeuw  r,   manfredini  d,  svensson  p,   winocur  e.  j  oral  rehabil.   2013  jan;40(1):2-­‐4.  doi:   10.1111/joor.12011.  epub   2012  nov  4.  pmid:   23121262   2. bruxism  physiology  and  pa-­‐ thology:  an  overview  for  cli-­‐ nicians.  lavigne  gj,  khoury   s,  abe  s,  yamaguchi  t,   raphael  k.  j  oral  rehabil.   2008  jul;35(7):476-­‐94.  doi:   10.1111/j.1365-­‐ 2842.2008.01881.x.  review.   pmid:  18557915   3. role  of  psychosocial  factors   in  the  etiology  of  bruxism.   manfredini  d,  lobbezoo  f.  j   orofac  pain.  2009   spring;23(2):153-­‐66.  re-­‐ view.  pmid:  19492540   4. genetics  of  pediatric  anxiety   disorders.  sakolsky   dj,  mccracken  jt,  nurmi  el.   child  adolesc  psychiatr  clin   n  am.  2012  jul;21(3):479-­‐ 500.  doi:   10.1016/j.chc.2012.05.010.   review.  pmid:  22800990   5. a  review  and  meta-­‐analysis   of  the  genetic  epidemiology   of  anxiety  disorders.  hette-­‐ ma  jm,  neale  mc,  kendler   ks.  am  j  psychiatry.  2001   oct;158(10):1568-­‐78.  pmid:   11578982   6. first  genome-­‐wide  associa-­‐ tion  study  on  anxiety-­‐ related  behaviours  in  child-­‐ hood.  trzaskowski  m,  eley   tc,  davis  os,  doherty  sj,   hanscombe  kb,  meaburn   el,  haworth  cm,  price  t,   plomin  r.  plos  one.   2013;8(4):e58676.  doi:   10.1371/journal.pone.0058 676.  epub  2013  apr  2.   pmid:  23565138   7. symptoms  of  anxiety  and   depression  in  a  volunteer   twin  population.  the  etio-­‐ logic  role  of  genetic  and  en-­‐ vironmental  factors.   kendler  ks,  heath  a,  martin   ng,  eaves  lj.  arch  gen  psy-­‐ chiatry.  1986   mar;43(3):213-­‐21.  pmid:   3954540   8. bruxism:  a  literature  re-­‐ view.  reddy  sv,  kumar  mp,   sravanthi  d,  mohsin  ah,   anuhya  v.  j  int  oral  health.   2014  nov-­‐dec;6(6):105-­‐9.   review.  pmid:  25628497   9. the  anxiety  in  bruxer  child.   a  case-­‐control  study.  mona-­‐ co  a,  ciammella  nm,  marci   mc,  pirro  r,  giannoni  m.   minerva  stomatol.  2002   jun;51(6):247-­‐50.  pmid:   12147977   10. periodontal  disease  and   coronary  heart  disease  inci-­‐ dence:  a  systematic  review   and  meta-­‐analysis.  humph-­‐ rey  ll,  fu  r,  buckley  di,   freeman  m,  helfand  m.  j   gen  intern  med.  2008   dec;23(12):2079-­‐86.  doi:   10.1007/s11606-­‐008-­‐0787-­‐ 6.  epub  2008  sep  20.  re-­‐ view.  pmid:  18807098   11. diabetes  mellitus:  biochem-­‐ ical,  histological  and  micro-­‐ biological  aspects  in  perio-­‐ dontal  disease.  marigo   l,  cerreto  r,  giuliani  m,   somma  f,  lajolo  c,  cordaro   m.  eur  rev  med  pharmacol   sci.  2011  jul;15(7):751-­‐8.   review.  pmid:  21780542   12. suicide-­‐related  behaviors   and  anxiety  in  children  and   adolescents:  a  review.  hill   rm,  castellanos  d,  pettit  jw.   clin  psychol  rev.  2011   nov;31(7):1133-­‐44.  doi:   10.1016/j.cpr.2011.07.008.   epub  2011  jul  28.  review.   pmid:  21851804     microsoft word 57 2016.docx   vol  4,  no  1  (2016)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2016.57           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     association  of  axin2  gene  polymorphisms  with  nonsyndromic           oligodontia  in  turkish  families   nuriye  dinckan1,3,  zehra  oya  uyguner1,  hulya  kayserili2,  ariadne  letra3,4     1department  of  medical  gene+cs,  istanbul  medical  faculty,  istanbul  university,  istanbul,  34093,  turkey   2  department  of  medical  gene2cs,  koc  university,  school  of  medicine  (kusom),  istanbul,  34010,  turkey   3  department  of  diagnos/c  and  biomedical  sciences  and  center  for  craniofacial  research,  university  of  texas  health  science  center  at  houston  school  of  den:stry,   houston,  tx,  77054,  usa   4  pediatric  research  center,  university  of  texas  health  science  center  at  houston  mcgovern  medical  school,  houston,  tx,  77030,  usa   abstract   tooth   agenesis   is   the  most   common   developmental   abnormality   of   the   human   den''on   characterized  by   the   congenital   absence  of   one  or  more  permanent   teeth.  oligodon7a   is   the  term  used  to  describe  severe  tooth  agenesis,  where  six  or  more  permanent  teeth  are   missing.  the  wnt  gene  pathway  regulates  mul)ple  developmental  processes  during  cranio-­‐ facial  and  tooth  development,  and  varia2ons  in  wnt  pathway  genes  have  been  reported  in   individuals  with   tooth   agenesis.   in   this   study,  we   inves0gated   the   associa0on  of   37   snps   in/nearby   12   wnt   pathway   genes   (wnt3,   wnt3a,   wnt5a,   wnt8a,   wnt9b,   wnt10a,   wnt11,   axin1,   axin2,   apc,   lrp5,   lrp6)  with   oligodon,a   in   22  mul,plex   families.   geno-­‐ types  were  generated  using  taqman  chemistry  in  a  real-­‐!me  polymerase  chain  reac!on  as-­‐ say.    family-­‐based  associa*on  tests  were  performed  using  fbat.  pairwise-­‐haplotype  analy-­‐ sis  was  also  performed.  bonferroni  correc2on  was  used  to  adjust  for  mul+ple  tes+ng  and  p-­‐ values  ≤  0.001  were   considered   sta0s0cally   significant.  we   found  nominal   associa"on   for   axin2   rs7591,   located   in   the   3’   utr,   with   oligodon:a   (p=0.04).   in   silico   analysis   of   snp   func%on  predicted  a  binding   site   for  mir-­‐205  with  poten-al   impact  on  axin2  expression.   although  modest,  these  results  con1nue  to  support  a  role  for  axin2  in  the  e'ology  of  famil-­‐ ial  tooth  agenesis.       cita%on:   dinckan,   et   al.   (2016).   associa'on   of   axin2   gene   polymorphisms  with   nonsyndromic   oligodon'a   in   turkish   families.   den$stry   3000.   1:a001  doi:10.5195/d3000.2016.57   received:  july  19,  2016   accepted:    august  1,  2016   published:    october  3,  2016   copyright:  ©2016  dinckan,  et  al.  this  is  an  open   access   ar!cle   licensed   under   a   crea!ve   com-­‐ mons   a"ribu"on   work   4.0   united   states   li-­‐ cense.   email:  ariadne.m.letra@uth.tmc.edu   introduction   tooth  agenesis  is  the  most   common  craniofacial  congenital   malformation  in  humans  [1].  up  to   20%  of  the  general  population  has   agenesis  of  at  least  one  third  mo-­‐ lar.  agenesis  of  other  permanent   teeth,  excluding  third  molars,   ranges  from  ~1.6  to  9%,  depend-­‐ ing  on  the  population  studied,  and   in  70-­‐80%  of  these  cases  one  or   two  teeth  are  missing  [2,3].  tooth   agenesis  can  be  identified  as  hy-­‐ podontia  (up  to  5  teeth  missing,   excluding  third  molars),  or  oligo-­‐ dontia  (lack  of  more  than  6  teeth   missing,  excluding  third  molars)   [1],  in  sporadic  cases  or  segregat-­‐ ing  in  families.  in  most  of  the  fa-­‐ milial  cases,  inheritance  is  auto-­‐ somal  dominant,  however,  auto-­‐ somal  recessive  and  x-­‐linked  in-­‐ heritance  have  also  been  de-­‐ scribed  [4].    associa&on  of  axin2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  turkish  families   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   2   the  etiology  of   tooth  agenesis  is  com-­‐ plex  and  poorly  under-­‐ stood  [4].  studies  in   mice  have  allowed  the   identification  of  genes   directly  or  indirectly  in-­‐ volved  in  the  regulation   of  tooth  development,   and  have  been  funda-­‐ mental  to  the  under-­‐ standing  of  the  basic   genetic  principles  of   tooth  development  and   its  defects  [5].  never-­‐ theless,  very  few  hu-­‐ man  mutations  have   been  described  in  sev-­‐ eral  genes  known  to   arrest  tooth  develop-­‐ ment  in  mice.  this  may   reflect  basic  differences   in  agenesis  mechanisms   because  of  species-­‐ specific  characteristics,   such  as  tooth  type  (only   incisors  and  molars  in   mice)  and  number  of   dentitions  (one  denti-­‐ tion  in  mice  vs.  two   dentitions  -­‐  deciduous   and  permanent  -­‐  in  hu-­‐ mans)  [6].  mutations  in   pax9  (paired  box  9),   msx1  (msh  homeobox   1),  and  eda  (ectodys-­‐ plasin  a),  have  been   shown  to  cause  arrest   of  tooth  development   in  mice  and  humans,   and  these  genes  have   been  extensively  stud-­‐ ied  [1-­‐10].     table&1.!details!of!study!families.! family&no.& individual&no.& phenotype& relationship& no.&of& missing& teeth& type&of&missing& teeth& inheritance&& 1! 111! oligodontia! proband! 11! incisors,!premolars! complex! ! 112! unaffected! mother! 0! !! !! ! 113! unaffected! father! 0! !! !! ! 114! unaffected! brother! 0! !! !! ! 115! unaffected! brother! 0! !! !! ! 116! oligodontia! aunt! 10! !! !! ! 117! oligodontia! uncle! 7! !! !! ! 118! oligodontia! uncle! 7! !! !! 2! 211! oligodontia! proband! 28! incisors,!canines,! premolars,!molars! complex! ! 212! unaffected! mother! 0! !! !! ! 213! oligodontia! father! 8! !! !! ! 214! unaffected! brother! 0! !! !! ! 215! hypodontia! sister! 4! !! !! ! 216! hypodontia! brother! 4! !! !! ! 217! unaffected! brother! 0! !! !! ! 218! unaffected! sister! 0! !! !! ! 219! hypodontia! brother! 5! !! !! ! 2110! oligodontia! uncle! 7! !! !! ! 2111! hypodontia! aunt! 5! !! !! ! 2112! oligodontia! cousin! 21! !! !! ! 2113! oligodontia! cousin! 23! !! !! ! 2114! oligodontia! cousin! 19! !! !! ! 2115! oligodontia! cousin! unk! !! !! 3! 311! oligodontia! proband! 20! incisors,!premolars,! molars! ar! ! 312! unaffected! mother! 0! !! !! ! 313! oligodontia! father! unk! !! !! ! 314! hypodontia! sister! unk! !! !! ! 315! unaffected! sister! 0! !! !! ! 316! hypodontia! brother! unk! !! !! ! 317! oligodontia! sister! 17! !! !! ! 318! hypodontia! cousin! unk! !! !! 4! 411! oligodontia! proband! 8! lower!incisors,! molars! ad! ! 412! unaffected! mother! 0! !! !! ! 413! unaffected! father! 0! !! !! ! 414! hypodontia! brother! 4! !! !! ! 415! hypodontia! aunt! unk! !! !! ! 416! unaffected! cousin! 0! !! !! ! 417! hypodontia! grandmother! unk! !! !! unk,!unknown!missing!tooth!types! inheritance!patterns!(suspected):!ad,!autosomal!domimant;!ar,!autosomal!recessive.! !  associa&on  of  axin2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  turkish  families   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   3   the  wnt  gene  pathway   regulates  multiple  developmental   processes  during  craniofacial  and   tooth  development  [11-­‐12].  previ-­‐ ous  evidence  showing  the  expres-­‐ sion  of  several  wnt  genes  during   mouse  tooth  de-­‐ velopment   strongly  impli-­‐ cated  this  gene   family  in  the  eti-­‐ ology  of  tooth   agenesis  [11-­‐14].   in  recent  years,   mutations  in   wnt  pathway   genes,  namely   axin2  (axis  inhi-­‐ bition  protein  2),   wnt10a  (wing-­‐ less-­‐type  mmtv   integration  site   family,  member   10a),  lrp6  (low-­‐ density  lipopro-­‐ tein  receptor-­‐ related  protein   6),  and  recently   wnt10b  (wing-­‐ less-­‐type  mmtv   integration  site   family,  member   10b),  have  also   been  shown  to   cause  tooth   agenesis  in  hu-­‐ mans  [8,  10,  15-­‐ 18].  additional   studies  have  also   shown  the  asso-­‐ ciation  of  com-­‐ mon  single  nu-­‐ cleotide  poly-­‐ morphisms  in  a   few  wnt  path-­‐ way  genes  with   the  milder  form   of  tooth  agene-­‐ sis,  hypodontia   [19-­‐22].  howev-­‐ er,  the  associa-­‐ tion  of  wnt  pathway  gene  poly-­‐ morphisms  in  oligodontia  pheno-­‐ table&1&(continued).!details!of!study!families.! family&no.& individual&no.& phenotype& relationship& no.&of& missing& teeth& type&of&missing&teeth& inheritance&& 5! 511! oligodontia! proband! 13! incisors,!premolars! ar! ! 512! unaffected! mother! 0! !! !! ! 513! unaffected! father! 0! !! !! ! 514! unaffected! sister! 0! !! !! 6! 611! oligodontia! proband! 23! incisors,!premolars,!molars! complex! ! 612! hypodontia! mother! 2! upper!lateral!incisors! !! ! 613! unaffected! father! 0! !! !! ! 614! unaffected! brother! 0! !! !! 7! 711! oligodontia! proband! 17! incisors,!premolars,!molars! ar! ! 712! unaffected! mother! 0! !! !! ! 713! unaffected! father! 0! !! !! ! 714! unaffected! sister! 0! !! !! 8! 811! oligodontia! proband! 9! incisors,!premolars! ad! ! 812! unaffected! mother! 0! !! !! ! 813! hypodontia! father! 2! upper!lateral!incisors! !! 9! 911! oligodontia! proband! 15! incisors,!premolars,!molars! complex! ! 912! unaffected! mother! 0! !! !! ! 913! hypodontia! father! unk! !! !! ! 914! hypodontia! brother! unk! !! !! ! 915! hypodontia! brother! unk! !! !! ! 916! unaffected! sister! 0! !! !! ! 917! unaffected! brother! 0! !! !! ! 918! unaffected! sister! 0! !! !! 10! 1011! oligodontia! proband! 9! incisors,!premolars,!molars! x1linked! ! 1012! unaffected! mother! 0! !! !! ! 1013! hypodontia! father! 3! !! !! ! 1014! hypodontia! sister! 4! !! !! ! 1015! unaffected! brother! 0! !! !! 11! 1111! oligodontia! proband! 8! incisors,!canines,!premolars! ar! ! 1112! unaffected! mother! 0! !! !! ! 1113! unaffected! father! 0! !! !! ! 1114! unaffected! sister! 0! !! !! 12! 1211! oligodontia! proband! 12! incisors!and!premolars! ad! ! 1212! oligodontia! mother! unk! !! !! ! 1213! unaffected! father! 0! !! !! ! 1214! unaffected! brother! 0! !! !! ! 1215! oligodontia! uncle! 12! !! !! 13! 1311! oligodontia! proband! 12! incisors,!canines,!molars! ar! ! 1312! unaffected! mother! 0! !! !! ! 1313! oligodontia! father! 7! !! !! ! 1314! oligodontia! brother! 8! !! !! unk,!unknown!missing!tooth!types! inheritance!patterns!(suspected):!ad,!autosomal!domimant;!ar,!autosomal!recessive.!  associa&on  of  axin2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  turkish  families   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   4   types  is  still  unclear.  therefore,  in   this  study,  we  investigated  the  as-­‐ sociation  of  single  nucleotide  pol-­‐ ymorphisms  in  12  wnt  pathway   genes  with  oligodontia  in  multi-­‐ plex  families  from  turkey.     material  and   methods   sample  popula-­‐ tion   this   study  was  ap-­‐ proved  by  the   istanbul  univer-­‐ sity  institutional   ethical  review   board  and  the   committee  for   protection  of   human  subjects   at  the  university   of  texas  health   science  center   at  houston.  clin-­‐ ical  and  demo-­‐ graphic  infor-­‐ mation  and  dna   samples  from   peripheral  blood   were  obtained   from  the  crani-­‐ rare2  project,   an  european  un-­‐ ion-­‐funded  col-­‐ loborative  era-­‐ net  project  on   craniofacial  mal-­‐ formations  run   at  the  istanbul   university,  is-­‐ tanbul  medical   faculty,  medical   genetics  de-­‐ partment.  all   registry  partici-­‐ pants  had  signed   an  informed   consent  form  agreeing  to  partici-­‐ pate  in  genetic  studies  and  pro-­‐ table&1&(continued).!details!of!study!families.! family&no.& individual&no.& phenotype& relationship& no.&of& missing& teeth& type&of&missing&teeth& inheritance&& 14! 1421! oligodontia! proband! 12! incisors!and!premolars! complex! ! 1422! hypodontia! mother! 4! !! !! ! 1423! unaffected! father! 0! !! !! ! 1424! hypodontia! brother! 4! !! !! ! 1425! hypodontia! cousin! 2! upper!lateral!incisors! !! ! 1426! unaffected! uncle! 0! !! !! ! 1427! hypodontia! uncle’s!wife! 2! upper!lateral!incisors! !! 15! 1521! oligodontia! proband! 7! premolars,!molars! complex! ! 1522! unaffected! mother! 0! !! !! ! 1523! hypodontia! father! 2! upper!lateral!incisors! !! ! 1524! unaffected! sister! 0! !! !! ! 1525! hypodontia! uncle! 2! upper!lateral!incisors! !! ! 1526! hypodontia! uncle! 1! upper!lateral!incisor! !! ! 1527! hypodontia! uncle! 1! upper!lateral!incisor! !! 16! 1621! oligodontia! proband! 10! incisors,!canines,!premolars,! molars! ad! ! 1622! unaffected! mother! 0! !! !! ! 1623! unaffected! father! 0! !! !! ! 1624! unaffected! brother! 0! !! !! 17! 1721! oligodontia! proband! 6! upper!lateral!incisors,! premolars! ar! ! 1722! unaffected! mother! 0! !! !! ! 1723! hypodontia! father! 2! upper!lateral!incisors! !! ! 1724! unaffected! brother! 0! !! !! ! 1725! hypodontia! sister! 4! premolars! !! 18! 1821! oligodontia! proband! 18! incisors,!premolars,!molars! ad! ! 1822! unaffected! mother! 0! !! !! ! 1823! unaffected! father! 0! !! !! ! 1824! oligodontia! sister! 28! incisors,!canines,!premolars,! molars! !! 19! 1921! oligodontia! proband! 10! incisors!and!premolars! complex! ! 1922! hypodontia! mother! 2! upper!lateral!incisors! !! ! 1923! unaffected! father! 0! !! !! 20! 2021! oligodontia! proband! 13! !! ad! ! 2022! unaffected! mother! 0! !! !! 21! 2121! oligodontia! proband! 28! incisors,!canines,!premolars,! molars! complex! ! 2122! unaffected! mother! 0! !! !! ! 2123! unaffected! father! 0! !! !! 22! 2221! oligodontia! proband! 16! incisors,!canines,!premolars,! molars! complex! ! 2222! hypodontia! mother! 2! upper!lateral!incisors! !! ! 2223! unaffected! father! 0! !! !! unk,!unknown!missing!tooth!types! inheritance!patterns!(suspected):!ad,!autosomal!domimant;!ar,!autosomal!recessive.! !  associa&on  of  axin2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  turkish  families   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   5   vided  a  blood  samples  as  a  source   of  genomic  dna.     probands  were  selected   from  the  registry  based  on  their   radiographic  records  showing  con-­‐ genital  tooth  agenesis  and  were   invited  to  participate.  individuals   were  considered  to  have  oligodon-­‐ tia  when  six  or  more  permanent   teeth  were  missing  in  the  oral  cav-­‐ ity,  excluding  third  molars.  fami-­‐ lies  were  ascertained  through   probands,  and  additional  relatives   were  recruited.  all  probands  and   available  family  members  were   further  examined  clinically  to  con-­‐ firm  the  tooth  agenesis  status  and   exclude  syndromic  cases.  in  a  few   cases,  history  of  tooth  agenesis   was  available  by  self-­‐report  from   family  members,  or  from  state-­‐ ments  by  their  dental  provider.   our  sample  population  consisted   of  22  multiplex  oligodontia  fami-­‐ lies  (117  total  individuals,  67  af-­‐ fected,  50  unaffected),  in  which   tooth  agenesis  segregated  in  both   autosomal  dominant  and  autoso-­‐ mal  recessive  forms,  and  an  aver-­‐ age  of  2-­‐18  teeth  were  missing  in   each  affected  individual.  details  of   studied  families  are  presented  in   table  1.     selection  of  candidate  genes  and   single  nucleotide  polymorphisms   we  selected  37  single  nu-­‐ cleotide  polymorphisms  (snps)   in/nearby  apc,  axin1,  axin2,   lrp5,  lrp6,  wnt3,  wnt3a,   wnt5a,  wnt8a,  wnt9b,   wnt10a,  and  wnt11  genes  for   genotyping  in  our  families.  snps   were  selected  based  on  their  loca-­‐ tions  within  the  genes,  on  their   likelihood  to  have  functional  con-­‐ sequences  (i.e.,  located  in  the   promoters,  exons,  or  near  ex-­‐ on/intron  boundaries),  or  if  con-­‐ sidered  tag-­‐snps  for  the  linkage   disequilibrium  blocks  surrounding   the  respective  genes  [23].  we   used  information  available  at  the   ncbi  dbsnp   (http://www.ncbi.nlm.gov/snp/)   and  hapmap  project   (http://www.hapmap.org)  data-­‐ bases  to  select  polymorphisms.   details  of  studied  genes  and  pol-­‐ ymorphisms  are  presented  in  ta-­‐ ble  2.     genotyping   genomic  dna  was  extract-­‐ ed  from  blood  using  established   protocols.  genotypes  were  gener-­‐ ated  using  taqman  chemistry  [24].   reactions  were  carried  out  in  5-­‐μl   volumes  in  a  viia7  sequence  de-­‐ tection  system  (applied  biosys-­‐ tems,  foster  city,  ca).  assays  and   reagents  were  supplied  by  applied   biosystems.  the  results  were  ana-­‐ lyzed  using  eds  software  (applied   biosystems).  in  order  to  ensure   quality  control  of  genotyping  reac-­‐ tions,  we  included  a  non-­‐template   control  (water  instead  of  dna)  as   negative  control  and  a  dna  sam-­‐ ple  of  known  genotype  as  positive   control  in  each  reaction.   association  analyses   family-­‐based  association   tests  were  performed  using  fbat   software  version  1.06  [25].  we   used  bonferroni  correction  to  ad-­‐ just  for  multiple  testing  (0.05/37)   and  p-­‐values  ≤  0.001  were  consid-­‐ ered  significant.   in  silico  prediction  of  snp  function   we  performed  in  silico   analysis  of  snp  function  to  predict   the  effects  of  the  associated             axin2  rs7591  snps  function  using   mirbase  software  [26].       results   association  analyses   we  found  evidence  of  al-­‐ tered  allelic  transmission  for             axin2  rs7591,  in  the  gene  3’  utr,   with  oligodontia  (p=0.04).        associa&on  of  axin2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  turkish  families   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   6   in  silico  prediction  of  snp  function   in  silico  analysis  of  the  3’   utr  snp  in  axin2  rs7591  predict-­‐ ed  a  binding  site  for  the  microrna   mir-­‐205-­‐5p  with  potential  effects   on  gene  expression  (figure  1).     discussion   in  this  study,  we  investi-­‐ gated  the  association  of  12  wnt   pathway  genes  (apc,  axin1,             axin2,  lrp5,  lrp6,  wnt3,   wnt3a,  wnt5a,  wnt8a,  wnt9b,   wnt10a,  and  wnt11)  with  non-­‐ syndromic  oligodontia  in  22  well-­‐ characterized  turkish  caucasian   multiplex  families.  although  mod-­‐ est,  our  results  suggest  a  positive   association  between  axin2  and   oligodontia,  and  corroborate  the   results  of  previous  studies  [19-­‐21].   to  our  knowledge,  this  is  the  most   comprehensive  analysis  of  the  as-­‐ sociation  of  wnt/β-­‐catenin  path-­‐ way  genes  with  tooth  agenesis,   particularly  oligodontia.     over  the  years,  many  sig-­‐ naling  pathways  have  shown  to  be   involved  in  the  organogenesis  and   embryogenesis  of  teeth  [3,  5,  7,   12,  27].  individuals  with  oligodon-­‐ tia  constitute  approximately  1%  of   all  individuals  with  hypodontia,   and  both  conditions  can  be  found   in  the  same  family,  indicating  vari-­‐ able  expression  of  shared  genetic   factors  [2,  7].  the  importance  of   the  wnt/β-­‐catenin  signaling   pathway  during  tooth  develop-­‐ ment  has  been  reported  by  sever-­‐ al  authors  [11-­‐14].  many  studies   showed  that  wnt  pathway  plays  a   critical  role  in  tooth  morphogene-­‐ sis  and  several  wnt  genes  are  ex-­‐ pressed  in  craniofacial  and  dental   tissues  [11-­‐14].  wnt  pathway  acti-­‐ vation  has  roles  at  the  lamina-­‐ early  bud  stage  and  also  important   for  molar  cusps  development  [13].   during  tooth  development,  axin2   is  expressed  in  the  dental  mesen-­‐ chyme,  the  odontoblasts  and  the   enamel  knot,  and  it  is  needed  as  a   negative  regulator  of  wnt-­‐ signaling  at  specific  stages  [12,   13].   additional  common  vari-­‐ ants  in  axin2  have  also  been  asso-­‐ ciated  with  increased  susceptibil-­‐ ity  to  hypodontia  in  eastern  euro-­‐ peans  [19].  however,  the  snp  as-­‐ sociated  in  the  present  study,   rs7591,  located  in  the  3’  utr,  has   not  been  previously  reported  in   association  with  tooth  agenesis   and  warrants  additional  confirma-­‐ tory  studies.  previously,  this  same   snp  was  reported  in  association   with  oral  clefts  in  families  with  in-­‐ creased  susceptibility  to  colon   cancer  [28,  29].  interestingly,  in   silico  analyses  predicted  that  this   snp  harbors  a  binding  site  for  the   mir-­‐205-­‐5p,  with  a  potential  regu-­‐ latory  role  in  gene  expression.  re-­‐ cent  evidence  has  shown  that  a   number  of  cellular  functions,  in-­‐ cluding  development,  differentia-­‐ tion,  growth,  metabolism,  anabo-­‐ lism,  and  carcinogenesis  can  be   affected  by  mirna  functions  [30].   although  the  role  of  mir-­‐205-­‐5p  in   craniofacial  development  is  yet   unknown,  it  has  been  suggested  to   play  a  role  in  cancer  development   and  parkinson’s  disease  [31].  fur-­‐ ther,  the  level  of  mir-­‐205-­‐5p  ex-­‐ pression  was  found  to  be  down-­‐ regulated  in  various  cancer  cells,   including  breast,  oral,  prostate   cancer  cells,  and  melanoma  [32].   additional  studies  on  mir-­‐205-­‐5p   and  its  effect  on  the  regulation  of   axin2  might  elucidate  the  role  of   these  molecules  in  tooth  agenesis.   in  addition  to  a  critical  role   in  embryonic  development,  the   wnt/β-­‐catenin  signaling  pathway   is  also  associated  with  tumorigen-­‐ esis  events  [9].  mutations  in  axin2   were  found  segregating  with  auto-­‐ somal  dominant  tooth  agenesis   and  colorectal  cancer  in  a  large   multiplex  family,  suggesting  that  a   same  gene  may  be  involved  in   congenital  anomalies  and  cancer   later  in  life  [15].  axin2  mutations   were  also  detected  segregating  in   autosomal  dominant  pattern  with   oligodontia  and  other  findings  in-­‐ cluding  colonic  polyposis,  gastric   polyps,  a  mild  ectodermal  dyspla-­‐ sia  phenotype  with  sparse  hair  and   eyebrows,  and  early  onset  colo-­‐ rectal  and  breast  cancers  [33].  the   axin2  gene  encodes  the  axis  inhi-­‐ bition  protein  2  that  regulates  the   stability  of  beta-­‐catenin  and  early   organ  differentiation  and  devel-­‐ opment  and  plays  a  key  role  in   many  basic  cell  functions,  like  cell   homeostasis  [9].  since  the  report   by  lammi  et  al.  [15],  numerous   human  genetic  studies  have  fo-­‐ cused  on  identifying  variants  in   axin2  in  association  with  tooth   agenesis  or  other  birth  defects   such  as  cleft  lip/palate  [8-­‐10,  19-­‐ 21,  28],  due  to  the  previously  sug-­‐ gested  hypothesis  that  cancer-­‐ related  genes  may  have  a  role  in   tooth  agenesis.  notwithstanding,   despite  the  positive  associations   reported,  additional  studies  are   needed  to  determine  if  potential   correlations  exist  between  axin2,   birth  defects  and  cancer.    associa&on  of  axin2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  turkish  families   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   7   recently,  wnt10a  has   been  suggested  as  a  major   candidate  gene  for  tooth   agenesis,  and  oligodontia  in   particular,  and  rare  variants   in  this  gene  have  been  found   in  individuals  with  tooth   agenesis  from  multiple  popu-­‐ lations  [33].  interestingly,  in   the  present  study,  we  did  not   identify  any  association  be-­‐ tween  common  variants  in   wnt10a  and  oligodontia  in   our  turkish  families.     in  summary,  although   modest,  our  results  continue   to  support  a  role  for  axin2   and  the  wnt/β-­‐catenin  sig-­‐ naling  pathway  in  human   tooth  agenesis.  discrepancies   between  the  present  and   previous  studies  may  be  due   to  heterogeneity  of  the  con-­‐ dition  across  distinct  popula-­‐ tions,  and/or  the  common   variant-­‐common  disease  ap-­‐ proach  used  in  this  associa-­‐ tion  study,  when  rare  variants   in  relevant  genes  could  be   the  cause  of  the  phenotype.   future  studies  should  focus   on  the  identification  of  po-­‐ tentially  functional  variants  in   axin2  and  additional  wnt   pathway  genes  to  further  es-­‐ tablish  a  biological  role  of  this   pathway  in  tooth  agenesis   phenotypes.   acknowledgements   we  would  like  to  thank  all   of  the  participating  patients  and   their  families  for  their  support  in   this  research.  this  work  partially   supported  by  research  fund  of   istanbul  university;  project  num-­‐ ber  48398,  and  from  the  scientific   and  technological  research  coun-­‐ cil  of  turkey  (tubitak),  grant   number  112s398  [cranirare-­‐2].     references   1.   genetic  basis  of  tooth   agenesis.  nieminen  p.  j  exp  zool  b   mol  dev  evol.  2009  jun   15;312b(4):320-­‐42.   pmid:19219933.     2.   a  meta-­‐analysis  of  the   prevalence  of  dental  agenesis  of   permanent  teeth.  polder  bj,  van't   hof  ma,  van  der  linden  fp,  kui-­‐ jpers-­‐jagtman  am.  community   table&2.&details(of&snps(genotyped(in(this(study(and(association(results.( snp& locus& gene& function& alleles*& maf& informative&families**& p>value***& rs861674( chr.5:(112064475( apc$ intron( a/t( 0.454( 17( 0.67( rs2431238( chr.5:(112124369( apc$ intron( c/t( 0.343( 14( 1( rs454886( chr.5:(112146117( apc$ intron( c/t( 0.348( 18( 0.65( rs351771( chr.5:(112164561( apc$ synonymous( c/t( 0.009( 1( iiii( rs448475( chr.5:(112181379( apc$ 3’(utr( c/g( 0.482( 18( 0.67( rs2301522( chr.16:(359953( axin1$ intron( a/g( 0.4( 16( 0.5( rs7591( chr.17:(63525082( axin2$ 3’(utr( a/t( 0.143( 11( 0.04$ rs7224837( chr.17:(63528123( axin2$ intron( a/g( 0.491( 23( 0.91( rs11867417( chr.17:(63537898( axin2$ intron( c/t( 0.201( 13( 0.84( rs3923086( chr.17:(63549488( axin2$ intron( g/t( 0.438( 18( 0.43( rs2240307( chr.17:(63554307( axin2$ intron( a/g( 0.179( 7( 0.28( rs740026( chr.17:(63561681( axin2$ intergenic( a/g( 0.232( 14( 0.86( rs634008( chr.11:(68094741( lrp5$ intron( c/t( 0.402( 20( 0.46( rs667126( chr.11:(68177728( lrp5$ intron( c/t( 0.267( 16( 0.29( rs312788( chr.11:(68122295( lrp5$ intron( g/t( 0.384( 20( 0.57( rs312014( chr.11:(68084962( lrp5$ intron( c/g( 0.438( 17( 0.77( rs10743980( chr.12:(12412795( lrp6$ intron( c/t( 0.438( 17( 0.45( rs4477532( chr.12:(12279361( lrp6$ intron( a/g( 0.037( 4( iiii( rs7294695( chr.12:(12323618( lrp6$ intron( c/g( 0.393( 20( 0.65( rs121908120( chr.2:(218890289( wnt10a$ missense( a/t( 1( 0( iiii( rs3806557( chr.2:(218879152( wnt10a$ missense( a/g( 0.009( 1( iiii( rs199980023( chr.2:(218882196( wnt10a$ missense( c/t( 0.5( 22( 0.81( rs116998555( chr.2:(218890118( wnt10a$ missense( c/t( 0.009( 1( iiii( rs4574113( chr.2:(219762662( wnt10a$ intron( a/t( 0.194( 11( 1( rs10177996( chr.2:(219746561( wnt10a$ intron( c/t( 0.223( 12( 0.41( rs3806557( chr.2:(219743874( wnt10a$ intron( g/a( 0.221( 12( 0.33( rs1533767( chr.11:(75905800( wnt11$ intron( a/g( 0.009( 1( iiii( rs199498( chr.17:(44865603( wnt3$ intergenic( c/t( 1( 0( iiii( rs111769( chr.17:(44871987( wnt3$ intergenic( c/t( 0.356( 19( 0.31( rs9890413( chr.17:(44901449( wnt3$ intergenic( a/g( 0.356( 14( 0.6( rs708111( chr.1:(228191365( wnt3a$ intergenic( c/t( 0.143( 13( 0.5( rs3094912( chr.1:(228209815( wnt3a$ intron( a/t( 0.38( 18( 0.24( rs752107( chr.1:(228247351( wnt3a$ 3’(utr( c/t( 0.256( 13( 0.23( rs1745420( chr.1:(228251732( wnt3a$ intergenic( c/g( 0.393( 22( 0.15( rs566926( chr.3:(55520778( wnt5a$ intron( a/c( 0.348( 18( 0.41( rs2040862( chr.5:(137419989( wnt8a$ intron( c/t( 0.446( 23( 0.9( rs2165846( chr.17:(44941366( wnt9b$ intron( a/g( 0.5( 18( 0.64( *(ancestral(allele(listed(first,(ncbi(dbsnp(build(147( **(snps(with(less(than(5(informative(families(were(excluded(from(further(analysis( ***fbat,(p<0.001(denotes(statistical(significance.(pivalues(<0.05(are(shown(in(italic(font. !  associa&on  of  axin2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  turkish  families   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   8   dent  oral  epidemiol.  2004   jun;32(3):217-­‐26.  pmid:   15151692.   3.   tooth  agenesis:  from  mo-­‐ lecular  genetics  to  molecular  den-­‐ tistry.  matalova  e,  fleischmannova   j,  sharpe  pt,  tucker  as.  j  dent   res.  2008  jul;87(7):617-­‐23.  pmid:   18573979.   4.   anomalies  associated  with   hypodontia  of  the  permanent  lat-­‐ eral  incisor  and  second  premolar.   symons  al,  stritzel  f,  stamation  j.   j  clin  pediatr  dent.  1993  win-­‐ ter;17(2):109-­‐11.  pmid:  8466838.   5.   tooth  morphogenesis  and   cell  differentiation.  thesleff  i,   nieminen  p.  curr  opin  cell  biol.   1996  dec;8(6):844-­‐50.  pmid:   8939666.   6.   two  genes  for  missing   teeth.  thesleff  i.  nat  genet.  1996   aug;13(4):379-­‐80.  pmid:  8696323.   7.   the  genetic  basis  of  inher-­‐ ited  anomalies  of  the  teeth.  part  1:   clinical  and  molecular  aspects  of   non-­‐syndromic  dental  disorders.   bailleul-­‐forestier  i,  molla  m,  ver-­‐ loes  a,  berdal  a.  eur  j  med  genet.   2008  jul-­‐aug;51(4):273-­‐91.  pmid:   18499550.   8.   isolated  oligodontia  asso-­‐ ciated  with  mutations  in   edaradd,  axin2,  msx1,  and  pax9   genes.  bergendal  b,  klar  j,   stecksén-­‐blicks  c,  norderyd  j,  dahl   n.  am  j  med  genet  a.  2011   jul;155a(7):1616-­‐22.  pmid:   21626677.   9.   exclusion  of  coding  region   mutations  in  msx1,  pax9  and  ax-­‐ in2  in  eight  patients  with  severe   oligodontia  phenotype.  gerits  a,   nieminen  p,  de  muynck  s,  carels   c.  orthod  craniofac  res.  2006   aug;9(3):129-­‐36.  pmid:  16918677.   10.   mutational  analysis  of  ax-­‐ in2,  msx1,  and  pax9  in  two  mexi-­‐ can  oligodontia  families.  mu  yd,   xu  z,  contreras  ci,  mcdaniel  js,   donly  kj,  chen  s.  genet  mol  res.   2013  oct  10;12(4):4446-­‐58.  pmid:   24222224.   11.   expression  of  wnt  signaling   pathway  genes  during  tooth  de-­‐ velopment.  sarkar  l,  sharpe  pt.   mech  dev.  1999  jul;85(1-­‐2):197-­‐ 200.  pmid:  10415363.   12.   expression  patterns  of   wnt/β-­‐catenin  signaling  mole-­‐ cules  during  human  tooth  devel-­‐ opment.  wang  b,  li  h,  liu  y,  lin  x,   lin  y,  wang  y,  hu  x,  zhang  y.  j  mol   histol.  2014  oct;45(5):487-­‐96.   pmid:  24647585.     13.   wnt/beta-­‐catenin  signaling   directs  multiple  stages  of  tooth   morphogenesis.  liu  f,  chu  ey,   watt  b,  zhang  y,  gallant  nm,  andl   t,  yang  sh,  lu  mm,  piccolo  s,   schmidt-­‐ullrich  r,  taketo  mm,   morrisey  ee,  atit  r,  dlugosz  aa,   millar  se.  dev  biol.  2008  jan   1;313(1):210-­‐24.  pmid:  18022614.   14.   wnt5a  plays  a  crucial  role   in  determining  tooth  size  during   murine  tooth  development.  cai  j,   mutoh  n,  shin  jo,  tani-­‐ishii  n,   ohshima  h,  cho  sw,  jung  hs.  cell   tissue  res.  2011  sep;345(3):367-­‐ 77.  pmid:  21879290.   15.   mutations  in  axin2  cause   familial  tooth  agenesis  and  predis-­‐ pose  to  colorectal  cancer.  lammi   l,  arte  s,  somer  m,  jarvinen  h,   lahermo  p,  thesleff  i,  pirinen  s,   nieminen  p.  am  j  hum  genet.   2004  may;74(5):1043-­‐50.  pmid:   15042511.   16.   mutations  in  wnt10a  are   present  in  more  than  half  of  iso-­‐ lated  hypodontia  cases.  van  den   boogaard  mj,  créton  m,  bronk-­‐ horst  y,  van  der  hout  a,   hennekam  e,  lindhout  d,  cune  m,   ploos  van  amstel  hk.  j  med  genet.   2012  may;49(5):327-­‐31.  pmid:   22581971.   17.   loss-­‐of-­‐function  mutations   in  the  wnt  co-­‐receptor  lrp6   cause  autosomal-­‐dominant  oli-­‐ godontia.  massink  mp,  créton  ma,   spanevello  f,  fennis  wm,  cune   ms,  savelberg  sm,  nijman  ij,  mau-­‐ rice  mm,  van  den  boogaard  mj,   van  haaften  g.  am  j  hum  genet.   2015  oct  1;97(4):621-­‐6.  pmid:   26387593.   18.   mutations  in  wnt10b  are   identified  in  individuals  with  oli-­‐ godontia.  yu  p,  yang  w,  han  d,   wang  x,  guo  s,  li  j,  li  f,  zhang  x,   wong  sw,  bai  b,  liu  y,  du  j,  sun   zs,  shi  s,  feng  h,  cai  t.  am  j  hum   genet.  2016  jul  7;99(1):195-­‐201.   pmid:  27321946.   19.   axis  inhibition  protein  2   (axin2)  polymorphisms  may  be  a   risk  factor  for  selective  tooth   agenesis.  mostowska  a,  biedziak   b,  jagodzinski  pp.  j  hum  genet.   2006;51(3):262-­‐6.  pmid:   16432638.   20.   axin2  and  cdh1  polymor-­‐ phisms,  tooth  agenesis,  and  oral   clefts.  letra  a,  menezes  r,  gran-­‐ jeiro  jm,  vieira  ar.  birth  defects   res  a  clin  mol  teratol.  2009    associa&on  of  axin2  gene  polymorphisms  with  nonsyndromic  oligodon0a  in  turkish  families   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.57    http://dentistry3000.pitt.edu   9   feb;85(2):169-­‐73.  pmid:   18683894.   21.   axis  inhibition  protein  2   (axin2)  polymorphisms  and  tooth   agenesis.  callahan  n,  modesto  a,   meira  r,  seymen  f,  patir  a,  vieira   ar.  arch  oral  biol.  2009   jan;54(1):45-­‐9.  pmid:  18790474.   22.   nucleotide  variants  of   genes  encoding  components  of   the  wnt  signaling  pathway  and  the   risk  of  non-­‐syndromic  tooth  agen-­‐ esis.  mostowska  a,  biedziak  b,  za-­‐ durska  m,  dunin-­‐wilczynska  i,  li-­‐ aneri  m,  jagodzinski  pp.  clin   genet.  2013  nov;84(5):429-­‐40.   pmid:  23167694.   23.   selecting  a  maximally  in-­‐ formative  set  of  single-­‐nucleotide   polymorphisms  for  association   analyses  using  linkage  disequilibri-­‐ um.  carlson  cs,  eberle  ma,  rieder   mj,  yi  q,  kruglyak  l,  nickerson  da.   am  j  hum  genet.  2004   jan;74(1):106-­‐20.  pmid:   14681826.   24.   high-­‐throughput  genotyp-­‐ ing  with  single  nucleotide  poly-­‐ morphisms.  ranade  k,  chang  ms,   ting  ct,  pei  d,  hsiao  cf,  olivier  m,   pesich  r,  hebert  j,  chen  yd,  dzau   vj,  curb  d,  olshen  r,  risch  n,  cox   dr,  botstein  d.  genome  res.  2001   jul;11(7):1262-­‐8.  pmid:  11435409.   25.   family-­‐based  tests  of  asso-­‐ ciation  in  the  presence  of  linkage.   lake  sl,  blacker  d,  laird  nm.  am  j   hum  genet.  2000  dec;67(6):1515-­‐ 25.  pmid:  11058432.   26.    mirbase:  the  microrna   sequence  database.  griffiths-­‐jones   s.  methods  mol  biol.   2006;342:129-­‐38.  pmid:   16957372.   27.   genetic  basis  for  tooth   malformations:  from  mice  to  men   and  back  again.  mitsiadis  ta,   luder  hu.  clin  genet.  2011   oct;80(4):319-­‐29.  pmid:   21819395.   28.   axis  inhibition  protein  2,   orofacial  clefts  and  a  family  history   of  cancer.  menezes  r,  marazita   ml,  goldstein  mchenry  t,  cooper   me,  bardi  k,  brandon  c,  letra  a,   martin  ra,  vieira  ar.  j  am  dent   assoc.  2009  jan;140(1):80-­‐4.   pmid:19119171.     29.   the  axis  inhibition  protein   2  polymorphisms  and  non-­‐ syndromic  orofacial  clefts  suscep-­‐ tibility  in  a  chinese  han  popula-­‐ tion.  han  y,  zhou  l,  ma  l,  li  d,  xu   m,  yuan  h,  ma  j,  zhang  w,  jiang   h,  wu  y,  wang  l,  pan  y.  j  oral   pathol  med.  2014  aug;43(7):554-­‐ 60.  pmid:  24484320.   30.   micrornas  in  human  dis-­‐ eases:  from  cancer  to  cardiovas-­‐ cular  disease.  ha  ty.  immune   netw.  2011  jun;11(3):135-­‐54.   pmid:  21860607.   31.   a  program  of  micrornas   controls  osteogenic  lineage  pro-­‐ gression  by  targeting  transcription   factor  runx2.  zhang  y,  xie  rl,  cro-­‐ ce  cm,  stein  jl,  lian  jb,  van   wijnen  aj,  stein  gs.  proc  natl   acad  sci  u  s  a.  2011  jun   14;108(24):9863-­‐8.  pmid:   21628588.   32.   microrna-­‐205  suppresses   the  oral  carcinoma  oncogenic  ac-­‐ tivity  via  down-­‐regulation  of  axin-­‐ 2  in  kb  human  oral  cancer  cell.   kim  js,  park  sy,  lee  sa,  park  mg,   yu  sk,  lee  mh,  park  mr,  kim  sg,   oh  js,  lee  sy,  kim  cs,  kim  hj,   chun  hs,  kim  js,  moon  sm,  kim   dk.  mol  cell  biochem.  2014   feb;387(1-­‐2):71-­‐9.  pmid:   24166197.   33.   axin2-­‐associated  autoso-­‐ mal  dominant  ectodermal  dyspla-­‐ sia  and  neoplastic  syndrome.   marvin  ml,  mazzoni  sm,  herron   cm,  edwards  s,  gruber  sb,  petty   em.  am  j  med  genet  a.  2011   apr;155a(4):898-­‐902.  pmid:   21416598.   34.   wnt10a  variants  are  asso-­‐ ciated  with  non-­‐syndromic  tooth   agenesis  in  the  general  popula-­‐ tion.  song  s,  zhao  r,  he  h,  zhang  j,   feng  h,  lin  l.  hum  genet.  2014   jan;133(1):117-­‐24.  pmid:   24043634.     microsoft word 60 2017.docx   vol  5,  no  1  (2017)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2017.60           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     assessment  of  knowledge  among  public  and  private  elementary   school  teachers  in  dental  trauma  management   merve  bayram1,  mine  koruyucu2,  figen  seymen2      1istanbul  medipol  university,  school  of  den3stry,  department  of  pedodon3cs,  istanbul,  turkey   2istanbul  university,  faculty  of  den6stry,  department  of  pedodon6cs,  istanbul,  turkey abstract   purpose:  trauma&c  dental  injuries,  majority  of  which  occur  at  school  and  primary  care  given  by  teacher,  should  man-­‐ aged  as   soon  as  possible.  the  knowledge  and  a2tudes  of   teachers   is   cri8cal   for   the  best  prognosis  of   trauma8zed   teeth.   the   purpose   of   this   study   was   to   evaluate   knowledge   and   a7tudes   among   elementary   public   and   private   school   teachers   in  dental   trauma  management.  materials  and  methods:  a  modified   three-­‐part  ques*onnaire  com-­‐ prised  of  ques,ons  regarding  demographic  data,  a5tude  and  knowledge  about  dental  trauma  was  distributed  to  328   teachers  (public  schools:164/private  schools:164).  the  results  of  the  ques:onnaire  were  expressed  as  frequency  dis-­‐ tribu&ons.  sta&s&cal  analysis  was  performed  by  version  20.0  of  the  spss  sta&s&cs  so<ware.  results:  the  average  cor-­‐ rect   knowledge   score   was   4.96   out   of   10.   two   individual   predictors   significantly   improved   the   respondents’   knowledge:  being  more  than  50  years  old  age  (p=0.001)  and  more  than  15  years  teaching  experience  group  (p<0.001).   while  84.5%  of  public,  79%  of  private  school  teachers  found  their  knowledge  poor;  and  85%  of  public,  76%  of  private   school  teachers  state  they're  not  sa.sfied  with  their  level  of  knowledge.  private  school  teachers  had  given  significant-­‐ ly  more  correct  answers   to   the  ques1ons  about  dental  avulsion  management   than  public   school   teachers   (p=0.01).   there  was  no  significant  difference  between  public  and  private  school  teachers'  knowledge  regarding  dental  trauma   management.  conclusion:  the   level  of  knowledge   in  dental   trauma  management  determined   in   this   study  was  un-­‐ sa!sfying.  programs  on  increasing  the  awareness  and  mo1va1on  of  teachers  on  dental  trauma  management  in  both   types  of  schools  are  recommended.     cita%on:  bayram,  et  al.  (2017)  assessment  of   knowledge  among  public  and  private  elemen-­‐ tary  school  teachers  in  dental  trauma  manage-­‐ ment.  den$stry  3000.  1:a001   doi:10.5195/d3000.2017.60   received:    october  4,  2016   accepted:    may  25,  2017   published:    june  ,  2017   copyright:  ©2017  bayram,  et  al.  this  is  an  open   access   ar!cle   licensed   under   a   crea!ve   com-­‐ mons   a"ribu"on   work   4.0   united   states   li-­‐ cense.   email:  mine.yildirim@istanbul.edu.tr   introduction   traumatic  dental  injuries   (tdi)  are  prevalent  in  schoolchil-­‐ dren  and  is  a  public  health  con-­‐ cern  [1,2].  the  prevalence  rate  of   injuries  to  permanent  teeth  varies   between  6.1%  and  58.6%  [3-­‐16]   depending  on  type  of  study,  trau-­‐ ma  classification,  methodology,   study  size  and  population,  geo-­‐ graphical  location  and  culture  [17].   a  tdi  has  several  consequences   such  as  physiological,  psychologi-­‐ cal,  financial  and  affect  the  quality   of  life  of  both  parents  and  children   [18].     severe  tdi  can  damage  the   pulp  and  periodontal  ligament  and   lead  to  tooth  loss.  optimally,  for   long-­‐term  success,  the  manage-­‐ ment  or  treatment  of  traumatized   teeth  should  be  carried  out  as   soon  as  possible  [1].  immediate   care  is  particularly  important  for   the  avulsed  permanent  tooth  as   the  replantation  takes  place  as   soon  as  possible  after  avulsion  and   preferably  with  30  minutes  [19-­‐ 21].  avulsion  of  permanent  teeth   is  the  most  serious  of  all  types  of   traumatic  tooth  injuries  because   the  complete  dislodgement  of  the   tooth  from  its  socket  causes  se-­‐ vere  damage  to  the  supporting   tissues  and  vascular  and  nerve   structures.  the  ideal  treatment  for   an  avulsed  permanent  tooth  is  its   immediate  replantation  into  the   socket  [19].  immediate  tooth  re-­‐ plantation  leads  to  a  better  pdl   repair  and  reduces  significantly   the  occurrence  of  root  resorption.   therefore,  shortening  as  much  as   possible  the  time  elapsed  between   trauma  and  tooth  replantation   and  maintaining  the  avulsed  tooth   in  a  suitable  transport  medium   may  attenuate  the  deleterious  ef-­‐ fects  of  the  extrabuccal  period  on   root  surface  and  increase  the   prognosis  considerably  [1,  19].   most  incidences  happen   during  school  time  where  the  pri-­‐ mary  care  is  given  by  a  teacher.   reports  also  show,  in  some  in-­‐ stances  that  more  than  half  of  the   children  interviewed  are  affected   by  tdi’s  in  school  [22].  since  most   tdi’s  occur  during  school  time,  it   follows  that  suitable  and  timely   assessment  of  knowledge  among  public  and  private  elementary  school  teachers  in  dental  trauma  management   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.60    http://dentistry3000.pitt.edu   action  is  imperative  to  the  long-­‐ term  prognosis  of  an  affected  child   by  a  teacher  or  school  staff  mem-­‐ ber  [23].     the  purpose  of  this  study   was  to  (i)  evaluate  the  knowledge   of  elementary  school  teachers’   toward  common  dental  trauma   management,  (ii)  compare  the   knowledge  of  public  and  private   elementary  school  teachers’  atti-­‐ tudes  toward  dental  trauma  man-­‐ agement  for  crown  fracture  and   avulsion  injuries.     materials  and  methods   a  questionnaire  consisting   of  three  parts  was  applied  to   teachers  (164  public  and  164  pri-­‐ vate  school  teachers)  in  15  ran-­‐ domly  chosen  elementary  schools   in  istanbul,  turkey.  part  1  consists   of  questions  on  gender,  age,   teaching  experience,  first  aid  train-­‐ ing  and  dental  trauma  experience.   part  2  consists  of  questions  on   subjective  self-­‐assessment  of  atti-­‐ tude.  part  3  consists  of  questions   about  dental  trauma  manage-­‐ ment.  introduction  of  the  ques-­‐ tionnaire  included  consent  of  con-­‐ fidentiality  information.  the   teachers  and  the  school  admin-­‐ istrations  who  agreed  to  take  part   in  the  study  were  assured  of  strict   confidentially.  similar  question-­‐ naires  were  used  with  oliveira  et   al  [24].      this  questionnaire  is  in-­‐ cluded  in  table  1.   teachers  received  a  grade   according  to  their  correct  answers   for  part  iii.  the  grades  were  then   normalized  by  10.  the  results  of   table  1.  questionnaire   part  i   1.  gender:   male       female   2.  age:       20-­‐34  years     35-­‐49  years     ≥50  years   3.  length  of  teaching  experience?        <  1       1-­‐5   5-­‐10   10-­‐15     >15     4.  do  you  have  kids?         yes     no   5.  have  you  had  first  aid  training  in  your  own  organization?   yes     no   6.  have  you  had  first  aid  training  in  another  organization?   yes     no   7.  if  you  did,  have  it  included  dental  trauma  first  aid?   yes     no   8.  did  you  have  personal  experience  (yourself/your  student)  in  dental  trauma?        yes     no       part  ii   1.  self  assessed  knowledge  in  dental  trauma  management:   a-­‐none     b-­‐little     c-­‐moderate     d-­‐high   2.  satisfaction  with  self  assessed  knowledge:   a-­‐very  low                      b-­‐low     c-­‐moderate     d-­‐high:     3.  enthusiasm  for  more  education  in  the  subject:   a-­‐not  interested   b-­‐little  interested   c-­‐very  interested:       part  iii   1.  an  8  year  old  boy  was  hit  in  the  face  with  a  football  during  the  school  time  and  his  upper  front  tooth  was   broken.  he  was  otherwise  unhurt  and  didn’t  lose  consciousness.     1(a)  is  the  damaged  front  tooth  likely  to  be  primary  or  permanent?     a.primary     b.permanent   1(b)  which  of  the  following  actions  would  you  considered  as  the  most  appropriate?   1. a.a$er  class,  contact  his  parents  to  explain  what  had  happened.   2. b.give  him  a  warm  drink  and  contact  his  parents   3. c.send  him  immediately  to  the  doctors  or  infirmary   4. d.send  him  immediately  to  the  den/st   5.   6. 2.  a  12  year  old  girl  fell  from  the  stairs  and  hit  in  the  mouth  during  the  school  0me.  her  mouth  is  bleeding  and   an  upper  front  tooth  is  found  to  be  missing.     7.   8. 2(a)  which  of  the  following  would  you  do?   9. a.sideline  the  injured  girl,  ge1ng  her  to  bite  on  a  hankerchief  to  control  the  bleeding.   10. b.look  for  the  tooth,  wash  it  and  give  it  to  the  girl  to  take  home.   11. c.look  for  the  tooth  and  put  it  back  into  the  socket.   12. d.put  the  tooth  in  liquid  and  send  the  girl  home  straight  away.   13. e.find  the  tooth  and  put  it  in  liquid  and  send  the  girl  to  the  nearest  den.st.   14. 2(b)  in  case  of  this  condi.on,  which  would  be  the  first  place  you  would  contact  to?   15. a.doctor   b.den&st   c.hospital   d.  i  would  tried  to  treat   16. 2(c)  in  case  of  this  condi.on,  how  urgent  do  you  think  it  is  to  seek  professional  help?   17. a.  immediately  -­‐  within  30  min   b.  within  1  hour   18. c.  within  a  few  hours       d.  within  1  day     3.would  you  replant  the  primary  tooth  that  has  been  knocked  out?   a.yes     b.no     4.would  you  replant  the  permanent  tooth  back  into  socket  yourself?   a.yes,  i  would     b.no,  i  would  get  professional  help     4(a)  if  your  answer  is  “yes”,  when  you  get  the  tooth  from  the  ground  you  saw  the  tooth  was  dirty  what  would   you  do?   a.scrub  the  tooth  gently  with  a  toothbrush.   b.rinse  the  tooth  under  tap  water.   c.put  the  tooth  straight  back  into  the  socket  without  any  pretreatment.   d.rinse  the  tooth  with  detergant  or  soap.       4(b)if  your  answer  is  “no”,  how  would  you  transport  the  tooth  to  the  dentist?   a.in  tap  water         b.in  milk       c.in  alcohol     d.in  physiologic  water  (saline)   e.in  paper  tissue     f.in  ice       5.would  you  search  for  if  the  child  had  tetanus  vaccine?   a.yes     b.no     assessment  of  knowledge  among  public  and  private  elementary  school  teachers  in  dental  trauma  management   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.60    http://dentistry3000.pitt.edu   the  questionnaire  were  expressed   as  frequency  distributions.  statis-­‐ tical  analysis  was  performed  using   chi-­‐square  test  with  a  significance               level  of  .05  by  version  20.0   of  the  spss  statistics  software.   results   the  results  from  part  i  on   teachers’  from  public  and  private   schools  demographic  information   are  shown  in  table  2.     in  part  ii,  while  138   (84.5%)  teachers  in  public  school,   129  (79%)  teachers  in  private   school  claimed  they  have  poor   knowledge  of  the  subject;  and   139  (85%)  teachers  in  public   school,  124  (76%)  teachers  in  pri-­‐ vate  school  were  low  satisfied   with  their  knowledge  only  33   (20%)  were  interested  to  learn   more  in  both  school  type.  teach-­‐ ers  specified  that  the  main  source   for  information  was  a  dentist.   part  iii  of  the  question-­‐ naire  was  evaluated  the   knowledge  of  elementary  school   teachers  about  dental  trauma   management.  the  average  score   for  all  respondents  was  4.96  (of   10).  two  groups  significantly  im-­‐ proved  the  respondents’   knowledge:  being  above  50  year   old  age  group  (p=0.001)  and  hav-­‐ ing  teaching  experience  above  15   years  (p<0.001).     table  3  summarizes  the   following  data:  for  question  1(a),   54.9%  public  school  teachers,   69.5%  private  school  teachers  rec-­‐ ognized  that  the  upper  incisor  is  a   permanent  tooth  in  an  8-­‐year-­‐old   child.  there  was  a  significantly  as-­‐ sociation  between  public  and  pri-­‐ vate  schoolteachers  knowledge   (p=0.003).  for  case  1(b)  52.4%   public  school  teachers,  39.6%  pri-­‐ vate  school  teachers  would  ur-­‐ gently  take  her  to  the  dentist.   there  was  a  significantly  associa-­‐ tion  between  public  and  private   schoolteachers  knowledge   (p=0.01).  for  case  2(a)  54,9%  pub-­‐ lic  schoolteachers  and  51,8%  pri-­‐ vate  schoolteachers  would  find   the  tooth  and  put  it  in  liquid  and   send  the  girl  to  the  nearest  den-­‐ tist.  for  question  2(b)  79.3%  public   schoolteachers  and  67.7%  private   schoolteachers  knew  that  in  case   of  dental  trauma,  first  place  of   contact  is  a  dentist.  for  question   2(c)  only  5%  public  schoolteachers   and  10.9%  private  schoolteachers   were  aware  of  getting  early  pro-­‐ fessional  help.  private  school-­‐ teachers  statistically  had  more   correct  answers  on  ideal  replanta-­‐ tion  time  than  public  schoolteach-­‐ ers  (p=0.02).  for  question  3,  100%   public  schoolteachers  and  93.3%   private  schoolteachers  chose  that   replantation  of  primary  tooth  is   not  recommended.  there  was  a   significantly  association  between   public  and  private  schoolteachers   knowledge  (p=0.001).   private  schoolteachers  had   significantly  more  correct  answers   on  trauma  management  of   avulsed  teeth  than  public  school-­‐ teachers  (p=0.01).   discussion   table   2.   demographic   characteristics   of   15   randomly   chosen   public   and  private  elementary  school  teachers     demographic  characterictics   public   private   n   %   n   %   gender             male   76   46.3   46   28.0     female   88   53.7   118   72.0   age             20-­‐34   72   43.9   80   48.8     35-­‐49   61   37.2   67   40.9     ≥50   31   18.9   17   10.4   length  of  teaching  experience   (year)             <1     5   3.0   2   1.2     1-­‐5   24   14.6   36   22.0     5-­‐10   32   19.5   58   35.4     10-­‐15   23   14.0   26   15.9     >15   80   48.8   42   25.6   having  children             yes   109   66.5   94   57.3     no   55   33.5   70   42.7   received  first  aid  training  in  own   organization             yes   50   30.5   23   14.0     no   114   69.5   141   86.0   received  first  aid  training  in   another  organization             yes   61   37.2   53   32.3     no   97   59.1   108   65.9                              no  answer   6     3     content  of  first  aid  training     (including  dental  trauma)               yes   8   5   3   1,8     no   120   73.2   108   65.9   no  answer   36     53     dental  trauma  experience   (yourself/  your  student)             yes   40   24.4   29   17.7     no   118   72.0   128   78.0   no  answer   6     7     assessment  of  knowledge  among  public  and  private  elementary  school  teachers  in  dental  trauma  management   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.60    http://dentistry3000.pitt.edu   the  level  of  knowledge  of   dental  trauma  management  as   found  in  this  survey  was  low,  av-­‐ erage  score  being  4.96  of  10;  fux-­‐ noy  et  al  found  similar  results   (4.59)  [25].  in  addition,  only  20%   were  interested  in  additional   training  to  dental  trauma  man-­‐ agement.  these  results  show  the   lack  of  awareness  among  teachers   to  the  importance  of  immediate   and  optimum  treatment  of  dental   injuries.     teachers  who  were  above   50  year  old  had  better  knowledge   about  the  subject.  it  is  a  fact  that   this  age  group  had  more  experi-­‐ ence  than  the  younger  age  groups.   having  teaching  experience  above   15  years  was  also  found  to  be  a   related  factor  to  better   knowledge  on  the  subject  (ta-­‐ ble  2).   private  school  teachers   aren’t  good  at  referring  an  8-­‐ year-­‐old  child  with  broken  up-­‐ per  incisor  tooth  get  suitable   health  service.  possible  reason  –   we  concluded  subjectively  due   to  oral  discussions  -­‐  for  this  dif-­‐ ference  can  be  the  fact  that  pri-­‐ vate  schools  have  their  own   school  doctors  and  they  choose   to  take  the  child  to  the  school   doctor  in  case  of  dental  trauma.     primary  teeth  may  also   be  avulsed,  but  they  should  not   be  replanted  because  this  ma-­‐ nipulation  may  result  in  injury   to  the  underlying  permanent   teeth  germ  [1].     in  this  survey,  only  5.1%   teachers  replant  the  tooth   themselves.  while  chan  et  al.   [26]  and  fux-­‐noy  et  al.  [25]   found  similar  results;  hamilton  et   al.  [27]  and  pacheco  et  al.  [28]   found  higher  percentage.  more   than  94%  of  teachers  did  not  feel   the  capable  of  performing  tooth   replantation.  other  studies  have   reported  70%  to  80%  respondents   wouldn’t  replant  the  tooth  or   wouldn’t  be  comfortable  to  do  so   [29-­‐31].   an  avulsed  tooth  should  be   replanted  in  its  socket  as  soon  as   possible  to  avoid  further  damage   to  the  periodontal  membrane  [32-­‐ 35].  before  replantation,  the  tooth   should  be  cleaned  with  saline  so-­‐ lution  if  the  tooth  would  be  ob-­‐ served  dirty  [28,34].  the  replanta-­‐ tion  procedure  of  the  avulsed   teeth  has  been  recommended  to   be  performed  immediately  -­‐  with-­‐ in  30  minutes  after  trauma   [26,30,36].  if  the  tooth  can’t  be   replanted,  until  replantation  time,   the  teeth  must  be  kept  in  a  proper   media  [30].  hank‘s  balanced  salt   solution  (hbss)  has  been  especial-­‐ ly  developed  for  cell  maintenance   and  thus,  theoretically,  it  allows  a   better  conservation  of  tissues  for   long  time  periods.  it  has  been   widely  employed  as  a  reference   solution  in  studies  on  dental  avul-­‐ sion  as  it  has  the  ideal  osmolality   and  ph  for  preserving  the  vitality   of  cells  [37].   milk  has  a  favorable  osmo-­‐ lality  and  composition  for  the  via-­‐ bility  of  periodontal  ligament  cells   and  has  therefore  been  widely   recommended  to  dentists  and   general  population  for  keeping   avulsed  teeth  to  be  replanted,  be-­‐ ing  the  second  or  third  best  trans-­‐ portation  media  for  avulsed  teeth   include  (in  order  of  preference),   after  viaspan®  and/or  hank’s  bal-­‐ anced  salt  solution®,  according  to   the  international  association  of   dental  traumatology  [19],  and  the   american  academy  of  pediatric   dentistry  [38]  due  to  its  beneficial   effects  and  characteristics,  and  its   ease  of  access  at  the  moment  of   trauma  [37].   while  53.3%  teachers   choose  to  put  the  avulsed  tooth  in   liquid,  only  14.9%  teachers  knew   that  the  proper  media  is  milk.  sim-­‐ ilar  results  were  found  in  other   studies  among  teachers  [25-­‐ 27,36].  the  popular  wrong  an-­‐ swers  are  paper  tissue,  ice  and  tap   water  respectively.  private  school   table   3.   assessment   of   teachers’   knowledge   on   dental   trauma   management   in   15   randomly   chosen   elementary   schools   compared   by  school  type.   questions   public  school   private   school   p   value     n   %   n   %     recognizing  that   the  upper  incisor  is   a  permanent  tooth   in  an  8-­‐year-­‐old   child   90   54.9   114   69.5   0.003   suitable  approach   of  a  8-­‐year-­‐old   child  with  upper   incisor  tooth   broken   86   52.4   65   39.6   0.01   suitable  direction   of  a  12-­‐year-­‐old   child  with  avulsed   teeth   90   54.9   85   51.8   >0.05   suitable  affiliation   direction  in  case  of   dental  avulsion     130   79.3   111   67.7   >0.05   right  answer  about   ideal  replantation   time   9   5   18   10.9   0.02   suitable  approach   for  replantation  of   primary  tooth   164   100   153   93.3   0.001   suitable  approach   for  trauma  mana-­‐ gement  of  avulsion   22   13.4   39   23.8   0.01   remember  to   check  tetanus   vaccine  control   76   46.3   90   54.9   >0.05   assessment  of  knowledge  among  public  and  private  elementary  school  teachers  in  dental  trauma  management   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.60    http://dentistry3000.pitt.edu   teachers  gave  better  answers   about  ‘suitable  approach  for   trauma  management  of  avulsion’   than  the  public  school  teachers.   in  case  of  dental  trauma   especially  intra-­‐oral  wounds  teta-­‐ nus  vaccine  is  necessary  to  be   checked  out  [39].  half  of  both   public  and  private  school  teachers   knew  the  importance  of  tetanus   vaccine  control  in  dental  trauma.       conclusion   the  level  of  knowledge  of   dental  trauma  management  both   public  and  private  school  teachers   from  istanbul,  turkey,  as  found  in   this  study  was  unsatisfying.  it  ap-­‐ pears  that  additional  education   programs  are  critical  to  improve   teachers’  awareness  of  the  im-­‐ portance  of  dental  trauma  man-­‐ agement  in  both  types  of  schools.   first  aid  training  should  involve   ‘dental  trauma  management’   courses  given  in  elementary   schools.     references   1. andreasen  jo,  andreasen   fm,  andersson  l.  (2007)   textbook  and  color  atlas   of  traumatic  injuries  to   the  teeth,  4th  edition.  co-­‐ penhagen:  munksgaard.   217–54  p,  444–88  p,  516– 41  p.   2. traumatic  dental  injuries   in  children  presenting  for   treatment  at  the  depart-­‐ ment  of  pediatric  den-­‐ tistry,  faculty  of  den-­‐ tistry,  university  of  jor-­‐ dan,  1997–2000.  rajab   ld.  dent  traumatol  2003;   feb;19(1):6-­‐11.  pmid:   12656848   3. a  retrospective  study  of   dento-­‐alveolar  injuries  of   children  in  ankara,  tur-­‐ key.  altay  n,  güngör  hc.   dent  traumatol.  2001;   oct;17(5):  201-­‐4.  pmid:   11678537   4. traumatic  injuries  to   permanent  teeth  in  turk-­‐ ish  children,  ankara.  dent   traumatol.  altun  c,  ozen   b,  esenlik  e,  guven  g,   gürbüz  t,  acikel  c,  basak   f,  akbulut  e.  2009;  jun;25   (3):  309-­‐13.  pmid:   19583580   5. prevalence  of  traumatic   injuries  to  the  permanent   incisors  in  candidates  for   orthodontic  treatment.   dent  traumatol.  bauss  o,   röhling  j,  schwestka-­‐ polly  r.  2004;apr  20(2):   61-­‐6.  pmid:15025687   6. prevalence  and  handed-­‐ ness  correlates  of  trau-­‐ matic  injuries  to  the  per-­‐ manent  incisors  in  13-­‐17-­‐ year-­‐old  adolescents  in   erzurum,  turkey.  canakci   v,  akgül  hm,  akgül  n,   canakci  cf.  dent  trau-­‐ matol.  2003;  oct  19(5):   248-­‐54.  pmid:14708648   7. factors  associated  with   traumatic  dental  injuries   among  12-­‐year-­‐old   schoolchildren  in  south   india.  david  j,  astrøm  an,   wang  nj.  dent  traumatol.   2009;  oct  25(5):  500-­‐5.   pmid:19614932   8. fractured  permanent  in-­‐ cisors  among  nigerian   school  children.  falomo  b.   asdc  j  dent  child.  1986;   mar-­‐apr  53(2):  119-­‐21.   pmid:3457032   9. traumatic  dental  injuries   in  schoolchildren  from   santo  domingo.  garcia-­‐ godoy  f,  morbán-­‐laucer   f,  corominas  lr,  franjul   ra,  noyola  m.  community   dent  oral  epidemiol.   1985;  jun  13(3):  177-­‐9.   pmid:3860343   10. dental  trauma  in  children   and  young  adults  visiting   a  university  dental  clinic.   ivancic  jokic  n,  bakarcic   d,  fugosic  v,  majstorovic   m,  skrinjaric  i.  dent   traumatol.  2009;  feb   25(1):  84-­‐7.   pmid:19208016   11. epidemiologic  survey  of   traumatic  dental  injuries   in  children  seen  at  the   federal  university  of  rio   de  janeiro,  brazil.  jesus   ma,  antunes  la,  risso   pde  a,  freire  mv,  maia  lc.   braz  oral  res.  2010;jan-­‐ mar  24  (1):  89-­‐94.   pmid:20339720   12. dental  trauma  in  turkish   children,  istanbul.  kargul   b,  cağlar  e,  tanboga  i.   dent  traumatol.  2003;   apr  19(2):  72-­‐5.   pmid:12656836   13. prevalence  of  traumatic   injuries  to  maxillary  per-­‐ manent  teeth  in  9-­‐  to  14-­‐ year-­‐old  school  children   in  yazd,  iran.  navabazam   a,  farahani  ss.  dent   traumatol.  2010;apr   26(2):  154-­‐7.   pmid:20089070   assessment  of  knowledge  among  public  and  private  elementary  school  teachers  in  dental  trauma  management   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.60    http://dentistry3000.pitt.edu   14. traumatic  injuries  to  the   anterior  teeth  among   south  kanara  school  chil-­‐ dren-­‐-­‐a  prevalence  study.   rai  sb,  munshi  ak.  j  indi-­‐ an  soc  pedod  prev  dent.   1998;  jun  16(2):  44-­‐51.   pmid:11813754   15. prevalence  and  risk  fac-­‐ tors  related  to  traumatic   dental  injuries  in  brazili-­‐ an  schoolchildren.  so-­‐ riano  ep,  caldas  ade  f  jr,   diniz  de  carvalho  mv,   amorim  filho  hde  a.  dent   traumatol.  2007;aug  23   (4):  232-­‐40.   pmid:17635357   16. dental  trauma  in  school-­‐ children  six  to  twelve   years  of  age.  zaragoza  aa,   catalá  m,  colmena  ml,   valdemoro  c.  asdc  j  dent   child.  1998;  nov-­‐dec  65   (6):  492-­‐4,  439.   pmid:9883326   17. the  caries  decline:  a  re-­‐ view  of  reviews.  peters-­‐ son  gh,  bratthall  d.  eur  j   oral  sci.  1996;  aug  104   (4):  436-­‐43.   pmid:8930595   18. andreasen  jo,  andreasen   fm,  andersson  l.  (2007)   socio-­‐psychological  as-­‐ pects  of  traumatic  dental   injuries.  in:  textbook  and   color  atlas  of  traumatic   injuries  to  the  teeth.  4th   edition.  copenhagen:   munksgaard  pp  201.   19. guidelines  for  the  man-­‐ agement  of  traumatic   dental  injuries.  ii.  avul-­‐ sion  of  permanent  teeth.   flores  mt,  andersson  l,   andreasen  jo,  bakland   lk,  malmgren  b,  barnett   f,  bourguignon  c,  diange-­‐ lis  a,  hicks  l,  sigurdsson   a,  trope  m,  tsukiboshi  m,   von  arx  t.  dent  trau-­‐ matol  2007;  jun  23(3):   130–6.  pmid:17511833   20. tooth  avulsion  and  re-­‐ plantation  –  a  review.   hammarström  l,  pierce  a,   blomlöf  l,  feiglin  b,  lind-­‐ skog  s.  endod  dent   traumatol  1986;  feb   2(1):  1–8.  pmid:3516666   21. treatment  of  the  avulsed   tooth.  trope  m.  pediatr   dent  2000;  mar-­‐apr   22(2):  145–7.   pmid:10769860   22. oral  trauma  in  children:  a   hospital  survey.  o’neil   dw,  clark  mv,  lowe  jw,   harrington  ms.  oral  surg   oral  med  oral  pathol   1989;  dec  68  (6):  691–6.   pmid:2574434   23. the  prevalence  of  trau-­‐ matic  injuries  treated  in   the  pedodontic  clinic  of   ankara  university,  tur-­‐ key,  during  18  months.   saroğlu  i,  sönmez  h.  dent   traumatol  2002;  dec  18   (6):  299–303.   pmid:12656862   24. knowledge  and  attitude   of  mothers  with  regards   to  emergency  manage-­‐ ment  of  dental  avulsion.   oliveira  tm,  sakai  vt,   moretti  abs,  thiago  cs,   santos  cf,  machado  ma.  j   dent  child  2007  sep-­‐dec;   74  (3):  200-­‐2.   pmid:18482514   25. knowledge  of  elementary   school  teachers  in  tel-­‐ aviv,  isreal,  regarding   emergency  care  of  dental   injuries.  fux-­‐noy  a,  sar-­‐ nat  h,  amir  e.  dent   traumatol.  2011;  aug   27(4):  252-­‐6.   pmid:21535404   26. lay  knowledge  of  physi-­‐ cal  education  teachers   about  the  emergency   management  of  dental   trauma  in  hong  kong.   chan  awk,  wong  tks,   cheung  gsp.  dent  trau-­‐ matol  2001;  apr   17(2):77–85.   pmid:11475950   27. investigation  of  lay   knowledge  of  the  man-­‐ agement  of  avulsed  per-­‐ manent  incisors.  hamil-­‐ ton  fa,  hill  fj,  mackie  ic.   endod  dent  traumatol   1997;feb.13(1):19-­‐23.   pmid:9206384   28. evaluation  of  the   knowledge  of  the  treat-­‐ ment  of  avulsion  in  ele-­‐ mentary  school  teachers   in  rio  de  janeiro,  brazil.   pacheco  lf,  filho  pfg,   letra  a,  menezes  r,  vil-­‐ loria  gem,  ferreira  sm.   dent  traumatol  2003;apr   19(2):  76–8.   pmid:12656837   29. avulsed  permanent  inci-­‐ sors:  knowledge  and  atti-­‐ tudes  of  primary  school   teachers  with  regard  to   emergency  management.   blakytny  c,  surbuts  c,   thomas  a,  hunter  ml.  int   j  paediatr  dent  2001;  sep   11(5):  327–32.   pmid:11572262   assessment  of  knowledge  among  public  and  private  elementary  school  teachers  in  dental  trauma  management   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.60    http://dentistry3000.pitt.edu   30. tooth  reimplantation  af-­‐ ter  traumatic  avulsion:  a   report  of  10  cases.  ki-­‐ noshita  s,  kojima  r,   taguchi  y,  noda  t.  dent   traumatol  2002;  jun   18(3):  153-­‐6.   pmid:12154771   31. elementary  school  staff   knowledge  about  dental   injury.  mcintyre  jd,  lee   jy,  trope  m,  vann  wf.   dent  traumatol  2008;  jun   24(3):  189–98.   pmid:18410391   32. american  association  of   endodontists.  treatment   of  the  avulsed  permanent   tooth.  recommended   guidelines  of  the  ameri-­‐ can  association  of  endo-­‐ dontists.  dent  clin  north   am  1995;  jan  39(1):  221– 5.  pmid:7890106   33. effects  of  treatment  delay   upon  pulp  and  periodon-­‐ tal  healing  of  traumatic   dental  injuries-­‐  a  review   article.  andreasen  jo,  an-­‐ dreasen  fm,  skeie  a,  hjör-­‐ ting-­‐hansen  e,  schwartz   o.  dent  traumatol  2002;   jun  18(3):  116–28.   pmid:12110104   34. replantation  of  400   avulsed  permanent  inci-­‐ sors.  part  1.  diagnosis  of   healing  complications.   andreasen  jo,  borum  mk,   jacobsen  hl,  andreasen   fm.  endod  dent  trau-­‐ matol  1995;apr  11  (2):   51–8.  pmid:7641619   35. avulsed  permanent  teeth:   a  review  of  literature  and   treatment  guidelines.   barrett  ej,  kenny  dj.  en-­‐ dod  dent  traumatol   1997;aug  13(4):  153–63.   pmid:9550040   36. dental  trauma  manage-­‐ ment  awareness  of  singa-­‐ pore  pre-­‐school  teachers.   sae-­‐lim  v,  lim  lp.  dent   traumatol  2001;apr   17(2):  71–6.   pmid:11475949   37. storage  media  for  avulsed   teeth:  a  literature  review.   poi  wr,  sonoda   ck,  martins  cm,  melo   me,  pellizzer  ep,  de  men-­‐ donça  mr,  panzarini  sr.   braz  dent  j  2013;sep-­‐oct   24(5):437-­‐45.   pmid:24474282   38. guideline  on  management   of  acute  dental  trauma.   pediatr  dent.  2008-­‐ 2009;30(7):175-­‐83   pmid:19216418       39. tetanus  prophylaxis  in  in-­‐ juries.  guidelines  for  the   dental  practice.  becking   ag.  ned  tijdschr   tandheelkd  1995;jul  102   (7):  266–8.   pmid:11837111     microsoft word 82 2018.docx   vol  6,  no  1  (2018)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2018.82         http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     maximum  mouth  opening  in  healthy  children  and  adolescents  in             istanbul   mine  koruyucu1,  derya  tabakcilar2,  figen  seymen1,  koray  gencay1     1istanbul  university,  faculty  of  den6stry,  department  of  pedodon(cs,  istanbul,  turkey   2yeditepe  university,  faculty  of  den7stry,  department  of  pedodon(cs,  istanbul,  turkey     abstract   objec&ves:  mouth  opening  capacity  is  o0en  regarded  as  one  of  the  important  parameters  for  evalua7ng  the  func7on   of  the  temporomandibular  joint  (tmj)  and  mas,catory  muscle  status.  a  reduced  mouth  opening  capacity  may  be  one   of  the  first  clinical  signs  of  tmj  involvement.  the  purpose  of  this  study  was  to  create  age  related  percen;les  for  the   maximal  interincisal  distance  (mid)  of  healthy  children.  methods:  the  pa'ents  admi.ed  for  rou'ne  dental  examina-­‐ !ons  to  istanbul  university  faculty  of  den!stry,  department  of  pedodon!cs  were  included  in  this  study.  the  interin-­‐ cisal   measurements   were   performed   with   metallic   calliper   and   also   malocclusions  were   recorded   for   all   children.   oneway  anova  test,  tukey  hds  test,  tamhane’s  t2  test  and  student  t  test  were  used  for  sta;s;cal  analysis.  results:   the  study  popula.on  comprised  of  1059  (569  males,  490  females),  3-­‐to  15-­‐year-­‐old  (mean  age  8.82±3.06)  children.   the  mean  score  of  maximal  inter-­‐incisal  distance  was  found  33.24±5.54  for  females;  33.32±5.71  for  males.  there  was   no  sta's'cally  significant  difference  according  to  gender  (p=0.815;  p>0.05).  the  mean  score  of  maximal   inter-­‐incisal   distance  was  found  28.63±4.34  for  3-­‐5  years;  33.52±4.84  for  6-­‐11  years;  37.35±5.52  for  12-­‐15  years  children.  sta2s2-­‐ cally   significant   differences  were   found   between   age   groups   (p:   0.001;   p<0.01).  the  mean   score   of  maximal   inter-­‐ incisal  distance  was  found  32.9±5.6  for  class  i;  34.92±5.51  for  class  ii;  35.2±5.36  for  class  iii  malocclusions.  sta:s:cally   significant   differences   were   found   between   malocclusion   groups   (p:0.001;   p<0.01).   conclusion:   the   result   of   this   study  indicated  that  posi/ve  rela/onship  between  the  maximum  mouth  opening  and  age  and  malocclusion.     cita%on:  koruyucu  m,  et  al.  (2018)  maximum   mouth  opening  in  healthy  children  and  adoles-­‐ cents  in  istanbul.  den$stry  3000.  1:a001   doi:10.5195/d3000.2018.82   received:    december  11,  2017   accepted:    february  21,  2018   published:    july  13,  2018   copyright:  ©2018  koruyucu  m,  et  al.  this   is   an   open   access   ar!cle   licensed   under   a   crea!ve   commons   a"ribu%on   work   4.0   united   states   license.   email:  mine.yildirim@istanbul.edu.tr         introduction   palpation  of  muscle  and   joint,  occlusal  and  radiographic   examination  are  performed  for   assessment  of  mandibular  func-­‐ tion.  to  be  able  to  assess  tem-­‐ poromandibular  joint  (tmj)  func-­‐ tion,  the  primary  value  to  be   known  is  how  much  joints  move   when  mouth  is  opened  fully  max-­‐ imum  mouth  opening  (mmo)   [1,2].    mmo  is  “  the  greatest  dis-­‐ tance  between  two  central  inci-­‐ sors  (maxillary  and  mandibular)  at   the  midline  when  measured  from   their  incisal  edges  during  the  pos-­‐ sible  wildest  opening  of  the   mouth”  according  to  many  re-­‐ searchers  [3,4].   mouth  opening  limitation   may  be  associated  with  some  clin-­‐ ical  situations  like  temporoman-­‐ dibular  disorders,  odontogenic  in-­‐ fections,  oral  malignancies,  sub-­‐ mucous  fibrosis,  mandibular  frac-­‐ tures,  myopathies,  and  trauma  [5].     sex,  age  and  height  have   an  impact  on  how  much  a  person   can  open  their  mouth.  as  an  im-­‐ portant  step,  before  diagnosing   that  a  person  is  suffering  from  lim-­‐ ited  mouth  opening,  it  is  necessary   to  acknowledge  normal  opening  of   the  population  [6].  some  studies   researched  children  and  adoles-­‐ cents  mmo  values  among  differ-­‐ ent  populations  (table  1)  [6–15].   also,  most  of  these  studies  re-­‐ vealed  as  age  increases,  mmo  in-­‐ creases  as  well.  moreover,  girls   have  a  decreased  mmo  compared   to  boys.  for  this  reason,  it  is  im-­‐ portant  to  define  normal  mmo   values  for  each  specific  popula-­‐ tion,  so  that  it  is  possible  to  diag-­‐ nose  whether  a  person  suffers   from  reduced  mouth  opening.   the  aim  of  this  study  was   to  evaluate  age  related  percentiles   for  the  maximal  inter-­‐incisal  dis-­‐ tance  of  healthy  children,  based   on  sex  and  malocclusion.         maximum  mouth  opening  in  healthy  children  and  adolescents  in  istanbul       vol  6,  no  1  (2018)        doi  10.5195/d3000.2018.82    http://dentistry3000.pitt.edu   material  and  methods   the  study  was  approved  by   the  ethics  committee  of  the  istan-­‐ bul  university,  medical  faculty       (no:2013/105)  and  was  carried   out  in  agreement  with  the  decla-­‐ ration  of  helsinki  principles.  the   study  consisted  of  1059  turkish   children  who  were  attending  to   istanbul  university  faculty  of  den-­‐ tistry,  clinics  of  pedodontics,  for   routine  dental  examinations  in   2013.  569  boys  and  490  girls  be-­‐ tween  the  ages  of  between  3-­‐15   years  were  included.   medical  and  dental  anam-­‐ nesis  was  taken  and  a  question-­‐ naire  filled  for  each  patient.  the   children  were  examined  by  one   experienced  pediatric  dentist   (mk).  they  were  positioned  in  a   way  that  they  would  stay  standing   up  and  were  soothed  to  be  com-­‐ fortable  by  the  dentist  by  support-­‐ ing  their  head.  the  mmo  meas-­‐ urement  was  recorded  by  measur-­‐ ing  the  maximum  distance  be-­‐ tween  the  incisal  edges  of  the   maxillary  central  incisor  and  man-­‐ dibular  central  incisor  at  the  mid-­‐ line  while  the  mouth  of  the  sub-­‐ ject  was  open  at  its  widest.    the   interincisal  measurements  were   performed  with  metallic  calliper   (seitz  &  haag  munchner  modell).   inclusion  criteria   this  study  includes  the  fol-­‐ lowing  criteria:  healthy  and  im-­‐ mobile  primary  maxillary  and   mandibular  central  incisors,  no   dental  trauma  history,  no  anterior   open-­‐bite,  no  caries  and  no  re-­‐ storative  materials  that  influenced   the  incisal  edges,  presence  of  fully   erupted  maxillary  and  mandibular   central  incisors  and  no  orthodon-­‐ tic  treatment  that  could  influence   the  position  of  the  central  incisors.   exclusion  criteria   the  exclusion  criteria  were   subjects  with  temporomandibular   disorders,  neurological  disorders,   craniofacial  deformities,  systemic   diseases  (juvenile  rheumatoid  ar-­‐ thritis),  congenital  abnormalities   and  neck  pain,  because  these   problems  might  cause  limited   mouth  opening  as  in  the  previous   cases  reported.     statistical  analysis   all  statistical  analyses  were   performed  using  the  ibm  spss  sta   table  1.  children  and  adolescent  mmo  values  from  different  studies.   studies   country   age  group   sample  size   mmo  (mm)   rothenberg  [7]   usa   4-­‐14   189   43.99   sousa  et  al.  [8]   brazil   6-­‐14   303   43.79   müller  et  al.  [9]   switzerland   4-­‐17   20719   45   kumar  et  al.  [6]   india   6-­‐8   8-­‐10   10-­‐12     856   45.95  (girls),  46.04  (boys)   47.27  (girls),  48.53  (boys)   52.05  (girls),  52.38  (boys)   abou  atme  et  al.  [10]   lebanon   4-­‐15   102   45.8   feteih  [11]   saudi  arabia   12-­‐16   385   46.5  (girls),  50.2  (boys)   chen  et  al  [12]   taiwan   3-­‐5   518   36.93  (girls),  37.47  (boys)     benevides  et  al  [13]   brasil   8-­‐12   181   49.06  (girls),  49.59  (boys)     al-­‐dlaigan  &  asiry  [14]   saudi  arabia   12-­‐16   1825   35.5  (girls),  43.5  (boys)     choi  [15]   korea   2-­‐6   151   37.72  ±  5.10   maximum  mouth  opening  in  healthy  children  and  adolescents  in  istanbul       vol  6,  no  1  (2018)        doi  10.5195/d3000.2018.82    http://dentistry3000.pitt.edu   tistics  22  (ibm  spss,  tur-­‐ key).  the  assumption  of  normal   distribution  was  confirmed  using   the  shapiro  wilk  test  and  mmo   was  found  appropriate  to  normal   distribution.  one-­‐way  anova  fol-­‐ lowed  by  the  post  tukey  hds  and   tamhane’s  t2  tests  were  used  to   examine  differences  in  mmo  rela-­‐ tive  to  sex  and  age  groups.  the   statistical  analyses  were  per-­‐ formed  using  the  student’s  t-­‐test   for  independent  samples  of  males   and  females.  p  <  0.05  values  were   considered  statistically  significant.   results   descriptive  statistics  of  the   subjects  are  shown  in  table  2.  the   study  population  comprised  of   1059  (569  males,  490  females),  3-­‐ to  15-­‐year-­‐old  (mean  age   8.82±3.06)  children.  the  mmo  in   relation  to  gender  and  age  is   shown  in  table  2.  the  maximal   inter-­‐incisal  distance  was  observed   with  the  mean  score  of   33.24±5.54  for  the  female  group   and  33.32±5.71  for  the  male   group.  statistically  significant  dif-­‐ ference  was  not  found  according   to  sex  (p=0.815;  p>0.05).  there   were  significant  rises  in  mmo  with   increasing  age,  regardless  of  sex.   the  highest  mean  mmo  according   to  age  was  in  those  aged  12-­‐15   years.  the  mean  score  of  maximal   inter-­‐incisal  distance  was  found   28.63±4.34  for  3-­‐5  years;   33.52±4.84  for  6-­‐11  years;   37.35±5.52  for  12-­‐15  years  chil-­‐ dren  (table  2,  figure  1,  figure  2).   statistically  significant  differences   were  found  between  age  groups   (p:  0.001;  p<0.01)  (table  2).   the  maximal  inter-­‐incisal   distance  was  observed  with  the   mean  score  of  32.9±5.6  for  class  i;   34.92±5.51  for  class  ii;  35.2±5.36   for  class  iii  malocclusions  (figure   3).  statistically  significant  differ-­‐ ences  were  found  between  mal-­‐ occlusion  groups  (p:  0.001;   p<0.01)  (table  2).   discussion   mmo  has  been  described   as  the  inter-­‐  incisal  distance  or  as   overbite  added  inter-­‐incisal  dis-­‐ tance  [3,16–18].  to  measure   overbite  added  inter-­‐incisal  dis-­‐ tance,  the  distance  that  mandible   travels  vertically  should  be  meas-­‐ ured,  but,  as  pointed  out  by   mezitis  et  al.  [16],  the  functional   opening  of  the  mouth  is  more  im-­‐ portant,  because  this  is  the  value   that  actually  affects  chewing  and   dental  treatment.  therefore,  the   mmo  in  this  study  was  defined  as   table  2.  distribu(on  of  maximal  mouth  opening  according  to  gender,  age,  and  mal-­‐ occlusion.         maximal  mouth  opening  (mm)   p   n  (%)   mean  ±  sd   1gender         girls   490  (%46,3)   33,24  ±  5,54   0,815   boys   569  (%53,7)   33,32  ±  5,71   2age         3-­‐5  year   220  (%20,8)   28,63  ±  4,34   0,001**   6-­‐11  year   625  (%59,0)   33,52  ±  4,84   12-­‐15  year   214  (%20,2)   37,35  ±  5,52   2malocclusion         class  i   872  (%82,3)   32,9  ±  5,6   0,001**   class  ii   98  (%9,3)   34,92  ±  5,51   class  iii   89  (%8,4)   35,2  ±  5,36   1student  t  test       2oneway  anova                                          **p<0.01   maximum  mouth  opening  in  healthy  children  and  adolescents  in  istanbul       vol  6,  no  1  (2018)        doi  10.5195/d3000.2018.82    http://dentistry3000.pitt.edu   the  maximal  inter-­‐incisal  distance   (mid)  [5].   there  are  various  methods   to  measure  mmo  like  directly  by   using  a  calibrated  fiber  ruler   [9,14,19–21],  simple  ruler   [7,22,23],  scale  and  divider  [20],   vernier  caliper  or  wiley’s  bite   gauge  [12,13,24–26],  boley  gauge   [27,28],  calibrated  boley  gauge  [8],   modified  vernier  calliper  [6],  sub-­‐ ject’s  finger  [4,10],  optoelectric   jaw-­‐  tracking  system  [29,30],  jaw   motion  analyzer  system  [31]  and   therabite  range  of  motion  scales   [4,32].  wood  and  branco  [33]   compared  different  measurement   types  and  suggested  that  direct   measurements  using  a  ruler  or  cal-­‐ liper  were  more  accurate  and  pre-­‐ cise.  the  present  study  was  done   using  metallic  calliper.   the  most  important  factor   in  measuring  mmo  is  the  head   position  [30,34].  higbie  et  al.  [34]   described  short-­‐term  alterations  in   head  position  have  a  significant   effect  on  the   amount  of  mmo  in   a  normal  popula-­‐ tion.  in  this  study,   all  subjects  were   placed  in  a  vertical   position  for  measur-­‐ ing  in  order  to  elim-­‐ inate  the  possible   influence  of  differ-­‐ ent  head  positions.   a  wide  range   of  mmo  has  been   reported  from  all   over  the  world  in   different  studies.   studies  of  mmo   values  of  children  and  adolescents   from  different  countries  with  dif-­‐ ferent  age  range  are  described  in   table  1.  we  could  not  compare   our  study  with  them  about  racial   differences  because  of  the  wide   age  range.  longitudinal  studies  are   required  to  compare  racial  differ-­‐ ences  with  same  age.  the  only   study  done  on  turkish  children  in   this  regard  [35];  tmj  movements   were  examined  with  the  tem-­‐ poromandibular  opening  index   (toi)  and  its  own  formula  in  all   dentition  types   with  and  with-­‐ out  temporo-­‐ mandibular   joint  dysfunc-­‐ tion  syndrome.   toi  showed   that  there   were  no  signifi-­‐ cant  differ-­‐ ences  between   different  denti-­‐ tions  or  genders.   children  and  adolescents   do  not  grow  constantly  and  stably,   their  growth  show  different  phas-­‐ es  from  birth  to  adulthood.  their   body  parts  do  not  develop  at  the   same  speed.    however,  some  stud-­‐ ies  have  shown  that  mmo  con-­‐ stantly  rises  after  birth  until  adult-­‐ hood,  and  then  gradually  decreas-­‐ es  during  aging.  our  research  has   shown  that  mid  has  increased   with  age  among  the  subjects.  the-­‐ se  measurements  are  similar  to   reported  by  many  researchers  that   aged  among  3  to  15  [6– 10,14,15,27,31,32].  these  results   were  divergent  with  rothenberg     [7],  sousa  [8]  and  ingervall  [36].   age  may  be  an  important  predic-­‐ tor  of  mmo  measurements,  but   the  relationship  between  age  and   mmo  has  not  yet  been  estab-­‐ lished.  in  our  study,  mean  value  of   mmo  was  28,63  ±  4,34  mm  in   primary  dentition.  these  meas-­‐ urements  are  lower  from  to  the   ones  reported  by  choi  et  al    [15].   chen  et  al  [12]  and  ying  et  al  [32]   among  two  to  six  year-­‐old  chil-­‐ dren.  the  mean  score  of  mmo   was  found  33.52±4.84  for  6-­‐11   years;  37.35±5.52  for  12-­‐15  years   figure  1.  scatterplot  of  the  correlation  between  inter-­‐ incisal  distance  and  age  of  all  children  (girls  and  boys).   figure  2.  mean  maximum  opening(mm)  by  age  groups.   maximum  mouth  opening  in  healthy  children  and  adolescents  in  istanbul       vol  6,  no  1  (2018)        doi  10.5195/d3000.2018.82    http://dentistry3000.pitt.edu   children.  these  measurements  are   lower  from  to  the  many  studies   [6–11,13–15,28].   there  was  no  statistically   significant  difference  of  mmo  ac-­‐ cording  to  gender  in  this  study.   these  results  were  compatible   with  many  studies  [6–10,32,37].   only  few  researches  report  sex   difference  in  mmo  [7,24].  there-­‐ fore,  it  is  suggested  that  the  gen-­‐ der  effect  on  mmo  in  adults  is  not   observed  in  children  and  adoles-­‐ cents  because  of  they  have  in-­‐ complete  sexual  maturity.  howev-­‐ er,  ingervall  [36]  observed  mmo   values  of  10-­‐year  old  females  were   similar  to  the  adult  females;  but  it   was  not  similar  to  male  adults.   in  our  study,  the  mean   score  of  maximal  inter-­‐incisal  dis-­‐ tance  was  found  32.9±5.6  for  class   i;  34.92±5.51  for  class  ii;  35.2±5.36   for  class  iii  malocclusions.  maloc-­‐ clusion  groups  showed  statistically   significant  differences.  we  hy-­‐ pothesized  like  ying  [32],  that  a   class  ii  molar  relationship,  due  to   a  retrognathic  mandible,  would   demonstrate  a  smaller  mmo.  but   the  largest  mmo  value  was  ob-­‐ served  in  class  iii   malocclusion.  we   thought  that  rea-­‐ son  of  this  situa-­‐ tion  was  our  study   contained  chil-­‐ dren  adolescents   who  have  not  yet   completed   growth.  but  ying   et  al  [32]  found   occlusion  type   were  statistically   insignificant  in   affecting  mmo.  they  found  larg-­‐ est  mmo  value  at  class  ii  in  left   molar  classification.   in  this  study,  we  estab-­‐ lished  basic  standard  values  of   mmo  in  turkish  children  and  ado-­‐ lescents  for  detect  the  mandibular   and  tmj  functions.  in  general,   mmo  was  lower  than  that  report-­‐ ed  in  similar  studies  that  conduct-­‐ ed  in  many  countries  all  over  the   world.  however,  additional  studies   are  needed  to  establish  certain   average  values  of  mmo  in  turkish   population  for  all  ages.  the  result   of  this  study  reported  the  positive   relationship  between  the  maxi-­‐ mum  mouth  opening  and  age  and   malocclusion.   references   1.  on  the  development,  morphol-­‐ ogy  and  function  of  the  temporo-­‐ mandibular  joint  in  the  light  of  the   orofacial  system.  fanghanel  j,   gedrange  t.  annals  of  anatomy.   2007;189:314–9.  pmid:17695983   2.  the  face-­‐  physiognomic  expres-­‐ siveness  and  human  identity.   fanghanel  j,  gedrange  t,  proff  p.   annals  of  anatomy.   2006;188:261–6.  pmid:16711165   3.  normal  mouth  opening  in  the   adult  french  population.  placko  g,   bellot-­‐samson  v,  brunet  s.  revue   de  stomatologie  et  de  chirurgie   maxillo-­‐faciale.  2005;106:267–71.   pmid:16292219   4.  an  index  for  the  measurement   of  normal  maximum  mouth  open-­‐ ing.  zawawi  kh,  al-­‐badawi  ea,  lo-­‐ bo  sl,  melis  m,  mehta  nr.  journal   canadian  dental  association.   2003;69:737–41.  pmid:14653940   5.  research  diagnostic  criteria  for   temporomandibular  disorders:   review,  criteria,  examinations  and   specifications,  critique.  dworkin  s,   leresche  l.  craniomandibular   disorders.  1992;6:300–55.   pmid:1298767   6.  maximal  mouth  opening  in  indi-­‐ an  children  using  a  new  method.   kumar  a,  mehta  r,  goel  m,  dutta   s,  hooda  a.  journal  of  cranio-­‐ maxillary  diseases.  2012;1:79–86.     7.  an  analysis  of  maximum  man-­‐ dibular  movements,  craniofacal   relationships  and  temporoman-­‐ dibular  joint  awareness  in  chil-­‐ dren.  rothenberg  lh.  the  angle   orthodontist.  1991;61:103–12.     pmid:2064068   8.  evaluation  of  mandibular  range   of  motion  in  brazilian  children  and   its  correlation  to  age,  height,   weight,  and  gender.  sousa  lm  de,   nagamine  hm,  chaves  tc,  grossi   db,  regalo  sch,  oliveira  as  de.   brazilian  oral  research.   2008;22:61–6.  pmid:18425247   figure  3.    mean  maximum  opening  (mm)  in  different  occlusion   types.   maximum  mouth  opening  in  healthy  children  and  adolescents  in  istanbul       vol  6,  no  1  (2018)        doi  10.5195/d3000.2018.82    http://dentistry3000.pitt.edu   9.  maximal  mouth  opening  capaci-­‐ ty :  percentiles  for  healthy  chil-­‐ dren  4  –  17  years  of  age.  müller  l,   waes  h  van,  langerweger  c,  mo-­‐ linari  l,  saurenmann  rk.  paedita-­‐ tric  rheumatology.  2013;22:  11-­‐ 17.  pmid:23607797     10.  clinical  measurement  of  nor-­‐ mal  maximum  mouth  opening  in   children.  abou-­‐atme  ys,  chedid  n,   melis  m,  zawawi  kh.  journal  of   craniomandibular  practice.   2008;26:191–6.  pmid:18686495   11.  signs  and  symptoms  of  tem-­‐ poromandibular  disorders  and  oral   parafunctions  in  urban  saudi  ara-­‐ bian  adolescents:  a  research  re-­‐ port.  feteih  rm.  head  &  face  med-­‐ icine.  2006;2:25.  pmid:16914032   12.  analysis  of  maximum  mouth   opening  and  its  related  factors  in   3-­‐  to  5-­‐year-­‐old  taiwanese  chil-­‐ dren.  chen  h  sen,  yang  pl,  lee  cy,   chen  kk,  lee  kt.  odontology.   2013;1–5.  pmid:24174163     13.  determining  the  mandibular   range  of  motions  in  children  from   bahia  state.  benevides  sd,  araujo   rp,  ribeiro  c  de  o,  mello  smf.  re-­‐ vista  cefac.  2016;18:95–103.     14.  maximum  mouth  opening  in   saudi  adolescents.  al-­‐dlaigan  yh,   asiry  m.  journal  of  international   oral  health.  2014;6:45–9.   pmid:25628483   15.  measurement  of  maximum   mouth  opening  in  2  to  6  year-­‐old   korean  children.  choi  h,  kim  c,   lee  d,  yang  y,  kim  j.  journal  of   the  korean  academy  of  pediatric   dentistry.  2015;42:242–3.     16.  the  normal  range  of  mouth   opening.  mezitis  m,  rallis  g,  zach-­‐ ariades  n.  journal  of  oral  maxillo-­‐ facial  surgery.  1989;47:1028−9.   pmid:2795295   17.  maximal  incisal  opening:  a  di-­‐ agnostic  index?  sheppard  i,  shep-­‐ pard  s.  the  journal  of  dental  med-­‐ icine.  1965;20:13–5.   pmid:14232340   18.  signs  of  temporomandibular   disorders  in  girls  receiving  ortho-­‐ dontic  treatment  .  a  prospective   and  longitudinal  comparison  with   untreated  class  ii  malocclusions   and  normal  occlusion  subjects.   henrikson  t,  nilner  m,  kurol  j.  eu-­‐ ropean  journal  of  orthodontics.   2000;22:271–81.  pmid:10920559   19.  normal  mouth  opening  in  an   adult  indian  population.  khare  n,   patil  s,  kale  s,  sumeet  j,  sonali  i,   sumeet  b.  journal  maxillofacial   oral  surgery.  2012;11:309–13.   pmid:23997482   20.  normal  range  of  maximal   mouth  opening  amongst  north   indian  population :  a  cross  sec-­‐ tional  study  in  meerut.  gupta  s,   sreenisavan  v,  goel  s.  indian   journal  of  research.  2016;5:534– 6.     21.  clinical  characterization  of   mouth  opening  among  mexican   adolescents  and  young  adults.   casanova-­‐rosado  j,  medina-­‐solis   c,  casanova-­‐rosado  a,  vallejos-­‐ sanchez  a,  patino-­‐marin  n,  mau-­‐ pome  g,  et  al.  journal  of  dental   sciences.  2012;7:81–4.     22.  discriminant  validity  of  tem-­‐ poromandibular  joint  range  of  mo-­‐ tion  measurements  obtained  with   a  ruler.  walker  n,  bohannon  rw,   cameron  d.  the  journal  of  ortho-­‐ paedic  and  sports  physical  thera-­‐ py.  2000;30:484–92.   pmid:10949505     23.  the  range  of  inter-­‐incisal  open-­‐ inig  among  university  students  of   ajman,  uae.  sohail  a,  amjad  a.   pakistan  oral  &  dental  journal.   2011;31:37–42.     24.  the  normal  range  of  mouth   opening  in  an  irish  population.   gallagher  c,  gallagher  v,  whelton   h,  cronin  m.  journal  of  oral  reha-­‐ bilitation.  2004;31:110–6.   pmid:15009593     25.  maximum  mouth  opening.   sawair  fa,  hassoneh  ym,  al-­‐ zawawi  bm,  baqain  zh.  saudi   medical  journal.  2010;962:369– 73.  pmid:20383412   26.  signs  and  symptoms  of  tem-­‐ poromandibular  joint  disorders   related  to  the  degree  of  mouth   opening  and  hearing  loss.  kitsoulis   p,  marini  a,  iliou  k,  galani  v,  zim-­‐ pis  a,  kanavaros  p,  et  al.  bmc  ear,   nose  and  throat  disorders.   2011;11:1–8.  pmid:21612586   27.  maximum  mouth  opening  of   ethnic  chinese  in  taiwan.  yao  kt,   lin  cc,  hung  ch.  journal  of  dental   sciences.  association  for  dental   sciences  of  the  republic  of  china;   2009;4:40–4.     28.  the  normal  range  of  maximum   mouth  opening  and  its  correlation   with  height  or  weight  in  the  young   maximum  mouth  opening  in  healthy  children  and  adolescents  in  istanbul       vol  6,  no  1  (2018)        doi  10.5195/d3000.2018.82    http://dentistry3000.pitt.edu   adult  chinese  population.  li  x,  jia   c,  zhang  z.  journal  of  dental  sci-­‐ ences;  2017;12:56–9.     29.  sex  differences  in  mandibular   movements  during  opening  and   closing.  lewis  rp,  buschang  ph,   throckmorton  gs.  american  jour-­‐ nal  of  orthodontics  &  dentofacial   orthopedics.  2001;120:294–303.   pmid:11552129   30.  concomitant  mandibular  and   head-­‐neck  movements  during  jaw   opening  –  closing  in  man.  eriksson   p,  zafar  h,  nordh  e.  journal  of  oral   rehabilitation.  1998;25:859–70.   pmid:9846906   31.  comparison  of  maximum   mouth-­‐opening  capacity  and  con-­‐ dylar  path  length  in  adults  and   children  during  the  growth  period.   reicheneder  c,  proff  p,  baumert   u,  gedrange  t.  annals  of  anato-­‐ my.  2008;190:344–50.   pmid:18595675     32.  cross  sectional:  normal  maxi-­‐ mal  incisal  opening  and  associa-­‐ tions  with  physical  variables  in   children.  ying  qv,  bacic  j,   abramowicz  s,  sonis  a.  pediatric   dentistry.  2013;35:61–6.   pmid:23635901   33.  a  comparison  of  three  meth-­‐ ods  of  measuring  maximal  opening   of  the  mouth.  wood  g,  branco  j.   journal  of  oral  surgery.   1979;37:175–7.  pmid:283218   34.  effect  of  head  position  on  ver-­‐ tical  mandibular  opening.  higbie   ej,  seidel-­‐cobb  d,  taylor  lf,   cummings  gs.  journal  of  ortho-­‐ paedic  &  sports  physical  therapy.   1999;29:127–30.  pmid:10322587   35.  evaluation  of  the  applicability   of  temporomandibular  opening   index  in  turkish  children  with  and   without  signs  and  symptoms  of   temporomandibular  joint  disor-­‐ ders.  sari  s,  kucukesmen  c,  son-­‐ mez  h.  journal  of  craniomandibu-­‐ lar  practice.  2014;26:197–201.   pmid:18686496   36.  range  of  movement  of  mandi-­‐ ble  in  children.  ingervall  b.  scandi-­‐ navian  journal  of  dental  research.   1970;78:311–22.pmid:5274221     37.  analysis  of  maximum  mouth   opening  and  its  related  factors  in   3-­‐  to  5-­‐year-­‐old  taiwanese  chil-­‐ dren.  chen  hs,  yang  pl,  lee  cy,   chen  kk,  lee  kt.  odontology.   2013;1–5.  pmid:24174163     psychological impact of covid-19 vol 10 no 1 (2022) doi 10.5195/d3000.2022.185 http://dentistry3000.pitt.edu social isolation in covid-19: impact on mental health of young adults aditi verma1, abhishek mehta1 1 department of public health dentistry, faculty of dentistry, jamia millia islamia, new-delhi, india abstract background: the coronavirus disease (covid-19) is profoundly affecting life around the globe. social isolation, contact restrictions and economic shutdown impose a complete change to the psychosocial environment threatening the mental health of young adults significantly. objectives: the present study aims to assess the impact of social isolation on the mental health of young adults during the pandemic. methods: a web based research was conducted on a sample of 438 college students of age 18-24 years. the participants were asked for responding to a standardized online questionnaire that included questions related to their anxiety levels and the 7-item generalized anxiety disorder scale (gad-7). results: the results revealed 78.4% of the study populations were suffering with mild (35.2%), moderate (23.7%), and severe anxiety (19.4%) respectively. univariate and logistic regression analysis revealed gender and course/year of study had a significant effect on the level of anxiety (p<0.05). also, students living alone and having a relative or an acquaintance infected with covid-19 were likely to be more anxious (p<0.05). spearman correlation analysis depicted social isolation as the main covid-19 related stressor which was positively correlated and highly significant (p=0.0001) to the level of anxiety of young adults. conclusions: the present study revealed approximately 43% of young adults is suffering from moderate to severe anxiety levels which require urgent attention. keywords: social isolation, mental health, young adults, india, covid-19 pandemic. citation: verma a and mehta a. (2022) social isolation in covid-19: impact on mental health of young adults. dentistry 3000. 1:a001 doi:10.5195/d3000.2022.185 received: may 13, 2021 accepted: may 27, 2021 published: april 30, 2022 copyright: ©2022 verma a and mehta a. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: averma1@jmi.ac.in introduction the world is going through a major health and socio-economic crisis due to the emergence of novel corona virus disease (covid-19). the best option available for breaking the chain of transmission of this viral infection is with social isolation. as of 23rd march, 2020, all schools, colleges and educational institutions have been suspended under nationwide lockdown by the government of india. the continuous rise in cases coupled with social isolation measures such as the delay in the opening of schools, colleges/ universities or educational institutions across the country, closure of shopping malls and restrictions on social gatherings it is expected that the mental health of young adults is influenced [1]. these measures resulted in the major disruptions in the daily routines of socializing in schools, colleges and educational institutions which were important stress coping mechanisms for young adults [2]. research suggests that this social isolation could lead to various psychologicalrelated disorders if they are left unattended [3, 4]. several studies have already reported the negative psychological impact of pandemic on different age-groups [1, 3-5]. according to a review, young adults who feel lonely might be as much as three times more likely to develop depression in the future and that the impact of this loneliness on mental health could last for at least 9 years [3]. http://dentistry3000.pitt.edu/ psychological impact of covid-19 vol 10 no 1 (2022) doi 10.5195/d3000.2022.185 http://dentistry3000.pitt.edu however, we found only a single indian published literature focusing on understanding the mental state of youth during the current crisis [6]. hence, the present study was conducted with an aim to assess the psychological impact of social isolation during covid-19 pandemic on the mental health of young adults with the objectives: a. evaluate the level of anxiety among young adults during this pandemic period; b. evaluate the relationship between the covid related stressors and anxiety. materials and methods a cross-sectional web-based study was conducted on a sample of 438 college students of age group 18-24 years (young adults) irrespective of courses enrolled from jamia millia islamia university, new-delhi, india. the snow-ball sampling method was utilized to achieve desired study sample. the minimum sample size of this study ‘n’ was estimated based on earlier study [6]. ethical issues and consent the study proposal was reviewed and approved faculty of dentistry, jamia millia islamia institutional review board (no. fod/irrc/40/2020/f/2072020) prior to the commencement of the study. the research was conducted in concordance with the provisions of the declaration of helsinki regarding research on human participants. informed consent was obtained from the study participants prior to the commencement of study after being informed about the study purpose. eligibility criteria students enrolled in various courses of jamia millia islamia university and of age less than 25 years were eligible for participation in this study. students on medications for any psychiatric disorder or anxiety issues or were unwilling to provide informed consent were excluded from the study. data collection a structured online questionnaire was prepared on google forms referenced from an earlier study [1] with a consent form appended to it. the link of the questionnaire was sent through emails and whatsapp to the university students 4 months postlockdown. the data collection was initiated on 1st august 2020 at 10 am ist and closed on 5th august 2020 at 10am ist. the students who are willing to participate voluntarily in this study were invited to fill the questionnaire after taking their due informed consent. the participants were encouraged to roll out the survey to as many students as possible. thus, the link was forwarded to students apart from the first point of contact and so on. on receiving and clicking the link the participants got auto directed to the information about the study and informed consent. participants with access to the internet could participate in the study. after inquiring about the general demographic information such as age, gender, place of residence, the participants were inquired about the covid-19 related stress factors such as its economic influence, academic delays and social isolation along with the availability of social support. a standardized generalized anxiety disorder scale (gad-7) [2] was used to measure the anxiety levels of the respondents. the gad-7 includes seven items based on seven core symptoms and inquires the frequency with which respondents suffered from these symptoms within the last two weeks. respondents report their symptoms using a 4-item likert rating scale ranging from 0 (not at all) to 3 (almost every day), such that the total score ranges from 0 to 21. the gad-7 is a well-validated screening instrument, and it has demonstrated excellent internal consistency (cronbach's ɑ = 0.911) [3]. the level of anxiety and its probable causal factors (covid-19 stressors) among university students during the pandemic were investigated. statistical analysis the data obtained were analyzed with a statistical package for the social sciences v22 (spss inc; http://www.spss.com). descriptive statistics were performed for illustrating the baseline characteristics of the respondents while univariate analysis was employed to check for the association between the respondent’s characteristics and their level of anxiety. the association between http://dentistry3000.pitt.edu/ psychological impact of covid-19 vol 10 no 1 (2022) doi 10.5195/d3000.2022.185 http://dentistry3000.pitt.edu covid-19 related stressors and anxiety levels were evaluated by spearman’s correlation coefficient. results a total of 438 study participants were included in the present study comprising of 131 (30%) males and 307 (70%) females. mean age of the study participants was 22.15 ±1.93 years. the majority of the participants were living in urban areas (70%); 82.9% (n = 363) were staying with their parents and have a steady source of income while 17% (n = 75) of the parents of students did not have a steady income. more than ¾ (78.8%) of the participants reported that none of their relatives or acquaintances are or were infected with covid-19 while 21% (n = 93) of the participants had relatives or acquaintances who were infected with covid-19 [table 1]. level of anxiety among students the mental health of the participants was affected during the pandemic to varying extent. the total of 438 study participants, only 21.6% reported no symptoms of anxiety whereas the proportions of students with mild, moderate, and severe anxiety were 35.2%, 23.7%, and 19.4%, respectively [table 2]. factors influencing students’ anxiety univariate analysis revealed gender and course/year of study had a significant effect on the level of anxiety among the study participants whereas place of residence and families with steady source of income table 1. comparison of different independent variables with severity of anxiety levels of study participants. variables total no/ minimal mild moderate severe p gender male 131 38 42 31 20 0.038a female 307 57 112 73 65 place of residence urban 345 73 120 79 73 0.483b rural 34 7 15 9 3 urban-rural 59 15 19 16 9 course/year of study bds 186 41 61 48 36 0.003a others than bds 252 54 93 56 49 steady family income yes 363 81 126 88 68 0.535a no 75 14 28 16 17 social support/ live with parents yes 363 82 130 95 56 0.009a no 75 13 24 9 29 relative or acquintaince got covid-19 yes 93 14 32 24 23 0.042a no 345 81 122 80 62 aman-whitney u – test, bkruskal wallis test http://dentistry3000.pitt.edu/ psychological impact of covid-19 vol 10 no 1 (2022) doi 10.5195/d3000.2022.185 http://dentistry3000.pitt.edu had no significant effect on the anxiety levels. moreover, students living alone and having a relative or an acquaintance infected with covid19 were more likely to be more anxious [table 1]. table 2. frequency distribution of study participants with different anxiety levels (n=438). anxiety level number ratio minimal anxiety 95 21.6 mild anxiety 154 35.2 moderate anxiety 104 23.7 severe anxiety 85 19.4 significant factors from the univariate analysis were included in the ordinal logistic regression analysis. in the model test, p<0.05, indicating that the or value of at least one variable was statistically significant. therefore, χ2 = 27.197, p>0.05, obtained in the test of parallel lines indicated a good model fit with the observed values. the results showed that having a social support, i.e. staying with their parents was one of the significant protective factors against students’ anxiety (or = 0.246, 95%ci = 0.387 1.352) while gender, course/year of study were other significant risk factors for anxiety [table 3]. correlation between the covid-19related stressors and levels of students’ anxiety worrying about the economic influences of the epidemic and academic delays were positively related to the levels of anxiety in college students but was not statistically significant. moreover, the influence of social isolation/lockdown in terms of affecting their daily life, feeling of limitation of identity and anxiousness were positively correlated and were highly significant with the levels of anxiety [table 4]. table 3. frequency distribution of study participants with different anxiety levels (n=438). factors number se or p 95%ci gender female 307 0.207 0.459 0.027 (0.053, 0.864) malea 131 courses/ year of study 1st year bds 38 0.407 1.218 0.003 (0.421, 2.015) 2nd year bds 35 0.405 1.073 0.008 (0.280, 1.867) 3rd year bds 31 0.428 1.392 0.001 (0.554, 2.231) final year bds 46 0.382 0.915 0.017 (0.166, 1.664) internship 36 0.333 0.757 0.023 (0.105, 1.410) other than bdsa 252 social support/live with parents yes 363 0.870 0.246 0.000 (0.387, 1.352) noa 75 relative or acquintaince got covid-19 no 345 0.222 -0.217 0.327 (-0.652, 0.217) yesa 93 *ordinal regression analysis, aman-whitney u test, bkruskal wallis test http://dentistry3000.pitt.edu/ psychological impact of covid-19 vol 10 no 1 (2022) doi 10.5195/d3000.2022.185 http://dentistry3000.pitt.edu table 4: correlation analysis between covid related stressors and students anxiety. covid stressors anxiety levels p worried about economic influence 0.064 0.114 worried about academic delays 0.084 0.078 social isolation/ lockdown influences on my daily life 0.242 0.000 social isolation/ lockdown is my limiting identity 0.273 0.000 feeling anxious because of social isolation/lockdown 0.174 0.000 *spearman’s co-relation discussion the anxiety and other psychological concerns of society are globally affecting every individual to variable extents. recent evidence suggests that individuals who are kept in isolation experience significant distress in the form of anxiety, anger, confusion and post-traumatic stress symptoms [7]. the aim of this study was to evaluate the psychological impact of the current pandemic on the college students and explore underlying factors influencing their anxiety. the present study revealed that the majority of the college students (nearly 80%) were tormented with higher anxiety levels because of the covid-19 outbreak. the anxiety levels reported in the present study were higher than in the previous studies done by cao w et al (24.9%) and gaikwad s (48%). the probable reason for reporting such higher anxiety levels may be because of their stronger association with the duration of the social isolation [3]. in india, the educational institutions and colleges have been closed since midmarch and we conducted this research after 4 months of social isolation. it is known anxiety disorders are more likely to occur and worsen in the absence of interpersonal communication [8]. another factor may be students in india are still uncertain about the impact of pandemic on their academic year and future employment opportunities. the results also suggested that students' anxiety regarding the epidemic was associated with gender, course/year of study, whether living with parents and whether a relative or an acquaintance was infected with covid-19. this difference in anxiety levels on the basis of gender predilection is in line with the previous studies [3, 9] but is in contrast with the study done in china [1]. the difference indicates that male and female students experienced the difference in levels of stresses and negative emotions as a result of the pandemic. however, no significant difference was reported in source of parental income or place of residence, which was different from previous findings [1]. the year/course of study were also reported to be another variable associated with student’s anxiety. the present study reported significant levels of anxiety among the dental students in comparison to other courses. the probable reason could be the possibility of acquiring the disease while treating the patients which is a part of their undergraduate curriculum is higher in comparison to the other nonspecialized courses. previous studies [1, 10] have also indicated that the risk factors associated with emotional and anxiety disorders in adults include not living with parents which are consistent with the results of this study. among the covid related stressors the social isolation/ lockdown reported to be positively associated and had a significant impact among the anxiety levels of students. this is in line with the previous study [3]. although economic influence and academic delays were also reported to be positively associated with anxiety but were not statistically significant. this is in contrast to the previous study done in china [1]. this suggests that the current social isolation measures enforced because of covid-19 could lead to an increase in mental health problems among college students and is particularly becoming problematic for young adults. some strengths and limitations of this study can be noted. the use of the robust methodology and the standardized anxiety scale for detecting small correlations is the major strength of this study. whilst the limitations include the crosshttp://dentistry3000.pitt.edu/ psychological impact of covid-19 vol 10 no 1 (2022) doi 10.5195/d3000.2022.185 http://dentistry3000.pitt.edu sectional nature of the study, selfreport nature of the questionnaire may exhibit social desirability bias and may limit the generalizability of our results to a wider population. conclusion of the total study participants, 19.4% experienced severe anxiety and 23.7% experienced moderate anxiety levels which needs psychological interventions. thus, there are numerous mental health threats associated with the current pandemic and subsequent restrictions. finding ways to give young adults a sense of belonging within the family and to feel that they are part of a wider community should be a priority. helping them to identify valued alternative activities and build structures and purpose into periods of involuntary social isolation may help to provide a wider range of rewards. addressing negative thoughts about social encounters (e.g. self-blame, self-devaluation) may also be effective. digital technology that provides evidencebased interventions to help young adults to reappraise their thoughts and change their behavior within the confines of the home setting should be [3]. hence, it is pertinent to understand and investigate the psychological states of our young minds during these hard times and provide them with appropriate help and support from the family, institutions/colleges and society at large. acknowledgement none. conflicts of interest the authors have no conflict of interest to disclose. funding this research did not receive any specific grants from funding agencies in the public, commercial or not-forprofit sectors. references 1. cao w, fang z, hou g et al. the psychological impact of the covid-19 epidemic on college students in china. psychiatry research. 2020; 287:112934. 2. spitzer rl, kroenke k, williams jb, lowe b. a brief measure for assessing generalized anxiety disorder. arch intern med. 2006; 166:1092-1097. 3. loades me, chatburn e, higson-sweeney n et al. rapid systematic review: the impact of social isolation and loneliness on the mental health of children and adolescents in the context of covid19. j am acad of child adolesc psychiatry. available online 3 june 2020. 4. hiremath p, suhas-kowshik cs, manjunath m, shettar m. covid 19: impact of lock-down on mental health and tips to overcome. asian journal of psychiatry. 2020; 51:102088. 5. fegert jm, vitiello b, plener pl, clemens v. challenges and burden of the coronavirus 2019 (covid-19) pandemic for child and adolescent mental health: a narrative review to highlight clinical and research needs in the acute phase and the long return to normality. child adolesc psychiatry ment health 2020; 14:20. 6. gaikwad s. psychological impact of covid-19 on adolescents. purakala. 2020 may; 31(37):639-643. 7. brooks sk, webster rk, smith le et al. the psychological impact of quarantine and how to reduce it: rapid review of the evidence. the lancet. 2020 february; 395 (10227):912-920. 8. xiao c. a novel approach of consultation on 2019 novel coronavirus (covid-19)-related psychological and mental problems: structured letter therapy. psychiatry investig. 2020; 17 (2), 175–176. 9. moreno e, muñoz-navarro r, medrano la et al. factorial in variance of a computerized version of the gad-7 across various demographic groups and over time in primary care patients. j. affect disord. 2019; 252, 114–121. 10. woodgate rl, tailor k, tennent p et al. the experience of the self in canadian youth living with anxiety: a qualitative study. plos one. 2020; 15 (1),e0228193. http://dentistry3000.pitt.edu/ microsoft word rosa proof may 6.docx vol 2, no 1 (2014) issn 2167-8677 (online) doi 10.5195/d3000.2014.19 http://dentistry3000.pitt.edu this work is licensed under a creative commonsattribution 3.0 united states license. this site is published by the university library system, university of pittsburgh as part of its d-scribe digital publishing program and is cosponsored by the university of pittsburgh press. manual and rotary instrumentation techniques for root canal preparation in primary molars francinne m. rosa1, adriana modesto2, italo m. faraco-junior3 1private practice, porto alegre, rs, brazil 2university of pittsburgh, school of dental medicine, pittsburgh, pa, usa 3private practice, porto alegre, rs, brazil abstract introduction: although rotary instrumentation has been widely studied in permanent dentition, it is a rather new field of study concerning primary teeth. purpose: we aimed to evaluate apical displacement and time needed for instrumentation of root canals of primary molars by manual and rotary techniques. materials and methods: root canals of 144 extracted first and second primary maxillary molars were randomly divided into 2 groups: i manual instrumentation (k-files); ii rotary instrumentation (k3 rotary system®). the canals were radiographed with pathfinding files in place, prepared by both techniques, and instrumentation time was recorded. after preparation, root canals were radiographed again with pathfinding files in place. to analyze the degree of apical displacement, digital images were superimposed using the adobe photoshop® software. results: mean apical displacement (0.70 mm) in the manual instrumentation group was not statistically different from that in the rotary instrumentation group (0.79 mm). however, mean time for root canal preparation was significantly shorter using the rotary system (128.0 s) than using the manual system (174.0 s) (p<0.05). conclusions: the use of rotary instrumentation in pediatric dentistry is feasible, offering time-saving advantages in root canal preparation. citation: rosa fm, modesto a, and faraco-junior im. (2014) manual and rotary instrumentation techniques for root canal preparation in primary molars. dentistry 3000. 1:a001 doi:10.5195/d3000.2014.19 received: january 14, 2014 accepted: april 24, 2014 published: may 27, 2014 copyright: ©2013 rosa et al. this is an open access article licensed under a creative commons attribution work 3.0 united states license. email: faraco@globo.com introduction the use of rotary instrumentation in pediatric dentistry is a rather new field of study, although it has been widely studied in permanent dentition. studies have compared rotary with manual instrumentation performance for root canal preparation in permanent teeth. the results have shown no significant differences in the cleaning capacity between techniques, but rotary instrumentation prepared canals more rapidly and uniformly [1,2]. in addition, the rotary instruments can reach the entire length of the root canal, causing little or no displacement, particularly in the apical region [35]. rotary instrumentation characteristics become significantly relevant when dealing with endodontic preparation of primary teeth, since appointment length is a crucial factor for pediatric patient compliance, and maintaining the original path of the root canal is essential to ensure the integrity of the germ of the permanent successor [6,7]. considering the reduced number of studies on this topic in pediatric dentistry, this study aimed to elucidate the apical displacement produced in root canals and the time needed for root canal preparation in primary teeth by the rotary instrumentation system in comparison with the manual instrumentation technique. materials and methods sixty-four primary maxillary molars extracted with no previous endodontic treatment, internal and/or external root resorption, and over two-thirds of resorbed root were selected for the study. after sample size calculation (power of 90%, confidence level of 95%), the sample was composed of 144 roots (table 1): 24 mesiobuccal, 24 distobuccal, and 24 palatal roots of the first primary maxillary molars; and 24 mesiobuccal, 24 distobuccal, and 24 palatal roots of the second maxillary molars. these roots were cleaned with periodontal curettes, decontaminated with 1% sodium hypochlorite and sterilized in 10% formalin. all teeth were previously embedded in clear acrylic-resin blocks. for this purpose, a thin layer of wax was placed on dental apex to seal the inlet channels, and the teeth were sunken until the table 1: distribution of the primary molars. tooth mesiobuccal root distobuccal root palatal root first primary maxillary molar 24 24 24 second primary maxillary molar 24 24 24 total number of each root 48 48 48 total number of roots=144 http://dentistry3000.pitt.edu manual and rotary instrumentation techniques for root canal preparation in primary molars vol 2, no 1 (2014) doi 10.5195/d3000.2014.19 http://dentistry3000.pitt.edu 2 figure 1: tooth embedded in clear acrylic-resin block. figure 3: manual instrumentation with k-files. level of acrylic-resin covered the roots (figure 1). after crown access, the teeth were irrigated with 1% sodium hypochlorite and the working length was determined by passive insertion of a k-file with a rubber stop. when the file tip was at the level of the dental apex, the rubber stop was leveled with the respective cusp tip and the length of each root canal was recorded. the working length was determined by subtracting 2 (two) mm from the total length of the root canals. the root canals were then placed on a radiographic platform and radiographed in proximal (mesial/distal) and frontal (palatal/buccal) directions, with pathfinding files in place, using a direct digital imaging sensor (cygnus mpd®,cygnusinc., redwood city, ca, usa), in order to record pathfinding file position in the apical region of root canals. radiographs were obtained with gnatus x-ray equipment (gnatus equipamentos médico-odontológicos ltda, ribeirão preto, são paulo, brazil), 70x, 70 kvp and 7 ma, at 30 cm focal distance, 90° core radius, and 0.08 seconds exposure time. biomechanical preparation of all root canals was performed by a single operator, with resin blocks fixed on a device that secured blocks in the same position (figure 2). instrumentation time for each root canal was recorded, and each instrument, in both groups, was used in 10 samples. the roots were randomly divided into two groups: group i (n=72): manual instrumentation. (1) coronal opening; (2) irrigation with 1% sodium hypochlorite (performed throughout the preparation); (3) location of root canals (4) root canal instrumentation using k-files: utilization of initial instrument (better fit in the root canal) and three sequential instruments (figure 3). group ii (n=72): rotary instrumentation. (1) coronal opening; (2) irrigation with 1% sodium hypochlorite (performed throughout the preparation); (3) location of root canals (4) root canal instrumentation using with k3 rotary system® (sybron endo, orange, ca, usa) in the sequences # 25/0.8, # 30/0.6, # 25/0.4, and # 25/0.2 (crown-apex direction), set in motion by an endo standard pro torque® motor (dentsply, rio de janeiro, rj, brazil) at 250 rpm speed (figure 4). after biomechanical preparation, root canals were placed again on the radiographic platform and radiographed in proximal and frontal directions, with pathfinding files in place, being careful to maintain the same standard conditions (position and other exposure factors). post-biomechanical preparation radiographs were digitally superimposed over their prepreparation counterparts using the adobe photoshop® software (figure 5). a straight line was drawn to connect the tips of the instruments inside the canals, thus generating two displacement measurements: one for frontal and one for proximal view. real apical displacement was measured (in millimeters) by a pre-calibrated examiner, blinded to technique assignment, by calculating the third side of a triangle constructed by the measurement of the displacement obtained from frontal and proximal view. reproducibility of the results was verified every five images analyzed. statistical analysis was performed with the statistical package for the social sciences (spss) software, version 14.0, and significance level of 5%. data regarding apical displacement were compared using the kruskal-wallis test and the mannwhitney test. differences in instrumentation time were statistically analyzed with anova, post hoc tukey s test, and the student s t test. results in the manual group, the lowest apical displacement was observed in the distobuccal root of the first primary maxillary molar (0.15 mm) and the greatest apical displacement was found in the mesiobuccal roots of the first and second primary maxillary molars (1.64 mm). in regard to the time, palatal root of the second maxillary molar showed the lowest value (121 s), and the highest value was observed in the mesiobuccal root of the second primary maxillary molar (416 s). in the rotary group, the lowest value for apical displacement was observed in the palatal root of the first primary maxillary molar (0.10 mm) and the greatest value in the distobuccal root of the first primary maxillary molar (3.09 mm). the shortest instrumentation time in the rotary technique was observed in the palatal root of the second primary maxillary molar (78 table 2: analysis of apical displacement (in millimeters) comparing the techniques used (rotary vs. manual instrumentation). tooth root rotary manual p* 1pmm mesiobuccal 0.27 0.43 0.184 1pmm distobuccal 0.85 0.63 0.175 1pmm palatal 0.24 0.14 0.839 2pmm mesiobuccal 0.28 0.56 0.599 2pmm distobuccal 0.49 0.35 0.166 2pmm palatal 0.38 0.18 0.049 1pmm=first primary maxillary molar; 2pmm=secondary primary maxillary molar. *statistically significant p values (p<0.05). mann-whitney nonparametric test. figure 2: acrylic-resin block positioned for instrumentation. http://dentistry3000.pitt.edu manual and rotary instrumentation techniques for root canal preparation in primary molars vol 2, no 1 (2014) doi 10.5195/d3000.2014.19 http://dentistry3000.pitt.edu 3 figure 4: rotary instrumentation with k3 rotary system ®. figure 5: (a) initial radiograph; (b) final radiograph; (c) overlay of digital images. s) and the longest time in the mesiobuccal root of the first primary maxillary molar (296s). the evaluation of apical displacement for each root type, after post hoc analysis for means, comparing manual and rotary techniques, showed no statistically significant differences (table 2). with regard to the time needed for instrumentation using manual and rotary techniques, within the same root type group, statistically significant differences were observed in all root types, except for the mesiobuccal root of the first primary maxillary molar. the shortest time for instrumentation was achieved with the rotary instrumentation technique (table 3). an overall data analysis, regardless of root type, showed no significant differences when comparing the degree of apical displacement within manual and rotary techniques. however, considering instrumentation time, the rotary technique needed a significantly shorter time than the manual technique. discussion this study sample was composed of extracted primary teeth, aiming to reproduce preparation conditions as similar as possible to in vivo conditions, since standardized root canals exclude some variables found in natural teeth [8]. furthermore, the reduced number of studies using rotary instrumentation in primary teeth [6,7,9-19] justifies the comparison of our findings with some studies in permanent teeth [1,2,20-25]. with regard to apical displacement, there were no statistically significant differences between techniques, when root types and groups were analyzed altogether, although displacements occurred in all root types and in several ways. these results are consistent with those found by nazari moghaddam et al. [9], azar, mokhtare [10], and igbal et al. [20], but disagree with the results found by loizides et al. [21], which may be explained by the fact that simulated root canals were used, as well as a rotary system different than the one employed in the present study. furthermore, the highest values for apical displacement produced in mesiobuccal roots, which usually show a higher degree of curvature [8], occurred using the manual instrumentation technique. in distobuccal and palatal roots, however, the highest values were obtained with rotary instrumentation. elmsallati et al. [22] showed that k3 rotary system® produces minimum wear of root canal walls, which is an interesting aspect in the endodontic preparation of primary teeth. moreover, crespo et al. [11] and musale, mujawar [12] stated that rotary files prepared more conical canals in primary teeth than manual instruments. when analyzing canal curvature, it is well known that the degree of curvature does not affect the performance of k3 rotary system® [23,24] validating the good performance of this system in mesiobuccal roots observed in this study. in the present study, two instrument fractures were observed, both with k3 rotary system®, although this is considered one of the safest systems [23,25]. nagaratna et al. [13], working on primary teeth, already pointed out as a disadvantage of the rotary system its higher fracture rate. two root perforations occurred in the present study, both in the rotary instrumentation group. only one [14] of the previous studies performed in primary teeth [6,7,9-13,15-18] reported such event, which might be attributed to the operator s level of skill in using the system. moreover, kummer et al. [14] stated that in some specimens, root perforations were observed in areas coinciding with largest root resorption. in regard to instrumentation time, when the groups were analyzed altogether, there was a significant difference between techniques, and the shortest preparation time was achieved with the rotary technique. the present results corroborate those findings in the existing literature, on instrumentation performed in primary teeth, which describe quick preparation as the main advantage of using rotary instruments in pediatric dentistry [6,7,9-19]. our data showed significant differences in time needed for instrumentation between roots within the same instrumentation group (manual or rotary). the palatal root of the maxillary second molar needed the shortest time for instrumentation, in both techniques, because it is straight and wide, whereas the longest instrumentation time was observed in the preparation of mesial roots, because they are curved and slender [8]. we also observed that the highest values in time needed for instrumentation were obtable 3: analysis of time (in seconds) comparing the techniques used (rotary vs. manual instrumentation). tooth root rotary manual p* 1pmm mesiobuccal 163.3 181.8 0.410 1pmm distobuccal 149.3 244.6 0.007* 1pmm palatal 131.7 151.7 0.042* 2pmm mesiobuccal 123.7 256.0 0.001* 2pmm distobuccal 133.0 206.2 <0.001* 2pmm palatal 111.1 145.9 0.001* 1pmm=first primary maxillary molar; 2pmm=secondary primary maxillary molar. *statistically significant p values (p<0.05). student t test. http://dentistry3000.pitt.edu manual and rotary instrumentation techniques for root canal preparation in primary molars vol 2, no 1 (2014) doi 10.5195/d3000.2014.19 http://dentistry3000.pitt.edu 4 tained with the manual technique, in all root types, except for the mesial root of the first primary maxillary molar. therefore, we can state that root anatomy affects instrumentation time, since deviant root anatomy leads to longer instrumentation time for both the manual and rotary technique. the results obtained in the present study are quite satisfactory concerning the reduction of instrumentation time by the rotary technique, a fact already reported in previous studies in primary teeth. however, there was no statistical significant difference between techniques concerning apical displacement when root types and groups were analyzed altogether, although displacements occurred in all root types and in several ways. pinheiro et al. [19] stated that endodontic treatment in children may be challenging and time consuming, especially during root canal preparation, which is one of the most important stages of endodontic therapy. considering that the rotary instruments provide similar root canal cleaning compared to manual instruments with a shorter instrumentation time, their utilization is well indicated in pediatric dentistry. in conclusion, rotary instrumentation is feasible and an important tool to be used in the endodontic preparation of primary teeth since it requires a shorter clinical time from the pediatric patient. acknowledgements none of the authors have a direct financial relation with any commercial identities mentioned in this paper, nor have any other conflict of interests to declare. we thank dr. carlos alberto feldens for helping us in the statistical analysis. references 1. comparing apical preparations of root canals shaped by nickeltitanium rotary instruments and nickel-titanium hand instruments. deplazes p, peters o, barbakow f.j endod. 2001 mar;27(3):196-202. pmid: 11487151 2. effectiveness of manual and rotary instrumentation techniques for cleaning flattened root canals. barbizam jv, fariniuk lf, marchesan ma, pecora jd, sousa-neto md. j endod. 2002 may;28(5):365-6. pmid: 12026920 3. efficiency of rotarynickel-titaniumk3 instruments compared with stainless steel hand k-flexofile. part 2. cleaning effectiveness and shaping ability in severely curved root canals of extracted teeth. schäfer e, schlingemann r. int endod j. 2003 mar;36(3):208-17. pmid: 12657147 4. a comparison of profile, hero642, and k3 instrumentation systems in teeth using digital imaging analysis. gonzález-rodrguez mp, ferrerluque cm. oral surg oral med oral pathol oral radiol endod. 2004 jan;97(1):112-5. pmid: 14716266 5. in vitro evaluation of the thermal alterations on the root surface during preparation with different ni-ti rotary instruments. capelli a, guerisoli dm, barbin el, spanó jc, pécora jd. braz dent j. 2004;15(2):115-8. epub 2005 mar 11. pmid: 15776193 6. use of nickel-titanium rotary files for root canal preparation in primary teeth. barr es, kleier dj, barr nv. pediatr dent. 1999 novdec;21(7):453-4. pmid: 10633522 7. use of nickel-titanium rotary files for root canal preparation in primary teeth. barr es, kleier dj, barr nv. pediatr dent. 2000 janfeb;22(1):77-8. pmid: 10730297 8. a three-dimensional study of canalcurvatures in the mesial roots of mandibular molars. cunningham cj, senia es. j endod. 1992 jun;18(6):294-300. pmid: 1402588 9. root canal cleaning efficacy of rotary and handfiles instrumentation in primary molars. nazari moghaddam k, mehran m, farajian zadeh h. iran endod j. 2009 spring;4(2):53-7. epub 2009 apr 17. pmid: 23940486 10. rotary mtwo system versus manual k-file instruments: efficacy in preparing primary and permanent molar root canals. azar mr, mokhtare m. indian j dent res. 2011 marapr;22(2):363. doi: 10.4103/09709290.84283. pmid: 21891918 11. comparison between rotary and manual instrumentation in primary teeth. crespo s, cortes o, garcia c, perez l. j clin pediatr dent. 2008 summer;32(4):295-8. pmid: 18767460 12. evaluation of the efficacy of rotary vs. hand files in root canal preparation of primary teeth in vitro using cbct. musale pk, mujawar sa. eur arch paediatr dent. 2014 apr;15(2):113-20. doi: 10.1007/s40368-013-0072-1. epub 2013 jul 27. pmid: 23893606 13. in vitro comparison of niti rotary instruments and stainless steel hand instruments in root canal preparations of primary and permanent molar. nagaratna pj, shashikiran nd, subbareddy vv. j indian soc pedod prev dent. 2006 dec;24(4):186-91. pmid: 17183182 14. ex vivo study of manual and rotary instrumentation techniques in human primary teeth. kummer tr, calvo mc, cordeiro mm, de sousa vieira r, de carvalho rocha mj. oral surg oral med oral pathol oral radiol endod. 2008 apr;105(4):e8492. doi:10.1016/j.tripleo.2007.12.008. pmid: 18329573 15. comparison of rotary and manual instrumentation techniques on cleaning capacity and instrumentation time in deciduous molars. silva la, leonardo mr, nelson-filho p, tanomaru jm. j dent child (chic). 2004 jan-apr;71(1):45-7. pmid: 15272656 16. comparison of conventional, rotary, and ultrasonic preparation, different final irrigation regimens, and 2 sealers in primary molar root canal therapy. canoglu h, tekcicek mu, cehreli zc. pediatr dent. 2006 novdec;28(6):518-23. pmid: 17249433 17. comparison between rotary and manual techniques on duration of instrumentation and obturation times in primary teeth. ochoaromero t, mendez-gonzalez v, flores-reyes h, pozos-guillen aj. j clin pediatr dent. 2011 summer;35(4):359-63. pmid: 22046692 18. k-file vs profiles in cleaning capacity and instrumentation time in primary molar root canals: an in vitro study. madan n, rathnam a, shigli al, indushekar kr. j indian soc pedod prev dent. 2011 jan-mar;29(1):2-6. doi: 10.4103/0970-4388.79907. pmid: 21521910 19. evaluation of cleaning capacity and instrumentation time of manual, hybrid and rotary instrumentation techniques in primary molars. pinheiro sl, araujo g, bincelli i, cunha r, bueno c. int endod j. 2012 apr;45(4):379-85. doi: 10.1111/ j.1365 2591.2011.01987.x. epub 2011 dec 22. pmid: 22188162 20. comparison of apical transportation in four ni-ti rotary instrumentation http://dentistry3000.pitt.edu manual and rotary instrumentation techniques for root canal preparation in primary molars vol 2, no 1 (2014) doi 10.5195/d3000.2014.19 http://dentistry3000.pitt.edu 5 techniques. iqbal mk, maggiore f, suh b, edwards kr, kang j, kim s. j endod. 2003 sep;29(9):587-91. pmid: 14503833 21. root canal transportation with a niti rotary file system and stainless steel hand files in simulated root canals. loizides a, eliopoulos d, kontakiotis e. quintessence int. 2006 may;37(5):369-74. pmid: 16683684 22. debris retention and wear in three different nickel-titanium rotary instruments. elmsallati ea, wadachi r, ebrahim ak, suda h. aust endod j. 2006 dec;32(3):107-11. pmid: 17201751 23. comparative study of six rotary nickel-titanium systems and hand instrumentation for root canal preparation. guelzow a, stamm o, martus p, kielbassa am. int endod j. 2005 oct;38(10):743-52. pmid: 16164689 24. a comparative study of root canal preparation with niti-tee and k3 rotary ni-ti instruments. jodway b, hülsmann m. int endod j. 2006 jan;39(1):71-80. pmid: 16409331 25. deformation and fracture of race and k3 endodontic instruments according to the number of uses. troian ch, só mv, figueiredo ja, oliveira ep. int endod j. 2006 aug;39(8):616-25. pmid: 16872456 http://dentistry3000.pitt.edu selective root retreatment: a randomized clinical trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.339 http://dentistry3000.pitt.edu selective root retreatment: a randomized clinical trial mohamed nagy¹, ahmed ghobashy² ¹associate professor of endodontics, faculty of dentistry, ain shams university, cairo, egypt ²associate professor of endodontics, department of conservative dentistry, faculty of dentistry, misr international university, cairo, egypt abstract objectives: post-treatment apical periodontitis is an inflammatory disorder of peri-radicular tissues caused by either persistent or secondary intra-radicular infection. the present study compared the prognosis of cases treated with the “selective root retreatment” concept to the traditional retreatment approach. material and methods: forty patients complaining of failed root canal treatment having a periapical lesion in one root were selected. the cases were randomly divided into two groups; group (s): selective root canal retreatment in which only the affected root was retreated and group (t): traditional root canal retreatment in which all canals were retreated. the peri-radicular condition was evaluated radiographically using the periapical index (pai) for one year. the chi-square test statistical tests analyzed the outcome data. results: at the baseline examination, there was no significant difference between the two groups regarding the patients’ baseline mean periapical indices. after one year, we found that the mean periapical index declined without a significant difference between the two groups. conclusions: the present study points to selective root retreatment as a successful “minimally invasive alternative” in cases of an independently diseased root. the remaining treated roots can be retained without intervention provided that they are confirmed: “healthy”. clinical relevance: selective root retreatment is a promising conservative option in the treatment of failed endodontic cases with periapical pathosis related to an individual root. citation: nagy & ghobashy (2022). selective root retreatment: a randomized clinical trial dentistry 3000. 1:a001 doi:10.5195/d3000.2022.339 received: april 24, 2022 accepted: june 23, 2022 published: 13 july, 2022 copyright: ©2022 nagy & ghobashy. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: mohamed.nagy@dent.asu.edu.eg introduction post-treatment apical periodontitis is an inflammatory disorder of periradicular tissues caused by either persistent or secondary intra-radicular infection [1, 2]. persistent infections are caused by microorganisms that persisted and survived after root canal obturation. secondary infections are usually caused by microorganisms introduced in the canal via coronal or apical leakage in obturated root canals. these microorganisms usually remain in a biofilm state [3]. the decision-making in such cases is influenced by the endodontist’s ability to identify and eradicate the underlying etiology responsible for the established disease. management of such cases includes nonsurgical retreatment, surgical treatment, or a combination of both procedures [4]. conventionally, nonsurgical retreatment has been believed to be an ‘‘all or none’’ treatment approach [5] in which the endodontist has to remove all restorations from the tooth and remove the obturation materials from all the canals in order to perform thorough disinfection of the entire root canal system. such an approach involves unwanted removal of dentin and, consequently, more weakening of the tooth structure, besides the increased possibility of potential iatrogenic errors [6], which might endanger the outcome of the case. nowadays, all operations in the medical field are aimed basically at the conservation of the human body [7]. in endodontics, the integrity of the tooth structure is a crucial factor that influences the case prognosis as it is directly related to the functional requirements of the tooth after restoration [8, 9]. in the past, treatment planning has been limited by two-dimensional radiographic imaging [10–12]. nowadays, with the introduction of cone-beam computed tomographic (cbct) imaging, it is possible to detect http://dentistry3000.pitt.edu/ selective root retreatment: a randomized clinical trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.339 http://dentistry3000.pitt.edu initial apical periodontitis before significant cortical bone demineralization [13]; consequently, the presence of periapical pathosis can be more precisely detected via cbct compared with conventional two-dimensional radiographic imaging techniques [14]. this alternative concept allows the endodontist to formulate clinical treatment decisions with respect to the presence of periapical pathosis related to individual roots rather than the tooth as a whole; therefore, the term “selective root retreatment” is advocated [15], i.e., to retreat the diseased root only while retaining the remaining treated roots without intervention provided that they are confirmed, “healthy.” up to date, the literature is lacking the clinical outcome studies dealing with the prognosis of cases treated with the “selective root retreatment” concept, so the aim of this study was to compare the prognosis of such cases in comparison to the traditional retreatment approach. materials and methods this double-blind, multi-center, with a parallel design randomized controlled trial was registered on www.clinicaltrials.gov (clinicaltrials.gov id: nct04737863). the study protocol was approved by fdasu-rec (faculty of dentistry, ain shams university-research ethical committee) institutional review board, and the study was performed in accordance with all applicable laws and regulations, including the declaration of helsinki. power calculation was performed using g*power 3.1 software [16] (heinrich heine university, dusseldorf, germany) . the calculation indicated that the sample size for each group should be a minimum of 15 cases forty study subjects were recruited from 2 private endodontic clinics between march 2019 and march 2021 complaining of failed root canal treatment. patients were selected according to the inclusion and exclusion criteria. inclusion criteria • patients who had an intact restoration at follow-up; • teeth having a baseline periapical lesion (pai score ≥3) in one root. exclusion criteria • patients who refused to undergo radiographic follow-up; • pregnant females; • patients suffered from a systemic disease; • teeth having a periapical lesion in two or more roots; • teeth with lesions connected to adjacent teeth; • teeth with root fractures or perforations; • teeth with lesion communicating with the alveolar crest. all teeth included in the analysis were selected by two blinded, independent evaluators based on the preoperative parallel periapical radiograph. all participants signed a written consent after an oral explanation of the aim of the study, the procedures, benefits, and the potential risks. the treatment options presented to the patient included traditional nonsurgical retreatment (removal of all restorative materials), or selective root canal retreatment (isolating the necessary procedures to the affected root), or extraction. two endodontists participated as investigators. the investigators ensured that subject names and data were kept confidential. the clinical examination involved periodontal pocket depths measurement, mobility testing, the presence or absence of swelling, percussion, and palpation. cbct evaluation was done for each study tooth before the procedure and at the 12-month follow-up visit. standardized parallel periapical radiographs were taken preoperative, postoperative, then every three months up to one year of follow-up. the cases were randomly divided into two groups; group (s): selective root canal retreatment and group (t): traditional root canal retreatment. randomization was done using http://www.random.org. group (s): for each case, profound anesthesia was administrated, dental dam isolation was performed. under a dental operating microscope (opmi pico; carl zeiss, gottingen, germany), the subject tooth was accessed using a suitable-sized round diamond stone under copious water irrigation. the affected root canal was identified. the old root canal filling was removed using hand and rotary instruments aided solvents. working length was http://dentistry3000.pitt.edu/ http://www.clinicaltrials.gov/ selective root retreatment: a randomized clinical trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.339 http://dentistry3000.pitt.edu re-established electronically and confirmed radiographically. a patent reproducible glide path was created to size #20/.02 and enlarged using protaper next (dentsply maillefer) x1, x2, and x3 at a rotational speed of 300 rpm and 200 g/cm torque. each file was used with a brushing motion. an irrigation protocol consisting of 2.6% naocl in conjunction with ultrasonic activation was used for canal disinfection. the canal was dried using paper points and obturated using the continuous wave condensation technique. the access was restored, and a final postoperative radiograph was taken. postoperative instructions were provided, and the patient was left for follow-up for one year. standardized parallel periapical radiographs were taken every three months. group (t): the same procedure was applied to all root canals including the unaffected root or roots . the periapical radiography was performed with a paralleling technique using an x-ray film holder (rinn xcp; dentsply, elgin, il, usa). the x-ray machine (cs2200; carestream dental, atlanta, ga, usa) was set at 60 kv and 7 ma, and exposure time for the periapical radiography ranged from 0.08 to 0.125 s (0.08 s for adult incisors and canines; 0.1 s for adult premolars; 0.125 s for adult molars). cbct images were obtained using care stream cs9300 (carestream dental llc, atlanta, ga, usa) with a voxel size of 0.09mm. we conducted follow up using standardized radiographs every three months for one year. the peri-radicular condition was evaluated using the periapical index (pai) created by ørstavik et al. (1986)[17]. the pai includes five grades represented on an ordinal scale as follows: (1) normal periapical structures; (2) small changes in the periapical bone or bone structure; (3) changes in the periapical bone structure with mineral loss, characteristic of apical periodontitis; (4) demineralization of the periapical bone within a well-defined radiolucent area; and (5) demineralization of the periapical bone with exacerbations and expansion in bone structure. the radiographs were evaluated independently by two examiners. any disagreement within the evaluation of the cases was resolved by taking the average. the following cutoff points were defined to categorize the pai values into health or diseased: 1 denoted no disease, and values from 2 to 5 indicated the presence of periapical disease. any disagreement cases were resolved by discussion until an agreement was reached. complete healing and incomplete healing were classified as a success, and uncertain healing and unsatisfactory healing were classified as a failure based on molven’s criteria. 3d radiographic healing assessment 3d radiographic healing assessment was performed using cbct scans by two examiners. the two examiners discussed and reached an agreement in any disagreement cases. statistical analysis data were collected, tabulated, and statistically analyzed using spss software 20.0 (spss inc, chicago, il, usa). frequencies of qualitative variables were calculated using the chi-square test. results figure 1 shows the consort flow diagram for this study. fifty four study subjects were initially selected. fourteen cases were excluded because 8 cases don’t meet the criteria and 6 cases declined to participate. forty cases were eventually included in the study and were randomly allocated to group s (20 teeth) and group t (20 teeth). the overall recall rate was 77.5% (31 of 40). basic patients’ demographics were collected and tabulated in table 1. periapical index distribution and mean periapical index recorded initially and after one year are tabulated in tables 2 and 3. statistical analysis of patients’ basic data confirmed that there was no significant difference between the two groups regarding sex (p=0.594), age (p=0.721), and type of teeth subjected to treatment (p=0.792). there was no significant difference between the two groups regarding the patients’ baseline mean periapical indices (p=0.579). our results showed that the mean periapical index declined after one year without a significant difference between the two groups (p=0.853). representative case of group s is shown in figure 2. http://dentistry3000.pitt.edu/ selective root retreatment: a randomized clinical trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.339 http://dentistry3000.pitt.edu figure 1. consort flow diagram table 1. demographic data of participants in both groups group (s) group (t) p value sex males 9 (56%) 7 (47%) 0.594 females 7 (44%) 8 (53%) mean age (years) 43.9±10.5 42.5±11.2 0.721 teeth maxillary premolars 2 (12.5%) 1 (6.7%) 0.792 maxillary molars 5 (31.3%) 4 (26.7%) mandibular molars 9 (56.2%) 10 (62.6%) *p<0.05 is considered significant figure 2. cbct views of a representative case of group (s); “selective retreatment”, a: preoperative views showing separate periapical lesion related to the mesial root. b: one-year follow-up views showing marked healing of the lesion. table 2. periapical index distribution initially and after one year of follow-up among the two tested groups. group (s) group (t) initial score final score initial score final score stage (1) 0 (0%) 11 (68.75%) 0 (0%) 11 (73.3%) stage (2) 0 (0%) 4 (25%) 0 (0%) 3 (20%) stage (3) 3 (18.75%) 1 (6.25%) 3 (20%) 1 (6.67%) stage (4) 11 (68.75%) 0 (0%) 8 (53.3%) 0 (0%) stage (5) 2 (12.5%) 0 (0%) 4 (26.67%) 0 (0%) total cases n=16 n=15 http://dentistry3000.pitt.edu/ selective root retreatment: a randomized clinical trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.339 http://dentistry3000.pitt.edu table 3. mean periapical index initially and after one year of follow-up among the two tested groups. group (s) group (t) p-value mean initial periapical index 3.94±0.574 4.07±0.704 0.579 mean final periapical index 1.38±0.619 1.33±0.617 0.853 *p<0.05 is considered significant discussion endodontically treated teeth with secondary apical periodontitis constitute a great challenge facing the endodontist. the complexity of the retreatment procedure, besides the added damage to the remaining tooth structure, remains the main issue influencing the treatment plan of such cases. the current options are limited to nonsurgical root canal retreatment, surgical root treatment, or extraction [18]. although a successful outcome can be achieved with these alternatives, yet current advances may allow for a more conservative approach, which may yield a similarly successful outcome. the integration between cbct and magnification has revolutionized the field of minimally invasive endodontics. cbct imaging has drastically improved endodontic diagnosis and treatment planning by providing a three-dimensional view of the teeth and the supporting structures [10]. the 3-planar cuts eliminate the superimposition of the overlying structures, which is always a major flaw of the regular 2dimensional radiograph. this advantage allows for accurate and early detection of the periapical pathosis related to the apex of a single root or roots [11]. the assessment also includes the location, size, and shape of the pulp chamber, root lengths, degree of canal curvature, and threedimensional analysis of the causative mishaps. this capability allows for formulating a more conservative treatment plan for teeth with secondary apical periodontitis. the acquisition of a cbct scan is thus a prerequisite during the assessment of tooth candidates for selective root retreatment procedure. the benefit to the patients of a positive outcome of this minimally invasive technique necessitated using cbct imaging with a slight increase in total ionizing radiation. when the selective root retreatment technique gains scientific evidence as a routine technique in daily clinical use, the outcomes can be monitored using conventional radiographs with less radiation exposure. according to the current consensus, when contamination occurs during intracanal procedures, apical periodontitis often develops quickly within one year of the initial treatment. in these instances, healing should not be expected, so one-year follow-up to evaluate the healing of intervention method was used in the management of the retreatment cases [19, 20]. the use of a dental operating microscope has another necessity within the field of minimally invasive endodontics. conservation of the remaining tooth structure is an essential factor influencing the prognosis and the survival rate of the tooth [21]. the ability to perform the selective retreatment procedures at high precision prevents the occurrence of additional iatrogenic damage; precise motor skills can be improved with higher magnification and in addition to a considerable reduction in treatment time for the whole procedure [22]. the literature shows scarce evidence evaluating the selective retreatment idea or concept. up to our knowledge, few case reports were done using this technique [15]. on the contrary, many publications support the removal of all obturation material from all canals within the tooth being retreated. the decision to selectively retreat one or more roots of a previously roottreated tooth is exclusively based on the treating clinician assessment of the case with patient acceptance. the decision must also be based on a thorough clinical examination. http://dentistry3000.pitt.edu/ selective root retreatment: a randomized clinical trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.339 http://dentistry3000.pitt.edu restorations with unsatisfactory marginal integrity or recurrent caries are not candidates for that procedure as the other roots might also be infected though they appear normal. coronal restoration works as a ‘‘barrier’’ to reinfection of the root canal system after endodontic treatment. that variable has been reported in the literature as an important factor related to the presence or absence of apical radiolucency. a high success rate for cases with the presence of coronal restoration was confirmed in multiple studies [23–25]. this high success rate shows that coronal restoration should be considered an important step to be taken after endodontic treatment. in our study, only cases with the intact coronal restoration were enrolled to guarantee the absence of reinfection of the other canals of the pulp system. the decision of retreatment of the whole root canal system was made to guarantee complete eradication of the bacteria and allow for complete disinfection of the root selected for retreatment. there is a high incidence of the isthmus in molars that have been reported in many studies, the reported incidence in the mesiobuccal root of maxillary first molars is from 76%–100% and in the mesial root of mandibular first molars being approximately 83% [26–28] so in this study, the decision of retreatment of the whole root canal system was taken. the significant limitations of the research described herein were the small sample size after dropouts and the short length of the evaluation period, but to our knowledge, it is the first study to compare between selective root retreatment and the traditional non-conservative technique. within the limitation of this study, it can be concluded that selective root retreatment is a successful “minimally invasive alternative” in cases of an independently diseased root. the remaining treated roots can be retained without intervention provided that they are confirmed: “healthy”. more extended follow-up periods are recommended to validate the outcome of such cases. references 1. ricucci d, siqueira jf. recurrent apical periodontitis and late endodontic treatment failure related to coronal leakage: a case report. j endod. 201l; 37:1171–1175. pmid: 21763916 2. siqueira jf, rôças in, ricucci d, hülsmann m. causes and management of post-treatment apical periodontitis. br dent j. 2014; 216:305–312. pmid: 24651336 3. costerton w, veeh r, shirtliff m, pasmore m, post c, ehrlich g. the application of biofilm science to the study and control of chronic bacterial infections. j. clin. invest. 2003; 112:1466–1477. pmid: 14617746 4. yan mt. the management of periapical lesions in endodontically treated teeth. aust endod j 2006; 32:2–15. pmid: 16603040 5. roda rs, gettleman bh. nonsurgical retreatment. in: cohen’s pathways of the pulp. 2011; pp 890–952 6. ruddle cj. nonsurgical retreatment. j endod. 2004; 30:827–845. pmid: 15564860 7. murdoch-kinch ca, mclean me. minimally invasive dentistry. j am dent assoc. 2003;134:87– 95. pmid: 12555961 8. nagasiri r, chitmongkolsuk s. long-term survival of endodontically treated molars without crown coverage: a retrospective cohort study. j prosthet dent. 2005; 93:164– 170. pmid: 15674228 9. tang w, wu y, smales rj. identifying and reducing risks for potential fractures in endodontically treated teeth. j. endod. 2010; 36:609–617. pmid: 20307732 10. ee j, fayad mi, johnson br. comparison of endodontic diagnosis and treatment planning decisions using cone-beam volumetric tomography versus periapical radiography. j endod 2014; 40:910–916. pmid: 24935534 11. patel s, wilson r, dawood a, foschi f, mannocci f. the detection of periapical pathosis using digital periapical radiography and cone beam computed tomography part 2: a 1-year post-treatment follow-up. int endod j. 2012; 45:711–723. pmid: 22775142 12. lo giudice r, nicita f, puleio f, alibrandi a, cervino g, lizio as, pantaleo g. accuracy of periapical radiography and cbct in endodontic evaluation. int j dent 2018;2018 pmid: 30410540 http://dentistry3000.pitt.edu/ selective root retreatment: a randomized clinical trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.339 http://dentistry3000.pitt.edu 13. venskutonis t, plotino g, juodzbalys g, mickevičiene l. the importance of cone-beam computed tomography in the management of endodontic problems: a review of the literature. j. endod. 2014; 40:1895–1901. pmid: 25287321 14. liang yh, li g, wesselink pr, wu mk. endodontic outcome predictors identified with periapical radiographs and conebeam computed tomography scans. j endod. 2011; 37:326– 331. pmid: 21329816 15. nudera wj. selective root retreatment: a novel approach. j endod 2015; 41:1382–1388. pmid: 25906919 16. faul f, erdfelder e, lang ag, buchner a. g*power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. in: behavior research methods. psychonomic society inc. 2007;39:175–191. pmid: 17695343 17. ørstavik d, kerekes k, eriksen hm. the periapical index: a scoring system for radiographic assessment of apical periodontitis. dent traumatol 1986; 2:20–34. pmid: 29326509 18. aryanpour s, van nieuwenhuysen jp, d’hoore w. endodontic retreatment decisions: no consensus. int endod j. 2000; 33:208–218. pmid: 11307437 19. ørstavik d. time-course and risk analyses of the development and healing of chronic apical periodontitis in man. int endod j. 1996;29:150–155. pmid: 9206419 20. anjum as, hegde s, mathew s, graduate student p. minimally invasive endodontics-a review. j dent oro-facial res. 2019;15:7788.pmid: 34513652 21. wong aw, zhu x, zhang s, li sk, zhang c, chu ch. treatment time for non-surgical endodontic therapy with or without a magnifying loupe. bmc oral health. 2015; 15:1–6. pmid: 25887978 22. siqueira jf, rôças in, alves frf, campos lc. periradicular status related to the quality of coronal restorations and root canal fillings in a brazilian population. oral surgery, oral med oral pathol oral radiol endodontology. 2005; 100:369– 374. pmid: 16122668 23. kirkevang ll, væth m, hörsted-bindslev p, wenzel a. longitudinal study of periapical and endodontic status in a danish population. int endod j 2006; 39:100–107. pmid: 16454789 24. segura-egea jj, jiménezpinzón a, poyato-ferrera m, velasco-ortega e, ríos-santos jv. periapical status and quality of root fillings and coronal restorations in an adult spanish population. int endod j. 2004;37:525–530. pmid: 15230905 25. borlina sc, de souza v, holland r, murata ss, gomesfilho je, junior ed, de carvalho marion jj, dos anjos neto d. influence of apical foramen widening and sealer on the healing of chronic periapical lesions induced in dogs’ teeth. oral surgery, oral med oral pathol oral radiol endodontology 2010; 109:932– 940. pmid: 20451847 26. norman weller r, niemczyk sp, kim s. incidence and position of the canal isthmus. part 1. mesiobuccal root of the maxillary first molar. j endod. 1995; 21:380–383. pmid: 7499980 27. cleghorn bm, christie wh, dong ccs. root and root canal morphology of the human permanent maxillary first molar: a literature review. j endod. 2006; 32:813–821. pmid: 16934622 28. de pablo óv, estevez r, sánchez mp, heilborn c, cohenca n. root anatomy and canal configuration of the permanent mandibular first molar: a systematic review. j endod. 2010; 36:1919–1931. pmid: 21092807 http://dentistry3000.pitt.edu/ microsoft word ann 2016.docx   vol  4,  no  1  (2016)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2016.47           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     orthodontic  treatment  as  a  possible  trigger  of  periodontal  disease     ann  lin1     1  university  of  pi.sburgh  school  of  dental  medicine,  pi.sburgh  pa,  usa abstract   background:  periodontal  disease  leads  to  the  deteriora.on  of  oral  health,  includ-­‐ ing   loss   of   teeth   and   its   suppor1ng   structures.   it   has   been   established   that   the   cause  of  this  disease  is  mul/factorial,  indica/ng  that  both  genes  and  environmen-­‐ tal  factors  are  intertwined  with  the  forma1on  and  progression  of  periodontal  dis-­‐ ease.  case  descrip+on:  here  we  discuss   the  possibility  of  orthodon4c   treatment   serving   as   a   trigger   to   an   early   development   of   periodontal   disease   in   a   pa2ent   who  has   a   family   history   of   periodon22s.   between  debonding   at   age   17.5   years   and   age   28,   the   pa-ent   experienced   gradual   general   loss   of   a2achment   on   the   mandibular  arch  with   the   le0  mandibular  canine  having  exaggerated  a4achment   loss.   between  age  28  and  29   years,   the  pa6ent  experienced  exacerbated   loss  of   a!achment  at  both  mandibular  canines.  prac#cal  implica#ons:  the  occurrence  of   a"achment  loss  during  and  a2er  orthodon3c  treatment  should  not  be  overlooked.   it  is  crucial  for  orthodon0sts  to  obtain  a  family  history  of  periodontal  disease  and   carefully  monitor  pa0ent’s  periodontal  condi0ons  throughout  the  treatment.       cita%on:  lin,  a.  (2016)  orthodon(c  treatment   as  a  possible  trigger  of  periodontal  disease.   den$stry  3000.  1:a001   doi:10.5195/d3000.2016.47   received:  may  1,  2016   accepted:    may  5,  2016   published:    june  7,  2016   copyright:  ©2016  lin  a.  this   is   an  open  access   ar!cle   licensed   under   a   crea+ve   commons   a!ribu%on  work  4.0  united  states  license.   email:  apl22@pi'.edu   introduction   it  has  been  shown  that  per-­‐ iodontal  disease,  to  be  more  spe-­‐ cific,  periodontitis,  affects  30-­‐50%   of  the  population  in  the  united   states  [1].  this  disease  is  triggered   by  the  imbalance  of  local  factors,   i.e.  bacteria  in  the  oral  cavity,  and   systemic  conditions  of  the  individ-­‐ ual.  despite  its  etiology  being  bac-­‐ terial  infection  such  as  aggregati-­‐ bacter  actinomycetemcomitans,   porphyromonas  gingivalis,   prevotella  intermedius,  etc.,  other   factors  such  as  familial  aggrega-­‐ tion  including  genetic  and  envi-­‐ ronmental  elements  play  a  major   role  in  the  development  of  perio-­‐ dontal  disease.   signs  and  symptoms  of  ear-­‐ ly  stage  periodontal  disease  (i.e.   gingivitis)  will  progress  to  the   more  advanced  periodontitis  if  a   patient  is  susceptible  to  periodon-­‐ tal  disease  in  aspects  of  genetics,   local  factors  in  the  oral  cavity,  oral   hygiene,  environment,  systemic   health  conditions,  etc.  alveolar   bone  and  teeth  loss  are  the  end   results  of  periodontal  disease  if   left  untreated  or  unattended.   teeth  loss  due  to  periodontal  dis-­‐ ease  has  detrimental  effects  be-­‐ cause  the  patient  may  not  be  the   best  candidate  for  complete  den-­‐ tures  due  to  lack  of  mechanical   support  and  retention  from  the   resorbed  alveolar  ridges.  dental   implants  placed  in  periodontal  pa-­‐ tients  may  not  have  the  desired   success  or  survival  rate  [2].     periodontal  disease  has  se-­‐ rious  implications  not  only  be-­‐ cause  a  continuous  bone  loss  will   eventually  lead  tooth  loss  but  also   because  it  is  a  risk  factor  for  cardi-­‐ ovascular  disease  and  other  major   systemic  diseases  such  as  diabetes   mellitus.  it  has  been  investigated   that  periodontal  patients  may  be   low  in  their  neutrophil  cell  counts   and/or  dysfunction  of  neutrophils   so  that  the  body  cannot  provide   protection  to  the  periodontium   against  bacteria  [1].        orthodon(c  treatment  as  a  possible  trigger  of  periodontal  disease   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.47    http://dentistry3000.pitt.edu   2   having  atherosclerotic   problems  already,  cardiovascular   patients  are  further  in  danger   since  bacteria  contributing  to  per-­‐ iodontal  disease  such  as  strepto-­‐ coccus  sanguis  and  porphyromo-­‐ nas  gingivalis  can  enter  the  blood   stream  and  can  adhere  to  athero-­‐ sclerotic  plaques  inducing  blood   clots.  patients  with  diabetes  are  at   an  increased  risk  of  developing   periodontal  disease  because  of  the   increase  in  pro-­‐inflammatory  cy-­‐ tokines  such  as  tnf-­‐α    that  will   increase  the  host  response  to  per-­‐ iodontal  pathogens  and  conse-­‐ quently,  the  host  tissue  itself.       given  the  inconveniences   and  detrimental  life-­‐changing  ef-­‐ fects  this  disease  brings  towards   its  patients,  more  emphasis  in  the   research  to  elucidate  the  mecha-­‐ nisms  and  treatments  of  periodon-­‐ tal  disease  should  be  given.  genet-­‐ ic  testing  at  an  early  age  could   help  patients  with  their  choice  of   lifestyle.  if  done  appropriately,   genetic  testing  can  also  help  pa-­‐ tients  avoid  potential  triggers  of   periodontal  disease  in  life,  which   we  discuss  in  this  report.     orthodontic  treatent  triggering   localized  bone  loss   here  we  describe  a  case  of   a  female  patient  who  presented   with  generalized  mandibular  bone   loss  in  her  late  20s.  the  patient   had  bilateral  maxillary  canines   slightly  overlapping  lateral  incisors   creating  crowding  of  the  four   teeth  after  the  eruption  of  perma-­‐ nent  teeth  and  therefore  received   orthodontic  treatment  at  age  16   when  she  was  in  a  taiwanese  high   school.  prior  to  the  orthodontic   treatment,  the  patient  had  no   signs  of  periodontal  disease;  how-­‐ ever,  her  father  began  to  manifest   signs  of  bone  loss  during  his  col-­‐ lege  years,  suggesting  a  familial   susceptibility.   the  first  trigger   the  patient  had  orthodon-­‐ tic  brackets  for  16  months  and   during  the  course  of  the  treat-­‐ ment,  she  reported  to  her  ortho-­‐ dontist  that  she  was  aware  that   her  gum  lines  had  receded  slightly   on  her  right  maxillary  central  inci-­‐ sor  and  the  mandibular  anterior   teeth.  her  orthodontist  confirmed   her  observation  but  did  not  men-­‐ tion  how  he  would  proceed  on   managing  the  receding  gingiva.  he   simply  stated  that  there  is  nothing   he  can  do  and  “gums  do  not  grow   back,  once  they  are  lost,  they  are   gone  forever.”  disheartened,  the   patient  tried  to  move  past  the  de-­‐ pressing  news.  after  debonding  of   orthodontic  brackets,  the  patient   was  instructed  to  wear  upper  and   lower  arch  removable  retainers   during  sleep.  she  was  never  given   an  option  of  placing  a  lingual  bar   on  her  mandibular  anterior  teeth.     the  patient  noticed  that  af-­‐ ter  debonding  and  a  period  of   time  wearing  her  lower  retainer,   her  mandibular  left  canine  be-­‐ came  more  tipped  towards  the   lingual.  in  addition,  whenever  she   worn  her  mandibular  retainer,   pain  and  strain  on  teeth  occurred   in  her  entire  arch,  with  the  dis-­‐ comfort  exaggerated  on  the  left   mandibular  canine.  she  reported   this  to  her  orthodontist;  however,   reliefing  the  wires  that  rested  on   the  left  mandibular  canine  did  not   show  any  improvement.  the  pa-­‐ tient  still  had  discomfort  and   eventually  discontinued  the  use  of   retainers  after  5  years.  over  the   course  of  10  years  after  debond-­‐ ing,  the  patient  noticed  the  slow      orthodon(c  treatment  as  a  possible  trigger  of  periodontal  disease   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.47    http://dentistry3000.pitt.edu   3   progression  of  gingival  recession   on  the  mandibular  anterior  teeth.   the  second  trigger:  stress   at  age  28,  the  patient  be-­‐ gan  professional  school  and  during   the  intense  and  stressful  first  year   of  courses,  she  noticed  that  her   entire  mandibular  arch  had  gingi-­‐ val  recession  in  addition  to  pro-­‐ gressive  attachment  loss  on  the   mandibular  canines.  at  age  29  and   second  year  of  studies,  she  decid-­‐ ed  that  she  would  be  seeking  per-­‐ iodontal  treatment.   family  history   the  patient’s  father,  now   59  years  old,  had  begun  develop-­‐ ing  periodontal  problems  since   college.  his  father  (the  patient’s   grandfather)  had  complete  den-­‐ tures  by  age  65.  he  got  his  teeth   cleaned  for  the  first  time  in  col-­‐ lege,  at  age  20,  and  reported  “af-­‐ ter  the  hygienist  got  rid  of  my  cal-­‐ culus,  i  felt  that  my  teeth  became   loose  and  a  little  wobbly.”  later  in   graduate  school,  at  age  22,  he   picked  up  the  habit  of  smoking   because  of  the  stress  to  which  he   was  subjected.  he  reports  that  he   does  not  remember  if  he  began  to   notice  bone  loss  around  his  teeth   but  at  age  of  35  during  a  visit  to  a   dental  office,  he  was  diagnosed   with  moderate  periodontitis.  the   patient’s  father  kept  his  condition   under  control  and  has  not  lost   teeth  since  his  diagnosis.  the  pa-­‐ tient’s  mother  does  not  have  peri-­‐ odontal  disease.   discussion   this  case  illustrates  perio-­‐ dontal  bone  loss  triggered  by  or-­‐ thodontic  movement  and/or   stress  aggregating  in  the  same   family.  periodontitis  aggregates  in   this  family  (figure  1)  in  an  appar-­‐ ent  autosomal  dominant  mode  of   inheritance  since  the  father  (and   possibly  the  grandfather)  poten-­‐ tially  had  periodontitis  and  the   patient,  a  potential  third  genera-­‐ tion  case,  also  manifests  signs  of   bone  and  attachment  loss.   previous  studies  [3,4]  have   suggested  familial  aggregation  in   early  onset  periodontitis.  it  was   also  suggested  that  autosomal   recessive  genes  and  environmen-­‐ tal  factors  were  responsible  for   the  transmission  of  the  disease  in   early  onset  periodontitis  due  to   the  shared  genes  among  biologic   family  and  possible  saliva  trans-­‐ mission  of  bacteria  such  as  ag-­‐ gregatibacter  actinomycetem-­‐ comitans  [3].  a  report  of  monozy-­‐ gotic  twins  [4]    strongly  suggested   that  adult  chronic  periodontitis  is   influenced  by  genetic  and  envi-­‐ ronment  factors,  although  there  is   the  notion  that  early  family  envi-­‐ ronment  plays  less  of  a  significant   role  [5].  recent  studies  have  indi-­‐ cated  that  the  interleukin-­‐1  (il1)   gene  serves  as  an  important  regu-­‐ lator  for  the  expression  of  perio-­‐ dontitis.  both  the  t  allele  of   rs17561  of  il1-­‐αα  and  il1-­‐βββββ     rs1143634  t  allele  are  positively   associated  with  periodontitis  [6].   other  polymorphisms  of  il1-­‐α     were  also  shown  to  have  an  asso-­‐ ciation  with  moderate  to  severe   periodontitis  across  different  eth-­‐ nicities  [7].     this  case  report  substanti-­‐ ates  that  periodontal  disease  is  of      orthodon(c  treatment  as  a  possible  trigger  of  periodontal  disease   vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.47    http://dentistry3000.pitt.edu   4   multifactorial  inheritance.  since   one  of  the  risks  of  orthodontic   treatment  is  the  development  of   periodontal  loss,  it  is  not  possible   to  determine  exactly  whether  it   was  the  treatment  or  the  genetic   factor  that  caused  bone  loss.  a   synergistic  effect  of  the  two  fac-­‐ tors  could  be  a  possible  factor  that   contributed  to  the  onset  of  the   periodontal  problems  the  patient   presented  here  had.  however,  it  is   worth  noticing  that  there  was  only   localized  bone  or  attachment  loss   of  buccal  of  the  left  mandibular   canine  after  debonding  and  no   signs  of  generalized  attachment   loss  in  the  mandible  or  maxilla.   between  debonding  and  entering   professional  school,  a  10-­‐year  gap,   the  patient  reported  remembering   gradual  loss  of  attachment  on  the   posterior  mandibular  teeth.     during  the  first  year  in  pro-­‐ fessional  school,  the  patient  began   to  notice  rapid  attachment  loss  on   the  mandibular  incisors  showing   cementum  on  the  buccal  aspect  of   the  teeth  and  the  anterior  teeth   became  sensitive  to  cold  where   the  cementum-­‐enamel  junction   was  exposed  (figure  2).  these   signs  and  symptoms  all  indicate   that  the  environment  factor  (i.e.,   orthodontic  treatment  in  adoles-­‐ cence  and  stressful  life)  deter-­‐ mined  the  course  of  the  disease  in   addition  to  the  possible  variable   il1  gene  expression  of  the  patient.   references   1.  newman  mg,  takei  hh,   klokkevold  pr,  carranza  fa.   (2016).  carranza’s  clinical  perio-­‐ dontology.   2.  dental  implants  in  the  perio-­‐ dontal  patient.  greenstein  g,  ca-­‐ vallaro  j  jr,  tarnow  d.  dent  clin   north  am.  2010  jan;54(1):113-­‐28.   doi:  10.1016/j.cden.2009.08.008.   review.  pmid:20103475   3.  familial  aggregation  of  perio-­‐ dontal  indices.  beaty  th,  colyer   cr,  chang  yc,  liang  ky,  graybeal   jc,  muhammad  nk,  levin  ls.  j   dent  res.  1993  feb;72(2):544-­‐51.   pmid:8423252   4.  chronic  periodontitis  with  famil-­‐ ial  aggregation  and  discordant   identical  twins.  grech  sc.  dentis-­‐ try  3000  2015;  3(1).  doi:   10.5195/d3000.2015.30   5.  genetic  and  inheritance  consid-­‐ erations  in  periodontal  disease.   michalowicz  bs.  curr  opin  perio-­‐ dontol.  1993:11-­‐7.  review.   pmid:8401833   6.  association  between  il-­‐1α   rs17561  and  il-­‐1β  rs1143634  pol-­‐ ymorphisms  and  periodontitis:  a   meta-­‐analysis.  yin  wt,  pan  yp,  lin   l.  genet  mol  res.  2016  feb   5;15(1).  doi:   10.4238/gmr.15017325.   pmid:26909953   7.  association  of  interleukin-­‐1   gene  variations  with  moderate  to   severe  chronic  periodontitis  in   multiple  ethnicities.  wu  x,  offen-­‐ bacher  s,  lόpez  nj,  chen  d,  wang   hy,  rogus  j,  zhou  j,  beck  j,  jiang  s,   bao  x,  wilkins  l,  doucette-­‐stamm   l,  kornman  k.  j  periodontal  res.   2015  feb;50(1):52-­‐61.  doi:   10.1111/jre.12181.  epub  2014  apr   2.  pmid:24690098     differential expression of the irf6 gene in the signs of van der woude syndrome: are distinct genetic modifiers operating? vol 9 no 1 (2021) doi 10.5195/d3000.2021.164 http://dentistry3000.pitt.edu differential expression of the irf6 gene in the signs of van der woude syndrome: are distinct genetic modifiers operating? rosany de oliveira lisboa1,7,9, cláudia maria da rocha martins5, maria elisabete silva santos1,8, danilo leôncio aguiar pereira1,8, flávia martinez de carvalho4, ieda maria orioli2,3, joão farias guerreiro6,8, alexandre rezende vieira7, luiz carlos santana-da-silva1,9,10 ¹ laboratory of inborn errors of metabolism, institute of biological sciences, federal university of pará, pará, brazil. 2 department of genetics, institute of biology, federal university of rio de janeiro, rio de janeiro, brazil. 3 eclamc (latin‐american collaborative study of congenital malformations) at inagemp (national institute of population medical genetics), rio de janeiro, brazil. 4 eclamc at lemc (laboratory of congenital malformation epidemiology), oswaldo cruz institute, fiocruz, rio de janeiro, brazil. 5 department of speech therapy, ophir loyola hospital, pará, brazil. 6 laboratory of human and medical genetics, federal university of pará, pará, brazil. 7 departments of oral biology and pediatric dentistry and center for craniofacial and dental genetics, school of dental medicine, university of pittsburgh, pittsburgh, pennsylvania, usa. 8 genetics and molecular biology postgraduate studies program, federal university of pará, pará, brazil. 9 oncology and medical sciences graduate program, federal university of pará, pará, brazil. 10national institute on population medical genetics, institute of biological sciences, federal university of pará, pará, brazil. abstract objective: the purpose of this study was to report a new variant in the interferon regulatory factor 6 gene (irf6) and to determine phenotype-genotype correlations in a family segregating van der woude syndrome. methods: a five-generation family of 80 individuals segregating vws was investigated using a tabulated pedigree but considering that three individuals registered in the pedigree died shortly after birth, the final sample size was 77 individuals. five individuals had a complete dental clinical examination and molecular analysis performed using direct sequencing of the exon 4 and an adjacent region with 23 base pairs of the irf6 gene. results: features of vws reported in family history were present in 36.4% (28/77) of all family members; of these 57% (16/28) had pits in the lower lip, 36% (10/28) had both pits and orofacial clefts and 7 % (2/28) had only orofacial clefts. developmental dental anomalies were observed in three individuals. the sequence analysis of exon 4 of the irf6 gene carried out for 4 family members revealed the presence of the snp rs7552506 (c.175-5c> g) located in five base pairs before the start of exon. the analysis of exon 4 of the irf6 gene also revealed a new variant c.269g>c (p.ser90thr) which causes exchange of the serine amino acid residue for the threonine residue. conclusion: the c.269g>c(p.ser90thr) can interfere with multimeric interactions and with protein conformation that will be slightly destabilized, because the mutant residue is bigger than the wild-type residue. the phenotypic variations in the cases studied, despite carrying the same genetic mutation, suggest that distinct genetic modifiers operate on the formation of clefts and dental development. keywords: van der woude syndrome; orofacial clefts; irf6 gene citation: lisboa ro, et al. (2021) differential expression of the irf6 gene in the signs of van der woude syndrome: are distinct genetic modifiers operating? dentistry 3000. 1:a001 doi:10.5195/d3000.2021.164 received: march 30, 2021 accepted: august 5, 2021 published: november 4, 2021 copyright: ©2021 lisboa ro, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: lcsantana-pa@hotmail.com http://dentistry3000.pitt.edu/ differential expression of the irf6 gene in the signs of van der woude syndrome: are distinct genetic modifiers operating? vol 9 no 1 (2021) doi 10.5195/d3000.2021.164 http://dentistry3000.pitt.edu introduction orofacial clefts may occur in association with other birth defects or be isolated. the van der woude syndrome (vws) (online mendelian inheritance in man [omim]119300) is the most common and well-known syndrome associated with orofacial clefts, being present in approximately 2% of orofacial cleft cases [1, 2]. vws consists of a rare congenital malformation and presents clinical features in addition to orofacial clefts, such as lower lip pits and occasionally, hypodontia. the frequency of vws in the population varies from 1: 100,000 to 1: 40,000, and there are no reports of differences between males and females [3,4]. the expression of vws is highly variable, both interfamilial and within the same family. in other words, there is a range of associated phenotypes, from the complete absence of visible abnormalities, only the presence of pits in the lower lip, to the combination of these pits with different types of clefts. these types may include aspects such as laterality (unilateral or bilateral), extension (complete and incomplete) and may involve lip, palate or both [2, 5-7]. the etiology of vws has been attributed to mutations in the irf6 gene (interferon regulatory factor 6). however, from a molecular point of view, mutations in the irf6 gene can explain 70% of the cases of vws. several studies also point to the possibility of other genes causing vws, despite the limitations of molecular biology techniques in identifying mutations in many of these genes [8-10]. the irf6 gene is located on the long arm of chromosome 1 (1q32.3 q41) and contains 10 exons; exons 1, 2 and 10 are not translated. the product encoded by the irf6 gene is a 467 amino acid polypeptide [8, 11, 12]. in contrast to the other members of the irf family (interferon regulating factors), irf6 does not participate in the innate immune response, however it is essential for embryonic development, can act as a tumor suppressor and participate in the differentiation of ectodermal tissues [13-15]. recently, a new role assigned to the irf6 ortholog gene is participation in the development of the exocrine gland. this information can be used to investigate the suspicion of disorders in the salivary glands of patients with the two forms of syndrome caused by mutations in the irf6 gene, the vws and popliteal pterygium syndrome (spp), as well as to understand the presence of dental problems, for example, caries, in patients with orofacial clefts [16]. the aim of this study was to report a new variant in the irf6 gene and to determine phenotype-genotype correlations in a family segregating van der woude syndrome. material and methods genealogical data the family participating in the study was located in the service of assistance to the patients with orofacial clefts at ophir loyola hospital (belém pará, brazil). at the time, five members of this family were present, a mother and four children (two girls and two boys). all five had congenital pits in the lower lip and only one of them did not present orofacial cleft but instead presented excess of lower lip. the research was carried out in accordance with the basic ethical principles of the guidelines and standards that regulate research in human beings: autonomy, beneficence, non-maleficence and justice. the project was previously approved by the ethics committee of the health science center of the http://dentistry3000.pitt.edu/ differential expression of the irf6 gene in the signs of van der woude syndrome: are distinct genetic modifiers operating? vol 9 no 1 (2021) doi 10.5195/d3000.2021.164 http://dentistry3000.pitt.edu federal university of pará (caae 4879.0.000.073-10, no140 /10). to obtain genealogical data, the tabulated pedigree method according to polleta et al. (2014) [17] was used. the graphical construction of the pedigree was performed using an online tool from the progeny program, available at: www.progenygenetics.com. information about other members of this family was obtained during three interviews at different times, resulting in the registration of 80 individuals in the pedigree, belonging to five generations of the index family. it was not possible to specify the gender and the presence of any manifestations of vws in only three cases, all of whom had died shortly after birth; of these three cases, two were siamese twin siblings. of the final sample size of 77 individuals analyzed, 36 (45%) were females and 41 (51%) were males (figure 1). phenotypic characteristics family members with one or more phenotypic characteristics of vws were considered affected individuals. these characteristics included cleft lip with or without palate, cleft palate, and the presence of pits, whether bilateral or unilateral. the types of orofacial clefts found were grouped into: cleft lip (cl), cleft lip with cleft palate (clp) and cleft palate (cp). dental information was gathered through a complete dental clinical examination. dna analysis four family members underwent molecular analysis of exon 4 of the irf6 gene. the samples of biological material, 3 to 4 ml of blood, were collected through venipuncture and transferred to test tubes containing edta. for the extraction of dna, the invirsob spin blood mini kit (invitek) was used. for the amplification of the sequence of interest in the polymerase chain (polymerase chain reaction, pcr), the primers used were forward – gctctgggcaatgataggac and reverse accttctccccagcacct. the pcr products were subjected to direct sequencing analysis of exon 4 of irf6 and its exon-intron boundaries based on capillary electrophoresis, using the abi prism bigdye kit, terminator cycle sequencing. the same primers were used for pcr reactions and the abi prism® 3500 genetic analyzer capillary sequencer by applied biosystems. data analysis the family history information obtained from the tabulated pedigree was inserted in the spreadsheet of the microsoft excel 2010 program and later analyzed with the aid of the pedinfo program available at s.a.g.e. (statistical analysis for genetic epidemiology) version 6.3 (2012) (http://darwin.cwru.edu). the analysis of the occurrence of polymorphic mutations or variations in exon 4 of the irf6 gene was performed using the bioedit program version 7.1.9 (http://www.mbio.ncsu.edu/bioed it/bioedit.html). for the in silico prediction of functional effects on the protein, the bioinformatics tools hope (http://www.cmbi.ru.nl/hope) [18], provean (available at: http: // provean.jcvi.org/index.php) [19], sift (available at: http://sift.jcvi.org/) [20] and polyphen-2 (available at: http://genetics.bwh.harvard.edu/ pph2/) [21] were utilized. the data obtained was analyzed using descriptive statistics. results the family did not present a history of consanguineous marriage and from the second generation on, where a family member has a cleft lip and pits, all subsequent generations have more than one affected relative. it is observed through the pedigree that the affected individuals belong only to the paternal, ascending and descending sides in relation to the index case http://dentistry3000.pitt.edu/ differential expression of the irf6 gene in the signs of van der woude syndrome: are distinct genetic modifiers operating? vol 9 no 1 (2021) doi 10.5195/d3000.2021.164 http://dentistry3000.pitt.edu (indicated with an arrow in the pedigree, figure 1). lip pits were present in 26 of the 28 affected individuals and 12 out of 28 individuals had orofacial clefts, with six of them having the lip and palate affected. ten individuals had both pits and orofacial clefts (table 1). out of the five individuals that had a dental examination (table 2), one had permanent dentition and four had primary dentition. three out of five had developmental dental anomalies. phenotype n % pits 16 57 pits and clef lip 4 14 pits and cleft lip with cleft palate 6 21 cleft palate 2 7 total 28 100 figure 1. pedigree showing family members affected by van der woude syndrome. table 1. phenotypic characteristics of van der woude syndrome present in affected family members. http://dentistry3000.pitt.edu/ differential expression of the irf6 gene in the signs of van der woude syndrome: are distinct genetic modifiers operating? vol 9 no 1 (2021) doi 10.5195/d3000.2021.164 http://dentistry3000.pitt.edu table 2. phenotype variation of individuals with irf6 ser90thr variant and van der woude syndrome. affected individuals pits orofacial clefts dental anomalies 27-year-old female, mother of 2, 3, 4, and 5 yes left cleft lip microdontia of right maxillary lateral incisor and agenesis of right mandibular lateral incisor 7-year-old female, halfsister of 3, 4, and 5* yes bilateral cleft lip with cleft palate fusion of mandibular primary left lateral incisor and canine 4-year-old male, brother of 4 and 5 yes no agenesis of mandibular primary left lateral incisor 2-year-old female, sister of 5 yes bilateral cleft lip with cleft palate none evident 1-year-old male yes bilateral cleft lip with cleft palate none evident *biological sample not collected. http://dentistry3000.pitt.edu/ differential expression of the irf6 gene in the signs of van der woude syndrome: are distinct genetic modifiers operating? vol 9 no 1 (2021) doi 10.5195/d3000.2021.164 http://dentistry3000.pitt.edu the sequencing analysis of exon 4 of the irf6 gene, performed for 4 family members (table 2), revealed the presence of the snp rs7552506 (nm_006147.3: c.1755c> g), located five base pairs before exon and revealed a new variant c.269g>c (p.ser90thr) present in heterozygosity in the 4 affected family members (figure 2). the in silico analysis using the provean tool, pointed out that the c.269g> c variant (p.ser90thr) should be considered as deleterious since this variant had a score of -2.704; according to the platform parameters, a variant below the score of -2.5 is considered deleterious. as for the prediction result obtained by the poliphen-2 tool, the new variant was considered potentially deleterious, with a score of 1,000 (sensitivity: 0.00; specificity: 1.00). according to the sift tool this variant is predicted as tolerated with a score of 0.37. one of the most informative prediction tools was hope. the analysis derived from it provided information about the structure of the mutant amino acid residue compared to the wild type of residue, showing that the mutant residue is structurally larger, a factor that can interfere in the multimeric interactions and in the conformation of the protein (figure 3). b a figure 2. electropherograms showing sequence without variant (a) and with the new mutation in exon 4 of the irf6 gene (b). an arrow indicates the altered nucleotide. a b c figure 3. images representing parts of the protein structure with the mutant residue. a. ribbon overview of the protein, in gray, with the mutant residue represented in purple. b and c. detailed view of the representation of the side chains of the mutant amino acid residue, in red compared to the wild residue, in green. source: generated by the hope tool report. http://dentistry3000.pitt.edu/ differential expression of the irf6 gene in the signs of van der woude syndrome: are distinct genetic modifiers operating? vol 9 no 1 (2021) doi 10.5195/d3000.2021.164 http://dentistry3000.pitt.edu discussion for the clinical diagnosis of vws, lower lip pits must be present in one or more members of a family, together with the presence of cleft lip with or without cleft palate and cleft palate [22]. the family investigated in this study presented both the classic vws triad and mixed types of clefts. compared to other families, the percentage of individuals affected in the studied family (36.4%, 28/77) differs little from the percentages found by martellijúnior et al. (2007) [23] who studied two brazilian families with vws, one with 54 and the other with 17 descendants, where the phenotypic characteristics of the syndrome were present in 22.23% and 47.06% of the family members, respectively. due to the fact that in each generation of the studied family there is at least one affected member and they also had at least one of their parents affected, it’s clear that this family follows the expected high penetrance that can vary from 80% to 100% according to previous studies in the literature [2, 24]. variable expression was observed in the studied family, with the main phenotypic manifestation of vws in the affected individuals being congenital lip pits. according to the literature, this type of pits occurs in around 85% of individuals affected by vws and in 64% of cases it is the only manifestation of the syndrome [4, 7]. regarding orofacial clefts, the studied family presented mixed types of clefts, with the most prevalent type (50%) being the clp, a finding similar to other studies [2, 7, 25]. the presence of mixed types of orofacial clefts in the same family is often observed in families with vws. in an attempt to explain this occurrence, it has been proposed that individuals with genetic components that cause vws are subject to some type of multifactorial influence, similar to the non-syndromic form of orofacial clefts [8, 26-28]. furthermore, another possible explanation could be the fact that another gene could be involved in this difference, for example variants in the gene grhl3 (grainyhead like transcription factor 3) are more likely to have cp and less likely to have cl or lip pits than individuals with an irf6 mutations [10] as for the molecular analysis in exon 4, the snp rs7552506 identified in the investigated family members is a polymorphism already described in the literature in studies with families with non-syndromic orofacial clefts and also in studies with families with vws [28, 29]. in the study by pegelow et al. (2008) [30] with 17 swedish families this snp showed an association with clp. in addition, in the same study it was found that when there was combined unilateral and bilateral clp in the same family, there was an association of this polymorphism. this information corroborates the findings in the present study. according to the analysis performed by the hope tool, the c.269g> c variant (p.ser90thr) found in the present study results in a mutant amino acid residue that is larger than the wild type residue, which can influence the conformation of the protein that will be slightly destabilized and, in addition, there may be disturbances in the multimeric interactions. the ser90 position of the polypeptide chain is located in the dna-binding domain (structural motif of the helix-gyrohelix type), which is important for the binding of other molecules and is in contact with the residue of a regulatory domain. therefore, this mutation can affect this interaction and disrupt protein regulation. variants that occur in the irf6 gene are mostly missense-type and occur mainly in http://dentistry3000.pitt.edu/ differential expression of the irf6 gene in the signs of van der woude syndrome: are distinct genetic modifiers operating? vol 9 no 1 (2021) doi 10.5195/d3000.2021.164 http://dentistry3000.pitt.edu the dna-binding or proteinbinding domains; variants in these domains are critical to the function of the irf6 gene [8, 29, 31]. in addition, the silico analysis to verify the effect on the protein conformation of irf6, suggests that this mutation is potentially pathogenic. more specifically, the wild-type residue interacts with the amino acid residue alanine at position 86 of the polypeptide chain; the size difference observed in the mutant residue prevents it from being in the correct position to perform the hydrogen bonding possible in the wild type of residue. only two mutations for position 90 of the protein chain encoded by the irf6 gene are described in the literature, one of which is mutation c. a268g (p.ser90gly) as described by kondo et al. (2002) [8] that causes an exchange of the amino acid residue serine for the amino acid residue glycine. the other mutation, c.g269t (p. ser90il2) as described by lima et al. (2009) [29], promotes the exchange of the amino acid residue serine for the amino acid residue isoleucine. among the dental anomalies associated with vws, the most common among patients is hypodontia [32]. in our study, individuals who presented dental anomalies, as tooth agenesis were in the mandibular arch, which is something already predicted in the literature. syndromic agenesis generally appears associated with other manifestations such as microdontia and the upper lateral incisor is the most frequently affected tooth in the cleft area [33, 34]. considering the data shown in table 2, it’s possible to see the phenotypic variation of the cases presented despite carrying the same genetic mutation, suggesting that distinct genetic modifiers operate on the formation of clefts and the dental development. despite being one of the most studied syndromic forms of orofacial clefts, this study reveals how much remains to be discovered in terms of how genetic factors act underlying the phenotypic expression of vws. in addition, we emphasize that families with this syndrome still go unnoticed in health systems. observationally, many families in turn see congenital lower lip pits as a family trait and therefore fail to seek specialized care and appropriate treatment. thus, it is also expected that this study will contribute in the future to the genetic counseling for these families and also will improve the knowledge and qualifications of health professionals who care for them. acknowledgment the authors would like to thank the national institute of population medical genetics, hospital ophir loyola and the graduate program in oncology and medical science (ppocm) of the federal university of pará (ufpa). this study was supported by the national institute of population medical genetics (465549/2014-4/conselho nacional de desenvolvimento científico e tecnológico cnpq 88887,136366/201700/coodernação de aperfeiçoamento de pessoal de nível superior capes 17/25510000521-0/fundação de amparo à pesquisa do estado do rio grande do sul – fapergs) and prorectory of research and graduate program ufpa (propesp). this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001. conflicts of interest the authors declare no competing interest. references [1] cleft lip a comprehensive review. shkoukani ma, chen m, vong a. http://dentistry3000.pitt.edu/ differential expression of the irf6 gene in the signs of van der woude syndrome: are distinct genetic modifiers operating? vol 9 no 1 (2021) doi 10.5195/d3000.2021.164 http://dentistry3000.pitt.edu front pediatr. 2013 dec. 53 (1): 1 10. pmid: 2440029. [2] van der woude syndrome: clinical presentation in 64 patients. huang jj, hou jw, tan yc, chen kt, lo lj, chen yr. cleft palate craniofac j. 2007 nov;44(6):649-52. pmid: 18177185. [3] van der woude syndrome: a review of 11 cases seen at the lagos university teaching hospital. james o, adeyemo wl, emeka ci, ogunlewe mo, ladeinde al, butali a. afr j paediatr surg. 2014 jan-mar;11(1):525. pmid: 24647295. [4] van der woude syndrome: a review. cardinal signs, epidemiology, associated features, differential diagnosis, expressivity, genetic counselling and treatment. rizos m, spyropoulos mn. eur j orthod. 2004 feb;26(1):17-24. pmid: 14994878. [5] the penetrance and variable expression of the van der woude syndrome: implications for genetic counseling. shprintzen rj, goldberg rb, sidoti ej. cleft palate j. 1980 jan;17(1):52-7. pmid: 6928118. [6] epidemiology of van der woude syndrome from mutational analyses in affected patients from pakistan. malik s, kakar n, hasnain s, ahmad j, wilcox er, naz s. clin genet. 2010 sep;78(3):247-56. pmid: 20184620. [7] van der woude syndrome: dentofacial features and implications for clinical practice. lam ak, david dj, townsend gc, anderson pj. aust dent j. 2010 mar;55(1):51-8. pmid: 20415912. [8] mutations in irf6 cause van der woude and popliteal pterygium syndromes. kondo s, schutte bc, richardson rj, bjork bc, knight as, watanabe y, howard e, de lima rl, daack-hirsch s, sander a, mcdonaldmcginn dm, zackai eh, lammer ej, aylsworth as, ardinger hh, lidral ac, pober br, moreno l, arcos-burgos m, valencia c, houdayer c, bahuau m, moretti-ferreira d, richieri-costa a, dixon mj, murray jc. nat genet. 2002 oct;32(2):285-9. pmid: 12219090. [9] an etiologic regulatory mutation in irf6 with lossand gain-of-function effects. fakhouri wd, rahimov f, attanasio c, kouwenhoven en, ferreira de lima rl, felix tm, nitschke l, huver d, barrons j, kousa ya, leslie e, pennacchio la, van bokhoven h, visel a, zhou h, murray jc, schutte bc. hum mol genet. 2014 may 15;23(10):2711-20. pmid: 24442519. [10] dominant mutations in grhl3 cause van der woude syndrome and disrupt oral periderm development. peyrard-janvid m, leslie ej, kousa ya, smith tl, dunnwald m, magnusson m, lentz ba, unneberg p, fransson i, koillinen hk, rautio j, pegelow m, karsten a, basel-vanagaite l, gordon w, andersen b, svensson t, murray jc, cornell ra, kere j, schutte bc. am j hum genet. 2014 jan 2;94(1):23-32. pmid: 24360809. [11] a preliminary gene map for the van der woude syndrome critical region derived from 900 kb of genomic sequence at 1q32-q41. schutte bc, bjork bc, coppage kb, malik mi, gregory sg, scott dj, brentzell lm, watanabe y, dixon mj, murray jc. genome res. 2000 jan;10(1):81-94. pmid: 10645953. [12] the irf family of transcription factors: inception, impact and implications in oncogenesis. yanai h, negishi h, taniguchi t. oncoimmunology. 2012 nov 1;1(8):1376-1386. pmid: 23243601. [13] maternal interferon regulatory factor 6 is required for the differentiation of primary superficial epithelia in danio and xenopus embryos. sabel jl, d'alençon c, o'brien ek, van otterloo e, lutz k, cuykendall tn, schutte bc, houston dw, cornell ra. dev biol. 2009 jan 1;325(1):249-62. pmid: 19013452. [14] abnormal skin, limb and craniofacial morphogenesis in mice deficient for interferon regulatory factor 6 (irf6). ingraham cr, kinoshita a, kondo s, yang b, sajan s, trout kj, malik mi, dunnwald m, goudy sl, lovett m, murray jc, schutte bc. nat genet. 2006 nov;38(11):1335-40. pmid: 17041601. [15] irf family of transcription factors as regulators of host defense. taniguchi t, ogasawara k, takaoka a, tanaka n. annu rev immunol. 2001;19: 623-55. pmid: 11244049. [16] interferon regulatory factor 6 is necessary for salivary glands and pancreas development. metwalli ka, do ma, nguyen k, mallick s, kin k, farokhnia n, jun g, fakhouri wd. j dent res. 2018 feb;97(2):226-236. pmid: 28898113. http://dentistry3000.pitt.edu/ differential expression of the irf6 gene in the signs of van der woude syndrome: are distinct genetic modifiers operating? vol 9 no 1 (2021) doi 10.5195/d3000.2021.164 http://dentistry3000.pitt.edu [17] genealogical data in population medical genetics: field guidelines.poletta fa, orioli im, castilla ee. genet mol biol. 2014 mar;37(1 suppl):171-85. pmid: 24764752. [18] protein structure analysis of mutations causing inheritable diseases. an e-science approach with life scientist friendly interfaces. venselaar h, te beek ta, kuipers rk, hekkelman ml, vriend g. bmc bioinformatics. 2010 nov 8;11:548. pmid: 21059217. [19] provean web server: a tool to predict the functional effect of amino acid substitutions and indels. choi y, chan ap. bioinformatics. 2015 aug 15;31(16):2745-7. pmid: 25851949. [20] sift web server: predicting effects of amino acid substitutions on proteins. sim nl, kumar p, hu j, henikoff s, schneider g, ng pc. nucleic acids res. 2012 jul;40(web server issue):w452-7. pmid: 22689647. [21] predicting functional effect of human missense mutations using polyphen-2. adzhubei i, jordan dm, sunyaev sr. curr protoc hum genet. 2013 jan; chapter 7:unit7.20. pmid: 23315928. [22] novel irf6 mutations in families with van der woude syndrome and popliteal pterygium syndrome from sub-saharan africa. butali a, mossey pa, adeyemo wl, eshete ma, gaines la, even d, braimah ro, aregbesola bs, rigdon jv, emeka ci, james o, ogunlewe mo, ladeinde al, abate f, hailu t, mohammed i, gravem pe, deribew m, gesses m, adeyemo aa, murray jc. mol genet genomic med. 2014 may;2(3):254-60. pmid: 24936515. [23] clinical and genetic features of van der woude syndrome in two large families in brazil. martelli-junior h, chaves mr, swerts ms, de miranda rt, bonan pr, coletta rd. cleft palate craniofac j. 2007 may;44(3):239-43. pmid: 17477759. [24] congenital symmetrical lower lip pits: van der woude syndrome. ibrahim a, ajike s. oman med j. 2015 jan;30(1):e081. pmid: 30838101. [25] wound complications after cleft repair in children with van der woude syndrome. jones jl, canady jw, brookes jt, wehby gl, l'heureux j, schutte bc, murray jc, dunnwald m. j craniofac surg. 2010 sep;21(5):1350-3. pmid: 20856020. [26] the genetics of isolated orofacial clefts: from genotypes to subphenotypes. jugessur a, farlie pg, kilpatrick n. oral dis. 2009 oct;15(7):437-53. pmid: 19583827. [27] gene/environment causes of cleft lip and/or palate. murray jc. clin genet. 2002 apr;61(4):248-56. pmid: 12030886. [28] identification of irf6 gene variants in three families with van der woude syndrome. tan ec, lim ec, yap sh, lee st, cheng j, por yc, yeow v. int j mol med. 2008 jun;21(6):74751. pmid: 18506368. [29] prevalence and nonrandom distribution of exonic mutations in interferon regulatory factor 6 in 307 families with van der woude syndrome and 37 families with popliteal pterygium syndrome. de lima rl, hoper sa, ghassibe m, cooper me, rorick nk, kondo s, katz l, marazita ml, compton j, bale s, hehr u, dixon mj, daack-hirsch s, boute o, bayet b, revencu n, verellen-dumoulin c, vikkula m, richieri-costa a, moretti-ferreira d, murray jc, schutte bc. genet med. 2009 apr;11(4):241-7. pmid: 19282774. [30] familial non-syndromic cleft lip and palate--analysis of the irf6 gene and clinical phenotypes. pegelow m, peyrard-janvid m, zucchelli m, fransson i, larson o, kere j, larsson c, karsten a. eur j orthod. 2008 apr;30(2):169-75. pmid: 18209213. [31] novel mutations in the irf6 gene for van der woude syndrome. wang x, liu j, zhang h, xiao m, li j, yang c, lin x, wu z, hu l, kong x. hum genet. 2003 oct;113(5):382-6. pmid: 12920575. [32] van der woude syndrome with short review of the literature. deshmukh pk, deshmukh k, mangalgi a, patil s, hugar d, kodangal sf. case rep dent. 2014; 2014:871460. pmid: 25050184. [33] hypoplasia and hypodontia in van der woude syndrome. oberoi s, vargervik k. cleft palate craniofac j. http://dentistry3000.pitt.edu/ differential expression of the irf6 gene in the signs of van der woude syndrome: are distinct genetic modifiers operating? vol 9 no 1 (2021) doi 10.5195/d3000.2021.164 http://dentistry3000.pitt.edu 2005 sep;42(5):459-66. pmid: 16149825. [34] dental agenesis: genetic and clinical perspectives. de coster pj, marks la, martens lc, huysseune a. j oral pathol med. 2009 jan;38(1):1-17. pmid: 18771513. http://dentistry3000.pitt.edu/ microsoft word grech proof 3-23.docx   vol  3,  no  1  (2015)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2015.30           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.   chronic  periodontitis  with  familial  aggregation  and  discordant  identi-­‐ cal  twins   sarah  chris)na  grech1     1  university  of  pittsburgh  school  of  dental  medicine,  pittsburgh  pa,  usa     abstract   chronic  periodon,,s   is  an  advancement  of  gingivi,s  that   involves  progressive  a5achment   loss,  pocke*ng,  and  bone  loss  around  the  teeth.    it  has  been  linked  to  both  gene*c  and  en-­‐ vironmental  factors,  making  it  a  mul3factorial  disease.    this  case  examines  a  family  of  eight   siblings  with   a   50%  prevalence   of   chronic   periodontitis.  a   strong   gene)c   influence   is   not   likely,  since  one  iden-cal  twin  in  this  family  has  chronic  periodon--s,  while  the  other  twin   is   unaffected.     environmental   risk   factors,   including   smoking,   emo-onal   stress,   and   poor   oral  hygiene,  are  present  in  all  affected  individuals,  thus  appearing  to  play  a  significant  role   in   the  development  of  periodon//s   in   this   family.     it   is   important   for   a  den/st   to  under-­‐ stand  the  e)ology  of  the  family’s  chronic  periodon22s  in  order  to  provide  proper  treatment   and  management  of  risk  factors  so  that  good  oral  health  can  be  maintained.       cita%on:  grech,  sc.  (2015)  chronic  periodon,,s   with   familial   aggrega-on   and   discordant   iden--­‐ cal   twins.   den$stry   3000.   1:a001   doi:10.5195/d3000.2015.30   received:  march  9,  2015   accepted:  march  22,  2015   published:  april  5,  2015   copyright:  ©2015  grech.  this  is  an  open  access   ar!cle   licensed   under   a   crea!ve   commons   a!ribu%on  work  4.0  united  states  license.   email:  scg27@pi).edu     background     periodontal   diseases   are   a   broad   category   of   in-lammatory   condi-­‐ tions   that  affect   the   tissue  and  sup-­‐ porting   bone   that   surround   the   teeth.     gingivitis   and   periodontitis   are   the   most   common   periodontal   diseases   [1].      gingivitis   is   a  milder   form,   marked   by   in/lammation   of   the   gingiva.     the   in,lammation   is   a   result  of  the  host  immune  response   to   bacteria   in   the   oral   cavity,   as   plaque  is  the  initiating  factor  of  per-­‐ iodontal   disease   [1].     numerous   other   factors   contribute   to   the   de-­‐ velopment   of   gingivitis,   including   a   genetic   predisposition,   use   of   cer-­‐ tain   medications,   smoking,   preg-­‐ nancy,  puberty,  and  the  presence  of   systemic  disease.    signs  of  gingivitis   can   progress   from   bleeding   on   probing  to  erythema  and  changes  in   texture   of   the   gingiva.     gingivitis   is   reversible,  but   if   left  untreated,   can   progress   to   periodontitis   [1].     peri-­‐ odontitis   has   multiple   forms.     this   study   will   focus   on   the   most   com-­‐ mon   form,   chronic   periodontitis.     chronic   periodontitis   is   de.ined   as   long   term   in+lammation  of   the   sup-­‐ porting   tissues   of   the   teeth   with   progressive   attachment   loss,   pock-­‐ eting,   and   bone   loss   around   the   teeth   [1,   2].     it   typically   occurs   in   adults   and   progresses   slowly,   and   treatment  is  crucial  otherwise  bone   loss   will   continue.     plaque   on   the   teeth  and  gingiva  is  the  primary  ini-­‐ tiating   agent,   and   speci-ic   bacteria   including  porphyromonas  gingivalis,   aggregatibacter   actinomycetem-­‐ comitans,   tannerella   forsythia,   prevotella   intermedia,  and   fusobac-­‐ terium   nucleatum  have   been   linked   to  chronic  periodontitis  [2].    genetic   factors  do  not  seem  to  play  as  much   of  a  role   in  chronic  periodontitis  as   in   other   forms   of   periodontal   dis-­‐ ease.    however,  there  is  still  genetic   in#luence   and   possible   heritable   risks  for  more  aggressive  periodon-­‐ tal   breakdown.       for   example,   ge-­‐ netic  variation  of  interleukin  -­‐1  (il-­‐ 1)   is  associated  with   increased  sus-­‐ ceptibility   to   aggressive   forms   of   chronic   periodontitis   [1,   3].     envi-­‐ ronmental   factors   appear   to   have   the   largest   role   in   the  development   and   progression   of   chronic   perio-­‐ dontitis.    these  factors  include  emo-­‐ tional   stress,   smoking,   diabetes,   in-­‐ creasing   age,   and   the   presence   of   other   systemic   disease   [2].     perio-­‐ dontal   therapy   aims   to   remove   plaque   and   manage   these   risk   fac-­‐ tors   in   order   to   halt   disease   pro-­‐ gression  and  restore  oral  health  [1].        chronic  periodon,,s  with  familial  aggrega,on  and  discordant  iden,cal  twins   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.30    http://dentistry3000.pitt.edu   2   case     this  case  examines  a  family  of  eight   siblings,   four   of   which   currently   have  or  have  had  chronic  periodon-­‐ titis.     the   siblings’   mother   had   chronic   periodontitis   during   her   lifetime.     she   was   a   heavy   smoker   until   age   60,   when   she   suffered   a   heart  attack  and  quit  smoking.      she   continued   to   suffer   from   chronic   obstructive   pulmonary   disease   (copd),   obesity,   and   other   health   issues  as  her  age  increased.  chronic   periodontitis   was   one   of   these   is-­‐ sues.    she  eventually   lost  or  had  all   of   her   teeth   extracted,   becoming   a   complete   denture   patient   around   age   65.     she  was   a  mother   to   eight   children,  including  one  set  of  identi-­‐ cal  twin  boys.      four  sons  are  affect-­‐ ed  with  chronic  periodontitis,  while   two  sons  and  two  daughters  remain   unaffected,  as  shown  in  the  figure  1   pedigree.     individual   iib   is  45  year-­‐ old  lifetime  smoker.    he  was  recent-­‐ ly  diagnosed  with  periodontitis  due   to   periodontal   pocketing   exceeding   8mm  and  mobility  of  multiple  teeth.     he  has  since  had  two  teeth  extract-­‐ ed   and   undergone   scaling   and   root   planing.    he   is   the   identical   twin  of   iic;  however,   individual   iic   remains   unaffected.     individual   iic   is   a   life-­‐ time   smoker  with   liver   disease   but   without   periodontitis.     individual   iie   is   52   year-­‐old   lifetime   smoker   with   a   history   of   colon   cancer   and   periodontal  disease.       individual   iig   is   a   58   year-­‐old   lifetime   smoker   currently   undergoing   treatment   for   chronic   periodontitis.     the   /inal   in-­‐ dividual   affected,   iih,   is   a   60   year-­‐ old  lifetime  smoker  with  a  history  of   drug   abuse   and   periodontitis.     he   had  full-­‐mouth  extractions  at  age  58   and   began   wearing   complete   den-­‐ tures.    he  no  longer  has  periodonti-­‐ tis   and   continues   to   maintain   his   oral  health.      individuals  iia,  iid,  and   iif   remain   unaffected;   they   are   all   non-­‐smokers   with   good   oral   hy-­‐ giene  and  minimal  emotional  stress.     discussion   this   case   demonstrates   the   multi-­‐ factorial   nature   of   chronic   perio-­‐ dontitis.     although   genetic   factors   may  make  the  children  of  this  fami-­‐ ly   more   susceptible   to   periodontal   disease,   they  do  not  seem  to  be  the   major   determinant.     the   set   of   twins,  iib  and  iic,  support  this  claim.     the   twins   are   identical,   meaning   they   share   the   same   dna,   yet   iib   has   chronic   periodontitis   and   iic   does  not.    thus,  there  must  be  addi-­‐ tional   factors   contributing   to   the   development  of   the  disease   in   indi-­‐ vidual   iib.     these   factors   are   most   likely  environmental  factors,  includ-­‐ ing   smoking,   poor   oral   care   and   emotional   stress.     since   all   four   af-­‐ fected   individuals   are   males   and   their  mother  was   affected,   x-­‐linked   inheritance   could   be   considered.     however,   this   has   not   been   shown   to   be   a   common   mode   of   inher-­‐ itance   of   chronic   periodontitis,   as   the   speci)ic   genetic   in)luences   of   chronic   periodontitis   still   remain   uncertain.     the   possible   environ-­‐ mental  risks  of  the  affected  individ-­‐ uals   of   this   case   have   been   speci1i-­‐ cally  linked  to  chronic  periodontitis.       furthermore,  three  of  the  four  unaf-­‐ fected   individuals   do   not   display   any   of   the   environmental   risk   fac-­‐ tors   of   chronic   periodontitis.     the   environmental   factors   thus  seem  to   play   a   signi*icant   role   in   the   devel-­‐ opment   of   periodontal   disease   in   this   family,   with   genetics   possibly   contributing   additional   susceptibil-­‐ ity   or   risks.     it   is   important   for   a   dentist   to   understand   the   etiology   of  the  family’s  chronic  periodontitis   in   order   to   provide   proper   treat-­‐ ment   and   management   of   risk   fac-­‐ tors  so  that  oral  health  can  be  main-­‐ tained.    if  the  environmental  factors   are   not   managed,   long-­‐term   treat-­‐ ment   will   not   be   successful.     bone   and   tooth   loss  will   continue   unless   the   periodontal   disease   is   success-­‐ fully   treated  [1].        successful   treat-­‐ ment   involves   mechanical   debride-­‐ ment  via  scaling  and  root  planing,  in   addition   to   managing   environmen-­‐ tal   factors  that  may  be  contributing   to   the   disease.     depending   on   the   severity,   some   effects   of   the   perio-­‐ dontal   destruction   may   be   irre-­‐ versible.    patient   speci0ic   treatment   planning  must   be   completed   in   or-­‐ der  to  determine  the  best  treatment   option,  which  may  include  tissue  or   bone   grafts,   extractions,   and   tooth   replacement.   ia   ib                                    iia        iib                            iic                                  iid                                iie                              iif                                  iig                              iih     figure  1.    pedigree  of  chronic  periodontitis  prevalence  over  two  generations.    affected  indi-­‐ viduals  marked  in  red.    iib  and  iic  represent  identical  twins;  iib  is  affected  with  periodonti-­‐ tis  and  iic  is  not.      chronic  periodon,,s  with  familial  aggrega,on  and  discordant  iden,cal  twins   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.30    http://dentistry3000.pitt.edu   3     references     1.     newman  m,   carranza   fa,   takei   h,   klokkevold   pr   (2006).     carran-­‐ za’s   clinical   periodontology:   tenth   edition.     philadelphia:   saunders.     494  p.     2.    genetic  and  heritable  risk  factors   in   periodontal   disease.   b.   s.   michalowicz.   journal   of   periodon-­‐ tology,   1994:may;65(5):   479–488.     pmid:  8046564.     3.    the  effect  of  il-­‐4  gene  polymor-­‐ phisms   on   cytokine   production   in   patients   with   chronic   periodontitis   and   in   healthy   controls.   bartoya   j,   linhartova   pb,   podzimek   s,   et   al.     mediators   of   in.lammation,   2014.   pmid:   25530681.   (epub   ahead   of   print).       microsoft word 254 2022.docx associa'on of sella turcica bridging and morphologic characteris'cs with palatally impacted canine in lateral cephalograms. vol 10 no 1 (2022) doi 10.5195/d3000.2022.254 h#p://den*stry3000.pi#.edu association of sella turcica bridging and morphologic characteristics with palatally impacted canine in lateral cephalograms. nikoo ghasemi1, soraya akhoondi2, zohre reyhani3, nima motamed4, azin nourian5 1dental student, school of dentistry, zanjan university of medical sciences, zanjan, iran 2dentist, private practice, zanjan, iran 3assistant professor, department of dental radiology, school of dentistry, zanjan university of medical sciences, zanjan, iran 4associate professor, department of social medicine, zanjan university of medical sciences, zanjan, iran 5assistant professor, orthodontic department, zanjan university of medical sciences, zanjan, iran abstract introduction: researchers have been interested in the rela'onship between sella turcica bridging and various dental anomalies. this study inves'gates the associa'on of sella turcica bridging and morphologic characteris'cs with the palatally impacted canine tooth. materials and methods: orthodon'c records with high-quality lateral cephalometric and panoramic images were chosen. the study comprised 30 pa'ents with palatally impacted canines (17 females and 13 males) and 30 controls with erupted canines (15 males and 15 females). the measurements were performed using the scanora 5.2.6 sokware. further, the relevant data was collected and analyzed using spss sokware version 22. descrip've sta's'cal analysis was used to inves'gate data aker their valida'on, with ttests and chi-square tests to compare the two groups. the regression test was used to lessen the effects of confounding variables. results: in the study group, 5 pa'ents had type iii sella turcica bridging, whereas 2 pa'ents in the control group had type iii sella turcica bridging. results showed a sta's'cally significant difference between the two groups (p-value=0.017). in the study group, the interclinoid distance and canine tooth angle were significantly lower than the control group (p-value=0.001, p-value=0.001). however, there were no significant differences in the depth and anterior-posterior diameter of sella turcica between the two groups (p-value=0.543, pvalue=0.766). in terms of age (p-value = 0.400) and gender (p-value = 0.605), there was no significant difference between the two groups. conclusion: the palatally canine impac'on may be associated with the bridging of the sella turcica. therefore, sella turcica bridging can be used as a diagnos'c parameter for palatally canine impac'on. keywords: tooth abnormali'es; orthodon'cs; sella turcica cita9on: ghasemi, n et al. (2022) associa9on of sella turcica bridging and morphologic characteris9cs with palatally impacted canine in lateral cephalograms. den9stry 3000. 1:a001 doi:10.5195/d3000.2022.254 received: october 16, 2021 accepted: april 23, 2022 published: june 23, 2022 copyright: ©2022 ghasemi, n et al. this is an open access ar9cle licensed under a crea9ve commons asribu9on work 4.0 united states license. email: azin.nourian@gmail.com introduction sella turcica, an important anatomical landmark in lateral cephalometric imaging, is an area in the form of a saddle that is based in the middle part of the cranial fossa[1]. it is also an important finding for orthodontists since many pathological processes are shown by associa'on of sella turcica bridging and morphologic characteris'cs with palatally impacted canine in lateral cephalograms. vol 10 no 1 (2022) doi 10.5195/d3000.2022.254 h#p://den*stry3000.pi#.edu changes in the form and dimensions of the sella turcica. sella turcica is classified into three groups: circular, oval and flat shapes. the most common among them is the circular, followed by oval and flat respectively. [2]. sella turcica bridging could be a common variation of sella turcica, possibly related to numerous systemic developmental syndromes[3]. according to the theories expressing that mutations of the hox hedgehog gene negatively affect the midface, teeth and part of the sella turcica development, any modifications in the developmental level could result in sella turcica bridging that might further cause dental anomalies[4]. dental anomalies can be caused by various factors, including genetic, epigenetic, and environmental influences[5]. although many studies have been conducted on the common occurrence of various dental irregularities, few have looked into the connection between bridging of the sella turcica and dental anomalies[6]. canine impaction is one of the types of dental anomalies. for many years, orthodontists have struggled with canine impaction. examinations can be an effective way to reduce the cost and treatment time of the patients[7]. excluding the third molars, the maxillary canine is the most commonly impacted[8]. furthermore, impaction is more common in women[9]. canine impaction in about 15% of cases is located buccally while in about 85% it is located palatally[10]. buccal canine impaction is originally caused by dental crowding[11]. while palatally canine impactions frequently, but not always, are found in dentitions with various anomalies[12]. some studies claim that sella turcica bridging is more common in patients with impacted canines[7, 13], while others claim there is no significant link between impacted canines and sella turcica bridging[14, 15]. as there are few studies about the association of sella turcica bridging and morphologic characteristics with canine impaction as well as contradictory comments. further studies are needed [16]. therefore, this study aims to determine sella turcica morphologic characteristics and the possible relationship between the sella turcica bridging and the palatally canine impaction in lateral cephalometric radiographs. material and methods this retrospective case-control study with convenience sampling evaluated 30 patients (17 females and 13 males) with at least one palatally impacted maxillary canine compared with 30 patients (15 females and 15 males) with normally erupted canines matched in terms of age and gender. this study was conducted to determine the association between palatally canine impaction and sella turcica bridging using orthodontic patients' pretreatment radiographic records referred to private oral and maxillofacial radiology centers in zanjan city (iran), from 2017 to 2020, with an equal racial background as iranian root. approval from the ethics committee of zanjan dental school was obtained (number ir.zums.rec.1398.417). all the patients who participated in the study signed written informed consent. the inclusion criteria were 13-30 years old orthodontic patients with good quality standardized panoramic and lateral cephalograms were included in the study. patients who had dental malformations and syndromes, significant illnesses or medical conditions as well as cases with blurred radiographic appearance and a history of orthognathic surgery, orthodontic treatment, cleft lip and palate, maxillofacial pathology, trauma were excluded from this study. the study group consisted of 30 patients (17 females and 13 males) with at least one palatally impacted maxillary canine and the control group of 30 patients (15 females and 15 males) with normally erupted canines no dental abnormalities. the two groups were matched in terms of age and sex. (table 1) the pretreatment orthodontic panoramic and lateral cephalometric radiographs were used to diagnose tooth impaction or eruption[18]. to determine the palatally impaction of canine teeth, panoramic and lateral cephalometry radiographs and all other sources of information available, such as cone beam associa'on of sella turcica bridging and morphologic characteris'cs with palatally impacted canine in lateral cephalograms. vol 10 no 1 (2022) doi 10.5195/d3000.2022.254 h#p://den*stry3000.pi#.edu computed tomography, occlusal view radiographs were used, in some cases referring to orthodontic diagnostic records. the radiographic parameters studied were the interclinoid distance (the distance between the tip of the tuberculum sellae and the dorsume sellae), sella turcica depth (the length of a line dropped perpendicularly from the line above to the deepest point on the sella floor), anteroposterior diameter of the sella turcica (the longest distance between the tip of the tuberculum sellae and the posterior contour of the sella), sella turcica bridging and canine angle. the scanora 5.2.6 software was used to conduct measurements. the line drawn from the highest point of the condyles was used to compute the canine angle. (figure 1) a standard scoring scale devised by leonardi et al was used to assess the degree of sella turcica bridging[19]. type i: normal sella turcica, interclinoid distance equal to or greater than three-quarters of the largest anteroposterior diameter. type ii: incomplete calcification, interclinoid distance equal to or lower than three-quarters of the largest anteroposterior diameter. type iii: complete calcification, only the sella turcica diaphragm is visible on radiography. (figure 2) lateral cephalometric radiographs were used to assess the sella turcica, performed by an expert oral and maxillofacial radiologist. to confirm the accuracy of the measures, 10 samples were re-examined at twoweek intervals to ensure the reliability and reproducibility of the assessments. no significant difference was found (p-value>0.05). the intraclass correlation coefficient (icc) is presented in the table 2. spss software version 22 was used to analyze data. descriptive statistical analysis was utilized and to compare the two groups, t-test and chi-square tests were performed. results a total of 60 radiographic records of patients were investigated. to assess data distribution, kolmogorovsmirnov test was employed. the results of the normality test showed normal distribution (p-value>0.05). we used t-test, two groups proportion and chi-square test to compare the data between two groups. the mean±sd of the age of control group and study group was 17.87±3.04 and 18.57±3.35, respectively. the study group consisted of 30 patients (17 females and 13 males) and the control group of 30 patients (15 females and 15 males). in terms of age (p value = 0.400) and gender (p value = 0.605), there was no significant difference between the variables intraclass correlation coefficient (icc) interclinoid distance 0.966 sella turcica depth 0.921 ant-post diameter of sella turcica 0.947 canine angle 0.928 figure 1. two views of scanora version 5.2.6 software showing how to measure variables of interclinoid distance, sella turcica depth, anteroposterior diameter of sella turcica bridging and canine angle figure 2. examples of different types of sella turcica bridging, a: type i, b: type ii, c: type iii table 2: method error according to intraclass correlation coefficient associa'on of sella turcica bridging and morphologic characteris'cs with palatally impacted canine in lateral cephalograms. vol 10 no 1 (2022) doi 10.5195/d3000.2022.254 h#p://den*stry3000.pi#.edu two groups of patients. in the study group 33.3%(n=10), 50%(n=15) and 16.7%(n=5) of patients had type i, type ii and type iii of sella turcica bridging, respectively whereas in the control group, 70%(n=21), 23.3%(n=7), 6.7%(n=2) of patients had type i, type ii and type iii of sella turcica bridging, respectively. this resulted in a statistically significant difference(p-value=0.017). (table 1) in the study group 20 patients had unilateral and 10 patients had bilateral canine impaction. while the total occurrence of canine impaction in maxillary left and right side was 54.5% (n=12) and 45.5% (n=10) in female respectively, this was 55.6% (n=10) and 44.4% (n=8) in male (z=0.06, p-value=0.949). the measures of study group's interclinoid distance was significantly shorter than the control group (pvalue = 0.001) as well as the canine angle which was significantly lower in the study group. (pvalue = 0.001). however, the depth of the sella turcica and anteroposterior diameter of sella was not significantly different between the two groups with p-value 0.543 and 0.766, respectively. (table 3) discussion the sella turcica is a structure that may be easily traced in cephalometric analysis and may be seen clearly on lateral cephalometric radiographs. the shape of the sella turcica is very important in orthodontics because it serves as a reference point in orthodontic analysis[20]. we opted to investigate the association between sella turcica bridging and canine impaction and compare it to normal canine eruption due to the importance of its characteristics, such as morphology and size, as factors that can alter the pituitary gland’s pathologic status. the results of this study revealed that patients with canine impaction had considerably higher sella turcica bridging. furthermore, the interclinoidal distance and canine angle of the study group were significantly lower than those of the control group. the depth of the sella turcica and the anteroposterior width of the sella turcica did not differ significantly. the findings of this investigation were in line with the majority of previous studies[7, 13, 19]. in line with the present study, dadgar et al.(2020) conducted a study in 46 patients with canine impaction and 46 patients with normal canine eruption. the results showed that palatal canine impaction is positively associated with both atlas arcuate foramen and sella turcica bridging. however, the arcuate foramen was not investigated, but morphological characteristics of sella turcica were defined in the present study[13]. in the baidas et al.(2018) study there was no difference in the morphology of the sella turcica between patients with and without canine impaction. the incidence of sella turcica bridging was observed to be considerably greater in patients with canine impaction (p-value 0.0001)[19]. the variables study group control group p-value age 18.57±3.35 17.87±3.04 0.400 male/female 13/17 15/15 0.605 sella turcica bridging type i 10(33.3%) 21(70%) 0.017 type ii 15(50%) 7(23.3%) type iii 5(16.7%) 2(6.7%) variables mean±sd p-value study group control group interclinoid distance 4.86±0.47 5.96±1.20 0.001 sella turcica depth 7.30±1.57 6.89±0.94 0.543 ant-post diameter of sella turcica 9.04±1.98 8.77±1.67 0.766 canine angle 57.40±12.6 3 89.66±5.28 0.001 table 1: demographic and types of sella turcica bridging in the two groups table 3: measures of sella turcica morphologic characteristics in two groups associa'on of sella turcica bridging and morphologic characteris'cs with palatally impacted canine in lateral cephalograms. vol 10 no 1 (2022) doi 10.5195/d3000.2022.254 h#p://den*stry3000.pi#.edu results are consistent with the present study. haji ghadimi et al. (2017) concluded that the impacted canine is related to the sella turcica bridging occurrence and severity in a case control study on lateral cephalometric records of 35 patients with canine impaction and 75 patients with normal canine eruption. the results are similar to the present study, although in the present study, morphological characteristics of sella turcica were also investigated [3]. in a study, batool ali et al. (2014) compared 31 patients with canine impaction to 70 patients with normal canine eruption. the study's findings were consistent with the current study, revealing that the incidence of sella turcica bridging was considerably higher in the study group than in the control group (p value = 0.001). in addition, the sagittal interclinoidal distance was significantly reduced. (p value=0.028) [7]. leonardi et al. (2006) also studied 34 individuals with maxillary impacted canine anomalies and determined that there is a relationship between impacted canine and sella turcica bridging, although there are no significant differences based on age or gender [6]. in this investigation as well, we observed no significant association between age and sex. unlike the current study, ortiz et al. (2018) discovered no significant relationship between maxillary palatally impacted canine and sella turcica bridging in 38 patients with maxillary palatally impacted canine and 38 patients without any dental anomaly. maybe their different results from our study are due to their diagnostic radiographic records (cone beam computed tomography images) [14]. in their investigation, el wak et al. found no significant link between sella turcica bridging and canine impaction, which contradicted our findings[15]. however, despite other research and our investigation revealing a significant relationship, these two studies with small sample sizes found no significant association between canine impaction and sella turcica bridging. there are several theories on the pathophysiologic cause of the association of canine impaction and sella turcica bridging. the sella turcica is the primary site for neural crest cell migration. mutations in the homebox, hox, or sonic hedgehog genes have a deleterious impact on the growth of the central section of the face, teeth, and sections of the sella turcica. because canine teeth and sella turcica originate from the same place, modifications throughout growth and development might result in sella turcica bridging and canine impaction[20, 21]. as a result, this anomaly could be used as a marker, allowing orthodontists and radiologists to detect and predict pituitary gland disorders in this critical location. the scope of our research was constrained by a few variables. the study used lateral cephalograms, which are a two-dimensional representation of a threedimensional subject with its own set of landmark recognition and tracing errors. due to the covid-19 pandemic, few patients were involved in our study, but although it resulted in a smaller sample size, this sample size was adequate to obtain proper test power in terms of most variables which were investigated in this study. further studies in larger communities with different ethnic backgrounds and more thorough examination of the patient's profile (e.g., height, bmi, family history, underlying condition, etc.) and using cone beam computed tomographic records instead of lateral cephalometric records are recommended. conclusion the results showed that the incidence of sella turcica bridging was greater in individuals with maxillary canine impaction than in the control group. in addition, the interclinoidal distance and canine angle were lower in the study group than in the control group, despite the fact that the depth of the sella turcica and the anteriorposterior diameter of the sella turcica were not substantially different. as a conclusion, palatally canine impaction may be related to the bridging of the sella turcica. therefore, sella turcica bridging can associa'on of sella turcica bridging and morphologic characteris'cs with palatally impacted canine in lateral cephalograms. vol 10 no 1 (2022) doi 10.5195/d3000.2022.254 h#p://den*stry3000.pi#.edu be used as a diagnostic parameter for palatally canine impaction. acknowledgement the present study was extracted from a thesis approved and funded by the faculty of dentistry of zanjan university of medical sciences (ethics code: ir.zums.rec.1398.417). the authors would like to express their gratitude to all staffs who cooperated in this research. references 1. bridging and dimensions of sella turcica in subjects treated by surgical-orthodon7c means or orthodon7cs only. jones r, faqir a, mille@ d, moos k, mchugh s. the angle orthodon7st. 2005;75(5):714-8. pmid: 16279819. 2. sella turcica-its importance in orthodon7cs and craniofacial morphology. sathyanarayana hp, kailasam v, chitharanjan ab. dental research journal. 2013 sep;10(5):571. pmid: 24348611 3. associa7ons among sella turcica bridging, atlas arcuate foramen (pon7culus pos7cus) development, atlas posterior arch deficiency, and the occurrence of palatally displaced canine impac7on. ghadimi mh, amini f, hamedi s, rakhshan v. american journal of orthodon7cs and dentofacial orthopedics. 2017;151(3):51320. pmid: 28257736 4. neural crest contribu7on to mammalian tooth forma7on. mile7ch i, sharpe pt. birth defects research part c: embryo today: reviews. 2004;72(2):20012. pmid: 15269893 5. mul7level complex interac7ons between gene7c, epigene7c and environmental factors in the ae7ology of anomalies of dental development. brook ah. archives of oral biology. 2009 dec 1;54:s3-17. pmid: 19913215 6. a sella turcica bridge in subjects with dental anomalies. leonardi r, barbato e, vichi m, caltabiano m. the european journal of orthodon7cs. 2006;28(6):580-5. pmid: 16954179 7. associa7on between sella turcica bridging and palatal canine impac7on. ali b, shaikh a, fida m. american journal of orthodon7cs and dentofacial orthopedics. 2014;146(4):43741. pmid: 25263146 8. frequency of impacted teeth and categoriza7on of impacted canines: a retrospec7ve radiographic study using orthopantomograms. al-zoubi h, alharbi aa, ferguson dj, zafar ms. european journal of den7stry. 2017 jan;11(01):11721. pmid: 28435377 9. a survey of 3,874 rou7ne fullmouth radiographs: ii. a study of impacted teeth. dachi sf, howell fv. oral surgery, oral medicine, oral pathology. 1961;14(10):1165-9. pmid: 13883048 10. the ectopic maxillary canine: a review. bri7sh journal of orthodon7cs. mcsherry pf. 1998;25(3):209-16. pmid: 9800020 11. is there a rela7onship between third-molar impac7on types and the dimensional/angular measurement values of posterior mandible according to pell & gregory/winter classifica7on? gümrükçü z, balaban e, karabağ m. oral radiology. 2020:1-7. pmid: 31925627 12. maxillary tooth size varia7on in den77ons with palatal canine displacement. becker a, sharabi s, chaushu s. the european journal of orthodon7cs. 2002;24(3):313-8. pmid: 12143095 13. associations among palatal impaction of canine, sella turcica bridging, and ponticulus posticus (atlas arcuate foramen). dadgar s, alimohamadi m, rajabi n, rakhshan v, sobouti f. surgical and radiologic anatomy. 2021;43(1):93-9. pmid: 32816070 14. a cbct inves7ga7on of the associa7on between sellaturcica bridging and maxillary palatal canine impac7on. or7z pm, tabbaa s, flores-mir c, aljewair t. biomed research interna7onal. 2018;2018. pmid: 29682544 15. associa7on between sella turcica bridging and palatal canine impac7on: evalua7on using lateral cephalograms and cbct. el wak t, akl r, ma7 m, khoury e, ghoubril j. interna7onal orthodon7cs. 2018;16(2):338-48. pmid: 29628424 16. sella turcica morphology on cephalometric radiographs and dental abnormali7es—is there any associa7on?—systema7c review. jankowski t, jedliński m, grocholewicz k, janiszewskaolszowska j. interna7onal journal of environmental research and public health. 2021;18(9):4456. pmid: 33922272 17. an associa7on between sella turcica bridging and dental transposi7on. leonardi r, farella m, cobourne mt. the european journal of orthodon7cs. 2011;33(4):461-5. pmid: 21212168 18. skeletal anomalies and normal variants in pa7ents with palatally displaced canines. leonardi r, barbato e, vichi m, associa'on of sella turcica bridging and morphologic characteris'cs with palatally impacted canine in lateral cephalograms. vol 10 no 1 (2022) doi 10.5195/d3000.2022.254 h#p://den*stry3000.pi#.edu caltabiano m. the angle orthodon7st. 2009;79(4):72732. pmid: 19537879 19. associa7on of sella turcica bridging with palatal canine impac7on in skeletal class i and class ii. baidas lf, al-kawari hm, al-obaidan z, al-marhoon a, alshahrani s. clinical, cosme7c and inves7ga7onal den7stry. 2018;10:179. pmid: 30154673 20. rela7onship between the maxillary transverse dimension and palatally displaced canines: a cone-beam computed tomographic study. hong w-h, radfar r, chung c-h. the angle orthodon7st. 2015;85(3):440-5. pmid: 25098187 21. role of homeobox genes in the pa@erning, specifica7on, and differen7a7on of ectodermal appendages in mammals. duverger o, morasso mi. journal of cellular physiology. 2008;216(2):337-46. pmid: 18459147 microsoft word 194 2022.docx impact of covid-19 on the frequency of dental appointments and types of dental health care services vol 10 no 1 (2022) doi 10.5195/d3000.2022.194 h#p://den*stry3000.pi#.edu impact of covid-19 on the frequency of dental appointments and types of dental health care services muhanad l. alshami1, ali a. abdulkareem2, sarhang s. gul3, yasir a. shareef4 1 department of dentistry, dijlah university college, baghdad, iraq 2 college of dentistry, university of baghdad, baghdad, iraq 3 college of dentistry, university of sulaimani, sulaimani, iraq 4 department of oral diagnosis, al-amiriya specialist dental centre, ministry of health, baghdad, iraq abstract objectives: to inves>gate the impact of the covid-19 pandemic on the frequency of provision of dental appointments and types of dental care services. materials and methods: the dental records of three dental health services, over 3 months, in the year before (2019) and a year during (2020) the pandemic were retrieved. the dental records were categorized according to the referral ajer diagnosis into prosthodon>cs, pedodon>cs, orthodon>cs, conserva>ve, endodon>cs, periodon>cs, and oral surgery. the demographic variables and types of treatment provided were recorded and analyzed. the frequency of appointments was determined from overall dental records and departmental dental records. chi square test was used to determine the effect of gender and age on dental appointments. results: according to the dental records for 2019, 7286 appointments were provided, and this number decreased by 2.4-fold to 3090 appointments in 2020. the records indicated that the highest decreases were in non-urgent dental appointments in the periodon>cs, orthodon>cs and conserva>ve departments (8, 8, and 4.6-fold, respec>vely). sta>s>cally significant differences in dental pre-pandemic and pandemic appointments were iden>fied for gender and age group. female and older (>64 years old) subjects appeared to be less willing to avend dental appointments (or=1.2 and 1.3, respec>vely) compared to their male and younger counterparts. conclusion: the covid-19 pandemic has had significant influence on decreasing avendance at dental appointments and the records show that the highest decreases were in nonurgent dental departments. keywords: covid-19; pandemics; impact; dental care; dental health services cita9on: abdulkareem a et al. (2022) impact of covid19 on the frequency of dental appointments and types of dental health care services den9stry 3000. 1:a001 doi:10.5195/d3000.2022.194 received: june 11, 2021 accepted: september 28, 2021 published: may 6, 2022 copyright: ©2022 abdulkareem a et al. this is an open access ar9cle licensed under a crea9ve commons awribu9on work 4.0 united states license. email: ali.abbas@codental.uobaghdad.edu.iq introduction the outbreak of covid-19 in december 2019 in wuhan, china has obliged many countries to take drastic measures to control the wide spread of infection, such as through surveillance and lockdown of cities [1]. given its novelty and rapid transmission, authorities have found it necessary to provide certain recommendations for public places and health sectors. health care workers and dentists are categorized as at high risk of infection [2] and evidence indicates that this could be due to the high risk of transmission posed by aerosol generating procedures (agp) [3, 4]. according to the world health organization (who) and american dental association (ada), during the outbreak, dental clinics should, in general, be closed. however, for emergency and urgent cases, dental treatment can be carried out with implementation of safety and precautionary measures provided by these two bodies [5, 6]. pain and swelling have been considered as the most common impact of covid-19 on the frequency of dental appointments and types of dental health care services vol 10 no 1 (2022) doi 10.5195/d3000.2022.194 h#p://den*stry3000.pi#.edu dental emergencies [7]. furthermore, in response to requests from several usa states, ada developed a guideline that would allow dental care providers to manage emergency works only [8]. the dental emergency cases include but are not limited to pain associated with pericoronitis, pulpitis, dry socket following tooth extraction, local swellings/abscesses, dental trauma, tooth fracture and failure of temporary restorations [9]. this means that provision of other dental procedures, such as routine checkups, professional cleaning, making and fitting dental prostheses, treating non-painful caries, orthodontic appointments, cosmetic works, among others, has had to cease. meanwhile, the emergence of covid19 has had a significant impact on both dentists and patients. for example, high levels of fear and anxiety have been reported by dental care providers during the outbreak [10, 11], and dentists have been obliged to take drastic actions including practice modification and optimizing their safety and precautionary measures [12]. in addition, as in many life sectors, the pandemic and lockdown have had a profound economic impact on dentistry and the economic consequences are suggested to be disproportional; thus, urgent action from authorities has been called for [12, 13]. similarly, high levels of fear and anxiety were reported amongst patients seeking dental treatment, which consequently decreased their willingness to undergo dental treatments and increased the risk of oral mucosal diseases [14, 15]. many countries, including iraq, adopted the who and ada guidelines for work carried out by dental professionals during the peak of the pandemic. the exact impacts of covid-19 on numbers of dental appointments and types of dental treatment provided needed to be explored. therefore, this study aimed to examine the overall impact of covid-19 on dental appointments and types of dental procedure performed via comparing the prepandemic and pandemic dental appointments records. material and methods study design the current study was a retrospective cross-sectional study based on the records from three main governmental primary dental care centers in the city of baghdad, iraq. the patients can attend these centers directly without referral or prior arrangement. the survey covered three months (june, july and august) of the year 2020, the period of the strictest lockdown measures, as compared to the same period in the previous year. access to the records was officially granted by the iraqi ministry of health (ref #72156 in 23/11/2020). furthermore, the study was approved by the ethical committee of the college of dentistry, university of sulaimani (ethical approval # 16/21) in accordance with helsinki declaration and its later amendments. eligibility criteria and data source records from the dental centers were organized according to patient referrals to different departments for the required treatment. all records for referring new patients were included while those with missing information or for patients with follow-up visits were excluded. the departments included diagnosis, prosthodontics, pedodontics, orthodontics, conservative dentistry, endodontics, periodontics, and oral surgery. the number of patients referred to each of the aforementioned departments was recorded. in addition, the sex and age of each patient was recorded. data entry was performed by one of the authors (m.l.a) using excel spreadsheets (microsoft excel 2016, microsoft corporation, usa) and checked by another author (y.a.s.). the primary outcome was to determine the fold changes in frequency and type of dental treatment during covid-19 crisis as compared to pre-pandemic period. statistical analysis statistically, the numbers of patients were expressed using frequency and percent. fold change in the number of patients in the two years was calculated by dividing the number of patients during 2019 by their number impact of covid-19 on the frequency of dental appointments and types of dental health care services vol 10 no 1 (2022) doi 10.5195/d3000.2022.194 h#p://den*stry3000.pi#.edu in 2020. association of the number of patients with different demographic variables was determined by using chi square test, while the strength of association was calculated by odds ratio (or). regarding the or for age groups, the age group 0 to 5 years was used as a reference for comparison with other age groups. statistical difference was considered significant at p <0.05. all statistical analyses were performed by using graphpad prism (version 8, usa) software. results analysis of records showed that the number of patient referrals in the three selected months during 2019 was 7286 (mean age= 27.8±16.2), while the number of patients during the same period in 2020 was 3090 (mean age= 29.4±15.1). during 2019, the highest frequency was associated with those referred for endodontic treatment (1385, 19%) and those seeking a diagnosis (1318, 19%), followed by patients needing surgical interventions (1310, 18%) (table 1). for the same year, the lowest number of patients was observed in referrals for prosthodontic treatment (204, 2.8%) (table 1). however, during the pandemic, the pattern changed as the highest number of patient referrals was seen in the oral and maxillofacial department (1016, 32.9%), followed by endodontics (751, 24.3%) (table 1). regarding the fold changes in numbers of patients, the lowest decreases were associated with the surgical (1.3-fold) and endodontic (1.8-fold) treatments, whereas the highest decreases were observed in periodontics and orthodontics (8folds each), followed by the conservative treatment (4.6-folds). other types of treatment recorded decreases ranging from 2.4 to 3.7fold. overall, the number of patients in 2020 decreased by 2.4-fold as compared to the same period in 2019 (figure 1). department 2019 2020 frequency % frequency % diagnosis 1381 19 584 18.9 prosthodontics 204 2.8 55 1.8 pedodontics 1225 16.8 378 12.2 orthodontics 456 6.3 57 1.8 conservative 908 12.5 197 6.4 endodontics 1385 19 751 24.3 periodontics 417 5.7 52 1.7 oral surgery 1310 18 1016 32.9 total 7286 100 3090 100 table 1: frequencies of patient referrals to dental departments during 2019 and 2020. impact of covid-19 on the frequency of dental appointments and types of dental health care services vol 10 no 1 (2022) doi 10.5195/d3000.2022.194 h#p://den*stry3000.pi#.edu regarding association of frequency of patient referrals with sex, a significant decrease was observed in the number of females seeking dental treatment as compared to males (p <0.05, or 1.279). similarly, a significant association was observed in relation to age group (p <0.05). the highest number of patients seeking dental treatment during the pandemic was recorded in the age range of 19 to 48 years (or 1.6) as compared to the younger and older age groups (table 2). discussion according to who and ada recommendations, dental practices should remain closed and only emergency dental treatments should be performed [5, 6]. the rationale behind this study was to determine the impact of covid-19 on frequency and types of dental services provision via comparing pre-pandemic with pandemic dental records. the study presents a descriptive analysis of 2019† 2020† p value* 95% ci or§ sex maleǂ 3365, 46.2 1617, 52.3 0.001 1 female 3921, 53.8 1473, 47.7 1.176 to 1.392 1.279 age group 0-5ǂ 358, 4.9 106, 3.4 1 6-13 1358, 18.6 368, 11.9 0.479 0.854 to 1.392 1.093 14-18 789, 10.8 321, 10.4 0.013 1.071 to 1.764 1.374 19-33 2288, 31.4 1149, 37.2 0.001 1.354 to 2.133 1.696 34-48 1531, 21.0 752, 24.3 0.001 1.317 to 2.092 1.659 49-64 813, 11.2 332, 10.7 0.012 1.077 to 1.767 1.379 65-78 137, 1.9 60, 1.9 0.039 1.019 to 2.147 1.479 79-98 12, 0.2 2, 0.1 0.451 0.062 to 2.260 0.563 total 7286, 100 3090, 100 figure 1: fold change of the patients referred to dental the departments in 2020 as compared to 2019. highest decrease was observed in number of patients seeking periodontal and orthodontic treatment (8-fold each). while the lowest decrease was associated with those referred to oral surgery (1.3-fold) and endodontic (1.8-fold) departments. table 2: associations of frequency of patient referrals with demographic variables † frequency, percent * significance at p <0.05 by chi square test § or: odds ratio ci: confidence interval ǂ reference variable impact of covid-19 on the frequency of dental appointments and types of dental health care services vol 10 no 1 (2022) doi 10.5195/d3000.2022.194 h#p://den*stry3000.pi#.edu dental records and meaningful findings of reduced frequency of dental patient appointments as well as a significant decrease in the number of non-urgent dental patients. there was overall a 2.4-fold decrease in the number of dental appointments (from 7268 to 3090), which can be explained by the fact that during the pandemic (june, july and august of 2020) most of the dental services were closed and according to the iraqi authorities only urgent treatment was to be provided. furthermore, fear and anxiety among patients was having a significant impact on their willingness to attend dental appointments unless the situation was urgent and the discomfort could not be tolerated [14, 15]. since this study was based on analysis of retrospective data, it was not possible to determine whether the referred patients received the intended treatment or if they complied to the given dental appointment. in addition, retrospective studies are prone to selection bias as the controls are recruited by convenience sampling which is unavoidable limitation in this type of studies. furthermore, it was not possible to obtain a feedback from the patients to evaluate the access to health care. emergency dental care includes treatment of pain and swelling which can be provided in oral surgery and endodontic and oral diagnosis departments [7]. this explains why the lowest fold of reductions was found in the dental records of these departments (oral surgery = 1.3-fold, endodontics = 1.8-fold and oral diagnosis = 2.4-fold). meanwhile, the non-urgent dental care departments, such as periodontics, orthodontics and conservative dentistry, recorded the highest reductions. again, this can be related to the authorities’ guidance to provide treatment for dental emergency cases only. moreover, the shortage of personal protection equipment in dental practices resulted in high levels of anxiety and fear among iraqi dentists; [12] thus, lengthy procedures and those which require the use of agp, such as filling teeth and scaling and polishing, were avoided. a statistically significant difference was identified between male and female dental records regarding the decrease in total patient numbers. under normal conditions, females have tended to be more compliant in attending dental appointments than males; [16] however, their fear and anxiety about the covid-19 pandemic had made them less willing to attend these appointments. this is in line with the data reported by other studies which indicates that females are more emotionally affected by difficult situations and consequently more likely to express feelings such as stress and anxiety [17]. perhaps, females were more anxious about the overall situation created by covid-19 pandemic from such as general health, economic and social distancing aspects, with dental appointments seen as nonessential and capable of being addressed when the pandemic is under control [18]. moreover, a statistically significant association was observed between reduction in dental appointment attendance and age group. it was to be expected that attendance would be more greatly reduced among older age groups as the covid-19 mortality rate has been higher in older age groups [19]. this is supported by the data of the present study as the dental records showed that the highest reduction was in the age group of >65 years old. similarly, the dental records for children younger than 13 years old showed a significant decrease in referrals. this could be explained by the uncertainty about the impact of covid-19 on children and their parents not wanting to risk attending dental appointments unless the condition required urgent treatment. on the other hand, the 19 to 48 years age group recorded the highest attendance at dental practices. this result could be related to the fact that the prevalence of dental caries and periodontal diseases is high among iraqi residents [20-22]. thus, despite the fear and anxiety surrounding covid-19, people still have to attend dental practices for treatment of urgent illnesses such as severe pulpitis, swelling and cellulitis. in addition, the impact of covid-19 on the frequency of dental appointments and types of dental health care services vol 10 no 1 (2022) doi 10.5195/d3000.2022.194 h#p://den*stry3000.pi#.edu incidence of severe symptoms and mortality due to covid-19 has been low among younger people [23, 24], which has made them less fearful of seeking dental treatment during the pandemic. in early 1980s, penchansky and thomas proposed a multidimensional-model to assess the fit between the patient and the health care system [25]. the criteria of this theory are based on five dimensions (affordability, availability, accessibility, accommodation, and acceptability). in the current study, accessibility seem to be compromised due to application of strict lock-down measures that limited the mobility of individuals and transportation or fear from infection which discouraged subjects from seeking dental services. in addition, acceptability was also affected by the precautions and preventive measures including longer operation time due to application of a fallow period, minimizing referrals, and reducing number of patients treated by the dentists. since these dental centers are supported by the government; therefore, other aspects were either not or minimally affected including the cost, quality of treatment, infrastructure, and supplies before or during the pandemic. while the focus during this pandemic has been more on stopping the spread of covid-19, the future health consequences, including consequences associated with dental health, should not be underestimated. analysis of the current situation is helpful for predicting the future dental needs. based on the results of the present study, people’s need for dental services can be expected to increase dramatically in the post pandemic period. dental care authorities should implement a comprehensive program to address the future dental care needs and prevent collapse of the dental care system, especially regarding departments managing non-urgent dental illnesses. caution should be considered for the generalization of results from the current survey due to its dependence on retrospective data analysis. therefore, further prospective studies are required to assess the long-term impact of covid-19 on dental services and health care providers. conclusion the results of this study suggested that covid-19 is having a strong influence on reducing the overall number of dental appointments, with emergency dental treatments showing the lowest numbers of decreases. references 1. the impact of the covid-19 epidemic on the utilization of emergency dental services. guo h, zhou y, liu x, tan j. journal of dental sciences. 2020;15(4):5647. 2. administration osah. dentistry workers and employers 2021 [available from: https://www.osha.gov/coronavir us/control-prevention/dentistry. 3. aerosols and splatter in dentistry: a brief review of the literature and infection control implications. harrel sk, molinari j. journal of the american dental association (1939). 2004;135(4):429-37. 4. risk and prevention of transmission of infectious diseases in dentistry. araujo mw, andreana s. quintessence international (berlin, germany : 1985). 2002;33(5):376-82. 5. who. considerations for the provision of essential oral health services in the context of covid19: interim guidance, 3 august 2020 2020 [available from: https://apps.who.int/iris/handle /10665/333625. 6. ada. ada urges dentists to heed april 30 interim postponement recommendation, maintain focus on urgent and emergency dental care only 2020 [available from: https://www.ada.org/en/pressroom/news-releases/2020archives/april/summary-of-adaguidance-during-the-covid-19crisis. 7. dentistry during the covid-19 epidemic: an italian workflow for the management of dental practice. peditto m, scapellato s, marcianò a, costa p, oteri g. international journal of environmental research and public health. 2020;17(9). 8. ada. covid-19 state mandates and recommendations 2021 [available from: https://success.ada.org/en/pract ice-management/patients/covidimpact of covid-19 on the frequency of dental appointments and types of dental health care services vol 10 no 1 (2022) doi 10.5195/d3000.2022.194 h#p://den*stry3000.pi#.edu 19-state-mandates-andrecommendations. 9. ada. ada develops guidance on dental emergency, nonemergency care 2020 [available from: https://www.ada.org/en/publica tions/ada-news/2020archive/march/ada-developsguidance-on-dental-emergencynonemergency-care. 10. fear and practice modifications among dentists to combat novel coronavirus disease (covid-19) outbreak. ahmed ma, jouhar r, ahmed n, adnan s, aftab m, zafar ms, et al. international journal of environmental research and public health. 2020;17(8). 11. covid-19 and the risk to health care workers: a case report. ng k, poon bh, kiat puar th, shan quah jl, loh wj, wong yj, et al. annals of internal medicine. 2020;172(11):766-7. 12. anxiety, practice modification, and economic impact among iraqi dentists during the covid19 outbreak. mahdee af, gul ss, abdulkareem aa, qasim ssb. frontiers in medicine. 2020;7:595028. 13. impact on dental economics and dental healthcare utilization in covid-19: an exploratory study. patel n. journal of advanced oral research. 2020;11(2):12836. 14. relationship between psychological factors and oral health status and behaviours. alkan a, cakmak o, yilmaz s, cebi t, gurgan c. oral health & preventive dentistry. 2015;13(4):331-9. 15. impact of covid-19 on dental education in the united states. iyer p, aziz k, ojcius dm. journal of dental education. 2020;84(6):718-22. 16. compliance with supportive periodontal treatment in private periodontal practice. a 14-year retrospective study. demetriou n, tsami-pandi a, parashis a. journal of periodontology. 1995;66(2):145-9. 17. sex moderates the association between symptoms of anxiety, but not obsessive compulsive disorder, and error-monitoring brain activity: a meta-analytic review. moser js, moran tp, kneip c, schroder hs, larson mj. psychophysiology. 2016;53(1):21-9. 18. covid-19-related anxiety disorder in iraq during the pandemic: an online crosssectional study. karim sk, taha ph, amin nmm, ahmed hs, yousif mk, hallumy am. middle east current psychiatry. 2020;27(1):55. 19. age-related morbidity and mortality among patients with covid-19. kang sj, jung si. infection & chemotherapy. 2020;52(2):154-64. 20. the prevalence of dental caries among students of dentistry colleges in holy kerbala governorate/ iraq in 2017. al mousawi a, al ali b, al mousawi z. journal of contemporary medical sciences. 2019;5(3). 21. dental caries prevalence and risk factors among 12-year old schoolchildren from baghdad, iraq: a post-war survey. ahmed na, astrøm an, skaug n, petersen pe. international dental journal. 2007;57(1):3644. 22. association of overweight/obesity with the severity of periodontitis using bpe code in an iraqi population. gul ss, imran nk, al-sharqi ajb, abdulkareem aa. clinical epidemiology and global health. 2021;9:21-5. 23. estimation of risk factors for covid-19 mortality preliminary results. caramelo f, ferreira n, oliveiros b. medrxiv. 2020:2020.02.24.20027268. 24. risk factors of critical & mortal covid-19 cases: a systematic literature review and metaanalysis. zheng z, peng f, xu b, zhao j, liu h, peng j, et al. the journal of infection. 2020;81(2):e16-e25. 25. the concept of access: definition and relationship to consumer satisfaction. penchansky r, thomas jw. medical care. 1981;19(2):127-40. restoration microhardness in contact with cscs vol 9 no 1 (2021) doi 10.5195/d3000.2021.159 http://dentistry3000.pitt.edu do different time intervals in placement of restorative materials over calcium silicate cements, affect interface microhardness of different restorative materials? behnam bolhari1, naghmeh meraji2, pegah khazaee3, sholeh ghabraei4, sara valizadeh5 1associate professor of endodontics, laser research center of dentistry, dental school, tehran university of medical sciences, tehran, iran 2assistant professor, department of endodontics, dental school, tehran university of medical sciences, tehran, iran 3dentist, tehran university of medical sciences, tehran, iran 4associate professor, department of endodontics, dental school/dental research center, tehran university of medical sciences, tehran, iran. 5associate professor, dental research center, dentistry research institute, restorative department, dental school, tehran university of medical sciences, tehran, iran. abstract objectives: the type of materials and application time of final restorations on calcium silicate cements (cscs) are important factors which influence the interfacial properties. the aim of this study was to investigate the effect of different placement time of rmgi (resin modified glass ionomer), composite resin and amalgam over different cscs on the surface microhardness of these restorative materials. methods: each cscs material (biodentine, mta, cem cement) was mixed and carried into a hole (6mm diameter ×4 mm thickness) in the center of 270 molds (n=90 /each csc). then these molds were randomly divided into three main experimental groups (n=30) in regard of restorative materials (amalgam, rmgi, composite) that were placed in the other molds with the same hole size to make restorative materials and cscs in contact together. afterwards, each experimental group was divided into three subgroups according to the time interval of restoration placement that was immediately, after 24h and after 72h (n=10). two molds were separated from each other after one week storage in incubator with 100% humidity in 37 °c, in order to evaluate the vickers microhardness of restorative materials in csc-restorative material interface. statistical analysis included two-way anova followed by post hoc dunnett t3 in cases with lack of homogeneity and tukey hsd in cases with homogeneity. (p=0.05) results: the microhardness of all restorative materials was neither significantly influenced by the cscs materials (p>0.05) nor by the timing of final restoration (p>0.05) except in rmgi in immediate contact with cem cement. (p<0.001) conclusion: restorative materials hardness in interface with studied cscs may not affect. this in vitro study found no evidence against immediate definitive restoration over cscs. keywords: calcium silicate cement; restorative materials; microhardness; time intervals. citation: bolhari b, et al. (2021) do different time intervals in placement of restorative materials over calcium silicate cements, affect interface microhardness of different restorative materials? dentistry 3000. 1:a001 doi:10.5195/d3000.2021.159 received: march, 6, 2021 accepted: april, 27, 2021 published: september, 10, 2021 copyright: ©2021 bolhari b, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: valizadeh_s@sina.tums.ac.ir http://dentistry3000.pitt.edu/ mailto:valizadeh_s@sina.tums.ac.ir restoration microhardness in contact with cscs vol 9 no 1 (2021) doi 10.5195/d3000.2021.159 http://dentistry3000.pitt.edu introduction in recent years, application of biomaterials in dentistry, has resulted in improvement of various restorative and endodontic procedures [1]. tooth preservation is an ultimate goal in modern dentistry. in this regard, vital pulp therapy (vpt) has gained considerable attention. the aim of vpt is keeping the dental pulp, vital and healthy following carious exposure or traumatic injuries. some biomaterials can be used as a pulp protective layer in this procedure [2]. ideally, these materials should be bioactive and biocompatible. low solubility, ability to bind to dental tissues, adequate setting time and antibacterial activity are some of their characteristics too [3]. cscs are bioactive materials which are used in many dental treatments like vital pulp therapy, repair of perforations and regenerative treatment. nevertheless they have many favorable properties, difficult handling and tooth discoloration are considered as their disadvantages [4]. mineral trioxide aggregate (mta) was the first csc introduced in endodontics [5]. biodentine and calcium-enriched mixture (cem) cement are known as cscs that are fast setting dentine replacement material and claim to have no discoloration potential respectively [6]. direct contact of cements with the final restoration happens in most clinical situations. the time span between the cscs placement and final restoration could have an important role in success rate of treatments [7]. however, it is better to place final coronal restoration immediately after pulp capping to promote coronal seal and less microleakage, this is not possible beacause of long setting time in some cscs for example mta [8]. resin-based composite, glass-ionomer cement (gic), resinmodified gi (rmgi) and amalgam are common materials that are used for coronal restorations. the effect of immediate coronal restoration on the physical properties of different cscs has been evaluated in few studies [9, 10, 11] but based on the authors’ search, it is assumed that the effect of the time of coronal restoration on the surface microhardness of restoration has not been assessed. therefore, the aim of the present study was to investigate the effect of different time placement of rmgi, composite resin and amalgam over different cscs on the surface microhardness of these restorative materials. it was hypothesized that the microhardness of restorative materials would not be affected by placement time. material and methods the materials used in the current study, manufacturer details, their composition and mode of application are shown in table 1. table 1 – materials composition and mode of application: material manufacture composition mode of application biodentine septodont, saint maurdes-fosses, france powder: tri-calcium silicate, di-calcium silicate, calcium carbonate and oxide, iron oxide, zirconium oxide liquid: calcium chloride, hydro soluble polymer one dose of liquid and powder mixed for 30 sec at 4000 rpm in an amalgamator http://dentistry3000.pitt.edu/ restoration microhardness in contact with cscs vol 9 no 1 (2021) doi 10.5195/d3000.2021.159 http://dentistry3000.pitt.edu microhardness test: in this in vitro experimental study, 540 cylindrical polymethylmethacrylate molds with a central hole with an internal diameter of 6 mm and height of 4 mm (according to astm e384 standard for microhardness tests) were fabricated by cnc laser cutting (laserproi, gcc, new taipei city, taiwan). half of the molds were considered for cscs which were placed on slabs and according to the csc materials randomly divided into three main experimental groups (n=90). csc material was prepared according to the manufactures instructions and placed in the molds. then the rest of the molds were placed on each mold filled with csc in a way that the two internal holes were positioned along each other. the second mold was used for restorative material placement. afterwards, each main experimental group was divided into three subgroups (n=30) according to the restorative material applied. the time interval of restoration placement was varied. the final restoration was cem cement bioniquedent, tehran, iran powder: calcium oxide, sulfur trioxide, phosphorous pentoxide, silicon dioxide, aluminum trioxide, sodium oxide, magnesium oxide, chloride liquid: water-based liquid mix powder/liquid ratio: 3/1 proroot mta dentsply maillefer, ballaigues, switzerland powder: calcium carbonate, silicon dioxide, aluminum oxide, calcium zirconia complex liquid: distilled water mix powder/liquid ratio: 3/1 adpertm single bond 2 3m espe, quadrantlc, cavex, haarlem, netherlands bisgma, hema, dimethacrylates, a methacrylate functional copolymer of polyacrylic and polyitaconic acids, ethanol, water, silica fillers, camphorquinone 1. apply 37% phosphoric acid etchant for 15 sec 2. rinse for 10 sec 3. apply 2 or 3 onsecutive coats of adhesive 4. allow gentle air stream for 5 seconds 5. light cure for 10 sec amalgam sdi, victoria, australia amalgam alloy and mercury in amalgamator filtekn z250 composite resin 3m espe; quadrantlc, cavex, haarlem, netherlands filler: zr/si (60 vol%) 0.01-3.5µm resin: bisgma, udma, bisema light cure for 40 s fuji ii lc light cure glass ionomer gc, tokyo, japan powder: fluoro-alumino-silicate glass liquid: poly-acrylic acid, hema, dimethacrylate, camphorquinone, water light cure for 40s http://dentistry3000.pitt.edu/ restoration microhardness in contact with cscs vol 9 no 1 (2021) doi 10.5195/d3000.2021.159 http://dentistry3000.pitt.edu placed a. immediately b. after 24h and c. after 72h (n=10). there was no pretreatment prior to amalgam or glass ionomer placement but before placing composite resin in molds, total etch and bond (adpertm single bond 2) preceded by etching with 37% phosphoric acid for 15 seconds was applied. light curing time for both rmgi and composite resin was 40 seconds at 800 mw/cm2 using a light-emitting diode (woodipecker, china) light source. two molds were separated from each other after one week storage in incubator with 100% humidity in 37 °c , in order to evaluate the microhardness of restorative materials in csc and restorative material interface. the side of restorative materials in contact with cscs were polished using silicon carbide sandpaper with varying particle sizes of 400, 500, 800, 1000, 1200, 1500 and 2000 grit respectively. all the procedures were done by one person. for the purpose of facilitating indentation and minimizing the influence of sample preparation on surface microhardness, wet polishing with minimal hand pressure was employed. the surface microhardness test was performed using a vickers tester (bareiss prufgeratebau gmbh, oberdischingen, germany) and a pyramid shaped diamond indenter with a load of 300 g for 10 s . according to the pilot study this load created a clear and reliable indent in all three materials. five indents were made on the polished surface of each sample at separate locations with a 2.5×d (2.5 times the mean diameter of each indent) distance between indentations and each indent from the edge of the sample (in accordance to astm e384 standard for vickers microhardness test). the vickers microhardness value (hv) was calculated by the testing machine based on the following equation: 𝐻𝑉 = 2fsin 136 ̊ 2 d2 𝐻𝑉 = 1.854 𝐹 d2 f= load in kgf d = mean of the two diagonals, d1 and d2 in mm hv= vickers microhardness value statistical analysis statistical analysis included twoway anova followed by post hoc dunnett t3 in cases with lack of homogeneity and tukey hsd in cases with homogeneity. results mean and standard deviation of vickers microhardness of different restorative materials in interface with verities of cscs after several intervals, are listed in table 2. amalgam hardness in contact with all of cscs increased insignificantly (p value = 0.32, 0.38 and 0.25 in contact with biodentin, cem cement and mta respectively) over time. hardness of composite in interface with different cscs had no significant changes in different times (p value = 0.11, 0.25 and 0.21 in contact with biodentin, cem cement and mta respectively). rmgi showed significantly decrease in hardness in contact with all of cscs over time (p value = 0.001). comparison between restorative materials represented that hardness of rmgi is the least among them and amalgam had highest level of hardness contacting with all of cscs. there was just one exception, rmgi had greatest hardness in contact with cem cement immediately. http://dentistry3000.pitt.edu/ restoration microhardness in contact with cscs vol 9 no 1 (2021) doi 10.5195/d3000.2021.159 http://dentistry3000.pitt.edu table 2 – mean±sd of the microhardness of different restorative materials in contact with cscs in different times: biodentin cem cement mta restorative materials immediate 24h 72h immediate 24h 72h immediate 24h 72h amalgam 186.60±9.09 a 188.86±17.79 a 199/00±28.63 a 144.20±17.34 a 172.46±5.02 a 174.86±10.87 a 153.23±11.83 a 189.76±18.18 a 202.00±23.61 a composite 136.20±9.77 b 116.13±13.75 b 122.90±3.01 b 123.26±9.63 b 130.76±2.25 b 140.76±16.89 b 127.87±5.27 b 125.60±9.18 b 148.57±23.99 b rmgi 125.90±6.66 c 97.37±5.26 d 99.00±28.63 d 322.70±5.73 f 111.90±9.66 d 85.23±9.02 e 118.87±6.64 c 110.43± 6.88 d 88.37± 13.39 e *different letters show significant differences between groups discussion there are some kinds of materials known as calcium silicate cements (cscs) that recommended for pulp capping process. they provide pulp vitality maintenance and stimulate making growth factors and lead to dentin formation in asymptomatic teeth [12]. some characteristics are considered for these pulp capping materials like biocompatibility; dimensional stability; insolubility in tissue fluids; easy manipulation; adhesion to the tooth structure; providing adequate seal and remaining in place under dislocating forces [13]. among different factors affected pulp therapy prognosis, the time of the placement of well-sealed permanent coronal restoration is critical. glass-ionomer cement (gic), resin-modified gi (rmgi) resin-based composite, and amalgam are common materials for coronal restorations [14]. when restorative materials are applied on cscs immediately or after different intervals, it may that setting reactions of each materials affect the other one setting in the interface between them. timing of coronal restoration placement on the physical properties of cscs has been evaluated in some studies [14, 15, 16] but there is no study about the effect of the coronal restoration placement time on the surface microhardness of restorative materials. therefore, the aim of the present study was to investigate the microhardness of rmgi, resin composite and amalgam restoration in interface with different cscs after different time intervals. hardness is a property of major importance for assessing the adequate setting of restorative materials [17] it not only represents deformation resistance but also shows stability of material crystalline structure. as there is an inverse relationship between hardness and porosity, adequate levels of hardness play an important role in achieving an ideal seal [18]. hardness is one of the determinants of the life time of dental materials and prevent restorative material displacement http://dentistry3000.pitt.edu/ restoration microhardness in contact with cscs vol 9 no 1 (2021) doi 10.5195/d3000.2021.159 http://dentistry3000.pitt.edu and disruption of the physical seal during masticatory occlusal loads [19]. as it is possible to perceive, amalgam hardness in contact with three cscs used in this study, had an acceptable measure that increased insignificantly with time. this finding showed that amalgam hardness in interface was not affected by the cscs’ hydration process even when immediately placed. increasing amalgam hardness over time could be attributed to continuous crystallization of amalgam over time because of the setting reaction rate which is somehow slow and takes several days to be completed [20]. so, it seems that setting conditions of cscs have no adverse effect on amalgam strength [17]. in contrast some factors like the ph value of the environment, the condensation pressure, humidity and temperature may affect microhardness of cscs that it has been studied in many researches [21]. in this study composite reached to optimum microhardness in interface and no adverse effect were seen in composite setting reaction in contact with cscs and no significant changes in hardness was seen in different placement times. correlation between the microhardness and the degree of conversion of composite resins have shown in several studies [22] the results of the present study showed that the degree of cross linking in the polymerized matrix reaches the maximum hardness immediately after light curing and it does not change in contact with cscs setting reaction in different intervals especially in immediate composite placement [23]. in case of immediate coronal restoration, clinical manipulations including the condensation pressure, etching, rinsing, and priming could affect the setting of cscs [15] rmgi showed significantly decrease in hardness in contact with all of cscs over time. the reason may be because of moisture absorption from cscs by rmgi until microhardness test was done so environment humidity result in water sorption in rmgi. amalgam and composite are somehow stable materials in presence of humidity, where their water sorption and degradation rate is very low compared with rmgi that present higher degradation and lower hardness values [12]. in a study by ambrosano it was shown that after rmgi degradation in moisture conditions, the hardness decreased significantly in this material [24]. comparison between restorative materials represented that hardness of rmgi is the least among them and amalgam had maximum hardness contacting with all cscs and in all time intervals. this is not unexpected results because of materials mechanical properties as shown in literature that amalgam, composite and rmgi have the highest measure of hardness respectively [25]. there was just one exception in the present study, rmgi had the greatest hardness in contact with cem cement immediately. in studies by cantekin and doozaneh was shown that cem cement consist of different calcium compounds that can bond chemically with rmgi especially when placed immediately over cem cement that have not been fully set [26,27]. it is supposed during separation of two molds, because of chemical bonding, pieces of cem cement would remain on the rmgi surface and the higher amount of microhardness was associated with that. this was an in vitro study; therefore, the condition of the oral cavity wouldn’t completely have simulated. in this study just microhardness was evaluated, therefore, it is suggested that other characteristics like microleakage and bond strength between http://dentistry3000.pitt.edu/ restoration microhardness in contact with cscs vol 9 no 1 (2021) doi 10.5195/d3000.2021.159 http://dentistry3000.pitt.edu different types of restorations and several cscs, would be studied [28]. it should be noted that in another study, done by the authors, the microhardness of cscs is evaluated and the results of these both studies will be useful in determination of placement time of restorative materials over cscs [29]. conclusion within the limitation of this laboratory study, it was concluded that there is no difference between placement time of restorative materials over studied cscs in regards of the restorative material microhardness. so immediate placement of a final restoration over cscs not only provide better coronal seal but also would be clinically beneficial for patients and dentists, decreasing time and cost. conflicts of interest the authors declare no competing interest. availability of data and materials the data that support the findings of this research are available from the corresponding author [s.v] upon request. references 1. patel e, pradeep p, kumar p, choonara ye, pillay v. oroactive dental biomaterials and their use in endodontic therapy. journal of biomedical materials research part b: applied biomaterials. 2019 apr 8. 2. da rosa wl, cocco ar, silva tm, mesquita lc, galarca ad, silva af, piva e. c urrent trends and future perspectives of dental pulp capping materials: a systematic review. journal of biomedical materials research part b: applied biomaterials. 2018 apr;106(3):1358-68. 3. zamparini f, siboni f, prati c, taddei p, gandolfi mg. properties of calcium silicatemonobasic calcium phosphate materials for endodontics containing tantalum pentoxide and zirconium oxide. clinical oral investigations. 2019 jan 29;23(1):445-57. 4. yazdi ka, ghabraei s, bolhari b, kafili m, meraji n, nekoofar mh, dummer pm. microstructure and chemical analysis of four calcium silicatebased cements in different environmental conditions. clinical oral investigations. 2019 jan 29;23(1):43-52. 5. parreira rm, andrade tl, luz ap, pandolfelli vc, oliveira ir. calcium aluminate cement-based compositions for biomaterial applications. ceramics international. 2016 aug 1;42(10):11732-8. 6. llaquet m, mercadé m, plotino g. regenerative endodontic procedures: a review of the literature and a case report of an immature central incisor. giornale italiano di endodonzia. 2017 nov 1;31(2):65-72. 7. torabinejad m, parirokh m, dummer pm. mineral trioxide aggregate and other bioactive endodontic cements: an updated overview–part ii: other clinical applications and complications. international endodontic journal. 2018 mar;51(3):284-317. 8. palma p, marques j, falacho r, vinagre a, santos j, ramos j. does delayed restoration improve shear bond strength of different restorative protocols to calcium silicate-based cements? materials. 2018 nov;11(11):2216. 9. lipski m, nowicka a, kot k, postek-stefańska l, wysoczańskajankowicz i, borkowski l, andersz p, jarząbek a, grocholewicz k, sobolewska e, woźniak k. factors affecting the outcomes of direct pulp capping using biodentine. clinical oral investigations. 2018 jun 1;22(5):2021-9. 10. ha ht. the effect of the maturation time of calcium silicatebased cement (biodentine™) on http://dentistry3000.pitt.edu/ restoration microhardness in contact with cscs vol 9 no 1 (2021) doi 10.5195/d3000.2021.159 http://dentistry3000.pitt.edu resin bonding: an in vitro study. applied adhesion science. 2019 dec 1;7(1):1. 11. schmidt a, schäfer e, dammaschke t. shear bond strength of lining materials to calcium silicate cements at different time intervals. j adhes dent. 2017 mar 1;19(2):129-35. 12. napte bd, raghavendra ss. evaluation of shear bond strength of conventional glass ionomer cements bonded to mineral trioxide aggregate: an in vitro study. journal of dental and allied sciences. 2015 jul 1;4(2):73. 13. bayram hm. effect of acidic environment on dislodgment resistance of different silicatebased root repair materials. acta scientific dental sciences. 2018; 2:24-8. 14. nandini s, ballal s, kandaswamy d. influence of glassionomer cement on the interface and setting reaction of mineral trioxide aggregate when used as a furcal repair material using laser raman spectroscopic analysis. journal of endodontics. 2007;33(2):167-72. 15. tsujimoto m, tsujimoto y, ookubo a, shiraishi t, watanabe i, yamada s, et al. timing for composite resin placement on mineral trioxide aggregate. journal of endodontics. 2013;39(9):116770. 16. tunç eş, bayrak ş, eğilmez t. the evaluation of bond strength of a composite and a compomer to white mineral trioxide aggregate with two different bonding systems. journal of endodontics. 2008;34(5):603-5. 17. caronna v, himel v, yu q, zhang j-f, sabey k. comparison of the surface hardness among 3 materials used in an experimental apexification model under moist and dry environments. journal of endodontics. 2014;40(7):986-9. 18. kazemipoor m, azizi n, farahat f. evaluation of microhardness of mineral trioxide aggregate after immediate placement of different coronal restorations: an in vitro study. journal of dentistry (tehran, iran). 2018;15(2):116. 19. almuslet na, elamin h, elbashir b. hardness enhancement of amalgam teeth fillings using diode laser (675 nm). journal of analytical science and technology. 2011;2(2):64-8. 20. rajih ak, al-sultani kf, alkinani ma. mechanical properties improvement of dental amalgam using tio2 and zno. life sci j. 2015; 12:86-90. 21. kaur m, singh h, dhillon js, batra m, saini m. mta versus biodentine: review of literature with a comparative analysis. journal of clinical and diagnostic research: jcdr. 2017;11(8): zg01. 22. sahebalam r, boruziniat a, mohammadzadeh f, rangrazi a. effect of the time of salivary contamination during light curing on degree of conversion and microhardness of a restorative composite resin. biomimetics. 2018;3(3):23. 23. alzraikat h, taha na, qasrawi d, burrow mf. shear bond strength of a novel light cured calcium silicate based-cement to resin composite using different adhesive systems. dental materials journal. 2016;35(6):881-7. 24. ambrosano gmb, puppin-rontani rm. biomechanical degradation of the nano-filled resin-modified glassionomer surface. american journal of dentistry. 2012;25(6). 25. elmi m, ehsani m, esmaeili b, khafri s. comparison of bond strength of a composite resin with two different adhesive systems and a resin modified glass ionomer to calcium enriched mixture. journal of conservative dentistry: jcd. 2018;21(4):369. 26. doozaneh m, koohpeima f, firouzmandi m, abbassiyan f. shear bond strength of selfadhering flowable composite and resin-modified glass ionomer to two pulp capping materials. iranian http://dentistry3000.pitt.edu/ restoration microhardness in contact with cscs vol 9 no 1 (2021) doi 10.5195/d3000.2021.159 http://dentistry3000.pitt.edu endodontic journal. 2017;12(1):103. 27. cantek n k, avc s. evaluation of shear bond strength of two resin-based composites and glass ionomer cement to pure tricalcium silicate-based cement (biodentine®). journal of applied oral science. 2014; 22:302-6. 28. gandolfi m, spagnuolo g, siboni f, procino a, rivieccio v, pelliccioni g, et al. calcium silicate/calcium phosphate biphasic cements for vital pulp therapy: chemical-physical properties and human pulp cells response. clinical oral investigations. 2015;19(8):207589. 29. bolhari b, yazdi ka, abbasi m, sanjari s, meraji n, özcan m. calcium silicate cement interface with restorative materials through layering after different time intervals. odontology. 2021;109(1):210-21. http://dentistry3000.pitt.edu/ evalua&ng child behavior and preference toward numbee and tradi&onal syringe (a randomized clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.589 h#p://den*stry3000.pi#.edu evaluating child behavior and preference toward numbee and traditional syringe (a randomized clinical trial) duaa kamal naji1, zainab juma jafar2 1master degree student, department of pedodontics and preventive dentistry, college of dentistry, university of baghdad 2professor (msc.), department of pedodontics and preventive dentistry, college of dentistry, university of baghdad abstract background: local anesthe&cs are the gold standard for pain control in den&stry. most pa&ents s&ll view the “needle” as a source of anxiety and disrup&ve behavior rather than relief. the pa&ent’s coopera&on and comfort level will increase with the administra&on of painless local anesthe&c injec&on using appropriate technique. the purpose of this study was to assess the pa&ent coopera&on and behavior aler receiving local anesthesia injec&on and to compare pa&ents’ acceptance and preference of needleless numbee anesthe&c delivery device to conven&onal dental syringe. materials and methods: 30 non-fearful 6-8-year-olds who had never been to the den&st were selected for the study using a split-mouth design. they underwent a simple class i restora&on on both mandibular permanent first molar teeth in two separated dental visits. the frankl behavior ra&ng scale was used to assess pa&ent behavior following injec&on. pa&ents expressed their preference and acceptance of the used syringes by answering a ques&onnaire. results: aler injec&on by either method, 25 pa&ents had frankl behavior ra&ng score of 4. pa&ents that had a nega&ve response with the tradi&onal method, showed a score of 2, following numbee injec&on (p=0.035). conclusion: pa&ents accepted both methods, and numbee may induce less fear, with minimum s&nging and unpleasant taste. keywords: syringes; anesthesia; local; mental health; child behavior; numbee cita:on: naji, dk et al. (2024) evalua:ng child behavior and preference toward numbee and tradi:onal syringe (a randomized clinical trial) den:stry 3000. 1:a001 doi:10.5195/d3000.2024.589 received: september 3, 2023 accepted: september 16, 2023 published: january 9, 2024 copyright: ©2024 naji, dk et al. this is an open access ar:cle licensed under a crea:ve commons ayribu:on work 4.0 united states license. email: doaa.kamal1202a@codental.uobaghdad.edu.iq introduction numerous pediatric dental services require the administration of local anesthetic, which acts to decrease the sympathetic activation brought on by the dental operation. local anesthetics are regarded as the foundation of pain control in children that need to undergo dental treatments such as extractions, pulpotomies, or pulpectomies [1]. the most distressing part of the dental anesthesia experience is the use of a needle, which is referred to as “needle phobia” or blenophobia and may be the reason for delaying dental treatments [1-3]. therefore, the dental practitioner aims to perform treatments with minimum of pain and discomfort [4]. feeling anxiety may also lead to more severe and prolonged discomfort during dental treatments [5,6]. there are some risks, particularly when administering inferior alveolar nerve blocks [7-10]. to minimize risks and discomfort, dental syringes have http://dentistry3000.pitt.edu/ evalua&ng child behavior and preference toward numbee and tradi&onal syringe (a randomized clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.589 h#p://den*stry3000.pi#.edu been redesigned to make dental injections easier and less painful. one of the redesigned systems is a device named as numbee (biodent, simi valley, ca). the manufacturer of this device claims to deliver local anesthetics painlessly and without the need for a hypodermic needle. it comprises a tiny metal cannula that is covered in a silicone-like material and utilized for intraligamentary injections that do not penetrate the periodontal ligament. for pediatric patients, this could be an enormous gain over the standard syringe [11]. the aim of this work was to study on an intraligamentary needleless syringe (numbee) in order to evaluate patients’ behavior, preferences, and acceptance of the intraligamentary device for dental anesthesia. material and methods sample collection about 800 primary schools aged children were surveyed after approval from al-karkh health directorate, and approximately 450 children had no previous dental experience. the clinical phase was held at the primary health care center in baghdad. a visual intraoral examination was completed for all the 450 children. children included in this study were healthy with no cognitive impairment who never been in the dentist and with “positive” or “definitely positive” behavioral rating according to the frankl behavior classification scale [12]. we selected children that had bilateral class i carious lesion on their mandibular permanent first molars with initial or moderate caries according to ada caries classification 2015 (icidas scores 2/3/4) [13]. all the details of the study and the address of the dental center where the restorative procedure would be performed were explained in detail to the school headmaster, and the phone number of the investigator was given to allow interested parents to have direct contact with the investigators. parents/guardians who showed initial interest were then contacted privately and provided written informed consent after a comprehensive explanation of the study's objectives. information about the participant's overall health and an assessment of the likely adherence to the study guidelines were obtained. a date and time for the dentist appointment were then scheduled accordingly. calculation of sample size and randomization to determine sample size, g power 3.1.9.7 (university of düsseldorf, germany) with partial eta square ŋ2 =0.06 (medium effect size) was utilized. the correlation between measures (sides and treatments) used was 0.5, and the effect size of f for two devices and two sides under these conditions used was 0.2526, producing a sample size of 27 subjects plus 10% as an error rate [14], resulting in a sample size of 30. the impact size distribution of partial eta squares varies between small (0.01-0.059), moderate (0.06-0.139), and big (>=0.14 13-16 d) [15-18]. a single-blinded design was employed for this study to identify which method was utilized (numbee or a conventional syringe). using the random number generator (rng) tool in microsoft excel, participants and interventions were randomized also for the allocation of the injection site and the order of anesthetic method was conducted using a block design. intervention at the waiting room, the dentist monitored the patient’s general behavior in accordance to frankl behavior scale (scale 3/4). administration of local anesthesia with 2% lidocaine and 1:80,000 epinephrine was employed. regarding the standard injector for inferior alveolar nerve block, a long needle with a 27-gauge was employed. when local anesthetic administration was carried out with numbee, the dentist used a plastic tip which is particularly purchased from the manufacturer (figure 1). before administering the injection, there was no topical anesthesia used at the injection site. http://dentistry3000.pitt.edu/ evalua&ng child behavior and preference toward numbee and tradi&onal syringe (a randomized clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.589 h#p://den*stry3000.pi#.edu while using numbee, the manufacturer's instructions were followed. they recommend injecting four sites opposite to the mandibular permanent first molar roots, which will administer 0.48 ml of anesthetic fluid over 80 seconds. after 40 seconds the anesthesia can take full effect [11]. the inferior alveolar nerve block involves diffusion of 1.5 ml of anesthetic solution over the course of 60 seconds [19], with waiting for five minutes before starting the restorative session [20]. after the patient received the anesthesia, a questionnaire regarding their own preference and the degree of their acceptance of both methods was filled out. the questionnaire consisted of two parts: one was about the symptoms and fear they had during injection and the second included some of the observations that were recorded by the dentist by asking parents about any problems endure postoperatively by contacting them via telephone [21]. a. symptoms during anesthesia: 1. annoyance or pain during administration of anesthesia. no□ yes □ 2. fear during administration. no□ yes □ b. symptoms immediately after anesthesia : 1. bleeding of the mucosa. no□ yes □ 2. pain. no□ yes □ 3. stinging. no□ yes □ 4. bad taste. no□ yes □ 5. annoyance or discomfort after recovery from anesthesia. no□ yes □ children underwent class i restoration on their mandibular permanent first molar teeth in two separate but consecutive sessions. a tooth-colored restorative material was used for cavity filling. following this, the dentist assessed the patient behavior and determines a score based on the frankl behavior rating scale. at the end of both visits, patients were interviewed and asked to give their own opinion of the most scared and most favorable method, what method to choose for the next dental session to numb their teeth. statistical analysis data analysis was performed by using statistical package for social science (spss version -22, chicago, illinois, usa). frequency, mean rank, and inferential statistics (marginal homogeneity test and mc nemare’s test) were determined. the level of significance was 0.05. results on comparing both modalities of injection of frankl behavior rating scale by using the marginal homogeneity test, frankl behavior rating scale score 4 was the most prevalent score among the participants after receiving the injection using either method (25 patients) as shown in (table 1), however, frankl behavior rating scale score 3 was reported in both methods but was more frequent after inferior alveolar nerve block injection. patients showed a score of less cooperative status (score 2) on frankl behavior rating scale for both methods but it was higher in numbee injection (p value=0.035). regarding the questions asked for the participants to express their acceptance and preference of both modalities, the mcnemar's test showed that the numbee injection induced less fear in participants, a less unpleasant state after injection with a lesser number of participants who experienced a stinging sensation in comparison with inferior alveolar nerve block method, however; with statistically non-significant difference (table 2). figure 1. the intraligamentary needle-free (numbee) http://dentistry3000.pitt.edu/ evalua&ng child behavior and preference toward numbee and tradi&onal syringe (a randomized clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.589 h#p://den*stry3000.pi#.edu technique time score sex m f total n. n. n. numbee baseline 4 13 17 30 after 2 0 2 2 3 2 1 3 4 11 14 25 block baseline 3 0 1 1 4 13 16 29 after 2 0 1 1 3 1 3 4 4 12 13 25 sex variable numbee inferior alveolar nerve block mcnemar's test p-value n. n. boys pain during injection 8 6 0.625 fear during injection 6 5 1.000 bleeding after injection 7 6 1.000 pain after injection 2 1 1.000 stinging after injection 3 3 1.000 bad taste after injection 9 8 0.453 annoyance post-op. 2 1 1.000 girls pain during injection 10 8 0.727 fear during injection 9 11 0.688 bleeding after injection 7 6 1.000 pain after injection 2 1 1.000 stinging after injection 4 7 0.453 bad taste after injection 13 10 1.000 annoyance post-op 5 5 1.000 total pain during injection 18 14 0.388 fear during injection 15 16 1.000 bleeding after injection 14 12 0.791 pain after injection 4 2 0.688 stinging after injection 7 10 0.508 bad taste after injection 22 18 0.424 annoyance post-op. 7 6 1.000 regarding the interview made at the end of the dental sessions, most children favored numbee (n=27), chose numbee when asked about the method they would choose to anaesthetize their tooth in the future table 1: distribution of frankl behavior scale scores among techniques and time. frnb-frbb (pre for both methods): p value=1, frn1-frbl1(after injection for both methods): p value=0.451, frbl0-frbl1(pre – post for ianb): p value=0.059, frn0-frn1, p value=0.035 table 2: acceptance and preference of children for both techniques. http://dentistry3000.pitt.edu/ evalua&ng child behavior and preference toward numbee and tradi&onal syringe (a randomized clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.589 h#p://den*stry3000.pi#.edu (n=25), and stated the conventional syringe frightened them the most (n=26). discussion being able to guide children pleasantly through their dental experiences and promote a positive dental attitude in order to improve their oral health is one of the cornerstones of the pediatric dentistry practice [22]. dental needle injections have long been a source of worry and anxiety for individuals needing to face the dentist [23]. as a logical consequence, it was strongly recommended that an anesthetic delivery system be created with a less frightening appearance and with a low level of anxiety arousal [24]. numbee was selected for this study because of its needle-free design as an attempt to reduce the painful injection as it eliminates the damage from a needle stick which in turn would improve the pediatric patients’ attitude in the dental clinic [11]. after a thorough reviewing for the published scientific literatures, it was found that delivering local anesthetic to pediatric patients with numbee has not been studied. the design of the split-mouth offered the possibility of a savings costs. this design eliminates any aspects of the treatment comparisons that are related to differences between subjects being tested, which leads to a reduction in the amount of error variance, and, as a consequence, a more accurate assessment. participants of 6 to 8 years of age were selected, as this age group are easier to deal with during treatment in comparison to younger children. moreover, this age group has the capacity to comprehend logical concepts such as discomfort and anxiety and express themselves properly [25]. no topical anesthetic was used to ane sthetize the injection sites and this was to ensure that the topical anesthesia would not affect the children's reactions or behavior and the patient would express their actual feeling. the reason for completing the dental procedures in two sessions rather than one was because the procedure would be time-consuming and could hinder the child's ability to participate owing to fatigue and exhaustion, resulting in false positive responses. children who have previously received dental treatment may retain memories of anxiety and pain, which can increase their likelihood of experiencing anxiety and fear during future visits. this may also lead to more disruptive behavior. additionally, the presence of a positive treatment history can also impact the results of the study. therefore, it was imperative to select children with lack of history of dental visits. a double-blinded design was not possible, as the operator would always be aware of the significant difference between the methods concept for local anesthesia administration, in addition to apparatus design that was obviously different. in the present study, the researcher chose to treat the mandibular dentition because the mandible's higher bone density in contrast to the maxilla, which hopefully allowed for a more accurate evaluation of the intraligamentary anesthetic efficacy of the numbee [26,27]. as behavior evaluation is the most essential tool in the pediatric dentistry to enable the dentist to execute the required treatment plan in the most effective manner, promoting a positive attitude toward dental care. the frankl’s behavior rating scale is a tool used to evaluate the patient’s behavior and considered to be the most reliable tool established for rating children's behavior in a dental setting [28]. in the present study, the level of positive behavior for the participants who anesthetized with numbee was reduced (p=0.035), and this could be attributed to a lesser degree of the profound anesthesia anticipated from numbee, as anesthesia is infused into the epithelium and connective tissue that lies between the gingival sulcus's http://dentistry3000.pitt.edu/ evalua&ng child behavior and preference toward numbee and tradi&onal syringe (a randomized clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.589 h#p://den*stry3000.pi#.edu base and the pdl according to the manufacturer [29,30]. this means, as compared to the conventional needle injection, that less local anesthetic is defused to the marrow spaces of the bone around the teeth, giving less chance to provide deep sensation of tooth numbness. this gave rise to lesser negative behavior in frankl behavior score towards numbee injection compared to the inferior alveolar nerve block. patients in this study disclosed their acceptance and preference equally for both methods; however, less fear was induced in patient who were injected by numbee. this was also clear when n participants declared conventional syringe frightened them during the interview. conclusion the patient’s preference and the acceptance regarding both methods were equivalent; however, patients who are phobic of needles may benefit from numbee as a lower number experience fear while receiving local anesthesia, less stinging sensation, and they had a minimum unpleasant taste. ethical consideration all procedures were performed according to the declaration of helsinki's ethical guidelines and approved by the central committee of ethics of the college of dentistry, university of baghdad (no. 576322, dated 02.06.2022). furthermore, scientific approval has obtained from the scientific committee in the department of pediatric and preventive dentistry in the college of dentistry at the university of baghdad. besides that, clinicaltrials.gov has registered this study and assigned it a registration code of (nct05899296). references 1. ara s, austermann t, may a, mekhemar m, conrad j, knitschke m, böxger s, howaldt hp, riad a. pain percep\on following computer-controlled versus conven\onal dental anesthesia: randomized controlled trial. bmc oral health. 2022 dec;22(1):1-3. doi: 10.1186/s12903-022-02454-1. pmid: 36138388; pmcid: pmc9502910. 2. aghababaie st, monteiro j, stra\gaki e, ashley pf. techniques for effec\ve local anaesthe\c administra\on for the paediatric pa\ent. bri\sh dental journal. 2020 dec;229(12):779-85. pmid: 33339924 3. birnie ka, noel m, chambers ct, uman ls, parker ja. psychological interven\ons for needle-related procedural pain and distress in children and adolescents. cochrane database syst rev. 2018;10(10):cd005179. published 2018 oct 4. doi:10.1002/14651858.cd00517 9.pub4. pmc6517234 4. al haidar a. influence of nonnutri\ve sucking habits on the oral carriage of escherichia coli. interna\onal journal of den\stry. 2022 nov 14;2022. pmcid: pmc9678441 5. diab bs, huda s. khiala. depression status in rela\on to caries experience and salivary physiochemical characteris\cs among 15 years old students in al-swera city–wassit governorate-iraq. j bagh college den\stry. 2015, june, vol. 27(2),158-162 6. bagherian a, sheikhfathollahi m. children's behavioral pain reac\ons during local anesthe\c injec\on using coxon-roll vibra\on method compared with rou\ne topical anesthesia: a randomized controlled trial. dental research journal. 2016 may;13(3):272. pmcid: pmc4878213 7. jamil fa, asmael hm, al-jarsha my. the success of using 2% lidocaine in pain removal during extrac\on of mandibular premolars: a prospec\ve clinical study. bmc oral health. 2020 dec;20(1):1-8. pmcid: pmc7457358 8. koja db, bede sy. evalua\on of buffered local anaesthesia in dental extrac\on: a randomized controlled study. oral surgery. 2022 nov;15(4):489-95. 9. kämmerer pw, palarie v, schiegnitz e, ziebart t, al-nawas b, daubländer m. clinical and histological comparison of pulp anaesthesia and local diffusion aper periodontal ligament injec\on and intrapapillary infiltra\on anaesthesia. j pain relief. 2012;1(10.4172):21670846. 10. klingberg g, broberg ag. dental fear/anxiety and dental behaviour management problems in children and adolescents: a review of prevalence and concomitant psychological factors. interna\onal journal of http://dentistry3000.pitt.edu/ evalua&ng child behavior and preference toward numbee and tradi&onal syringe (a randomized clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.589 h#p://den*stry3000.pi#.edu paediatric den\stry. 2007 nov;17(6):391-406. pmid: 17935593 11. christensen c, arnason sc, oates r, crabtree m, kersey jr jw, vandewalle ks. efficacy of pulpal anesthesia using a needle-less syringe. anesthesia progress. 2020 dec 1;67(4):2006. pmcid: pmc7780256 12. frankl sn. should the parent remain with the child in the dental operatory? j. dent. child. 1962;29:150-63. 13. young da, nový bb, zeller gg, hale r, hart tc, truelove el, ekstrand kr, featherstone jd, fontana m, ismail a, kuehne j. the american dental associa\on caries classifica\on system for clinical prac\ce: a report of the american dental associa\on council on scien\fic affairs. the journal of the american dental associa\on. 2015 feb 1;146(2):79-86. pmid: 25637205 14. patra p. sample size in clinical research, the number we need. int j med sci public health 2012;1:5-9. 15. chen h, cohen p, chen s. how big is a big odds ra\o? interpre\ng the magnitudes of odds ra\os in epidemiological studies. communica\ons in sta\s\cs—simula\on and computa\on®. 2010 mar 31;39(4):860-4. 16. ferguson cj. an effect size primer: a guide for clinicians and researchers. professional psychology research and prac\ce. october 2009. 40 (5), 532–538. 17. rosnow, r. l., & rosenthal, r. sta\s\cal procedures and the jus\fica\on of knowledge in psychological science. american psychologist, 1989, 44, 12761284. 18. cohen j. sta\s\cal power analysis for the behavioural sciences. hillsdale, new jersey: l. lea. 1988;56:102. 19. haghighat a, jafari z, hasheminia d, samandari mh, safarian v, davoudi a. comparisons of success rate and onset \me of two different anesthesia techniques. medicina oral, patologia oral y cirugia bucal. 2015 jul;20(4):e459. pmcid: pmc4523259 20. thiem dge, schnaith f, van aken cme, köntges a, kumar vv, al-nawas b, kämmerer pw. extrac\on of mandibular premolars and molars: comparison between local infiltra\on via pressure syringe and inferior alveolar nerve block anesthesia. clin oral inves\g. 2018;22(3):1523-1530. doi:10.1007/s00784-017-2251-7. pmid: 29043507 21. arapostathis kn, dabarakis nn, coolidge t, tsirlis a, kotsanos n. comparison of acceptance, preference, and efficacy between jet injec\on injex and local infiltra\on anesthesia in 6 to 11 year old dental pa\ents. anesthesia progress. 2010 mar 1;57(1):3-12. pmcid: pmc2844236 22. riba h, al-zahrani s, al-buqmi n, al-jundi a. a review of behavior evalua\on scales in pediatric den\stry and suggested modifica\on to the frankl scale. ec dental science. 2017 dec 30;16(6):269-75. 23. enad hh, al-mizraqchi as. salivary cor\sol as a stress biomarker and total viable count of salivary bacterial microbiome among covid-19 pa\ents. j bagh coll dent. 2021 sep.15.33(4):6-10. available from: hxps://jbcd.uobaghdad.edu.iq/i ndex.php/jbcd/ar\cle/view/301 3 24. jafar z. interac\on of dental anxiety and abo secretor status of blood group on dental caries in a group of children. braz. dent. sci.2022;25(1):e2928. doi:hxps://doi.org/10.4322/bds. 2022.e2928 25. singh b, deshmukh s. cogni\ve contemporary behaviour management outline. journal of evolu\on of medical and dental sciences. 2021 oct 4;10(40):3548-54. 26. khalaf ms, khalaf bs, abass sm. management of trauma to the anterior segment of the maxilla: alveolar fracture and primary incisors crown and root fracture. journal of baghdad college of den\stry. 2021 jun 11;33(2):1620. 27. kaufman e, epstein jb, naveh e, gorsky m, gross a, cohen g. a survey of pain, pressure, and discomfort induced by commonly used oral local anesthesia injec\ons. anesthesia progress. 2005 dec 1;52(4):1227. 28. narayan vk, samuel sr. appropriateness of various behavior ra\ng scales used in pediatric den\stry: a review. j global oral health 2019;2(2):112-7 29. hxps://www.researchsquare.co m/ar\cle/rs-2579302/v1. 30. hashim abd ali a, d. te, v. bo, v. kg, p. st. inves\ga\on of the associa\on of agtr1 a1166c rs5186 and fto rs9939609 polymorphisms with the obesity in children and adolescents. baghdad sci.j [internet]. 2022 http://dentistry3000.pitt.edu/ evalua&ng child behavior and preference toward numbee and tradi&onal syringe (a randomized clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.589 h#p://den*stry3000.pi#.edu dec. 1 [cited 2023 apr. 18];19(6):1228. available from: hxps://bsj.uobaghdad.edu.iq/in dex.php/bsj/ar\cle/view/6540 http://dentistry3000.pitt.edu/ microsoft word 348 2022.docx a randomized control trial for compara3ve evalua3on of 3-d versus 2-d miniplates for internal fixa3on of mandibular fractures vol 10 no 1 (2022) doi 10.5195/d3000.2022.348 h#p://den*stry3000.pi#.edu a randomized control trial for comparative evaluation of 3-d versus 2-d miniplates for internal fixation of mandibular fractures sanjay kumar, nageshwar iyer, mansi jain abstract fractures of the mandible are a common facial injury. for restora3on of normal jaw structure and func3on, proper bony healing is important; thus, stable plate osteosynthesis has become an indispensable component of cranio-maxillofacial surgery. aim: this study aims to evaluate the efficacy of 3d versus 2d mini bone plates used for internal fixa3on of mandibular fractures. materials and methods: a prospec3ve randomized control trial was conducted on 20 pa3ents with mandibular fractures in the symphysis and parasymphysis regions. in group 1, 3-d miniplates were used with a single, rectangular, 4-holed 3tanium plate with 4 screws and in group 2, 2-d miniplates were used with two 4-holed 3tanium plates fixed with 8 screws. time taken for fixa3on and operator comfort was analysed using the chi square test, whereas trismus and pain were analysed using anova. results: 1. the difference for mean 3me taken for fixa3on was sta3s3cally highly significant (p < 0.01) between 3-d (33.1 minutes) and 2-d plate (44.7 minutes). 2. fixa3on of 2-d plates was found more comfortable. 3. swelling in both the groups was generally comparable and lasted for about 1 week. 4. mouth opening in pa3ents of both the groups showed a gradual recovery 3ll 1 month a[er surgery, a[er which it stabilized. conclusion: 3-d miniplates were more economical, with less opera3ve 3me required and with be\er performance over 2-d miniplates. however there was difficulty in their adap3on and they were inappropriate when the fracture line was very close to the mental foramen. keywords: titanium 3-d miniplates; titanium 2-d miniplates; internal fixa3on; mandibular fractures; facial injuries cita:on: kumar, s et al. (2022) a randomized control trial for compara:ve evalua:on of 3-d versus 2-d miniplates for internal fixa:on of mandibular fractures den:stry 3000. 1:a001 doi:10.5195/d3000.2022.348 received: may 11, 2022 accepted: july 5, 2022 published: august 12, 2022 copyright: ©2022 kumar, s et al. this is an open access ar:cle licensed under a crea:ve commons ayribu:on work 4.0 united states license. email: mansijainsoni@gmail.com introduction in this age of technological progress, there has been a notable increase in trauma including the maxillofacial region. the face, being the most admired point in one’s body, any defacement due to trauma would greatly affect a person both physically and psychologically. hence, any injury to maxillofacial region needs urgent and skilled management [1]. fractures of the mandible are more common than middle-third facial injuries and are the second most common after nasal fractures [2]. a primary objective in the management of fractures in the maxillofacial region is the restoration of normal jaw structure and function which should be accomplished expeditiously and with least patient discomfort. union of the fracture segments will occur in the absence of gross mobility; hence, stability of the segments is key for proper hard and soft tissue healing in the injured area. therefore, the fracture site must be stabilized in a randomized control trial for compara3ve evalua3on of 3-d versus 2-d miniplates for internal fixa3on of mandibular fractures vol 10 no 1 (2022) doi 10.5195/d3000.2022.348 h#p://den*stry3000.pi#.edu order to guide the process toward normal bony healing. hippocrates (460-375 b.c.) was the first to mention bandages as a method to immobilize fractures of jaw using leather straps with a paste to adhere to the skin to apply direct traction [3]. schede (1888) is credited with first use of bone plates and secured with four screws [4]. champy m et. al. (1978) introduced a miniplate system to treat midface fracture [2]. bone plates are placed according to the champy’s line of osteosynthesis perpendicular to the fracture line [5]. stable plate osteosynthesis has become an indispensable component of cranio-maxillofacial surgery in the treatment of fractures and in osteotomies of the facial bones. farmand min (1990) developed a new miniplate system made of titanium known as three dimensional plating systems (3-d) that takes the advantages of bio-geometry to provide stable fixation [6]. the basic concept of three dimensional fixations is the geometrically closed quadrangular plate to create stability in all three dimensions [2]. hence this study was conducted to compare the efficacy of 3-d versus 2d bone plates used for internal fixation of mandibular fractures. material and methods a prospective randomized control trial was conducted after approval of institution ethical committee iec project number 1491 and carried out in accordance with the code of ethics of the world medical association (declaration of helsinki, 2013) on 20 subjects, serially selected for the study from the out-patient department of oral and maxillofacial surgery. all patients with fairly good general health (asai & ii), without any contraindication for surgery with fracture between the mental foramina in anterior region of mandible, with or without other associated fractures and indicated for internal rigid fixation were included in study. patients with neurological damage from head injury, with pre-existing motor paralytic disease, grossly displaced or comminuted fractures, facture lines passing through or very close to the mental foramina and edentulous patients in whom occlusion was not assessable were excluded from the study. preoperative treatment preoperative erich’s arch bar, maxillomandibular fixation screw or ivy eyelet loop wiring as indicated were placed with 26 gauge soft stainless steel wire on both the arches to achieve the pre morbid occlusion. for group 1: 1. titanium 3-d miniplates, 2 mm system • 2 x 2 holes 2. titanium screws • 2 x 6 mm • 2 x 8 mm • 2 x 10 mm for group 2: 3. titanium 2-d miniplates, 2 mm system and 2.5 mm system • 4 holes with extended bar 4. titanium screws • 2 x 6 mm • 2 x 8 mm • 2 x 10 mm • 2.5 x 8 mm • 2.5 x 10 mm surgical technique all the patients were operated intra orally except one, who had a lacerated wound near the mental region. de-gloving incisions in symphysis or parasymphysis region were made. reduction of fracture fracture reduction was done to achieve occlusion with the help of maxillo-mandibular fixation using arch bar, imf screw or eyelet wiring. reduction under direct supervision using two pronged reduction clamps were done in cases where the arch bar reduction could not be done. adaption & fixation of plate after fracture reduction, fixation was done using a single three dimensional plate in group 1 (3-d) patients and a randomized control trial for compara3ve evalua3on of 3-d versus 2-d miniplates for internal fixa3on of mandibular fractures vol 10 no 1 (2022) doi 10.5195/d3000.2022.348 h#p://den*stry3000.pi#.edu two 4holed with extended bar in group 2 (2-d) patients. group 1 titanium 3-d 2 x 2 hole bone plate was adapted to confirm to the bone contour and lie passive against the bone surfaces. the horizontal crossbars were positioned perpendicular to the fracture line and vertical struts were placed parallel to fracture line. in oblique fracture, the plates were placed parallel to the lower border of mandible and in parasymphyseal fractures, the upper crossbar was placed in the subapical position. the drilling was performed through the hole in the plate perpendicular to the bone surface. the first screw was inserted in the drilled hole using a 2 mm screw holding device and the second hole was placed diagonally. layer wise suturing of the soft tissue was done and pre-operative maxillomandibular fixation was removed after the completion of the procedure (fig 1, 2). group 2bone plates were positioned perpendicular to the fracture line according to the champy’s line of osteosynthesis. the plate was adapted according to the contour of bone and held firmly with hand instruments. drilling was performed through the hole in the plate perpendicular to the bone surface. two parallel plates were used to neutralize torsional forces in the symphysis region between the two mental foramina. the lower plate was fixed first, followed by the sub-apical plate (fig 3, 4). figure 1. case 1 – preoperative view showing right parasymphysis fracture figure 2. case 1 – placement of 3-d plates a randomized control trial for compara3ve evalua3on of 3-d versus 2-d miniplates for internal fixa3on of mandibular fractures vol 10 no 1 (2022) doi 10.5195/d3000.2022.348 h#p://den*stry3000.pi#.edu post-operative follow up clinical and radiological follow up for 3 months postoperatively regarding the restoration of function, occlusion, stability of system used, ease of application and cost of treatment, incidence of operative & postoperative complication i.e. infection, dehiscence of wound, loosening of screw, disturbance in occlusion, nonunion and malunion was done. statistical analysis time taken for fixation and operator comfort was analysed using the chi square test. trismus and pain were analysed using anova. results all the patients were male with a mean age of 28 years (range 19 to 45 years). fractures on the right side predominated in the ratio of 3:2. all the fractures were due to road traffic accidents. following were the findings of the study: 1) mean bme taken for fixabon of 3-d plate was 33.1 minutes (ranging from 25 to 37 minutes); that for 2-d plate was 44.7 minutes (ranging from 40 to 50 minutes). the difference was highly significant (p < 0.01). (table 1) 2) fixabon of 2-d plate was found more comfortable by the operator. the difference was highly significant (p < 0.01). (table 2) 3) swelling in both the groups was generally comparable and lasted for about 1 week. (table 3) 4) neurosensory deficit associated with mental nerve was found in 3 pabents in group 2 (2-d plate) which lasted for a longer durabon (1 month) as compared to only in 1 pabent in group 1 (3-d plate) which lasted for 2 weeks. (table 4) figure 3. case 2 – preoperative view showing left parasymphysis fracture figure 4. case 2 – placement of two 2-d plates a randomized control trial for compara3ve evalua3on of 3-d versus 2-d miniplates for internal fixa3on of mandibular fractures vol 10 no 1 (2022) doi 10.5195/d3000.2022.348 h#p://den*stry3000.pi#.edu 5) there was no difference between the two groups in terms of reducbon of masbcatory efficiency. (table 5) 6) mouth opening in pabents of both the groups showed a gradual recovery during the first month aher which it stabilized. (table 6) 7) there was a gradual decrease in pain in pabents of both groups. none of the pabents complained of pain aher 1 week. (table 7) discussion injuries to the face can lead to devastating esthetic and functional sequelae. despite improved technology and fixation techniques for mandibular fractures, its management still remains challenging [7]. the anatomy of the mandible and vector of forces exerted by the masseter and temporalis muscles make symphysis/parasymphysis fractures exceedingly difficult. these vector forces tend to displace the segments of the mandible at the site of fracture. besides, the torsional forces, there are overlapping tensile and compressive loads in both the directions [1]. furthermore, the intra-operative and post-operative periods have become more comfortable for the surgeon as well as the patient. the 3d miniplates hold the fracture fragments rigidly by resisting the shearing, bending and torsional forces. farmand (1995) stated that a geometrically closed quadrangular plate secured with bone screws creates stability in all three dimensions. the stability is gained over a defined surface area and is achieved by its configuration, not by thickness or length. the large free areas between the plate arms and minimal dissection utilized permit good blood supply to the bone [6]. as both symphysis and parasymphysis fractures are under higher torsional strain than other areas of the mandible; 3d plates in this area provide superior stability [2]. in the present study, patients in group 1 had a mean age 32.8 years, and in group 2 a mean age of 23.2 years. this agrees with sawhney cp and ahuja rb, who observed that 75% of the cases were in the 16 – 45 year age group [8]. erdmann et al reported that road traffic accidents to be a common etiology for mandibular fractures [9]. lida s, kogo m et. al. (2001) in a retrospective analysis of 1,502 patients with facial fractures reported that male: female ratio was 2.8:1 with the largest subgroup being patients between 10 29 years of age [10]. abosadegh maher et al. reported that the largest group was in the age group of 20 – 39 years with a male: female ratio of 5:1 [11]. in group 1 patients, the total time taken for the procedure from the placement of incision till the completion of closure was in the range of 25–37 minutes (mean 33.1 minutes) whereas in group 2, it was in the range of 40 to 50 minutes(mean 44.7 minutes) which was a highly significant difference (p < 0.01). (table 1) it was in accordance with those who reported mean intraoperative times of 54.8 min for 2d miniplates and 40.6 for 3d miniplates [12]. group time taken range (min) mean (min) s.d (min) i (3d – plate) 25 37 33.1 3.41 ii (2d – plate) 40 – 50 44.7 3.52 total 25 50 38.9 6.84 table 1: mean time taken for fixation in minutes *p < 0.01 highly significant a randomized control trial for compara3ve evalua3on of 3-d versus 2-d miniplates for internal fixa3on of mandibular fractures vol 10 no 1 (2022) doi 10.5195/d3000.2022.348 h#p://den*stry3000.pi#.edu operator comfort for the placement of miniplates was judged as good, moderate or bad. it was observed that in group 1 it was moderate for 6 patient and good for 4 patients. in group 2 it was found to be good for all the 10 patients (table 2). the difference was highly significant (p < 0.01). hence, this shows that placement of conventional miniplates is easier as compared to 3-d plate. group total (no. of pts) good (no. of pts) moderate (no. of pts.) bad (no. of pts.) 1(3dplate) 10 4 6 0 ii(2dplate) 10 10 0 0 total 20 14 6 0 swelling was graded in all cases included in the study as none, mild, moderate or severe. it was observed that swelling reduced progressively with time. immediately postop, group 1 swelling was graded as mild in 9 patients and moderate in 1 patient. after 1 week, mild swelling was present in 4 patients. after follow up of two weeks none of patients reported with swelling (table 3). in group 2 swelling postoperatively was graded as mild in 8 patients and moderate in 2 patients. after 2 weeks 1 patient reported with swelling. on regular follow up none of the patient reported with swelling (table 3). neither method of fixation induces much swelling after placement. group swelling no: of patients pre tt immd postop. 1wk 2wk 1mon. 2 mon. 3mon i (3d – plate) mild 9 9 4 0 0 0 0 moderate 1 1 0 0 0 0 0 severe 0 0 0 0 0 0 0 total 10 10 4 0 0 0 0 ii (2d– plate) mild 8 8 1 1 0 0 0 moderate 2 2 0 0 0 0 0 table 2: operator’s comfort *p < 0.01 highly significant table 3: swelling in group a randomized control trial for compara3ve evalua3on of 3-d versus 2-d miniplates for internal fixa3on of mandibular fractures vol 10 no 1 (2022) doi 10.5195/d3000.2022.348 h#p://den*stry3000.pi#.edu severe 0 0 0 0 0 0 0 total 10 10 1 1 0 0 0 grand total 20 20 5 1 0 0 0 sensory changes were also observed throughout the period of study as paresthesia in the region of miniplates fixation. in group 1, 10% patient reported with neurosensory deficit post-operatively which recovered fully after 2 weeks follow up. in group 2, 30% reported with post-operative neurosensory deficit. after follow up of 1 month, recovery was complete in all the patients. none of the patients reported with permanent neurosensory deficit (table 4). nilima j. budhraja et al (2018), also reported that no patients treated with three dimensional miniplates had neurosensory deficit [13]. group total no: of patients no. of patients with neurosensory deficit pre t/t immd post-op. 1wk 2wk 1mon 2mon 3mon i (3d– plate) 10 1 1 1 1 0 0 0 ii (2d– plate) 10 3 3 2 1 1 0 0 total 20 4 4 4 2 1 0 0 masticatory efficiency was observed in all the patients throughout the study. patients of both groups were kept on soft diet for a period of two weeks. after follow up of 1 month all the patients were able to chew on medium hard food and after 2 months all the patients in both groups were able to chew hard food (table 5). table 4: number of patients with neurosensory deficit a randomized control trial for compara3ve evalua3on of 3-d versus 2-d miniplates for internal fixa3on of mandibular fractures vol 10 no 1 (2022) doi 10.5195/d3000.2022.348 h#p://den*stry3000.pi#.edu inter-incisal mouth opening also increased progressively with passage of time. mean mouth opening pretreatment was 20.1 mm in group 1 and 19.5 mm in group 2, which increased to 23.1 mm and 20.2 mm for group 1 and group 2 respectively immediately postoperatively. no statistically significant difference (p > 0.05) was found between the two groups at 3 months post operatively (table 6). group total no: of patients mean trismus index (mm) pre t/t immediately post t/t 1wk 2wk 1mon 2mon 3mon i (3d– plate) 10 20.1 23.1 25.9 29.4 36.5 37.2 37.4 ii (2d– plate) 10 19.5 20.2 23.6 26.6 33.9 36.1 36.8 total 20 19.8 21.65 24.75 28 35.2 36.6 37.1 group masticatory efficiency score no. of pts pre t/t 1 day post t/t 1wk post t/t 2weeks post t/t 1month post t/t 2month post t/t 3month post t/t i (3d– plate) 0 10 0 0 0 0 0 0 1 0 10 10 4 0 0 0 2 0 0 0 6 10 0 0 3 0 0 0 0 0 10 10 ii (2d– plate) 0 10 0 0 0 0 0 0 1 0 10 10 6 0 0 0 2 0 0 0 4 10 0 0 3 0 0 0 0 0 10 10 table 5: masticatory efficiency *key: 0 = unable to chew 1 = able to chew soft food 2 = able to chew medium hard food 3 = able to chew hard food table 6: mean trismus index (inter incisal opening in mm) *p > 0.05 a randomized control trial for compara3ve evalua3on of 3-d versus 2-d miniplates for internal fixa3on of mandibular fractures vol 10 no 1 (2022) doi 10.5195/d3000.2022.348 h#p://den*stry3000.pi#.edu pain was assessed in all the patients on a visual analogue scale (vas) of 0100. pretreatment mean value for group 1 was 78 and for group 2 was 85 (table 7). this gradually reduced to 0 by the end of two weeks. statistical analysis (anova) was not significant (p > 0.05) between the groups. group total no: of patients visual analogue scale pre t/t 1 day post t/t 1week post t/t 2weeks post t/t 1month post t/t 2month post t/t 3month post t/t i (3d– plate) 10 78 37 5 0 0 0 0 ii (2d– plate) 10 85 31 6.5 0 0 0 0 total 20 81.5 34 5.75 0 0 0 0 other complications studied were infection, exposure of plate, fracture of the hardware, and any other complication associated with placement of miniplates. none of the patients reported with infection postoperatively. 1 patient in group 2 reported with wound dehiscence and exposure of the root of right mandibular lateral incisor. none of the patients in either groups reported with fracture or exposure of the hardware or any other complication. it was concluded from the present study that 3d plates were easy and simple to use except in cases involving the mental nerve [14]. presence of connecting arms made the plate placement comfortable without displacement. the quadrangular geometry of plate assures a 3d stability at the fracture site and offers good resistance against torque forces, thereby avoiding the need for maxillomandibular fixation. further, there was no major postoperative complications such as nonunion or hardware failure. conclusion 3-d miniplates have the advantage of low cost and less operative time required. further no fracture or exposure of hardware was seen. however they were difficult to adapt and inappropriate when the fracture line is very close to the mental foramen. on the other hand 2-d miniplates were easier to adapt, but were harder to place, time consuming, with higher cost. clinical significance being more economical, requiring less intraoperative time and with equally good postoperative results, 3-d miniplates are preferable over 2-d miniplates. ethical statement the study was approved by the institutional ethical committee. conflict of interest there is no "conflict of interest" between the authors. references 1. comparison of 2 mm single locking miniplates versus 2 mm two non-locking miniplates in symphysis and parasymphysis fracture of mandible. vashistha a, singh m, chaudhary m, agarwal n, kaur g. j oral biol craniofac res. 2017 jan-apr; 7(1):42-48. doi: 10.1016/j.jobcr.2016.01.00 1, pmcid: pmc5343153 2. three-dimensional miniplate: for the management of mandibular parasymphysis fractures. mohd yq, reddy s, sinha r, agarwal a, fatima u, abidullah m. ann maxillofac table 7: pain on visual analogue scale *p > 0.01not significant a randomized control trial for compara3ve evalua3on of 3-d versus 2-d miniplates for internal fixa3on of mandibular fractures vol 10 no 1 (2022) doi 10.5195/d3000.2022.348 h#p://den*stry3000.pi#.edu surg. 2019; 9(2):333-339. doi:10.4103/ams.ams_172_17 pmid: 31909011 3. advancements in management of mandibular fractures. kanwaldeep singh soodan and pratiksha priyadarshni. acta scientific dental sciences.2018; 2.5: 29-31. 4. booth pw, stephen as, harisamen je.(1999) maxillofacial surgery.vol-1, edinburgh: churchill livingstone. 5. single miniplate fixation for mandibular symphysis and parasymphysis fracture as a viable alternative to conventional plating based on champy's principles: a prospective comparative clinical study. raut r, keerthi r, vaibhav n, ghosh a, kamath kateel s. j maxillofac oral surg. 2017;16(1):113-117. doi: 10.1007/s12663-016-09191, pmid: 28286395 6. a comparative study of 3dimensional titaniumversus 2dimensional titanium miniplates for open reduction and fixation of mandibular parasymphysis fracture. yogesh mittal, k. george varghese, s. mohan, n. jayakumar, and somil chhag. j maxillofac oral surg. 2016 mar; 15(1): 93–98. doi: 10.1007/s12663-015-07807, pmid: 26929559 7. mandible fractures. pickrell bb, serebrakian at, maricevich rs. semin plast surg. 2017 may;31(2):100-107. doi: 10.1055/s-0037-1601374, pmid: 28496390 8. faciomaxillary fractures in north india a stastical analysis and review of literature. sawhney cp and ahuja rb. br j oral maxillofac surg 1988;26:430-4. doi: 10.1016/02664356(88)90097-6, pmid: 3191093. 9. a retrospective analysis of facial fracture etiologies, annals of plastic surgery. erdmann detlev, follmar keith e., debruijn marlieke, bruno anthony d., jung sin-ho, edelman david, mukundan srinivasan md, marcus jeffrey r. april 2008 ; 60(4):398-403. doi: 10.1097/sap.0b013e31813 3a87b, pmid: 18362568 10. retrospective analysis of 1502 patients with facial fractures. lida s, kogo m, sugiura t, mima t, matsayu t. int j oral maxillofac surg 2001;30:286-90. doi: 10.1054/ijom.2001.0056, pmid: 11518349. 11. epidemiology and incidence of traumatic head injury associated with maxillofacial fractures: a global perspective. abosadegh maher m, rahman shaifulizan a. b. journal of international oral health. 2018; 10 :( 2):63-70. doi: 10.1111/j.13652842.2007.01799.x, pmid: 18034671 12. a comparahve evaluahon of 2.0mm two-dimensional miniplates versus 2.0mm threedimensional miniplates in mandibular fractures. godvine sarepally, swetcha seethamseiy, tanveer karpe, fazil a nasyam, umayra fahma, raia fatema. cureus. 2022 jan; 14(1): e21325. doi: 10.7759/cureus.21325, pmid: 35186584 13. three-dimensional locking plate and convenhonal miniplates in the treatment of mandibular anterior fractures. nilima j. budhraja, ramakrishna s. shenoi, samprah j. badjate, kshihj o. bang, pranav d. ingole, vrinda s. kolte. ann maxillofac surg. 2018 jan-jun; 8(1): 73–77. doi: 10.4103/ams.ams_175_17, pmid: 29963428 14. comparison of 3-dimensional and standard miniplate fixahon in the management of mandibular fractures. manoj kumar jain, k s manjunath, b k bhagwan, dipit k shah. j oral maxillofac surg. 2010 jul; 68(7):1568-72. doi: 10.1016/j.joms.2009.07.08 3, pmid: 20417012 vol 1, no 1 (2013) issn 2167-8677 (online) doi 10.5195/d3000.2013.16 http://dentistry3000.pitt.edu this work is licensed under a creative commons attribution 3.0 united states license. this site is published by the university library system, university of pittsburgh as part of its d-scribe digital publishing program and is cosponsored by the university of pittsburgh press. summary of the iadr cariology research, craniofacial biology, and mineralized tissue groups symposium, iguaçu falls, brazil, june 2012 adriana modesto 1 , ophir klein 2 , livia m.a. tenuta 3 , raquel f. gerlach 4 , alexandre r. vieira 1 1university of pittsburgh, school of dental medicine, pittsburgh, pa, usa 2university of california, school of dentistry, san francisco, ca, usa 3university of campinas, piracicaba dental school, piracicaba, sp, brazil 4university of são paulo, ribeirão preto, sp, brazil abstract characteristics of enamel may influence or modulate individual susceptibility to caries and erosion. these characteristics are defined during development, which is under strict genetic control, but can easily be modified in many ways by environmental factors. in the symposium, translational aspects of embryology, biochemistry, and genetics of amelogenesis were presented. the symposium provided unique insight into how basic sciences integrate with clinically relevant problems. the need for improved understanding of risks at the individual level, taking into consideration both environmental exposures and genetic background, was presented. the symposium was divided into four stepwise and interconnected topics as follows: 1) the many faces of enamel development; 2) enamel pathogenesis: biochemistry lessons; 3) environmental factors on enamel formation; and, 4) genetic variation in enamel formation genes. citation: modesto a, klein o, tenuta lma, gerlach rf, vieira ar. (2013) summary of the iadr cariology research, craniofacial biology and mineralized tissue groups symposium, iguaçu falls, brazil, june 2012. dentistry 3000 1:a005 doi: 10.5195/d3000.2013.16 received: november 18, 2013 accepted: november 25, 2013 published: december 12, 2013 copyright: ©2013 modesto et all. this is an openaccess article licensed under a creative commons attribution work 3.0 united states license. email: ams208@pitt.edu introduction oral diseases are progressive and cumulative, and they affect people throughout their life span. the etiology and pathogenesis of diseases and disorders affecting the craniofacial structures are multifactorial and complex, involving interplay among genetic, environmental, and behavioral factors. nearly every american has experienced dental caries, which is the single most common chronic childhood disease five times more common than asthma and seven times more common than hay fever. over 50 percent of 5to 9-year-old children have at least one carious lesion or restoration; that proportion increases to 78 percent among 17-year-olds [1]. for the rest of the world, caries is also a major public health problem with an estimated 60-90% of school children and a vast number of adults affected [2]. according to the report on the nih consensus development conference on diagnosis and management of dental caries throughout life, there is a need to improve methods of assessing risk and diagnosing dental caries to make progress in eliminating this disease. additionally, it was stated that there is a gap between research findings and oral disease prevention, health promotion practices, and education of the public and the health professions. research is needed to develop better measures of disease and health, to explain the differences among population groups, and to develop interventions targeted at eliminating disparities [3]. tooth enamel is unique because it has the highest mineral content of all mineralized tissues. it is also an active chemical system that participates in a variety of chemical reactions, including solute and ion transport from saliva to dentin and back, ionexchange reactions with saliva, and demineralization-remineralization processes. however, despite its high-level organization and outstanding physical properties, which make it the hardest tissue in the vertebrate body, tooth enamel can be destroyed fairly rapidly by dental caries [4-5]. current evidence supports the linkage of altered dental enamel development with increased susceptibility to dental caries. increased enamel porosity, decreased mineral content, and the presence of enamel crystal inhibitory proteins all are directly linked to dental caries risk [6]. gene-environment interactions and epigenetics in oral diseases: enamel formation and its clinical impact on tooth defects, caries, and erosion http://dentistry3000.pitt.edu/ http://creativecommons.org/licenses/by-nc-nd/3.0/us/ http://www.library.pitt.edu/ http://www.pitt.edu/ http://www.library.pitt.edu/articles/digpubtype/index.html http://upress.pitt.edu/ http://creativecommons.org/licenses/by-nc-nd/3.0/us/ summary of the iadr cariology research, craniofacial biology, and mineralized tissue groups symposium, iguaçu falls, brazil, june 2012 vol 1, no 1 (2013) doi 10.5195/d3000.2013.16 http://dentistry3000.pitt.edu 2 figure 1. perp localization in developing incisors at postnatal day 7. (a) h&e staining of the lower incisor and mandible in sagittal section. (a’) cartoon of the mandible. (b-e) h&e staining of the lower incisor. (f-i) perp immunofluorescence staining at presecretory, secretory, transitional, and mature stages. figure 2. scanning electronic microscope (sem) analysis of teeth at postnatal day 2. (a, c) lowmagnification images of the enamel surface of the first lower molars from wild-type (perp+/+) and perp-null (perp-/-) mice. (b, d) higher-magnification images of the boxed areas. therefore, the primary focus of the enamel formation and its clinical impact on tooth defects, caries, and erosion symposium was to discuss how diseases affecting the enamel result from imbalance between diseasedriving agents and protective factors, and we also discussed the importance of identifying tools to better diagnose, predict, and treat these diseases. the symposium was geared toward researchers and clinicians, in both academics and industry. the goal was to achieve a better understanding of how the interaction between environment and molecular biology underlie individual susceptibility, how this impacts oral health, and how to prevent or reduce the burden of disease. the many faces of enamel development (presenter o. klein) dental development involves many cellular processes, such as proliferation, differentiation, and apoptosis. hence, it is a complex mechanism that is still not fully understood. the outer layer of teeth is comprised of enamel, which is unique among mineralized tissues in its hardness and organization. enamel formation initiates as the epithelialderived enamel organ generates the inner enamel epithelium, which differentiates into the enamel-forming ameloblasts. presecretory ameloblasts differentiate into secretory ameloblasts, which deposit an extracellular matrix, comprised of proteins such as amelogenin, ameloblastin, enamelin, tuftelin, and matrix metalloproteinase 20 (mmp20) [7-9]. mineralization starts and a shift from matrix deposition to resorption occurs. the basal surface of ameloblasts is attached to the stratum intermedium, a layer of two or three cells between the inner enamel epithelium and the newly forming cells of the stellate reticulum. expression of mmp20, kallikrein 4, amelotin, and odam can be detected [8-11]. eventually, the matrix is replaced by secondary crystal growth, leading to complete mineralization of enamel. the adherens junction protein nectin-1 was previously shown to indirectly affect desmosomes at the ameloblast-stratum intermedium interface, resulting in enamel defects [12]. because perp (p53 apoptosis effector related to pmp-22) is required for desmosome assembly and for the integrity of stratified epithelia, our laboratory, in a project led by dr. andrew jheon, focused on determining whether perp has a role in the formation of enamel. perp is a tetraspan membrane protein that localizes to the desmosome, an important cell-cell junction structure. perp-/mice exhibit postnatal lethality and defects in stratified epithelia. perp is expressed at the ameloblast-stratum intermedium interface (figure 1), and because perp-/mice exhibit postnatal lethality, perp was conditionally inactivated in the dental epithelium. interestingly, perp conditional null mice had abnormal enamel (figure 2). in the abnormal enamel matrix in perp mutants, ameloblasts detach from the stratum intermedium (figures 3 and 4). http://dentistry3000.pitt.edu/ summary of the iadr cariology research, craniofacial biology, and mineralized tissue groups symposium, iguaçu falls, brazil, june 2012 vol 1, no 1 (2013) doi 10.5195/d3000.2013.16 http://dentistry3000.pitt.edu 3 perp-mutant incisors had fewer and smaller desmosomes. several genes involved in amelogenesis (ambn, enam, mmp20, and klk4) were downregulated in perp-mutants. these experiments point to a model in which perp is essential for amelogenesis involving cell-cell adhesion, through desmosome-mediated interactions between the ameloblasts and the stratum intermedium. these data and additional findings have been published [13]. enamel pathogenesis: biochemistry lessons (presenter l.m.a. tenuta) dental enamel is the hardest structure of the human body. it has a repeated structure of prisms in which hydroxyapatite crystals are tightly packed. at the ultrastructural level, the crystalline structure of enamel can be significantly altered, either during its formation, such as in fluorosis, or as a result of the interaction with the oral environment, such as in caries and erosion. the common fate of dental enamel and teeth has changed during human history. although in the past the enamel was submitted to abrasive forces due to the rough and hard nature of foods, in the last few centuries, it became more common to see dental enamel completely dissolving due to the action of acids. the big change in the pattern of enamel loss was the result of sugar, which was made highly accessible by man in the last centuries [14]. the problem with sugar is its metabolization by dental biofilm [15], which naturally forms on enamel surface exposed to the oral environment [16]. although saliva, and even the fluid phase of dental biofilm, are highly supersaturated with respect to dental minerals [17], upon exposure to fermentable sugars, fast and intense ph drop occurs [18]. the ph drop in the biofilm has a direct effect on the stability of hydroxyapatite in enamel. as hydroxyapatite is a phosphatecontaining mineral, its solubility increases as the ph decreases [19]. the reduction of the activity product of ions, which compose hydroxyapatite in the biofilm fluid during the low ph, results in the dissolution of solid minerals in order to maintain the solubility equilibrium [20]. the demineralization process is followed by a remineralizing period in which part of minerals dissolved can be replenished [21]. if this cycle frequently continues favoring demineralization, the resulting enamel crystals will gradually dissolve [22,23]. moreover, when the episodes of exposure to sugar are frequent during the day, acidogenic and aciduric bacteria will prevail in the biofilm [24]. if the sugar is sucrose, the resulting biofilm is even more cariogenic due to the presence of extracellular polysaccharides that will change the biofilm matrix to a more porous and cariogenic one [25,26]. in the dental caries process, this dissolution is controlled by pores which diffuse acid into enamel, resulting in more severe dissolution in the subsurface area of enamel [27]. this phenomenon has been attributed to the coupled diffusion of protons inward and of calcium and phosphate outward in enamel (faster for protons than for calcium and phosphate), to the reprecipitation of minerals dissolving from the subsurface at the surface, as well as the result of some protecting effects of fluoride on the surface [28]. the surface of a carious lesion, however, is not intact, but full of dissolution spaces, which gives the carious enamel a rough and opaque clinical appearance [29]. the great increase in caries rates in parallel to the availability of sucrose in occidental, modern diets culminated with the astonishing dental caries rates observed worldwide in the late 60’s. the recently-discovered physicochemical effect of fluoride on mineral loss, reducing enamel demineralization during a ph drop, and increasing remineralization rates by the precipitation of less soluble mineral phases [30] was responsible at that time for the reduction of caries progression even when the cariogenic challenge was maintained [31]. by using fluoride in various forms, the worldwide dental caries prevalence considerably declined [2]. fluoride use in water, as well as in toothpastes, has been systematically correlated with decreased dental caries prevalence [32,33]. however, the widespread use of fluoride to control caries has been followed by an increase in prevalence of dental fluorosis. dental fluorosis is a function of the overall chronic exposure to fluoride, and thus, fluoride in water has been related to fluorosis [32]. in regards to fluoride toothpaste use, the effect on fluorosis is still to be clearly determined [34,35]. most of all, fluorosis as a result of diet, and toothpaste exposure is mainly in the mild and very mild degrees, which were shown not to affect the oral health-related quality of life of those affected [36]. for this reason, fluoridebased strategies continue to help us to maintain caries at reduced levels considering our cariogenic diet. figure 3. h&e staining of developing mandibular incisors and maxillary molars in wild-type (perp+/+) and perp-null (perp-/-) mice at postnatal day 7 is sagittal sections. (a-d) low-magnification images of the lower incisor and first upper molar. (a’-d’) higher magnification images of the boxed areas. dentin (dn) and enamel (en) are denoted by yellow and red arrowheads, respectively. areas of defective enamel are denoted by red asterisks in the incisors and molars of perp-null mice. p, proximal; d, distal. http://dentistry3000.pitt.edu/ summary of the iadr cariology research, craniofacial biology, and mineralized tissue groups symposium, iguaçu falls, brazil, june 2012 vol 1, no 1 (2013) doi 10.5195/d3000.2013.16 http://dentistry3000.pitt.edu 4 figure 4. displacement of ameloblasts from the stratum intermedium (si). (a, b) h&e staining of the transitional stage in sagittal sections of incisors at postnatal day 7. detached cells between ameloblasts and the enamel matrix in perp-null mice are indicated (red arrowheads). (c, d) dapi staining of adjacent sections shows the presence of the detached cells (red arrowheads). (e-h) detached cells express ameloblast-specific proteins such as ameloblastin (ambn, red arrowheads) and amelogenin (amel; red arrowheads). matrix vesicles present in both wild-type and perp-null teeth are indicated (yellow arrowheads). en, enamel; am, ameloblasts. although fluoride has a history of great success on caries control, its effect on erosion is still to be proved. the erosive wear, as opposed to caries, occurs when teeth are exposed to very acidic solutions such as soft drinks, juices, or the acid juice from the stomach, which are highly undersaturated in respect to hydroxyapatite. therefore, minerals would be washed away from the surface of enamel, resulting in a softened etched surface [37]. the etched surface is susceptible to abrasive forces, and enamel loss happens from the surface, layer by layer. fluoride has little effect on preventing mineral loss from erosion, since fluoridecontaining minerals are also soluble at the ph levels at which erosion occurs. although fluoride might have an effect of enhancing remineralization of the eroded surface, the clinical significance is yet to be properly determined. in an overview of these processes, dental caries is considered a disease dependent of biofilm accumulation with a strong influence of the diet [38]. saliva and its remineralizing capacity and fluoride, which increase enamel remineralization, have positive effects in the reduction of the disease outcome. other factors, such as virulent bacterial species or the composition of the tooth structure, might also affect dental caries progression rates. for instance, it is well known that in the enamel of primary teeth, caries progresses faster as a result of the differential composition of both [39]. in the near future, perhaps we could identify other subtle differences in tooth composition that might also affect the disease. dental caries is also influenced by social modifying factors, which today make it a disease of the poor, who have an increasing vulnerability to diseases and less access to prevention policies. it is possible that besides the environmental influence on this subject, genetics also play a role on influencing the biological aspects of this disease. additionally, dental erosion has also been described by interplay of biological, chemical and behavioral factors, and some of these might be influenced by genetics as well. in summary, for thousands of years the cariogenic diet has been restricted to starchy products with low fermentable capacity and mineral loss was usually the result of abrasive forces. with the common availability of sugar since the colonial time, caries flourished as a widespread disease in our modern societies. our diet pattern (processed foods, highly available soft drinks) still supports diseases such as dental caries and erosion. hopefully, in the future, as long as we understand our intrinsic influences to these diseases, we can develop technologies to help us better modulate them. environmental factors on enamel formation (presenter r. gerlach) dental enamel is affected by many insults during its formation (called amelogenesis). the environmental factors most widely known to negatively interfere with amelogenesis are fevers, hypoxia, undernutrition, and exposure to certain substances that are toxic to enamel cells during enamel development. other factors that also influence amelogenesis are antibiotics, environmental pollutants, and socioeconomic status. such external factors may affect enamel formation when cells are secreting the enamel matrix and/or during the mineralizing process. molar-incisor hypomineralization is a clinical entity that exemplifies the possible effects of environmental factors on enamel formation. whereas a genetic component exists [40], medical issues during prenatal, perinatal, and postnatal stages also lead to enamel hypomineralization, as well as use of medications during the first year of life, and early life exposure to fluorides or environmental pollutants (dioxins and polychlorinated biphenyls or pcbs) [41]. in the continuously growing incisor of rats, fluorotic enamel clinically displays white discoloration that microscopically shows a http://dentistry3000.pitt.edu/ summary of the iadr cariology research, craniofacial biology, and mineralized tissue groups symposium, iguaçu falls, brazil, june 2012 vol 1, no 1 (2013) doi 10.5195/d3000.2013.16 http://dentistry3000.pitt.edu 5 pattern of repeated white and pigmented bands. these white bands represent hypomineralized superficial enamel, but no subsurface lesions exist [42]. in the presence of lead, clinical alterations due to the chronic exposure to higher levels of fluoride are exacerbated [43]. like other agents, if the attack on the enamel lasts for a short period of time, there will probably be no visible defects in the enamel. enamel maturation of permanent teeth lasts up to four years; hence, chronic exposures may lead to clinical alterations of enamel. this long maturation period probably explains why teeth harbor metal traces to which they were exposed (sodium, chloride, lead). this characteristic makes teeth useful for detecting metals in studies of samples from preindustrial ages, of hypoplasic and hypomineralized enamel, caries, and to test hypothesis involving the effect of the synergism of multiple environmental contaminants. genetic variation in enamel formation genes (presenter a.r. vieira) genes responsible for enamel formation have been proposed as potentially involved in caries susceptibility, and positive associations between genetic variation in amelogenin, tuftelin, and enamelin and higher caries experience have been reported by our group and others [44-46]. these results, however, are not consistent in all populations. in fact, the only consistent result is the lack of association between caries experience and variation in tuftelin interacting protein 11 (table 1). these latest data are published [47]. one big limitation of these studies is the definition of the phenotype of caries. dmft/dmfs scores provide a picture of the burden of the disease but little insight into the disease initiation and process. one approach we proposed was defining caries based on enamel microhardness. we tested enamel microhardness at baseline, after creation of artificial carious lesions, and after fluoride application. as expected, enamel microhardness decreased after creation of artificial caries lesions and then increased to levels similar to baseline after the one-time fluoride treatment and the phcycling protocol for 14 days. lower baseline microhardness was significantly associated with amelogenin (p=0.03 for buccal surface), tuftelin (p=0.03 for mesial and p=0.02 for buccal + lingual surfaces), and ameloblastin (p=0.04 for distal surface). after artificial caries creation, lower microhardness was significantly associated with tuftelin (p=0.02 for buccal + lingual surfaces and p=0.006 for distal surface), enamelin (p=0.02 for distal surface), and tuftelin interacting protein 11 (p=0.0006 for buccal + lingual and p=0.009 for occlusal surfaces). after fluoride treatment, microhardness was significantly associated with tuftelin (p=0.03 for occlusal surface). the ratio of change of microhardness after ph-cycling treatment was significantly associated with amelogenin (p=0.03 for buccal + lingual and p=0.03 for mesial surfaces), tuftelin (p=0.02 for occlusal surface), enamelin (p=0.01 for occlusal surface), and tuftelin interacting protein 11 (p=0.04 for buccal + lingual and p=0.009 for occlusal surfaces). to help overcome the limitations of using dmft/dmfs scores, we decided to design a series of functional assays to evaluate the response of enamel samples with known genotypes of the genes involved in enamel formation to simulated cariogenic challenges. despite the limitation of having samples from several different types of teeth (first, second, and third molars, premolars, canines, and incisors), the results of these experiments suggest that there may be some truth to the popular belief that some individuals may have “weaker” teeth, and hence, are more prone to caries development. another observation in our study is that enamel microhardness varies from individual to individual, sometimes substantially. traditional protocols avoid these variations by eliminating samples that are outside a specific range (i.e., limiting the study to specimens with knoop microhardness values between 350 and 380). although this methodological approach reduces interspecimen variation, it also eliminates the chance of interpreting the results in light of individual variation. our results suggest that the influence of genetic variation of enamel formation genes may influence the dynamic interactions between the enamel surface and the oral cavity. components not studied here include biofilm formation (both adhesion to the enamel and maturation), and the influence of salivary components. despite these limitations, the determination of the presence of specific genetic variants in patients holds the promise for allowing customized treatments that may better impact individual risks for caries. acknowledgements the authors are grateful to the iadr cariology research, craniofacial biology and mineralized tissue research groups. jaime a. cury helped plan the symposium. colgate provided financial support for the symposium. a.r. vieira is supported by the nih grant r01-de018914. references 1. genetic determinants of cranio-facial morphology: a twin study. nakata m, yu pl, davis b, nance we. ann hum genet 1974;37:431-43. 2. u.s. department of health and human services. oral health in america: a report of the surgeon general—executive summary. rockville, md: u.s. department of health and human services, national institute of dental and craniofacial research, national institutes of health, 2000. 3. world health organisation. the world oral health report 2003. geneva, switzerland: world health organisation, 2003. (accessed september 15, 2013, at http://www.who.int/oral_health/publications/r eport03/en/) 4. a report on the nih consensus development conference on diagnosis and management of dental caries throughout life. horowitz am.j dent res. 2004;83 spec no c:c157.pmid:15286115 5. dental enamel formation and its impact on clinical dentistry. simmer jp, hu jc. j dent educ. 2001 sep;65(9):896-905. review. pmid:11569606 6. molecular mechanisms of dental enamel formation. simmer jp, fincham ag. crit rev oral biol med. 1995;6(2):84-108. review. pmid:7548623 table 1. summary of association results between enamel formation gene variants and caries experience. the only consistent result is the lack of association between caries and tuftelin interacting protein 11. gene iowa, usa [44] tiquisate, guatemala [45] istanbul, turkey [46] cebu, philippines [47] patagonia, argentina [47] curitiba, brazil [47] rio de janeiro, brazil [47] ameloblastin + + amelogenin + + + enamelin +* + tuftelin +* +# + + + tuftelin interacting protein 11 * in the presence of streptococcus mutans; # only in less severely affected cases http://dentistry3000.pitt.edu/ http://www.who.int/oral_health/publications/report03/en/ http://www.who.int/oral_health/publications/report03/en/ http://www.ncbi.nlm.nih.gov/pubmed/15286115 http://www.ncbi.nlm.nih.gov/pubmed/15286115 http://www.ncbi.nlm.nih.gov/pubmed/15286115 http://www.ncbi.nlm.nih.gov/pubmed/11569606 http://www.ncbi.nlm.nih.gov/pubmed/11569606 http://www.ncbi.nlm.nih.gov/pubmed/7548623 http://www.ncbi.nlm.nih.gov/pubmed/7548623 summary of the iadr cariology research, craniofacial biology, and mineralized tissue groups symposium, iguaçu falls, brazil, june 2012 vol 1, no 1 (2013) doi 10.5195/d3000.2013.16 http://dentistry3000.pitt.edu 6 7. inherited risks for susceptibility to dental caries. shuler cf. j dent educ. 2001 oct;65(10):1038-45. pmid:11699975 8. cloning, characterization, and tissue expression pattern of mouse tuftelin cdna. macdougall m, simmons d, dodds a, knight c, luan x, zeichner-david m, zhang c, ryu oh, qian q, simmer jp, hu cc. j dent res. 1998 dec;77(12):1970-8. pmid: 9839784 9. genes and related proteins involved in amelogenesis imperfecta. stephanopoulos g, garefalaki me, lyroudia k. j dent res. 2005 dec;84(12):1117-26. review. pmid:16304440 10. enamel formation and amelogenesis imperfecta. hu jc, chun yh, al hazzazzi t, simmer jp. cells tissues organs. 2007;186(1):78-85. review. pmid:17627121 11. amelotin--a novel secreted, ameloblastspecific protein. iwasaki k, bajenova e, somogyi-ganss e, miller m, nguyen v, nourkeyhani h, gao y, wendel m, ganss b. j dent res. 2005 dec;84(12):1127-32. pmid:16304441 12. characterization of apin, a secreted protein highly expressed in tooth-associated epithelia. moffatt p, smith ce, st-arnaud r, nanci a. j cell biochem. 2008 feb 15;103(3):941-56. pmid:17647262 13. the cell adhesion molecule nectin-1 is critical for normal enamel formation in mice. barron mj, brookes sj, draper ce, garrod d, kirkham j, shore rc, dixon mj. hum mol genet. 2008 nov 15;17(22):3509-20. doi: 10.1093/hmg/ddn243. epub 2008 aug 14. pmid:18703497 14. perp regulates enamel formation via effects on cell-cell adhesion and gene expression. jheon ah, mostowfi p, snead ml, ihrie ra, sone e, pramparo t, attardi ld, klein od. j cell sci. 2011 mar 1;124(pt 5):745-54. doi: 10.1242/jcs.078071. epub 2011 feb 1. pmid:21285247 15. public health: the toxic truth about sugar. lustig rh, schmidt la, brindis cd. nature. 2012 feb 1;482(7383):27-9. doi: 10.1038/482027a. pmid:22297952 16. microbial ecology of dental plaque and its significance in health and disease. marsh pd. adv dent res. 1994;8:263-71. pmid:7865085 17. scanning electron microscopy of early microbial colonization of human enamel and root surfaces in vivo. nyvad b, fejerskov o. scand j dent res. 1987;95:287-96. pmid:3476984 18. degrees of saturation with respect to apatites in parotid saliva at various ph values. larsen mj. scand j dent res. 1975 jan;83(1):7-12. pmid:237316 19. intra-oral hydrogen-ion concentrations associated with dental caries activity. stephan rm. j dent res. 1944;23:257-66. 20. influence of fluoride in solution on tooth demineralization. i. chemical data. ten cate jm, duijsters pp. caries res. 1983;17:193-9. pmid:6573960 21. thermodynamic solubility product of human tooth enamel: powdered sample. patel pr, brown we. j dent res. 1975 jul-aug;54(4):72836. pmid:239974 22. alternating demineralization and remineralization of artificial enamel lesions. ten cate jm, duijsters pp. caries res. 1982;16(3):201-10. pmid:6953998 23. concepts of dental caries and their consequences for understanding the disease. fejerskov o. community dent oral epidemiol. 1997 feb;25(1):5-12. pmid:9088687 24. changing concepts in cariology: forty years on. kidd e, fejerskov o. dent update. 2013 may;40(4):277-8, 280-2, 285-6. pmid:23829008 25. effects of carbohydrate pulses and ph on population shifts within oral microbial communities in vitro. bradshaw dj, mckee as, marsh pd. j dent res. 1989 sep;68(9):1298-302. pmid:2674233 26. physical and biochemical studies of streptococcus mutans sediments suggest new factors linking the cariogenicity of plaque with its extracellular polysaccharide content. dibdin gh, shellis rp. j dent res. 1988 jun;67(6):890-5. pmid:3170900 27. biochemical composition and cariogenicity of dental plaque formed in the presence of sucrose or glucose and fructose. cury ja, rebelo ma, del bel cury aa, derbyshire mt, tabchoury cp. caries res 2000;34:491-7. pmid:11093024 28. electron microscopy of fissure caries in man. frank rm. arch oral biol. 1973 jan;18(1):9-25. pmid:4513117 29. assimilation of fluoride by enamel throughout the life of the tooth. weatherell ja, deutsch d, robinson c, hallsworth as. caries res. 1977;11(suppl 1):85-115. pmid:318575 30. thylstrup a, featherstone jdb, fredebo l. surface morphology and dynamics of early enamel caries development. in: leach sa, edgar wm. demineralization and remineralization of the teeth. london: irl press, 1983:165-84. 31. current concepts on the theories of the mechanism of action of fluoride. ten cate jm. acta odontol scand. 1999 dec;57(6):325-9. pmid:10777135 32. fluoride: its role in dentistry. tenuta lm, cury ja. braz oral res 2010; 24:suppl 1: 9-17. pmid:20857070 33. systematic review of water fluoridation. mcdonagh ms, whiting pf, wilson pm, sutton aj, chestnutt i, cooper j, misso k, bradley m, treasure e, kleijnen j. brit med j. 2000;321(7265):855-9. pmid:11021861 34. fluoride toothpastes for preventing dental caries in children and adolescents. marinho vcc, higgins jpt, logan s, sheiham a. cochrane database syst rev 2003, issue 1. art. no.: cd002278. pmid:12535435 35. topical fluoride as a cause of dental fluorosis in children. wong mc, glenny am, tsang bw, lo ec, worthington hv, marinho vc. cochrane database syst rev. 2010 jan 20;(1):cd007693. doi: 10.1002/14651858.cd007693.pub2. pmid:20091645 36. effects of low and standard fluoride toothpastes on caries and fluorosis: systematic review and meta-analysis. santos ap, oliveira bh, nadanovsky p. caries res. 2013;47(5):382-90. doi: 10.1159/000348492. pmid:23572031 37. a literature review of aesthetic perceptions of dental fluorosis and relationships with psychosocial aspects/oral health-related quality of life. chankanka o, levy sm, warren jj, chalmers jm. community dent oral epidemiol. 2010 apr;38(2):97-109. doi: 10.1111/j.16000528.2009.00507.x. pmid:20002631 38. initial erosion models. young a, tenuta lm. caries res. 2011;45(suppl 1):33-42. pmid:21625131 39. fejerskov o, manji f. risk assessment in dental caries. in: batler jd. ed. risk assesstnent in dentistry. chapel hill: university of north carolina dental ecology, 1990; 215-7. 40. quantitative determination of type a and type b carbonate in human deciduous and permanent enamel by means of fourier transform infrared spectrometry. sønju clasen ab, ruyter ie. adv dent res. 1997 nov;11(4):523-7. pmid:9470513 41. genes expressed in dental enamel development are associated with molar-incisor hypomineralization. jeremias f, koruyucu m, küchler ec, bayram m, tuna eb, deeley k, pierri ra, souza jf, fragelli cm, paschoal ma, gencay k, seymen f, caminaga rm, dos santos-pinto l, vieira ar. arch oral biol. 2013 oct;58(10):143442. doi: 10.1016/j.archoralbio.2013.05.005. epub 2013 jun 19. pmid:23790503 42. aetiology of molar-incisor hypomineralisation: a systematic review. alaluusua s. eur arch paediatr dent. 2010 apr;11(2):53-8. review. pmid:20403298 43. morphological characterization of rat incisor fluorotic lesions. saiani ra, porto im, marcantonio junior e, cury ja, de sousa fb, gerlach rf. arch oral biol. 2009 nov;54(11):1008-15. doi: 10.1016/j.archoralbio.2009.08.009. epub 2009 sep 24. pmid:19781688 44. exposure to lead exacerbates dental fluorosis. leite ga, sawan rm, teófilo jm, porto im, sousa fb, gerlach rf. arch oral biol. 2011 jul;56(7):695-702. doi: 10.1016/j.archoralbio.2010.12.011. epub 2011 jan 26. pmid:21269604 45. tuftelin, mutans streptococci, and dental caries susceptibility. slayton rl, cooper me, marazita ml. j dent res. 2005 aug;84(8):711-4. pmid:16040727 46. possible association of amelogenin to high caries experience in a guatemalan-mayan population. deeley k, letra a, rose ek, brandon ca, resick jm, marazita ml, vieira ar. caries res. 2008;42(1):8-13. epub 2007 nov 27. pmid:18042988 47. enamel formation genes are associated with high caries experience in turkish children. patir a, seymen f, yildirim m, deeley k, cooper me, marazita ml, vieira ar. caries res. 2008;42(5):394-400. doi: 10.1159/000154785. epub 2008 sep 10. pmid:18781068 48. enamel formation genes influence enamel microhardness before and after cariogenic challenge. shimizu t, ho b, deeley k, briseño-ruiz j, faraco im jr, schupack bi, brancher ja, pecharki gd, küchler ec, tannure pn, lips a, vieira tc, patir a, yildirim m, poletta fa, mereb jc, resick jm, brandon ca, orioli im, castilla ee, marazita ml, seymen f, costa mc, granjeiro jm, trevilatto pc,vieira ar. plos one. 2012;7(9):e45022. doi: 10.1371/journal.pone.0045022. epub 2012 sep 24. pmid:23028741 http://dentistry3000.pitt.edu/ http://www.ncbi.nlm.nih.gov/pubmed/11699975 http://www.ncbi.nlm.nih.gov/pubmed/11699975 http://www.ncbi.nlm.nih.gov/pubmed/9839784 http://www.ncbi.nlm.nih.gov/pubmed/9839784 http://www.ncbi.nlm.nih.gov/pubmed/16304440 http://www.ncbi.nlm.nih.gov/pubmed/16304440 http://www.ncbi.nlm.nih.gov/pubmed/17627121 http://www.ncbi.nlm.nih.gov/pubmed/17627121 http://www.ncbi.nlm.nih.gov/pubmed/16304441 http://www.ncbi.nlm.nih.gov/pubmed/16304441 http://www.ncbi.nlm.nih.gov/pubmed/17647262 http://www.ncbi.nlm.nih.gov/pubmed/17647262 http://www.ncbi.nlm.nih.gov/pubmed/18703497 http://www.ncbi.nlm.nih.gov/pubmed/18703497 http://www.ncbi.nlm.nih.gov/pubmed/21285247 http://www.ncbi.nlm.nih.gov/pubmed/21285247 http://www.ncbi.nlm.nih.gov/pubmed/23790503 http://www.ncbi.nlm.nih.gov/pubmed/23790503 http://www.ncbi.nlm.nih.gov/pubmed/23790503 http://www.ncbi.nlm.nih.gov/pubmed/20403298 http://www.ncbi.nlm.nih.gov/pubmed/20403298 http://www.ncbi.nlm.nih.gov/pubmed/19781688 http://www.ncbi.nlm.nih.gov/pubmed/19781688 http://www.ncbi.nlm.nih.gov/pubmed/21269604 http://www.ncbi.nlm.nih.gov/pubmed/16040727 http://www.ncbi.nlm.nih.gov/pubmed/16040727 http://www.ncbi.nlm.nih.gov/pubmed/18042988 http://www.ncbi.nlm.nih.gov/pubmed/18042988 http://www.ncbi.nlm.nih.gov/pubmed/18042988 http://www.ncbi.nlm.nih.gov/pubmed/18781068 http://www.ncbi.nlm.nih.gov/pubmed/18781068 http://www.ncbi.nlm.nih.gov/pubmed/23028741 http://www.ncbi.nlm.nih.gov/pubmed/23028741 http://www.ncbi.nlm.nih.gov/pubmed/23028741 microsoft word 311 2022.docx is oral candida associated with dental caries in children—a cross sec6onal analysis. vol 10 no 1 (2022) doi 10.5195/d3000.2022.311 h#p://den*stry3000.pi#.edu is oral candida associated with dental caries in children—a cross sectional analysis. mithun pai1, shweta yellapurkar2, srikant natarajan3, ramya shenoy4 1associate professor, department of public health dentistry, manipal college of dental sciences, mangalore. manipal academy of higher education. manipal. 2assistant professor, department of oral pathology and microbiology. manipal college of dental sciences, mangalore. manipal academy of higher education. manipal. 3professor and head, department of oral pathology and microbiology. manipal college of dental sciences, mangalore. manipal academy of higher education. manipal. 4associate professor, department of public health dentistry, manipal college of dental sciences, mangalore. manipal academy of higher education. manipal. abstract background: dental caries is a world-wide public health issue and is considered the most common of all non-communicable disease. there has been a growing interest in associa6on between oral fungal flora especially candida and dental caries. hence the aim of the study was to assess the correla6on between ac6ve caries status and oral candida carriage in children aged twelve years. materials and methods: this is a cross-sec6onal study where a mul6phase sampling was done and a total of thirty children were selected randomly among children screened previously. data was collected through clinical oral examina6ons and structured interviews. the oral rinse technique described by samaranayake et al. was used for extrac6ng candida spp. the correla6on between candida spp level and caries experience was performed by the pearsons correla6on coefficient. results: the mean dmfs and defs scores of the children were 3.70±4.61 and 5.83±5.51 respec6vely. significant associa6on with decay scores for deciduous teeth (p<0.05) and defs (p= <0.001) was demonstrated. candida species was associated highly with caries prevalence and significant posi6ve correla6on was found between candida (cfu/plate) and defs scores (r=0.790) (p= <0.001) conclusion: the study results suggested that there is a significant posi6ve correla6on between candida carriage and caries, especially in children in the age group of 12 years. keywords: candida; children; dental caries; dmft cita8on: pai, m et al. (2022) is oral candida associated with dental caries in children—a cross sec8onal analysis. den8stry 3000. 1:a001 doi:10.5195/d3000.2022.311 received: february 2, 2022 accepted: august 8, 2022 published: august 31, 2022 copyright: ©2022 pai, m et al. this is an open access ar8cle licensed under a crea8ve commons asribu8on work 4.0 united states license. email: shweta.y@manipal.edu introduction dental caries is a world-wide public health issue and is considered the most common of all noncommunicable disease. caries is ranked first among all diseases in global burden of disease study affecting 2.3 billion people for permanent teeth and 12th for deciduous teeth affecting 560 million children [1]. identifying high caries risk individuals and groups is a matter of prime importance for all public health programs worldwide. it is widely known that caries prevention and management would be more effective if individuals or groups under greatest risk are identified. hence, methods for predicting caries risk are being investigated in greater detail. c. albicans is a common oral commensal and can be commonly isolated from the oral environment of majority of individuals. it is noted that candidal carriage is common in many individuals and as much as 80 percent health individuals have candidal is oral candida associated with dental caries in children—a cross sec6onal analysis. vol 10 no 1 (2022) doi 10.5195/d3000.2022.311 h#p://den*stry3000.pi#.edu isolates in their oral cavity, but the fact that just candidal carriage does not mean actual infection as it is considered as a normal flora of the oral ecosystem. candida is a highly adaptable commensal organism and any alterations in the host micro ecosystem that favor its proliferation provide this commensal with the opportunity to essentially invade any site and proliferate causing infection. this invasion usually manifests from surface infections to invasive disseminated disease with multiple organ involvement [2]. oral microbiota consists of not only bacteria but also numerous other microorganisms, which cause mixed infections in the oral cavity. hence the establishment and maintenance of oral microbiota is related not only to interbacterial co aggregations but also interactions of these bacteria with yeasts such as candida albicans. fungi are isolated on several sites in the oral cavity, including tongue, buccal mucosa, palate, oral biofilm, carious lesions, and prosthetic appliances. it has been suggested that a possible relation may occur between c. albicans and periodontal disease, dentin and/or root caries. c. albicans also has capacity similar to that of s. mutans for colonizing hydroxyapatite and invade the tooth structure. the mode of action for such invasion may be adherence of candida to salivary proteins, acidproducing capability, penetration into the dentinal tubules, and its enzymatic activity to degrade collagen which in turn may be responsible for causation of dental caries; however, these may be entirely different to that to the mechanisms of s. mutans, in initiation and progression of dental caries [3,4]. studies have also demonstrated that candida species are more prevalent in saliva of caries-active subjects than that of subjects with no caries especially in children. enamel and dentin demineralization produced by organic fungal acids, as well as the presence of c. albicans hyphae invading dentinal tubules, prove the ability of fungi to invade and destroy inorganic and organic dental tissues [5]. there has been a growing interest in association between oral fungal flora especially candida and dental caries, this has resulted in research focusing on the possible direct role of c. albicans in etiology of dental caries. the presence of c. albicans in dental plaque of children have been associated with high dmf (decayed, missing, filled) index. reduced growth has been associated with increased filled component of the individuals and abundant growth is associated with a high decay component [6]. hence in this study we assessed the correlation between active caries status and oral candida carriage in children aged twelve years in the coastal city of karnataka as we had no previous studies conducted in the present geographical area. the objectives were, to assess the frequency of oral candida species in children aged 12 years, to evaluate the caries experience of children aged 12 years and to correlate the frequency of oral candidal species with the caries status of the children. material and methods the study subjects included children from a primary school in the city of mangalore, karnataka, india. a prior permission from the respective authorities were obtained before the start of the study process. this is a cross-sectional study conducted with the aim of detecting candida carriage in the oral cavity of healthy school children and to correlate that with candidal occurrence and dental caries in this population. the students in the school were screened previously and caries status of the children was obtained. a multiphase sampling was done, where a total of thirty children were selected randomly among children screened previously. this was a pilot study to know the prevalence of candida in dental caries. the sample size was calculated by using g*power version 3.1 with a statistical power of 0.8 and a significant level of 0.05 and estimating effect size of 0.5 a sample size of 27 was calculated, hence a sample of 30 was included in the study. ethical committee clearance was obtained prior to the study from the institutional ethics committee and is oral candida associated with dental caries in children—a cross sec6onal analysis. vol 10 no 1 (2022) doi 10.5195/d3000.2022.311 h#p://den*stry3000.pi#.edu voluntary informed consent was obtained from the parents or guardians of the subjects prior to the study process. all infection control protocols were strictly followed, and participants were referred for further treatment if necessary to the parent institute after the examination. the study group included children randomly selected from the sample frame who fulfilled the inclusion criteria. the inclusion criteria were children with caries who were selected through random multiphase sampling technique. children in the age group of 12 years with no clinical signs of oral candidiasis and free from any known systemic disease. exclusion criteria included children with special needs, antimicrobial medications one month prior to the study and children with orthodontic appliances. data was collected through clinical oral examinations and structured interviews. a single blinded examiner examined all the children and two supports who helped with the recording of data participated in the study. intra examiner reliability was assessed and was found to be good (cohen's kappa ҝ = 0.72) during calibration. the structured interview included questions on age, gender, dental hygiene (frequency of brushing, flossing, and the use of toothpaste or mouthwash use), and dietary habits. decayed missing filled surfaces (dmfs) index for permanent teeth and decayed, missing due to caries, or indicated for extraction, filled surfaces (defs) index for primary teeth given by grubbel ao was used to assess the caries experience in primary dentition. each child was examined in a chair using natural light. white spot decalcifications were not considered to be caries, and loss of mineral substance with cavitation was recorded as caries [6]. evaluations was done according to the criteria recommended by the world health organization. mycological examination the oral rinse technique described by samaranayake et al. [7] was used for extracting candida spp from the subjects. the procedure involved the participant holding and rinsing 10 ml of sterile phosphate buffered saline (pbs) in the mouth for one minute. the expectorate was then transported at room temperature to the laboratory where it was centrifuged for ten minutes as described by samaranayake et al [7]. then 30µl of concentrate was inoculated onto sabouraud’s agar medium. the plates were incubated at 37°c for 72 hours, and semiquantitative assessment of candida growth was made by counting the total number of yeast colonies developed on the plates at the end of 72 hours and were quantified by the visual methods. data were analysed using statistical package for social sciences (spss), version 16.0 (spss inc, chicago il). mean (x), median, inter quartile distribution and standard deviation (sd) was calculated for describing the data. the difference was considered to be significant when p < 0.05. the presence or absence of candida spp in each subject was compared with gender and caries prevalence. the correlation between candida spp level (cfu/plate) and caries experience was performed by the pearsons correlation coefficient. the mann-whitney u and kruskal-wallis h test was used to assess the significance between gender, caries, dental hygiene methods and candidal count. results out of 30 children selected for this study, the sample contained equal number of children from both genders. the subjects selected for the study had a mean age of 11.12±0.33 years. a total 73% (n= 22) of individuals reported to brush their teeth at least once a day using tooth paste and toothbrush, and only 11% brushed their teeth twice a day. all the individuals reported to brush their teeth with fluoridated toothpaste. the individuals who brushed once daily using finger had higher frequency of candidal occurrence which was statistically significant (table i (a)). pearson’s correlation between the colony forming units and demographic and dental caries experience in the sample is summarized in table i (b). is oral candida associated with dental caries in children—a cross sec6onal analysis. vol 10 no 1 (2022) doi 10.5195/d3000.2022.311 h#p://den*stry3000.pi#.edu n mean cfu(sd) range median mann whitney u / kruskal wallis p value gender male 15 202(455) 0-1335 0(0,0) 110 0.935 female 15 123(276) 0-847 0(0,0) type of cleaning brush 21 96(248) 0-847 0(0,0) 6.773* 0.036 finger 5 573(660) 0-1335 299(0,1229) both 4 0(0) 0-0 0(0,0) material paste 22 105(246) 0-847 0(0,0) 0.996* 0.608 other 7 366(626) 0-1335 0(0,1229) both 1 0(.) 0-0 0(0,0) frequency once 22 35(115) 0-462 0(0,0) 48 0.063 twice 8 514(584) 0-1335 350(0,1038) time of brushing morning 23 33(112) 0-462 0(0,0) 11.642* 0.003 night 5 513(703) 0-1335 0(0,1229) both 2 773(105) 699-847 773(699,847) diet vegetarian 16 29(116) 0-462 0(0,0) 77.5 0.154 mixed 14 315(496) 0-1335 0(0,699) *-kruskal -wallis test *(p < 0.05) significant difference between/among subgroups cfus= colony forming units pearson correlation gender type of cleaning material diet df score dmfs cfus gender pearson correlation 1 -.326 .000 .267 -.076 -.265 -.108 p .079 1.000 .153 .691 .157 .571 type of cleaning pearson correlation -.326 1 .056 -.193 .073 .115 .096 p .079 .768 .307 .702 .544 .614 material pearson correlation .000 .056 1 .040 .199 .281 .261 p 1.000 .768 .833 .292 .133 .164 diet pearson correlation .267 -.193 .040 1 .393* -.133 .390* p .153 .307 .833 .032 .482 .033 dfscore pearson correlation -.076 .073 .199 .393* 1 .497** .790** p .691 .702 .292 .032 .005 .000 dmfs pearson correlation -.265 .115 .281 -.133 .497** 1 .404* p .157 .544 .133 .482 .005 .027 cfus pearson correlation -.108 .096 .261 .390* .790** .404* 1 p .571 .614 .164 .033 .000 .027 p =*. correlation is significant at the 0.05 level (2-tailed). p= **. correlation is significant at the 0.01 level (2-tailed). dmfs: decayed, missing, and filled surfaces cfus= colony forming units dfs= decayed filled for deciduous tooth table i (a): demographic variables, oral hygiene habits and colony forming units for candida. table i (b): demographic correlation between dfs score and dmft scores and cfu of candida. is oral candida associated with dental caries in children—a cross sec6onal analysis. vol 10 no 1 (2022) doi 10.5195/d3000.2022.311 h#p://den*stry3000.pi#.edu of the 30 oral samples collected, 24 (80 %) did not present any fungal growth and 6 (20 %) presented fungal microbiota consisting of candida with an average of 162.3 cfu/plate. the mean dmfs and defs scores of the children with caries was 3.70±4.61 and 5.83±5.51, respectively. the individual characteristics of index is presented in table ii, other than the deft index, the results of the questionnaire and oral examination revealed no significant differences between the children and there was no association between candida carriage within gender for dmfs (p=0.436) and defs (p=0.389). association of microbial counts with caries status associations between caries status (dmfs and defs scores, dependent variables) and microbial counts (independent variables) were evaluated. significant association with ds decay scores for deciduous teeth (p<0.05) (table ii) and defs (p= <0.001) was demonstrated. candida species was associated highly with caries prevalence and significant positive correlation was found between candida (cfu/plate) and defs scores (r= 0.790) (p= <0.001), in the subjects but there was no positive correlation between dmfs and candida carriage in children (r=0.404) (p=0.027). discussion several predictors up till now have been analysed and discussed in relation to dental caries. so far none of the indicators alone can correctly predict caries ranging from clinical, microbiologic, and an array of host factors. combined information of several predictors showed some ability to distinguish subjects into high or low caries groups. past caries experience, plaque index and microbiological factors are easy to determine and show a reasonably good association with caries risk but still prediction of caries has remained an enigma and a conundrum spinning the web of confusion in clinician’s mind. the association of candida to caries in children is a topic of intense discussion in recent times. in the present study, the oral candidal carriage was assessed in children and an association was observed for the same. of all the subjects in the study, candida was present in 20 percent of the subjects and this finding is similar to that beena et al in india [8], but much lower to that of studies by moalic et al. in france [9], cortelli in brazil [10], akdeniz et al [11]. candida in turkey, the difference in the candida count can be attributed to the difference in number and age of the subjects, geographic locations, and methodology used by different authors. the study of akdeniz used imprint method for culturing candida hence may have had higher candida samples [11]. the imprint method though the most followed method may not be as accurate as that of oral rinse method used in the study as explained by samaranayake et al [7]. in the present study, the prevalence of candida was higher in sample group than the general population as all the subjects in the present study had dental caries. the results demonstrated a significant association between the presence of candida species and caries in children. but there was no statistically significant relationship between age, gender, type, and method of cleaning teeth. material used for cleaning teeth, frequency of cleaning teeth and diet as all these factors were similar among the sample population. studies suggest that candida species have high cariogenic potential in chisquare value df p value gender 6.00 6 0.423 ds 139.16 72 <0.001 dfs 150.0 72 <0.001 dmft 95.45 54 <0.001 tooth cleaning methods 22.067 12 0.037 frequency of brushing 12.95 6 0.044 diet 7.40 6 0.285 table ii: association (pearsons chi square) between the colony forming units of candida, brushing habits, and dental caries experience. p =*. significant at the 0.05 level (2tailed). df= degree of freedom dmft=decayed, missing, and filled teeth dfs= decayed filled for deciduous tooth is oral candida associated with dental caries in children—a cross sec6onal analysis. vol 10 no 1 (2022) doi 10.5195/d3000.2022.311 h#p://den*stry3000.pi#.edu vitro. carious dentine has shown to have a higher colonization of candida spp, providing a significant ecologic niche for the dissemination of this fungi [12,13]. the recent evidence also points at relationship between the presence of candida and caries in children specifically in children with poor oral hygiene, carious dentinal lesions and high sugar diet [14,15]. the relationship between candidal carriage, initiation or progression of caries lesions is unclear. to say the least, it is not evident that whether and how the disease process ante cedes or predates the adhesion of yeasts. the current insight suggests that higher frequency of sugar intake, with poor oral hygiene, open caries, and the extensive intake of sweet substances via the feeding-bottle in nursing bottle caries may be some of the important factors responsible for the high prevalence of this fungi in the mouths of children [11]. the biggest drawback of the study might be the absence of no cariesfree children in, as this was a pilot study to test the contemporary hypothesis. in other words, a relationship between caries and frequency of oral candida carriage was observed only in children with caries but a negative correlation in not obtained in non-carious age group hence further research should be directed in this direction. the results of this study are specific to children from a single location and a single cross-section. any generalizations hence must be confirmed by repeating the study in additional age groups and different geographic locales. conclusion the study results suggested that there is a significant positive correlation between candidal carriage and caries, especially in children in the age group of 12 years. evaluation of oral candida level may help in planning preventive programs for children in public health settings. further longer-term studies with an extended study group are needed to confirm our results in evaluating these indicators as risk predictors of caries in children. the relationship between colonization, secretor status and in-vitro adhesion of candida albicans in oral cavity should be the focus of such research. references 1. dental caries and associated factors in ethiopia: systemamc review and meta-analysis. zewdu t, abu d, agajie m, sahilu t. environ heal prev med 2021 261 [internet]. 2021 feb 12 [cited 2021 jul 24];26(1):1–11. available from: h\ps://environhealthprevmed.b iomedcentral.com/armcles/10.11 86/s12199-021-00943-3 2. oral candidiasis: a disease of opportunity. vila t, sultan as, montelongo-jauregui d, jabrarizk ma. j fungi. 2020;6(1):1– 28. 3. minimum inhibitory concentramon of adherence of punica granatum linn (pomegranate) gel against s. mutans, s. mims and c. albicans. vasconcelos lcds, sampaio fc, sampaio mcc, pereira mds v, higino js, peixoto mhp. braz dent j. 2006;17(3):223–7. 4. oral microbiome: possible harbinger for children’s health. xiao j, fiscella ka, gill sr. int j oral sci. 2020;12(1):1–13. 5. streptococcal interacmons in biofilm-associated oral diseases.pdf. koo h, andes d, krysan d. 2018. 6. oral candidal carriage in children with and without dental caries. uygun-can b, kadir t, akyüz s. quintessence int (berl). 2007;38(1):45–9. 7. a comparison of oral rinse and imprint sampling techniques for the detecmon of yeast, coliform and staphylococcus aureus carriage in the oral cavity. samaranayake lp, macfarlane tw, lamey p -j, ferguson mm. j oral pathol med. 1986;15(7):386–8. 8. comparison of candida species isolated from children with and without early childhood caries : a descripmve cross secmonal study. ms b, peedikayil fc, mb g, tp c, soni k, dhanesh n. j indian soc pedod prev dent. 2017;296–300. 9. the extent of oral fungal flora in 353 students and possible relamonships with dental caries. moalic e, gestalin a, quinio d, gest pe, zerilli a, flohic am le. caries res [internet]. 2001 mar [cited 2021 jul 26];35(2):149–55. available from: h\ps://www.karger.com/armcle /fulltext/47447 10. correlamon between candida spp. and dmft index in a rural populamon. cortelli sc, junqueira jc, faria i da s, ito cyk, cortelli jr. brazilian j oral sci. 2006;5(17):1007–11. is oral candida associated with dental caries in children—a cross sec6onal analysis. vol 10 no 1 (2022) doi 10.5195/d3000.2022.311 h#p://den*stry3000.pi#.edu 11. prevalence of candida albicans in oral cavimes and root canals of children. akdeniz bg, koparal e, şen bh, ateş m, denizci aa. j dent child. 2002;69(3):289–92. 12. synergism of streptococcus mutans and candida albicans reinforces biofilm maturamon and acidogenicity in saliva: an in vitro study. kim h-e, liu y, dhall a, bawazir m, koo h, hwang g. front cell infect microbiol. 2020 feb;10. 13. candida biome of severe early childhood caries (s-ecc) and its cariogenic virulence traits. fakhruddin ks, samaranayake lp, egusa h, ngo hc, panduwawala c, venkatachalam t, et al. h\ps://doi.org/101080/2000229 720201724484. 2020 jan;12(1). 14. associamon of streptococcus mutans, candida albicans and oral health pracmces with acmvity status of caries lesions among 5-year-old children with early childhood caries. sridhar s, baranya /, suprabha s, shenoy r, suman e, rao a. oral heal prev dent. 2020;18:911–9. 15. oral candidal carriage and its associamon with dental carious lesions in asymptomamc adults: a cross-secmonal study from the uae. al-amad sh, rahman b, khalifa n, awad ma. bmc oral heal 2021 211. 2021 apr;21(1):1–6. microsoft word carlini 37 10-8.docx   vol  3,  no  1  (2015)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2015.37           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.   modification  of  the  technique  of  osteotomy  for  surgical  maxillary   expansion   joão  luis  carlini1,  cris%na  rieth2,  danielle  del  santo  severo2,  kesia  keiko  asami2     1oral  and  maxillofacial  surgery,  chief  of  stomatology  departament,  professor  at  federal  university  of  paraná,  staff  of  caif  –  center  for  integral  assistance  of  cle1  lip   and  palate  pa)ents,  curi#ba,  paraná,  brazil   2dental  surgeon,  school  of  dental  medicine  at  federal  university  of  paraná,  curi*ba,  paraná,  brazil     abstract   surgically  assisted  maxillary  expansion   is  a   technique  used   to  correct   transverse  maxillary   deficiency,  which   is  a  dentofacial  anomaly  related  to  the  decrease  of   the  upper  arch  over   the  lower  arch.  it  is  applied  to  pa1ents  in  their  late  teens  and  adults  due  to  skeletal  maturi-­‐ ty,  causing  oblitera1on  of  the  intermaxillary  suture,  which  requires  orthodon1c  procedure,   associated  with  surgery.  the  purpose  of  this  study  is  to  report  the  clinical  case  of  a  pa6ent   admi%ed  for  surgical  maxillary  expansion  through  the  modified  technique  and  point  out  the   possible   advantages,   such   as   aesthe3cs  maintenence,   long-­‐term   stability,   faster   return   to   orthodon'c   treatment,  and   improved  bone  healing.  however,   there  are   some  contraindi-­‐ ca#ons  when  the  pa#ent  presents  severe  crowding,  roots  of  the  canine  and  lateral   incisor   are  converging  and  in  pa-ents  who  will  undergo  to  protrac-on  with  facial  mask.   cita%on:   carlini   j,   rieth   c,   severo   d,   asami   k.   (2015)  modifica(on  of   the   technique  of  osteot-­‐ omy   for   surgical   maxillary   expansion.   den$stry   3000.  1:a001  doi:10.5195/d3000.2015.37   received:  july  29,  2015   accepted:    september  17,  2015   published:    october  23,  2015   copyright:  ©2015   carlini   j,   severo   d,  asami   k.   this   is   an   open   access   ar!cle   licensed   under   a   crea%ve  commons  a"ribu%on  work  4.0  united   states  license.   email:  kka12@pi(.edu   introduction     the  transverse  maxillary   deficiency  has  a  multifactorial  etiol-­‐ ogy,  and  it  is  associated  with  func-­‐ tional  and  aesthetic  commitment.   the  diagnosis  is  established   through  clinical  exams,  x-­‐rays,  and   model  study  [1].     the  patients  who  need  or-­‐ thodontic  and/or  surgical  maxillary   expansion  frequently  present  a  typ-­‐ ical  vertical  pattern:  long  facial   height  anterior  inferior,  high  palate,   low  tongue  posture,  incompetent   lips,  and  mouth  breathing  [2],  uni-­‐ lateral  or  bilateral  cross  bite,  and   anterior  crowding  [3-­‐6].     the  maturity  of  the  patient’s   skeleton  and  the  amount  of  expan-­‐ sion  expected  are  crucial  to  choose   the  technique  to  be  performed  to   correct  the  transverse  discrepancy   jaw  [3,  7,  8].  another  relevant  as-­‐ pect  to  be  assessed  is  the  magnitude   of  the  transverse  jaw  discrepancy   [4-­‐6].     following  hass  (1980),  in   order  to  correct  the  atresia  skeletal   jaw,  it  is  necessary  a  treatment  that   does  not  only  move  teeth,  but   changes  the  position  of  the  alveolar   bone  processes,  teeth,  and  conse-­‐ quently  the  jaw  segments.     the  rapid  maxillary  expan-­‐ sion  (rme),  using  breakers  only,  is   one  of  the  techniques  indicated  to   treat  transversal  maxillary  deficien-­‐ cy  orthopedic  and  it  has  a  history   over  145  years.  introduced  by  an-­‐ gell  [10,  11],  it  allows  the  treatment   of  transversal  discrepancy  in  skele-­‐ tally  immature  patients  [3-­‐6,  12,  13]   trough  the  power  emitted  by  the   expander,  opening  the  palatal  su-­‐ ture  and  making  the  jaw  expand   [12].     in  adult  patients,  who  have   transverse  maxillary  hypoplasia   unilateral  or  bilateral,  the  effective   form  of  treatment  is  the  surgically   assisted  rapid  maxillary  expansion   (sarme),  a  combination  of  ortho-­‐ dontics  and  surgery.  because  of   skeletal  maturity,  there  is  an  in-­‐ crease  in  secondary  bone  interdigi-­‐ tation,  and  fusion  of  palatal  suture   [4-­‐8,  13,  14].  this  procedure  releas-­‐ es  the  resistance  sites  [4-­‐8,  10,  12,   15,  16].      modifica(on  of  the  technique  of  osteotomy  for  surgical  maxillary  expansion   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.37    http://dentistry3000.pitt.edu   2     the  purpose  of  this  paper  is   to  assess  the  efficiency  of  the  surgi-­‐ cal  technique  modification,  demon-­‐ strated  with  a  case  report,  in  which   the  osteotomy  is  performed  be-­‐ tween  the  lateral  incisors  and  ca-­‐ nines,  fig.  5.  performing  the  modi-­‐ fied  technique,  it  was  possible  to   analyze  several  positive  aspects,   such  as:  increased  stability  of  the   correction  in  long  term,  optimized   bone  healing  due  to  the  increased   production  of  calluses,  faster  return   to  orthodontics  treatment,  mini-­‐ mum  periodontal  hazard,  it  pre-­‐ vents  the  shift  of  the  midline,  and   the  aesthetic  is  maintained  by  split-­‐ ting  the  diastema  generated  in  two   points,  as  you  can  see  in  fig.  9  dif-­‐ ferent  from  the  conventional  tech-­‐ nique,  where  the  diastema  is  single   and  between  central  incisors,  as  it  is   shown  in  fig.  9.       case  report       female  patient,  23  years   old,  was  referred  by  her  orthodon-­‐ tist  with  transverse  maxillary  defi-­‐ ciency,  illustrated  in  fig.  1.  the  di-­‐ agnosis  was  based  on  x-­‐  rays,  fig.  8,   clinical  exam  and  evaluation  of  plas-­‐ ter  models.  a  hyrax  expander  was   used  for  one  week  to  try  to  open  the   palatal  bone  suture  without  sur-­‐ gery,  but  it  failed  (fig.  2).     the  patient  was  submitted   to  pre-­‐operative  routine  laboratory   tests;  blood  tests,  electrocardio-­‐ gram,  chest  x-­‐ray,  and  also  a  pre   anesthetic  consultation,  then  she   was  released.  she  was  admitted  on   the  day  of  surgery,  under  general   anesthesia  and  nasotracheal  intuba-­‐ tion.  the  surgery  went  as  follows:  in   the  back  of  the  vestibule,  it  was  in-­‐ cised  with  15  scalpel  blades,  ex-­‐ tending  from  the  second  pre  molar   until  the  second  pre  molar  contrala-­‐ teral.  after  detached  the  perioste-­‐ um,  the  pyriform  aperture  and  the   zygomatic  pillar  were  exposed.   then  the  nasal  mucous  membrane   was  detached  and  tunneled  to  the   posterior  region  until  the  maxillary   tuberosity,  where  the  reverse  re-­‐ tractor  langenbeck  was  adapted.   protecting  the  nasal  mucosa,  with   shifter  molt  the  osteotomy  with  re-­‐ ciprocating  saw  was  performed;   starting  from  the  tuberosity,  a  sec-­‐ tion  was  made  5  mm  above  the   roots  of  the  teeth.  the  osteotomy   was  performed  on  the  entire  side   wall  of  the  maxillary  sinus  and  the   region  of  the  piriform  aperture,   being  bicortical,  fig  4.  using  the   curved  chisel,  it  promoted  the   separation  of  the  blade  pterygoid   of  the  tuberosity  of  the  maxilla.   with  a  single  guide  chisel,  the  me-­‐ dial  wall  of  the  maxillary  sinus   was  broken  and  with  the  double   guide  chisel,  the  nasal  septum  was   separated.  then  the  flap  was  dis-­‐ placed  in  the  region  between  the   canine  and  the  lateral  incisor  until   the  inserted  gingiva,  tunneling  until   the  cervical  of  the  teeth  was  per-­‐ formed.  using  the  reciprocating   saw,  the  osteotomy  was  done  with  a   blade  of  0.5  mm  thick,  illustrated  in   fig.  5,  leaving  from  the  piriform  ap-­‐ erture  toward  the  cervical  of  the   teeth,  protecting  the  palatal  mucosa   with  the  index  finger.  the  osteoto-­‐ my  extended  about  5  mm  below  the   alveolar  crest  interdental,  seen  in   fig.  6.  for  subsequent  region,  after   detaching  the  nasal  mucosa,  the  re-­‐ ciprocating  saw  was  in  the  most   posterior  position,  then  the  osteot-­‐ omy  was  complemented  with  a   straight  chisel  to  the  posterior  edge   of  the  jaw.  similarly,  we  use  the   chisel  to  complete  the  osteotomy   between  the  teeth,  always  protect-­‐ ing  the  palatal  mucosa  and  then  it   was  taken  into  occlusion  to  observe   the  positioning  of  the  teeth.  the   breaker  was  activated  to  observe  if   the  expansion  occurred  symmetri-­‐ cally,  as  shown  in  fig.  7.  if  this  was   not  happening,  it  would  be  im-­‐ portant  go  over  the  chisel  in  all  os-­‐ teotomies.  the  screw  breaker  was   turned  off  and  we  sutured  the   wound  with  wire  vicryl  4-­‐0.       the  time  of  surgery  was  50   minutes,  and  the  patient  was  dis-­‐ charged  the  same  day.  the  postop-­‐ erative  care  consisted  of  ice  packs   on  the  face,  three  days  of  a  liquid   diet,  followed  by  pasty-­‐liquid,  saline   solution  for  cleaning,  and  anti-­‐    modifica(on  of  the  technique  of  osteotomy  for  surgical  maxillary  expansion   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.37    http://dentistry3000.pitt.edu   3   inflammatory  and  analgesic  for   three  days.  after  seven  days  the  pa-­‐ tient  was  re-­‐evaluated  in  the  clinic   and  she  started  activating  the   breaker,  with  activation  ¼  turn  in   the  morning  and  ¼  turn  in  the  af-­‐ ternoon.  after  a  week  the  patient   was  referred  to  the  orthodontist  to   control  the  extent  of  maxillary  ex-­‐ pansion.       the  control  of  bone  healing   was  performed  by  occlusal  radio-­‐ graphs.  after  30  days,  seen  in  fig.  8,   and  after  three  months,  which  cal-­‐ lus  formation  was  observed.  then   the  orthodontist  removed  the   breaker  and  began  the  orthodontic   treatment,  fig.  8  and  fig  9.    no  se-­‐ quel  was  observed  after  surgery,   such  as  gingival  retraction,  tooth   roots  injury,  no  bone  callus  for-­‐ mation  or  recurrence  that  could   compromise  the  result.     the  measurement  was  per-­‐ formed  on  plaster  models,  preoper-­‐ atively  and  postoperatively,  using   calibrated  caliper.  we  analyzed  the   interdental  distances  between  cen-­‐ tral  fossae  of  the  upper  first  molars   and  the  palatal  portion  of  the  upper   canines,  table  1.     discussion       the  success  of  treatment   depends  on  correct  diagnosis,  ana-­‐ tomical  knowledge  and  selection  of   the  correct  technique.  to  treat  adult   patients  with  maxillary  atresia,  sur-­‐ gically  assisted  disjunction  has   demonstrated  excellent  results  as  a   complementary  method  to  ortho-­‐ dontic  treatment,  where  the  surgery   is  to  relieve  the  resistance  of  bone   structure  to  the  expansive  forces.       there  are  several  authors   who  describe  the  techniques  for   rapid  maxillary  expansion.  these   techniques  are  diversified  according   to  age,  advocated  osteotomies,  local   aspects,  etc  [12].  age  is  a  factor  to   be  considered  before  performing   the  surgical  expansion  because  with   increasing  age,  the  probability  of   decrease  in  the  bone  support  is   higher  and  thus  the  power  emitted   by  the  expander  is  not  transmitted   to  the  intermaxillary  bone  or  other   skeletal  structure,  but  for  teeth,  that   may  cause  extrusion  of  teeth,  and   the  skeletal  expansion  is  reduced   [17].       although  there  is  no  con-­‐ sensus  regarding  the  extension  of   the  surgery,  the  vast  majority  of  au-­‐ thors  cite  the  nasomaxillary,  pala-­‐ tine,  and  zigomaticomaxilar  osteot-­‐ omies  as  indispensable.  however,   pterygomaxillary  disjunction  is  not   always  included  [16].  seeberger  et   al.  (2010)  concluded  in  his  work   that  even  without  the  pterygomaxil-­‐ lary  disjunction,  it  is  possible  to   achieve  an  expansion  with  stable   bite.  laudemann  et  al.  (2009,  2010,   2011)  mentioned  that  this  maneu-­‐ ver  can  be  potentially  dangerous   and  can  lead  intra-­‐  and  extracranial   complications  such  as  severe  bleed-­‐ ing  or  injury  of  the  internal  jugular   vein  or  the  internal  carotid  artery,   caused  by  displaced  fragments  of   the  pterygoid  plate  or  by  the  chisel   during  pterygomaxillary  disjunc-­‐ tion.       regarding  the  technique,  in   this  study  we  chose  the  modified   technique,  in  which  the  osteotomy   is  performed  between  the  lateral   incisors  and  canines,  instead  of  be-­‐ tween  the  central  incisors.  even   though  we  performed  the  ptery-­‐ gomaxillary  disjunction,  there  were   no  complications  regarding  this   maneuver.       in  the  study  of  landes  et  al.   (2009,  2012),  the  minimum  dis-­‐ tance  between  the  roots  of  the  lat-­‐ eral  incisors  and  canines  with  95%   reliability  to  perform  the  modified   technique  was  1.4mm,  however,  in   clinical  practice  it  has  been  ob-­‐ served  that  with  a  measure  of  1.2   mm  is  already  possible  to  perform   modified  technique  of  maxillary  ex-­‐ pansion  without  major  damage.  in   the  present  study  the  distance  of   1mm  was  considered  safe  to  per-­‐ form  the  procedure.  that  is  because   the  reciprocating  saw  blades  are   0.5mm,  and  the  visualization  of  the   root  protuberances,  especially  the   canines,  assist  in  the  location  of  the   osteotomy.      modifica(on  of  the  technique  of  osteotomy  for  surgical  maxillary  expansion   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.37    http://dentistry3000.pitt.edu   4     landes  et  al.  (2009,  2012)   used  the  piezo  device  in  their  study,   arguing  there  was  less  damage  to   the  tooth  roots,  compared  to  recip-­‐ rocating  saw  that  we  used  in  this   study.  we  believe  it  is  a  great  option   for  the  execution  of  this  technique   because  the  piezo  is  much  less   traumatic.  however  the  time  for  the   execution  is  much  higher  when   compared  with  the  reciprocating   saw,  but  this  depends  on  the  sur-­‐ geon's  skill.    according  to  gauthier   et  al.  (2001),  the  surgical  maxillary   expansion,  when  it  releases  the  re-­‐ sistance  between  the  intermaxillary   suture,  produces  minimum  perio-­‐ dontal  hazard.  in  the  present  study,   the  same  conclusion  was  found.  it  is   worth  remembering  that  the  pa-­‐ tients  who  underwent  surgery  had   a  healthy  periodontal  condition  and   sufficient  posterior  dental  support.   however,  sanromán  et  al.  (2010)   mentioned  that  tailored  devices  are   an  alternative  to  a  stable  expansion   achieved  in  adult  patients  who  have   poor  periodontal  quality  and  low   posterior  dental  support.   other  advantage  of  the  surgical   maxillary  expansion  is  that  it  pro-­‐ vides  the  patient  an  enlargement  of   the  nasal  air  passages,  due  to  lateral   repositioning  of  the  lateral  nasal   walls,  thereby  improving  nasal   breathing  [7,  8,14,18].  in  this  study   the  patient  had  significant  im-­‐ provement  in  breathing  after  being   submitted  to  maxillary  expansion   surgery.     similar  to  the  work  of   anttila  et  al.  (2004),  in  this  study   the  patient  succeeded  in  transverse   maxillary  expansion.  another  im-­‐ portant  observation  of  anttila  et  al.   (2004)  that  was  also  observed  in   this  study  is  the  fact  that  the  sup-­‐ port  bone  of  teeth  must  not  be   compromised,  this  being  so,  it  is  a   prerequisite  to  achieve  success  in   this  surgical  procedure.     another  factor  to  be  consid-­‐ ered  is  the  type  of  breaker.  hyrax   breaker  can  increase  the  pressure   on  the  teeth  because  the  support  is   only  on  the  teeth.  when  opting  for   hass  breaker,  where  there  is  also  a   support  in  the  palatal  mucosa,  this   issue  is  minimized.  in  this  case  we   used  the  hyrax  expander   for  ease  of  cleaning,  less   compression  of  the  palatal   mucosa,  and  the  fact  that   osteotomies  were  checked   to  observe  possible  points   of  resistance.     general  anesthesia,  as  cited   alfaro  et  al.  (2010),  is  clas-­‐ sically  advocated  for  per-­‐ forming  the  sarme,  since  ptery-­‐ gomaxillary  disjunction  becomes   potentially  traumatic  to  the  patient;   it  is  performed  only  with  sedation.   for  this  reason,  in  this  study,  all   surgical  procedures  were  per-­‐ formed  with  patients  under  general   anesthesia.       it  is  important  to  note  that   comparisons  between  dimensions   achieved  through  the  expansion  of  a   study  and  another  are  irrelevant,   since  the  requirements  may  vary   from  patient  to  patient.  what  is  rel-­‐ evant  is  to  obtain  an  adequate  ex-­‐ pansion  to  correct  a  cross  bite  or   any  other  occlusal  patient's  need  in   a  specific  case  [19].     conclusion       surgically  assisted  rapid   maxillary  expansion  has  proven  to   be  an  effective  and  reliable  tech-­‐ nique  to  treat  maxillary  transverse   deficiency  in  adolescent  and  adult   patients.  sarme  has  shown  several   advantages,  such  as  a  stable  proce-­‐ dure,  with  minimum  periodontal   hazard;  improved  aesthetics  seen  in   the  immediate  post-­‐operative,  by   splitting  the  diastema  generated  in   two  points;  early  return  for  ortho-­‐ dontic  treatment,  and  improved  na-­‐ sal  breathing.        modifica(on  of  the  technique  of  osteotomy  for  surgical  maxillary  expansion   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.37    http://dentistry3000.pitt.edu   5     references   1.   avaliação  clínica  dos  pro-­‐ cedimentos  de  expansão  cirurgi-­‐ camente  assistida  da  maxila   (ecam).  clinical  evaluation  of  sur-­‐ gically  assisted  maxillary  expansion   (same).  paulo  domingos  ribeiro   jr.i;  eduardo  sanches  gonçalesii;   paulo  césar  ulson  de  souzaiii;  hugo   nary  filhoiv;  joão  gualberto   cerqueira  luzv  .  rev.  dent.  press   ortodon.  ortop.  facial  vol.11  no.1   maringá  jan./feb.  2006.  paulo  do-­‐ mingos  ribeiro  jr.i;  eduardo   sanches  gonçalesii     2.   nasal  cavity  size,  airway   resistance,  and  subjective  sensation   after  surgically  assisted  rapid  maxil-­‐ lary  expansion:  a  prospective  longi-­‐ tudinal  study.  magnusson  a,  bjerk-­‐ lin  k,  nilsson  p,  jönsson  f,  mar-­‐ cusson  a.  am  j  orthod  dentofacial   orthop.  2011  nov;140(5):641-­‐51.   doi:  10.1016/j.ajodo.2010.11.024.   pmid:  22051484   3.   periodontal  effects  of  surgi-­‐ cally  assisted  rapid  palatal  expan-­‐ sion  evaluated  clinically  and  with   cone-­‐beam  computerized  tomogra-­‐ phy:  6-­‐month  preliminary  results.   gauthier  c,  voyer  r,  paquette  m,       rompré  p,  papadakis  a.  am  j  or-­‐ thod  dentofacial  orthop.  2011   apr;139(4  suppl):s117-­‐28.  doi:   10.1016/j.ajodo.2010.06.022.   pmid:  21435529   4.   evaluation  of  surgically  as-­‐ sisted  rapid  maxillary  expansion   with  or  without  pterygomaxillary   disjunction  based  upon  preopera-­‐ tive  and  post-­‐expansion  3d  com-­‐ puted  tomography  data.  laude-­‐ mann  k,  petruchin  o,  mack  mg,   kopp  s,  sader  r,  landes  ca.  oral   maxillofac  surg.  2009   sep;13(3):159-­‐69.  doi:   10.1007/s10006-­‐009-­‐0167-­‐3.   pmid:  19714376   5.   long-­‐term  3d  cast  model   study:  bone-­‐borne  vs.  tooth-­‐borne   surgically  assisted  rapid  maxillary   expansion  due  to  secondary  varia-­‐ bles.  laudemann  k,  petruchin  o,   nafzger  m,  ballon  a,  kopp  s,  sader   ra,  landes  ca.  oral  maxillofac  surg.   2010  jun;14(2):105-­‐14.  doi:   10.1007/s10006-­‐009-­‐0194-­‐0.   pmid:  20108107   6.   assessment  of  surgically   assisted  rapid  maxillary  expansion   regarding  pterygomaxillary  disjunc     tion  using  thin  volume-­‐rendering   technique:  in  variance  analysis  and   in  reliability,  accuracy,  and  validity.   laudemann  k,  santo  g,  revilla  c,   harth  m,  kopp  s,  sader  ra,  landes   ca.  j  oral  maxillofac  surg.  2011   oct;69(10):2631-­‐43.  doi:   10.1016/j.joms.2010.12.007.  epub   2011  apr  7.  pmid:  21474227   7.   comparison  of  bipartite   versus  tripartite  osteotomy  for   maxillary  transversal  expansion   using  3-­‐dimensional  preoperative   and  postexpansion  computed  to-­‐ mography  data.  landes  ca,  laude-­‐ mann  k,  petruchin  o,  mack  mg,   kopp  s,  ludwig  b,  sader  ra,  seitz  o.   j  oral  maxillofac  surg.  2009   oct;67(10):2287-­‐301.  doi:   10.1016/j.joms.2009.04.069.  pmid:   19761925   8.   advantages  and  limits  of  3-­‐ segment  (paramedian)  versus  2-­‐ segment  (median)  surgically  assist-­‐ ed  rapid  maxillary  expansion   (sarme).  landes  ca,  laudemann  k,   petruchin  o,  revilla  c,  seitz  o,  kopp   s,  ludwig  b,  sader  ra.  oral  surg   oral  med  oral  pathol  oral  radiol.   2012  jan;113(1):29-­‐40.  doi:   10.1016/j.tripleo.2011.01.013.   table  1.  canine  /  molar  relation     pre-operative post-operative canine 24 mm 29 mm molar 25 mm 34 mm    modifica(on  of  the  technique  of  osteotomy  for  surgical  maxillary  expansion   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.37    http://dentistry3000.pitt.edu   6   epub  2011  mar  31.  pmid:   22677689   9.   long-­‐term  posttreatment   evaluation  of  rapid  palatal  expan-­‐ sion.  haas  aj.  angle  orthod.  1980   jul;50(3):189-­‐217.  no  abstract   available.  pmid:  6996533   10.   stability  in  dental  changes   in  rme  and  sarme:  a  2-­‐year  follow-­‐ up.  sokucu  o,  kosger  hh,  bicakci   aa,  babacan  h.  angle  orthod.  2009   mar;79(2):207-­‐13.  doi:   10.2319/031808-­‐155.1.  pmid:   19216605   11.   surgically  assisted  rapid   maxillary  expansion:  a  comparison   of  technique,  response,  and  stabil-­‐ ity.  northway  wm,  meade  jb  jr.  an-­‐ gle  orthod.  1997;67(4):309-­‐20.   pmid:  9267580   12.   minimally  invasive  surgical-­‐ ly  assisted  rapid  palatal  expansion   with  limited  approach  under  seda-­‐ tion:  a  report  of  283  consecutive   cases.  hernandez-­‐alfaro  f,  mareque   bueno  j,  diaz  a,  pagés  cm.  j  oral   maxillofac  surg.  2010   sep;68(9):2154-­‐8.  doi:   10.1016/j.joms.2009.09.080.  epub   2010  jul  2.  pmid:  20584567   13.   surgically  assisted  rapid   palatal  expansion  (sarpe).  god-­‐ dard  r,  witherow  h.  br  j  oral  maxil-­‐ lofac  surg.  2011  jan;49(1):65-­‐6.  doi:   10.1016/j.bjoms.2009.11.013.  epub   2010  feb  18.  no  abstract  available.   pmid:  20170994   14.   transverse  maxillary  dis-­‐ traction  in  patients  with  periodon-­‐ tal  pathology  or  insufficient  tooth   anchorage  using  custom-­‐made  de-­‐ vices.  fernández-­‐sanromán  j,  do-­‐ nascimento  mg,  lópez  ac,  ferro   mf,  berrondo  ia.  j  oral  maxillofac   surg.  2010  jul;68(7):1530-­‐6.  doi:   10.1016/j.joms.2009.09.074.  epub   2010  apr  22.  pmid:  20417007   15.   long  term  effects  of  surgi-­‐ cally  assisted  rapid  maxillary  ex-­‐ pansion  without  performing  osteot-­‐ omy  of  the  pterygoid  plates.   seeberger  r,  kater  w,  davids  r,   thiele  oc.  j  craniomaxillofac  surg.   2010  apr;38(3):175-­‐8.  doi:   10.1016/j.jcms.2009.07.003.  epub   2009  aug  5.  pmid:  19660962   16.   changes  in  nasopharyngeal   airway  following  orthopedic  and   surgically  assisted  rapid  maxillary   expansion.  kurt  g,  altuğ-­‐ataç  at,   atac  ms,  karasu  ha.  j  craniofac   surg.  2010  mar;21(2):312-­‐7.  doi:   10.1097/scs.0b013e3181cf5f73.   pmid:  20186094   17.   feasibility  and  long-­‐term   stability  of  surgically  assisted  rapid   maxillary  expansion  with  lateral   osteotomy.  anttila  a,  finne  k,  keski-­‐ nisula  k,  somppi  m,  panula  k,  pel-­‐ tomäki  t.  eur  j  orthod.  2004   aug;26(4):391-­‐5.  pmid:  15366383   18.   changes  in  nasal  volume   after  surgically  assisted  bone-­‐borne   rapid  maxillary  expansion.  deeb  w,   hansen  l,  hotan  t,  hietschold  v,   harzer  w,  tausche  e.  am  j  orthod   dentofacial  orthop.  2010   jun;137(6):782-­‐9.  doi:   10.1016/j.ajodo.2009.03.042.   pmid:  20685533   19.   correção  das  deficiências   transversas  e  ântero-­‐posteriores  da   maxila  em  pacientes  adultos  /  max-­‐ illary  anteroposterior  and  trans-­‐ verse  problems  correction  in  adult   patients.  carlini,  joão  luiz;  biron,   cássia;  gomes,  kelston  ulbricht;   gebert,  andréa;  strujak,  guilherme.   rev.  dent.  press  ortodon.  ortopedi.   facial;  12(5):  92-­‐99,  set.-­‐out.  2007.   ilus,  tab.  artigo  em  português  |  li-­‐ lacs  |  id:  lil-­‐465909.     microsoft word 58 2016.docx   vol  4,  no  1  (2016)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2016.58           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     multilevel  modeling  for  dental  caries  among  adolescents  in  a  bra-­‐ zilian  large  city     giovana  daniela  pecharki  1,  joão  armando  brancher1,  márcia  olandoski1,  andrea  duarte  doetzer1,  samuel  jorge  moyses1,  paula  cris%na  trevi-­‐ la!o1     1  school  of  health  and  biosciences,  pon&'cia  universidade  católica  do  paraná  (pucpr),  rua  imaculada  conceição,  curi'ba,  pr,  brazil abstract   dental  caries  is  a  complex  disease,  which  needs  an  approach  that  considers  caries  influenc-­‐ ing  factors  at  different  levels  and  their  integra2on.  mul$level  modeling  is  a  clustered  analy-­‐ sis  of  variables  from  the  individual  to  the  community  level.  the  aim  of  this  study  was  to  in-­‐ ves$gate  the  associa$on  of  social  and  biological  factors  grouped  into  hierarchical  levels,  in   students   with   caries.   a   sample   of   687   students   was   evaluated   from   public   and   private   schools  of  curi,ba.  the  parameters  evaluated  were:   individual   level,   school   level  and  dis-­‐ trict  level.  individual  variables  had  a  highly  significant  associa6on  with  caries  experience,  al-­‐ so  in  the  presence  of  school  and  district   levels.  male  sex  nega4vely  associated  with  caries   experience.  however,  the  interac2on  between  male  sex  and  no  fluoride  use  was  posi2vely   associated  with  caries.  lower  socioeconomic  status,  dental  biofilm,  and  fluorosis  were  as-­‐ sociated  with  caries.  nevertheless,  the  interac3on  between  dental  biofilm  and  fluorosis  was   nega%vely  associated  with  caries  experience.  the   interac%on  between  no  flossing  and  use   of  public  dental  services  were  also  associated  with  caries  outcome.  individual  factors  were   associated   with   caries   experience   even   with   the   inclusion   of   contextual   variables   in   the   study  popula+on.     cita%on:  pecharki,  et  al.  (2016)     mul$level  modeling  for  dental  caries  among   adolescents  in  a  brazilian  large  city.     den$stry  3000.  1:a001   doi:10.5195/d3000.2016.58   received:  july  22,  2016   accepted:    july  29  ,  2016   published:    october  3,  2016   copyright:  ©2016  pecharki,  et  al.  this  is  an  open   access   ar!cle   licensed   under   a   crea!ve   com-­‐ mons   a!ribu%on   work   4.0   united   states   li-­‐ cense.   email:  paula.trevila+o@pucpr.br   introduction   dental  caries  is  an  infec-­‐ tious  and  multifactorial  disease.   the  prevalence  of  caries  has  re-­‐ duced  significantly,  including  in   latin  america  and  brazil   (dmft=2.07  for  12  years-­‐old)  [1,2]   possibly  due  to  the  increased   availability  of  fluoride  and  oral   health  programs  [3,2].   nevertheless,  groups  of   children  have  still  been  showing   high  caries  activity.  it  is  estimated   that  20  to  25%  of  children  and  ad-­‐ olescents  in  brazil  concentrate  60   to  80%  of  caries  prevalence  [2].   the  phenomenon  of  disease  con-­‐ centration  in  small  groups  is   termed  polarization  and  repre-­‐ sents  one  of  the  epidemiological   disease  aspects,  where  a  popula-­‐ tion  portion  focuses  most  needs   for  treatment  [2,4].  thus,  the  early   identification  of  caries  risk  indica-­‐ tors  is  fundamental  for  measures   of  prevention,  control  and  reduc-­‐ tion  of  damage,  with  obvious  epi-­‐ demiological,  human  and  econom-­‐ ical  consequences  [5].   socioeconomic  status  [6],   oral  health  behavior,  including  diet   [7],  gender,  ethnicity  [3]  and  bio-­‐ logical  factors,  such  as  biofilm   formation  and  saliva  properties   [8,9],  have  been  considered  the   main  risk  factors  influencing  indi-­‐ vidual  susceptibility  to  caries.   however,  there  has  been  a  shift   from  individual  to  population– level  approach  when  researching   the  risk  indicators  for  chronic  dis-­‐ eases  [10].  as  previously  pub-­‐ lished,  individual  factors  are  usual-­‐ ly  influenced  by  the  population   context  [7,10].  also,  a  higher  car-­‐ ies  experience  and  a  lower  dental   care  index  (the  ratio  between  the   number  of  filled  teeth  and  dmft)   were  observed  in  children  attend-­‐ ing  public  schools  than  for  those   enrolled  in  private  schools  [3]  and   in  areas  with  lower  levels  of  em-­‐ powerment  and  income  [11,  12].   there  has  been  seen  significant   mul$level  modeling  for  dental  caries  among  adolescents  in  a  brazilian  large  city     vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.58    http://dentistry3000.pitt.edu   2   variation  in  the  severity  of  caries   between  low-­‐income  neighbor-­‐ hood  clusters.  indeed,  neighbor-­‐ hood  features  were  seen  to  be  as-­‐ sociated  with  self-­‐reported  oral   health  [13].     caries  is  a  complex  disease   and  an  approach  that  can  evaluate   the  factors  influencing  the  disease   at  different  levels  and  their  inte-­‐ gration  is  suitable.  to  date  there   are  only  few  studies  evaluating   caries  in  a  multilevel  approach,   and  they  do  not  analyze  all  factors   that  may  be  contributing  to  its   complex  development  [7].    multi-­‐ level  analysis  is  a  model  that  con-­‐ siders  factors  related  to  the  indi-­‐ vidual  together  with  the  commu-­‐ nity  level,  organized  in  hierarchical   levels.  thus,  the  aim  of  this  study   was  to  investigate  the  association   of  individual  and  community  fac-­‐ tors,  grouped  into  hierarchical   levels  by  multilevel  modeling.     methods   six  districts  in  curitiba-­‐  pr,   brazil  with  similar  socioeconomic   aspects  were  assessed  in  this   study  (table  1).  in  brazil,  the  soci-­‐ oeconomic  status  generally  im-­‐ plies  in  the  type  of  school,  public   (lower  income)  or  private  (higher   income).  then,  two  larger  schools   from  each  district,  one  public  and   one  private,  with  a  minimum  of   1500  students,  were  chosen  using   a  table  of  random  numbers  totaliz-­‐ ing  twelve  schools.  all  12-­‐year-­‐old   students  or  those  which  would   complete  this  age  in  the  year  of   the  study  were  invited  to  partici-­‐ pate  on  each  school.  an  informed   consent  form  was  given  to  every   student  caregiver  in  each  school.   adolescents  whose  caregiv-­‐ ers/parents  did  not  return  the   consent  form  were  not  included   for  study  along  with  smokers,  or-­‐ thodontic  appliances  users,  and   table 1. districts demographic and socioeconomic aspects. ! districts' ! 1! 2! 3! 4! 5! 6! ! demographic! aspects! 248,698! inhabitants!or! 14.2%!of!the! total! municipality! 155,794! inhabitants! or!8.9%!of! total! municipality! ! 215!503! inhabitants!or! 12.3%!of!the! total! municipality! 197!346! inhabitants! or!11.3%!of! the!total! municipality! 168,425! inhabitants!or! 9.6%!of!total! municipality! ! 243!506! inhabitants!or! 13.9%!of!the! total! municipality! 47427! children!!!!0m 14!years!old! (19.1%!of!the! regional! population)! ! 42125! children! 0m14!years! old!(40.4%!of! the!regional! population)! ! 45711! children!!!!!!!! 0m14!years! old!(21.2%!of! the!regional! population)! 41297!m! children!!!!!!0m 14!years! (20.9%!of!the! regional! population)! ! 42087! children!!!!!!!!!!!!! 0m14!years!old! (25.0%!of!the! regional! population)! 42125! children!!!!!!!!!! 0m14!years! (17.3%!of!the! regional! population)! ! socioeconomic! aspects! !!!!!!!!!!!!!!income! per!capita!–! 2010*!!!!!!!!!!!(*1! minimum!wage,!! mw=!$130)! 45.9%! received!over! 1!to!3!mw! 42.5%! received!over! 1!to!3!mw! 44.3%! received!over! 1!to!3!mw! 50.4%! received!over! 1!to!3!mw! 44.9%! received!over! 1!to!3!mw! 40.0%! received!over! 1!to!3!mw! water!supply! 99.2%!of! households! connected!to! the!water! network! 98.8%!of! households! connected!to! the!water! network! 99.4%!of! households! connected!to! the!water! network! 99.7%!of! households! connected!to! the!water! network! 99.0%!of! households! connected!to! the!water! network! 98.9%!of! households! connected!to! the!water! network! sewage! network! 82.7%!of! households! connected!to! sewage! network! 86.6%!of! households! connected!to! sewage! network! 94.8%!!of! households! connected!to! sewage! network! 93.9%!of! households! connected!to! sewage! network! 89.4%!of! households! connected!to! sewage! network! 95.8%!of! households! connected!to! sewage! network! mul$level  modeling  for  dental  caries  among  adolescents  in  a  brazilian  large  city     vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.58    http://dentistry3000.pitt.edu   3   individuals  taking  antibiotics  in  the   last  three  months.  the  study  was   approved  by  the  ethical  commit-­‐ tee  on  research  of  pontifical   catholic  university  of  paraná   (pucpr)  under  register  n°  487.   they  were  distributed  about  1100   consent  forms,  and  687  12-­‐year-­‐ old  unrelated  adolescents  agreed   to  participate,  approximately  55   students  from  each  school,  both   sexes.   the  687  students   were  diagnosed  according  to   the  decayed,  missing  and   filled  teeth  index  (dmft).   white  spot  lesions  in  dental   surfaces  were  considered   decayed  according  to  clinical   criteria  described  by  assaf  et   al.  [14].     examinations  were   conducted  in  schoolrooms  in   accordance  with  interna-­‐ tional  standards  established   by  world  health  organiza-­‐ tion  (who)  [15].  all  evalua-­‐ tions  were  performed  by   two  calibrated  examiners.   inter-­‐  and  intra-­‐examiner   reproducibility  was  taken  on   10%  of  the  sample  and  the   kappa  test  was  used  to   measure  reliability.  the  ob-­‐ tained  values  for  kappa  test   were  0.93  for  inter-­‐  and  0.99   for  intra-­‐examiner.   a  multilevel  study   was  designed  to  assess  the   individual  and  contextual   effects  on  caries  experience   (dmft=0  or  dmft≥1).  data   were  hierarchically  struc-­‐ tured  in  three  levels:  indi-­‐ viduals  (level  1),  schools   (level  2),  and  districts  (level   3).  the  choice  of  variables   was  based  in  caries  multifac-­‐ torial  model  proposed  by   fejerskov  &  manji  [16].   individual  level  (level  1)   individual  demographic   variables  included  sex   (male/female)  and  ethnicity:   white/  light-­‐  and  dark-­‐ table&2.&individual)and)contextual)characteristics)of)students)(n=687))and)caries)status.) variables& n& frequency& (%)& proportion&of&dmft≥1&(%)& ! 687) 100.0) ) level!1:!student! ) ) ) sociodemografic) ) ) ) ethnic'group' ) ) ) ) white)) 606) 88.2) 48.5a) ) lightd)and)darkdskinned)black) 62) 9.0) 69.3b) ) yellow) 19) 2.8) 63.2a,b) sex' ) ) ) ) female)) 377) 54.9) 52.2ª) ) male) 310) 45.1) 49.0a) behavioural) ) ) ) toothbrushing'frequency) ) ) ) ) 2)or)more/day) 644) 93.7) 49.8ª) ) until)once/day) 43) 6.3) 65.1ª) flossing' ) ) ) ) yes) 443) 64.5) 47.2a) ) no) 244) 35.5) 57.4b)) fluoride'use'(solution,varnish,gel)' ) ) ) ) yes) 405) 59.0) 47.4a)) ) no) 282) 41.0) 55.7b) dental'visits'frequency' ) ) ) ) 2)times)or)more/year) 304) 44.2) 48.7a) ) once/year) 315) 45.9) 50.8a) ) no) 68) 9.9) 60.3a) sugar'consumption'between'meals' ) ) ) ) no) 69) 10.0) 50.7ª) ) yes) 618) 90.0) 50.8ª) socioeconomic) ) ) ) dental'access' ) ) ) ) private) 427) 62.2) 41.4ª) ) public) 260) 37.8) 66.1b) individual'socieconomic'status' ) ) ) ) a1/a2)(highest)) 175) 25.5) 32.6ª) ) b1/b2) 310) 45.1) 51.9b) ) c) 166) 24.2) 65.1c) ) d/e)(lowest)) clinical) 36) 5.2) 63.9b,c) plaque'index'(modified)' ) ) ) ) 0) 95) 13.8) 38.9ª) ) >0)and)<1) 475) 69.2) 50.1ª) ) ≥1) 117) 17.0) 63.2b) gengivitis' ) ) ) ) no) 404) 58.8) 47.0a) ) yes) 283) 41.2) 56.2b) fluorosis' ) ) ) ) no) 497) 72.3) 52.3ª) ) yes) 190) 27.7) 46.8a) stimulated'salivary'flow'rate' ) ) ) ) >0.5)ml/min) 522) 76.0) 51.0a) ) ≤0.5)ml/min) 165) 24.0) 50.3a) salivary'buffering'capacity' ) ) ) ) ph>3.9) 670) 97.5) 50.9ª) ) ph≤3.9) 17) 2.5) 47.1a) level!2:!school! ) ) ) type'of'school' ) ) ) )))private) 334) 48.6) 41.3ª) )))public) 353) 51.4) 59.8b) oral'health'education'' ) ) ) )))yes) 240) 34.9) 52.5a)) )))no) 447) 65.1)) 49.9a) permission'for'sweet'consumption'' ) ) ) )))non)permitted) 228) 33.2)) 37.3a) )))permitted) 459) 66.8) 57.5b) level!3:!district& ) ) ) fluoride'concentration'in'water'supply' ) ) ) )))≥0.7)ml/l) 333) 48.5) 49.2a) )))<0.7)ml/l) 354) 51.5) 52.3a) socioeconomic'position' ) ) ) )))better)living)conditions) 447) 65.1) 47.2a) )))worse)living)conditions) 240) 34.9) 57.5b) fisher’s)exact)or)chidsquare)test.)distinct)lowerdcase)superscript)letters)indicate)statistical)significance)(p<0.05).) mul$level  modeling  for  dental  caries  among  adolescents  in  a  brazilian  large  city     vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.58    http://dentistry3000.pitt.edu   4   skinned/black  and  yellow  (asian   descent).the  parent/caregiver  an-­‐ swered  self-­‐completed  question-­‐ naires  about  oral  health  behavior:   tooth  brushing  frequency,  dental   flossing,  topical  fluoride  (solution,   varnish,  gel),  dental  attendance   pattern  (frequency  of  dental   check-­‐ups  and  public/private  ac-­‐ cess)  and  sugar  consumption  be-­‐ tween  meals  [12,6].     a  standard  brazilian  socio-­‐ economic  classification  based  on   household  items  and  on  the  level   of  education  of  the  head  of   household  was  adopted  [12].  a  set   of  points  is  assigned  to  these  indi-­‐ cators  and  a  final  score  defines  the   socioeconomic  groups;  a  (highest),   b,  c,  d,  and  e  (lowest).  because  of   the  small  number  of  observations   in  class  e,  data  were  categorized   into  four  groups:  high  social  class   (class  a),  high-­‐middle  social  class   (class  b),  middle  social  class  (class   c)  and  low  social  class  (classes  d   and  e).     the  biofilm  accumulation   was  verified  by  the  plaque  index   (pi)  [17]  modified,  which  adopted   the  same  criteria,  but  evaluating  6   teeth  surfaces:  16  [buccal  (b)],  12   b,  26  [lingual  (l)],  36  b,  32  l,  and   46  l.  the  pi  was  categorized  as:  no   plaque  accumulation  (pi=0),  regu-­‐ lar  plaque  accumulation  (pi>0  and   <1,  up  to  a  third  of  the  surface   with  accumulated  biofilm)  and   high  plaque  accumulation  (pi≥1,   more  than  a  third  of  the  surface   with  accumulated  biofilm).  indi-­‐ viduals  were  considered  positive   for  gingivitis  when  all  teeth  from   at  least  one  sextant  were  affected,   with  bleeding  and  inflammation.   the  presence  or  not  of  any  degree   of  fluorosis  was  established  ac-­‐ cording  to  dean’s  index  (who)   [15]  .   the  stimulated  salivary   flow  rate  (ssfr)  was  measured  as   previously  described  [18].  to  clas-­‐ sify  the  ssfr  the  following  numer-­‐ ical  scores  were  attributed:  0  for   low  caries  risk  >0.5  ml/min  and  1   for  high  caries  risk  ≤0.5  ml/min,   because  the  cut  line  for  dichoto-­‐ mization  was  based  on  the  data   distribution.  the  buffering  capaci-­‐ ty  (bc)  was  performed  as  de-­‐ scribed  previously  [19].  final  ph  of   the  mixture  was  determined  using   a  ph-­‐meter  and  bc  was  consid-­‐ ered  good  if  the  final  ph  was  >3.9   and  deficient  for  ph≤3.9.     school  level  (level  2)   twelve  large  schools  were   randomly  chosen,  being  1  public   and  1  private  from  each  of  the  6   health  districts  studied.  besides   the  type  of  school,  it  was  also  veri-­‐ fied  permission  or  not  for  sweet-­‐ ies’  consumption  in  the  classroom   and  other  areas  in  the  institution   and  the  presence  or  not  of  oral   health  education  programs.   district  level  (level  3)   means  of  fluoride  concen-­‐ tration  in  water  supply  in  2006   were  obtained  for  6  health  dis-­‐ tricts  from  the  municipal  health   secretary  of  curitiba  and  dichot-­‐ omized  into  <0.7  and  ≥0.7  ml/l,   because  it  is  the  minimum  concen-­‐ tration  allowed  in  the  city.   the  classification  of  the  so-­‐ cioeconomic  conditions  in  the   health  districts  was  described  by   moraes  &  ribeiro  [20],  and  con-­‐ sidered  income  and  education  of   the  household  head.  the  score   was  then  organized  in  descending   order  and  grouped  into  5  socioec-­‐ onomic  categories.  the  first  cate-­‐ gory  was  a  (best  living  conditions)   and  the  last  category  was  e  (worst   living  conditions).  because  of  data   distribution  in  this  study,  it  was   chosen  to  join  b/c  (better  living   conditions),  and  d/e  (worse  living   conditions).  none  of  the  evaluated   districts  obtained  score  a.         mul$level  modeling  for  dental  caries  among  adolescents  in  a  brazilian  large  city     vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.58    http://dentistry3000.pitt.edu   5   firstly,  an  exploratory   study  was  performed  using  fisher   and  chi-­‐square  test.  the  multilevel   software  mlwin  version  2.01   (centre  for  multilevel  modeling,   bristol,  uk)  was  used  to  estimate   the  effects  of  individual,  school   and  district  variables  on  dmft.   the  multilevel  analyzes  were  used   to  determine  the  relative  size  of   the  variance  at  each  level  [21].  as   the  outcome  was  binary,  a  multi-­‐ level  logistic  model  was  used.  both   the  marginal  quasi  likelihood   (mql)  first-­‐order  approximation   procedures  and  predictive  quasi   likelihood  (pql)  second-­‐order  ap-­‐ proximation  procedures  were   used.  however,  because  they  pro-­‐ duced  similar  results,  only  the  re-­‐ sults  based  on  pql  second-­‐order   procedures  are  reported  and  dis-­‐ cussed  [12].  five  models  were   specified  for  the  outcome.  first,  a   null  model  (model  1),  which  took   into  account  the  structure  from   levels  1,  2  and  3.  the  null  model   was  followed  by  model  2  in  which   variables  from  level  1  were  includ-­‐ ed.  in  model  3,  it  was  included   variables  from  levels  1,  2  and  3.   there  were  adjustments  in  models   4  considering:  i)  all  the  school  and   district  variables;  ii)  all  the  varia-­‐ bles  at  the  individual  level,  which   presented  statistically  significant   table&3.&multilevel(modeling(of(121yr1old(students,(considering(individual,(school(and(district(hierarchical(levels(in(relation(to(caries(experience( & model&1&(null&model)& model&2& model&3& model&4& model&5& variables& p" value& or& ci& &&95%& p&value& or& ci& &&95%& p&value& or& ci& &&95%& p"" value& or& ci& &&95%& p&value& or& ci& &&95%& " level"1:"individual" ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( " ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( sociodemografic" ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ethnic&group& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( white((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( light1(and(dark1skinned( black( ( ( ( 0.096( 1.66( (0.911 3.02)( 0.080( 1.71( (0.94(–( 3.11)( 0.060( 1.82( (0.981 3.38)( ( ( ( ( yellow( ( ( ( 0.129( 2.21( (0.791 6.17)( 0.109( 2.32( (0.83(–( 6.49)( 0.137( 2.24( (0.771 6.52)( ( ( ( sex& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( female((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( male( ( ( ( 0.133( 0.77( (0.54(1( 1.08)( 0.107( 0.75( (0.53(–( 1.06)( 0.002*( 0.49( (0.311 0.77)( 0.003*( 0.51( (0.33(–( 0.79)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( behavioural" ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( toothbrushing&frequency& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( 2(or(more/day((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( until(once/day( ( ( ( 0.859( 1.07( (0.521 2.21)( 0.929( 1.03( (0.50(–( 2.14)( 0.685( 0.85( (0.401 1.83)( ( ( ( flossing& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( yes((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( no(( ( ( ( 0.161( 1.30( (0.901 1.87)( 0.215( 1.26( (0.87(–( 1.82)( 0.189( 0.59( (0.261 1.30)( 0.635( 0.89( (0.56(–( 1.42)( fluoride&use&(solution,& varnish,&gel)& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( yes((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( no( ( ( ( 0.652( 1.09( (0.761 1.55)( 0.698( 1.07( (0.75(–( 1.54)( 0.087( 0.66( (0.401 1.06)( 0.082( 0.66( (0.41(–( 1.05)( dental&visits&frequency& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( 2(times(or(more/year((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( once/year( ( ( ( 0.441( 0.87( (0.611 1.24)( 0.510( 0.87( (0.63(–( 1.26)( 0.922( 0.98( (0.681 1.41)( ( ( ( ( no( ( ( ( 0.614( 0.85( (0.461 1.59)( 0.736( 0.90( (0.48(–( 1.68)( 0.285( 0.70( (0.361 1.35)( ( ( ( sugar&consumption&between& meals& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( no((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( yes( ( ( ( 0.481( 1.22( (0.701 2.11)( 0.474( 1.22( (0.70(–( 2.13)( 0.425( 1.27( (0.711 2.26)( ( ( ( mul$level  modeling  for  dental  caries  among  adolescents  in  a  brazilian  large  city     vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.58    http://dentistry3000.pitt.edu   6   association  (ssa)  with  the  out-­‐ come,  and  iii)  interactions  at  level   1,  which  have  made  sense  as  hy-­‐ potheses  and  also  demonstrated   ssa.  interactions  at  second  and   third  levels  did  not  reach  statistical   significance  and  thus  were  not  in-­‐ cluded  in  this  model.  in  model  5,  it   was  included  i)  all  variables  at  lev-­‐ el  1  which  showed  significance  in   model  4  ii)  all  variables  at  level  2   and  3,  and  iii)  all  individual  inter-­‐ actions  that  have  made  sense  as   hypotheses  and  showed  signifi-­‐ cance  in  model  4.  the  results  were   presented  as  odds  ratios  (or)  and   their  95%  confidence  intervals  (ci).   for  each  logistic  model  the  intra-­‐ class  correlation  (icc)  was  calcu-­‐ lated  using  an  approach  described   by  hox  [21].   results   individuals  from  level  1   (n=687),  schools  from  level  2   (n=12)  and  districts  from  level  3   (n=6)  were  evaluated  in  this  study.   caries  status  according  to  individ-­‐ ual  and  contextual  aspects  is   shown  in  table  2.     out  of  the  students,  338   subjects  (49.2%)  were  caries-­‐free   (dmft=0)  and  349  (50.8%)  had   caries  experience  (dmft≥1).  the   mean  dmft  for  students  with  car-­‐ ies  experience  was  2.88±1.79  and   the  general  mean  dmft  was   1.46±1.92,  being  1.82±2.05  for   public  and  1.08±1.70  for  private   schools.   in  the  exploratory  study,  it   was  observed  an  association  of   caries  experience  with  the  varia-­‐ bles  i)  ethnic  group  (light-­‐  and   dark-­‐skinned  black),  no  flossing,   no  fluoride  use  (solution,  varnish,   gel),  public  dental  access,  lower   individual  socioeconomic  status,   higher  plaque  index,  presence  of   gingivitis  (level  1),  ii)  public  school,   permission  for  candy  or  gum  con-­‐ sumption  (level  2),  and  iii)  worse   living  conditions  in  the  district  so-­‐ cioeconomic  condition  (level  3).  3   presents  the  findings  of  a  multi-­‐ level  logistic  regression  with  the   variable  outcome   dmft=0⁄dmft≥1.     model  1  (null  model)   showed  that  the  variation  be-­‐ table&3&(continued).&multilevel(modeling(of(121yr1old(students,(considering(individual,(school(and(district(hierarchical(levels(in(relation(to(caries(experience( socioeconomic( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( dental&access& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( private((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( public( ( ( ( 0.003*( 1.91( (1.251 2.92)( 0.014*( 1.75( (1.12(–( 2.75)( 0.401( 1.26( (0.731 2.19)( 0.139( 1.47( (0.88(–( 2.46)( individual&socieconomic& status& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( a1/a2((ref)(highest( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( b1/b2( ( ( ( 0.002*( 1.92( (1.271 2.92)( 0.009*( 1.80( (1.16(–( 2.78)( 0.099( 1.54( (0.921 2.57)( 0.009*( 1.79( (1.15(–( 2.77)( ( c( ( ( ( 0.013*( 2.05( (1.171 3.61)( 0.038*( 1.88( (1.03(–( 3.41)( 0.045*( 2.13( (1.021 4.44)( 0.047*( 1.83( (1.01(–( 3.32)( ( d/e(lowest( ( ( ( 0.368( 1.50( (0.621 3.60)( 0.458( 0.40( (0.57(–( 3.45)( 0.155( 2.47( (0.711 8.59)( 0.402( 1.47( (0.60(–( 3.63)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( clinical( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( plaque&index&(modified)& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( 0((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( >0(and(<1( ( ( ( 0.151( 1.43( (0.881 2.34)( 0.130( 1.46( (0.89(–( 2.38)( 0.016*( 2.11( (1.151 3.88)( 0.008*( 2.21( (1.23(–( 3.96)( ( ≥1( ( ( ( 0.014*( 2.28( (1.181 4.40)( 0.011*( 2.35( (1.22(–( 4.56)( 0.000*( 4.42( (1.971 9.91)( 0.000*( 4.94( (2.36(–( 10.37)( gengivitis& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( no((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( yes( ( ( ( 0.482( 1.14( (0.791 1.65)( 0.442( 1.16( (0.80(–( 1.68)( 0.397( 1.18( (0.801 1.73)( ( ( ( fluorosis& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( no((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( yes( ( ( ( 0.554( 0.90( (0.621 1.29)( 0.565( 0.90( (0.62(–( 1.29)( 0.026*( 3.03( (1.141 8.01)( 0.019*( 3.12( (1.21(–( 8.08)( stimulated&salivary&flow& rate& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( >0.5(ml/min((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ≤0.5(ml/min( ( ( ( 0.732( 0.94( (0.641 1.37)( 0.729( 0.93( (0.64(–( 1.37)( 0.753( 0.94( (0.631 1.40)( ( ( ( salivary&buffering& capacity& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ph>3.9((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ph≤3.9( ( ( ( 0.484( 0.69( (0.251 1.94)( 0.543( 0.72( (0.25(–( 2.05)( 0.459( 0.65( (0.211 2.03)( ( ( ( mul$level  modeling  for  dental  caries  among  adolescents  in  a  brazilian  large  city     vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.58    http://dentistry3000.pitt.edu   7   tween  districts  (7.8%)  was  much   smaller  than  the  variation  at  the   school  level  (92.3%).  the  intraclass   correlation  coefficient  for  schools   was  0.073  and  for  districts  was   0.006.     in  model  2,  it  was  ob-­‐ served  that  socioeconomic  status   (class  b,  p=0.002  and  class  c,   p=0.013),  public  dental  access   (p=0.003),  and  high  pi  (p=0.014)   were  associated  with  caries  expe-­‐ rience.     in  model  3,  it  was  verified   that  the  characteristics  of  schools   and  districts  have  not  affected  car-­‐ ies  experience.  significant  findings   remained  similar  to  those  from   model  2.   in  model  4,  the  lack  of  as-­‐ sociation  between  community-­‐ level  variables  (schools  and  dis-­‐ tricts)  and  caries  experience  was   still  maintained.  socioeconomic   class  c  (p=0.045),  dental  plaque   accumulation  (regular  pi,  p=0.016   and  high  pi,  p=0.000),  and  fluoro-­‐ sis  (p=0.026)  were  significantly  as-­‐ sociated  with  caries  experience.   the  interaction  between  fluorosis   and  dental  biofilm  accumulation   was  negatively  associated  with   caries  experience  [regular  pi  (β=-­‐ 1.265,  p=0.020)  and  high  pi  (β=-­‐ 2.032,  p=0.003)].  in  addition,  the   interactions  between  no  flossing   and  use  of  public  dental  services   were  associated  with  caries  expe-­‐ rience  [β=+0.809;  p=0.007;   or=3.79;  95%  ci:1.45-­‐9.92]  (table   3).   all  these  findings  remained   significant  even  after  statistical   adjustment  in  the  final  model   (model  5).       discussion   multilevel  modeling  is  ap-­‐ propriate  for  analyzing  hierarchical   data  and  provides  an  efficient  way   to  link  the  conventionally  distinct   community  and  individual-­‐level   approaches  [21].    moreover,  it   could  avoid  a  loss  of  statistical   power  in  the  use  of  variables  from   different  levels,  and  the  risk  of   ecological  fallacy,  which  is  a  kind   of  error  when  only  the  population   level  is  considered  [22].     table&3&(continued).&multilevel(modeling(of(121yr1old(students,(considering(individual,(school(and(district(hierarchical(levels(in(relation(to(caries(experience( ! level!2:!school! ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( type&of&school& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( private((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( public( ( ( ( ( ( ( 0.894( 0.96( (0.54(–( 1.70)( 0.796( 0.92( (0.501 1.69)( 0.948( 1.02( (0.56(–( 1.85)( oral&health&education&& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( yes((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( no( ( ( ( ( ( ( 0.517( 0.85( (0.53(–( 1.38)( 0.850( 0.95( (0.561 1.61)( 0.676( 0.90( (0.54(–( 1.50)( permission&for&candy&or&gum& consumption&& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( non(permitted((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( permitted( ( ( ( ( ( ( 0.136( 1.51( (0.88(–( 2.61)( 0.193( 1.48( (0.821 2.68)( 0.228( 1.43( (0.80(–( 2.56)( ! level!3:!district& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( fluoride&concentration&in&water& supply& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ≥0.7(ml/l((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( <0.7(ml/l( ( ( ( ( ( ( 0.789( 1.06( (0.69(–( 1.64)( 0.609( 1.14( (0.691 1.89)( 0.624( 1.13( (0.69(–( 1.88)( socioeconomic&position& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( better(living(conditions((ref)( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( worse(living(conditions( ( ( ( ( ( ( 0.623( 1.14( (0.68(–( 1.89)( 0.575( 1.16( (0.691 1.97)( 0.562( 1.17( (0.69(–( 1.98)( & interactions& ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( male(sex(and(no(fluoride(use( ( ( ( ( ( ( ( ( ( 0.002*( ( ( 0.003*( ( ( pi(score(1(and(fluorosis( ( ( ( ( ( ( ( ( ( 0.020*( ( ( 0.018*( ( ( pi(score(2(and(fluorosis( ( ( ( ( ( ( ( ( ( 0.003*( ( ( 0.002*( ( ( class(b(and(no(flossing(use( ( ( ( ( ( ( ( ( ( 0.112( ( ( ( ( ( class(c(and(no(flossing(use( ( ( ( ( ( ( ( ( ( 0.826( ( ( ( ( ( classes(d/e(and(no(flossing(( ( ( ( ( ( ( ( ( ( 0.269( ( ( ( ( ( no(flossing(and(public(access( ( ( ( ( ( ( ( ( ( 0.007*( ( ( 0.028*( ( ( ( σ2(district((( ( 0.020( ( ( ( 0.075(( ( ( ( 0.020( ( ( ( 0.043( ( ( ( 0.045( ( ( σ2(school(( 0.238( ( ( 0( ( ( 0( ( ( 0( ( ( 0( ( ( ρ(district(( 0.006( ( ( 0.022( ( ( 0.006( ( ( 0.013( ( ( 0.013( ( ( ρ(school(( 0.073( ( ( 0.022( ( ( 0.006( ( ( 0.013( ( ( 0.013( ( ( * association with dental caries outcome. mul$level  modeling  for  dental  caries  among  adolescents  in  a  brazilian  large  city     vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.58    http://dentistry3000.pitt.edu   8   the  mean  dmft  in  the   study  population  was  1.46,  con-­‐ sidered  low  by  who  standards   and  similar  to  the  levels  observed   in  europe  [23],  where  the  dmft   indexes  among  12-­‐year-­‐old  chil-­‐ dren  range  from  1.2  to  2.6.  how-­‐ ever,  the  mean  dmft  for  students   with  caries  experience  was  2.88,   which  demonstrates  higher  needs   for  treatment  in  this  population,   evidencing  the  dental  caries  polar-­‐ ization  in  the  present  study.     regarding  variables  from   the  individual  level,  ethnicity   seems  to  be  less  relevant  than  so-­‐ cioeconomic  status  to  determine   caries  outcome  [24].  in  brazil,  eth-­‐ nic  differences  in  dental  health   may  be  further  due  to  socioeco-­‐ nomic  status  and  access  to  ser-­‐ vices  than  to  biological  back-­‐ ground  [25].   in  this  study,  male  sex  was   negatively  associated  with  caries   experience,  differently  from  the   findings  by  aida  et  al.  [7],  who  did   not  observe  any  association  be-­‐ tween  gender  and  dental  caries.   nevertheless,  our  results  corrobo-­‐ rate  the  study  by  antunes  et  al.   [3],  who  argued  that  an  early   chronology  of  permanent  tooth   eruption  in  women  might  deter-­‐ mine  the  higher  risk  to  tooth  de-­‐ cay  for  girls  among  brazilians.   higher  access  to  dental  care  by   girls  may  lead  to  an  increase  in   dmft  index,  especially  regarding   the  number  of  filled  teeth,  as  a   result  of  overtreatment.  the  in-­‐ teraction  between  male  sex  and   no  use  of  fluoride  was  positively   associated  with  caries  experience.   this  is  due  to  the  fact  that  boys,  in   general,  have  a  lower  care  index,   which  reflects  their  decreased   concern  about  health  and  aesthet-­‐ ics  [6].  in  relation  to  caries-­‐ inhibiting  effect  of  fluoride   sources,  cochrane  systematic  re-­‐ views  found  that  fluoride  rinses   are  responsible  for  26%,  fluoride   gel  for  28%,  and  fluoride  varnish   for  46%  of  the  reduction  in  the   dmfs  index  [26],  demonstrating   the  relevance  of  these  topical   methods.  lower  individual  socio-­‐ economic  status  was  associated   with  caries  experience.  indeed,   low  social  class  has  been  associat-­‐ ed  with  high  dmft  [27,12].  the   higher  socioeconomic  positions   have  shown  the  better  health  sta-­‐ tus  measurements.     the  present  study  also   showed  that  higher  degrees  of   plaque  index  were  positively  asso-­‐ ciated  with  caries  outcome.  caries   lesions  develop  where  oral  bio-­‐ films  are  allowed  to  mature  and   remain  on  teeth  for  long  periods   [9].     fluorosis  presence  was  also  asso-­‐ ciated  with  caries  experience,  in   accordance  to  other  studies  re-­‐ sults  [28,  29].  severe  fluorosis  has   been  reported  to  increase  suscep-­‐ tibility  to  dental  caries  by  hypo-­‐ mineralization  [30].  however,   there  has  been  some  controversy   on  whether  dental  fluorosis  in-­‐ creases,  decreases,  or  has  no  ef-­‐ fect  on  caries  experience  [31].   when  plaque  index  and  fluorosis   were  analyzed  in  interaction,  fluo-­‐ rosis  seemed  to  protect  against   caries  only  in  the  presence  of  den-­‐ tal  biofilm.  the  interaction  be-­‐ tween  fluorosis  and  dental  biofilm   with  caries  experience  had  never   been  carried  out  in  vivo.  one  hy-­‐ pothesis  is  that  fluorotic  teeth   could  release  some  fluoride  ions   to  the  dental  biofilm  in  a  low  ph   condition  which,  consequently,   reduce  demineralization  [32].  on   the  other  hand,  enamel  changes  in   experimental  teeth  with  signs  of   fluorosis  that  had  been  exposed  to   plaque  accumulation  were  exam-­‐ ined  in  vitro  and  specimens   showed  signs  of  surface  deminer-­‐ alization  [33].   other  significant  interac-­‐ tion  in  the  present  study  was   shown  between  no  flossing  and   use  of  public  dental  services,   which  means  an  increase  in  odds   of  caries  experience  if  these  two   socioeconomic-­‐dependent  varia-­‐ bles  act  together.  this  finding   points  at  a  role  of  health  services   on  improvement's  individual  be-­‐ havior,  which  has  also  been  re-­‐ ported  for  other  chronic  diseases   [33].  in  relation  to  school  level,  it   was  not  found  a  ssa  between  the   type  of  school  and  oral  health  ed-­‐ ucation  with  caries  experience.   the  type  of  school  has  been  con-­‐ sidered  a  risk  indicator  of  the  dis-­‐ ease  in  a  brazilian  study  [27]  and   not  in  another  one  [13].  however,   public  school  enrollment  was  de-­‐ terminant  in  having  one  or  more   untreated  decayed  permanent   teeth  [3].  the  lack  of  association   between  oral  health  education  in   schools  and  dental  caries  may  be   due  to  the  fact  that  programs  con-­‐ sidering  specific  individual  needs   are  similar  in  both  types  of  school.     with  regard  to  district  lev-­‐ el,  an  association  between  fluo-­‐ mul$level  modeling  for  dental  caries  among  adolescents  in  a  brazilian  large  city     vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.58    http://dentistry3000.pitt.edu   9   ride  concentration  in  water  supply   and  caries  experience  was  not   found.  curitiba  has  a  water  fluori-­‐ dation  scheme  which  covers  the   whole  population  for  decades.   this  homogeneity  in  the  fluoride   distribution  could  be  the  reason   for  the  lack  of  association  with   caries  experience.  besides,  the  6   evaluated  districts  present  a  ho-­‐ mogeneous  income  distribution,   which  might  contribute  for  the   lack  of  influence  in  caries  experi-­‐ ence.     the  results  from  a  few   multilevel  studies  on  dental  caries,   taking  into  account  two  levels,   have  been  reported.  neighbor-­‐ hood  aspects  influenced  oral   health  [34]  and  dental  caries  expe-­‐ rience  in  brazilians  [12].  moreo-­‐ ver,  contextual  and  individual  so-­‐ ciodemographic  characteristics   influenced  dental  caries  experi-­‐ ence  in  12-­‐year-­‐old  schoolchildren   from  brazil  when  using  a  conven-­‐ tional  multivariate  logistic  regres-­‐ sion  (1).  otherwise,  aida  [7]   showed  that  90.8%  of  variance  in   dmft  index  among  japanese  3-­‐ year-­‐old  children  occurred  at  the   individual  level  and  9.2%  of  the   variance  occurred  at  the  commu-­‐ nity  level.     the  main  findings  of  this   study  were  that  individual  varia-­‐ bles  had  a  highly  significant  asso-­‐ ciation  with  caries  experience   even  in  the  presence  of  school  and   district  levels  (contextual  varia-­‐ bles).     the  variables  which  de-­‐ termine  the  risk  indicators  for  car-­‐ ies  development  varies  significant-­‐ ly  for  different  groups.  the  moni-­‐ toring  of  contrasts  in  dental  health   outcomes  is  relevant  for  pro-­‐ gramming  socially  appropriate  in-­‐ terventions.  the  analysis  of  differ-­‐ ent  risk  indicators,  which  influence   dental  caries,  and  its  early  identifi-­‐ cation,  is  relevant  for  planning   suitable  interventions  for  target   population  groups  whom  could   present  higher  levels  of  disease,   re-­‐orienting  attention  on  dental   prevention  and  reducing  costs  in   dental  treatments  offered  by  the   health  systems.     overall  effective  health  in-­‐ terventions  might  be  more  effec-­‐ tive  in  that  part  of  a  population   which  concentrates  disease  [7].   on  the  other  hand,  groups  of  low-­‐ er  caries  risk  should  not  be  under-­‐ estimated,  considering  that  risk   indicators  may  not  be  easily  rec-­‐ ognized  [35].  maybe,  a  good  tool   to  preserve  equity  and  universality   principles  for  dental  caries  should   be  the  combination  of  both  polari-­‐ zation  and  global  attention  ap-­‐ proaches.           the  multilevel  analysis  al-­‐ lows  the  evaluation  of  different   risk  indicators  from  distinct  hierar-­‐ chical  levels,  which  makes  this  ap-­‐ proach  valuable  for  the  integration   of  variables  influencing  the  dis-­‐ ease  outcome,  especially  for  com-­‐ plex  diseases,  such  as  dental  car-­‐ ies.  it  was  observed  that  individual   factors,  such  as  female  sex,  the   interaction  of  male  sex  with  no   fluoride  use,  lower  socioeconomic   status,  dental  plaque  accumula-­‐ tion,  fluorosis,  and  the  integration   of  no  flossing  with  public  health   service  use,  even  in  the  presence   of  contextual  variables  (schools   and  districts),  were  associated   with  caries  experience  in  the  study   population.     acknowledgements   we  would  like  to  thank  the   municipal  health  secretary  of  cu-­‐ ritiba  for  their  cooperation  to  this   study.  this  work  was  based  on  a   thesis  submitted  by  the  first  au-­‐ thor  to  the  catholic  university  of   paraná  (pucpr),  brazil,  in  partial   fulfillment  of  the  requirements  for   the  doctor's  degree  in  health  sci-­‐ ences.  the  first  and  fourth  authors   were  supported  by  scholarship   from  the  brazilian  government  –   capes  (coordination  for  the  im-­‐ provement  of  higher  level  –  edu-­‐ cation  –  personnel).  we  would  al-­‐ so  like  to  thank  kamilla  gabriella   dos  santos  medeiros  and  carlos   alberto  cordeiro-­‐jr,  for  the  contri-­‐ bution  with  the  sample  collection.   references   1. brasil.  ministery  of  health.   oral  health  brasil  2010  (sb   brasil  2010).  national  oral   health  research:  main  re-­‐ sults.  2012.  available  at:   http://dab.saude.gov.br/c nsb/sbbrasil/  arquivos/   proje-­‐ je-­‐ to_sb2010_relatorio_final. pdf  (last  acess  12  february   2015).   2. decline,  polarization,  ine-­‐ quality  and  social  exclu-­‐ sion.  narvai  pc,  frazao  p,   roncalli  ag,  antunes  jl.   mul$level  modeling  for  dental  caries  among  adolescents  in  a  brazilian  large  city     vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.58    http://dentistry3000.pitt.edu   10   dental  caries  in  brazil:  rev   panam  salud  publica.  2006;   19:385-­‐93.  pmid:   16968593.   3. ethnic  disparities  in  the   prevalence  of  dental  caries   and  restorative  dental   treatment  in  brazilian  chil-­‐ dren.  antunes  jl,  pegoretti   t,  de  andrade  fp,  junquei-­‐ ra  sr,  frazao  p,  narvai  pc.   int  dent  j.  2003;  53:7-­‐12.   pmid:  12653333.   4. prevalence  and  polariza-­‐ tion  of  dental  caries  among   young,  healthy  adults:   cross-­‐sectional  epidemio-­‐ logical  study.  tanner  t1,   kämppi  a,  päkkilä  j,  pat-­‐ inen  p,  rosberg  j,  kar-­‐ jalainen  k.  acta  odontol   scand.  2013;  71(6):1436-­‐ 42.  doi:   10.3109/00016357.2013.7 67932.  epub  2013  apr  30.   pmid:  23627898.   5. factors  associated  with   dental  caries  experience   and  oral  health  status   among  new  south  wales   adolescents.  skinner  j1,   johnson  g,  blinkhorn  a,   byun  r.  aust  n  z  j  public   health.  2014:  oct;   38(5):485-­‐9.  doi:   10.1111/1753-­‐6405.12245.   epub  2014  aug  28.  pmid:   25169434.   6. the  relation  between  fami-­‐ ly  socioeconomic  trajecto-­‐ ries  from  childhood  to  ado-­‐ lescence  and  dental  caries   and  associated  oral  behav-­‐ iours.  peres  ma,  peres  kg,   de  barros  aj,  victora  cg.  j   epidemiol  community   health.  2007;  61:141-­‐5.   pmid:  17234873.   7. contributions  of  social  con-­‐ text  to  inequality  in  dental   caries:  a  multilevel  analysis   of  japanese  3-­‐year-­‐old   children.  aida  j,  ando  y,   oosaka  m,  niimi  k,  morita   m.  community  dent  oral   epidemiol.  2008;    36:149-­‐ 56.  pmid:  18333879.   8. risk  indicators  of  dental   caries  in  5-­‐year-­‐old  brazili-­‐ an  children.  cortellazzi  kl,   pereira  sm,  tagliaferro  ep,   tengan  c,  ambrosano  gm,   meneghim  mde  c,  et  al.   community  dent  health.   2008;  25:253-­‐6.  pmid:   21367823.   9. dental  caries.  selwitz  rh,   ismail  ai,  pitts  nb.  lancet.   2007;  369:51-­‐9.  pmid:   17208642.   10. area  characteristics,  indi-­‐ vidual-­‐level  socioeconomic   indicators,  and  smoking  in   young  adults:  the  coronary   artery  disease  risk  devel-­‐ opment  in  young  adults   study.  diez  roux  av,  mer-­‐ kin  ss,  hannan  p,  jacobs   dr,  kiefe  ci.  am  j  epidemi-­‐ ol.  2003;  157:315-­‐26.   pmid:  12578802.   11. measuring  inequalities  in   the  distribution  of  dental   caries.  antunes  jl,  narvai   pc,  nugent  zj.    community   dent  oral  epidemiol.  2004;   32:41-­‐8.  pmid:  14961839.   12. the  potential  impact  of   neighborhood  empower-­‐ ment  on  dental  caries   among  adolescents.  pat-­‐ tussi  mp,  hardy  r,  sheiham   a.  community  dent  oral   epidemiol.  2006;  34:344-­‐ 50.  pmid:  16948673.   13. women's  health:  beyond   reproductive  years.  vibha,   laskar  ar.  indian  j  public   health.  2011;  55(4):247-­‐51.   pmid:  22298132.   14. influence  of  initial  carious   lesions  on  oral  health   planning.  assaf  av,  tengan   c,  tagliaferro  eps,   meneghim  zp,  meneghim   mc,  pereira  ac,  ambro-­‐ sano  gmb.  braz  j  oral  sci.   2006;  5(19):1217-­‐1222.     15. who.  oral  health  survey.   basic  methods.  fourth  edi-­‐ tion.  geneva:  world  health   organization.  1997;66.   16. risk  assessment  in  dental   caries.  in:  risk  assessment   in  dentistry.  fejerskov  o,   manji  f.  bader  jd.  chapel   hill:  university  of  north  ca-­‐ rolina  dental  ecology.   1990;  215–217.     17. the  gingival  index,  the   plaque  index  and  the  re-­‐ tention  index  systems.  loe   h.  j  periodontol.  1967;  38   suppl:610-­‐6.  pmid:   5237684.   mul$level  modeling  for  dental  caries  among  adolescents  in  a  brazilian  large  city     vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.58    http://dentistry3000.pitt.edu   11   18. effects  of  antidepressants   and  benzodiazepines  on   stimulated  salivary  flow   rate  and  biochemistry   composition  of  the  saliva.   de  almeida  pdel  v,  gregio   am,  brancher  ja,  ignacio   sa,  machado  ma,  de  lima   aa,  et  al.  oral  surg  oral   med  oral  pathol  oral  radi-­‐ ol  endod.  2008;  106:58-­‐65.   pmid:  18329910.   19. clinical  investigations  of   the  salivary  buffering  ac-­‐ tion.  ericsson  y.  acta   odontol  scand.  1959;   17:133-­‐65.     20. social  inequalities  and  vac-­‐ cination  coverage:  utiliza-­‐ tion  of  household  surveys.   moraes  jcd,  ribeiro   mcsda.  braz  j  epidemiol.   2008;  11:113-­‐24.     21. introduction  to  multilevel   analysis.  hox  j:  multilevel   analysis,  techniques  and   applications.  hox  j.  mah-­‐ wah:  lawrence  erlbaum   associates.  2002;  1-­‐36.     22. the  social  determinants  of   oral  health:  new  ap-­‐ proaches  to  conceptualiz-­‐ ing  and  researching  com-­‐ plex  causal  networks.  new-­‐ ton  jt,  bower  ej.  commu-­‐ nity  dent  oral  epidemiol.   2005;  33:25-­‐34.  pmid:   15642044.   23. the  world  oral  health  re-­‐ port  2003:  continuous  im-­‐ provement  of  oral  health  in   the  21st  century-­‐-­‐the  ap-­‐ proach  of  the  who  global   oral  health  programme.   petersen  pe.  community   dent  oral  epidemiol.  2003  ;   31  suppl  1:3-­‐23.  pmid:   15015736.   24. inequalities  in  oral  health:   a  review  of  the  evidence   and  recommendations  for   action.  watt  r,  sheiham  a.   br  dent  j.  1999;  187:6-­‐12.   pmid:  10452185.   25. multilevel  assessment  of   determinants  of  dental  car-­‐ ies  experience  in  brazil.  an-­‐ tunes  jl,  peres  ma,  de   campos  mello  tr,  wald-­‐ man  ea.  community  dent   oral  epidemiol.  2006;   34:146-­‐52.  pmid:   16515679.   26. evidence-­‐based  effective-­‐ ness  of  topical  fluorides.   marinho  vc.  adv  dent  res.   2008;  20:3-­‐7.  pmid:   18694869.   27. factors  associated  with   dental  caries  in  brazilian   children:  a  multilevel  ap-­‐ proach.  martins  mt,  sar-­‐ denberg  f,  abreu  mh,  vale   mp,  paiva  sm,  pordeus  ia.   community  dent  oral  epi-­‐ demiol.  2014;  42:  289–299.   pmid:  24354434.   28. the  relationship  between   dental  caries  and  dental   fluorosis  in  areas  with   moderate-­‐  and  high-­‐ fluoride  drinking  water  in   ethiopia.  wondwossen  f,   astrom  an,  bjorvatn  k,   bardsen  a.  community   dent  oral  epidemiol.  2004;   32:337-­‐44.  pmid:   15341618.   29. prevalence  and  severity  of   dental  caries  in  adoles-­‐ cents  aged  12  and  15  living   in  communities  with  vari-­‐ ous  fluoride  concentra-­‐ tions.  pontigo-­‐loyola  ap,   medina-­‐solis  ce,  borges-­‐ yanez  sa,  patino-­‐marin  n,   islas-­‐marquez  a,  maupome   g.  j  public  health  dent.   2007;  67:8-­‐13.  pmid:   17436973.   30. caries  and  dental  fluorosis   in  a  western  saharan  popu-­‐ lation  of  refugee  children.   almerich-­‐silla  jm,  montiel-­‐ company  jm,  ruiz-­‐miravet   a.  eur  j  oral  sci.  2008;   116:512-­‐7.  pmid:   19049520.   31. dental  fluorosis  increases   caries  risk.  cunha-­‐cruz  j,   nadanovsky  p.  j  evid  based   dent  pract.  2005;  5:170-­‐1.   pmid:  26092033.   32. biochemical  composition   and  cariogenicity  of  dental   plaque  formed  in  the  pres-­‐ ence  of  sucrose  or  glucose   and  fructose.  cury  ja,   rebelo  ma,  del  bel  cury   aa,  derbyshire  mt,  tab-­‐ choury  cp.  caries  res.   2000;  34:491-­‐7.  pmid:   11093024.   33. a  light  and  scanning  elec-­‐ tron  microscopic  study  of   enamel  decalcification  in   children  living  in  a  water-­‐ fluoridated  area.  thylstrup   a,  boyar  rm,  holmen  l,   mul$level  modeling  for  dental  caries  among  adolescents  in  a  brazilian  large  city     vol  4,  no  1  (2016)        doi  10.5195/d3000.2016.58    http://dentistry3000.pitt.edu   12   bowden  gh.  j  dent  res.   1990;  69:1626-­‐33.  pmid:   2212207.   34. the  independent  contribu-­‐ tion  of  neighborhood  dis-­‐ advantage  and  individual-­‐ level  socioeconomic  posi-­‐ tion  to  self-­‐reported  oral   health:  a  multilevel  analy-­‐ sis.  turrell  g,  sanders  ae,   slade  gd,  spencer  aj,  mar-­‐ cenes  w.  community  dent   oral  epidemiol.  2007;   35:195-­‐206.  pmid:   17518966.   35. life  course  dental  caries   determinants  and  predic-­‐ tors  in  children  aged  12   years:  a  population-­‐based   birth  cohort.  peres  ma,   barros  aj,  peres  kg,  araujo   cl,  menezes  am.  commu-­‐ nity  dent  oral  epidemiol.   2009;  37:123-­‐33.  pmid:   19250294.   comparison of force decay pattern in orthodontic elastomeric chains and niti closed coil springs, affected by five different mouthwashes: an in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.158 http://dentistry3000.pitt.edu comparison of force decay pattern in orthodontic elastomeric chains and niti closed coil springs, affected by five different mouthwashes: an in vitro study amirhossein mirhashemi 1, niloofar habibi khameneh 2, keyvan shahpoorzadeh 3, atefe saffar shahroudi 4 1 associate professor, department of orthodontics, school of dentistry, tehran university of medical sciences, tehran, iran 2 dental student, school of dentistry, tehran university of medical sciences, tehran, iran 3dental student, school of dentistry, tehran university of medical sciences, tehran, iran 4 assistant professor, dental research center, dentistry research institute, and department of orthodontics, dental school, tehran university of medical sciences, tehran, iran abstract background: the aim of this study was to compare the force decay pattern of elastomeric chains and niti coil springs which were exposed to five different commercially available mouthwashes. methods: in this in vitro study, 60 pieces of elastomeric chain (ec) and 60 niti closed coil springs (cs) were divided into 6 groups. the specimens were exposed to one of these mouthwashes twice a day for 60 seconds: listerine, chlorhexidine, orthokin, persica, fluoride and artificial saliva as the control group. the elastomeric chains and niti springs were stretched so that they exert the initial force of 250 gr. their force was measured on the 1, 7, 14 and 28 following days by means of a digital gauge. results: elastomeric chains and coil springs had force decrease over time, but ec' force reduction was greater with the highest reduction rate in the first week. however, in the cs group, the force decrease wasn't statistically significant in the first two weeks. after 28 days in the control groups, 49.8% of the initial force was remained in ecs while the value was 93.3% for css. in comparison between mouthwashes, in ec groups, in all mouthwashes except persica, the remained force was statistically less than control group. conclusion: force degradation of elastomeric chains could be exacerbated by use of mouthwashes. about coil springs, force decay was also observed. however, it was not statistically significant. force reduction was detectable after four weeks of coil springs usage, but in ec groups, the greatest reduction was after the first week. keywords: force reduction; orthodontics; mouthwash; elastomeric chain; closed coil spring. citation: mirhashemi, ah, et al. (2021) comparison of force decay pattern in orthodontic elastomeric chains and niti closed coil springs, affected by five different mouthwashes: an in vitro study. dentistry 3000. 1:a001 doi:10.5195/d3000.2021.158 received: march 5, 2021 accepted: may 25, 2021 published: october 28, 2021 copyright: ©2021 mirhashemi a, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: a-shahroudi@tums.ac.ir introduction in orthodontic treatment different materials have been used as force delivery systems for space closure such as elastomeric chains and modules, and nickel titanium (niti) coil springs [1-3]. to obtain the most efficient orthodontic tooth movement, light continuous and constant forces are preferred [4,5]. elastomeric products are more frequently used force delivery system in orthodontics due to simplicity of handling, low chair time, low cost, patient comfort and being biocompatible [1,6,7]. however, these materials are affected by different factors such as oral environment, duration of force application and nutritional factors. they undergo deformation and thereby their force decreases which results in an http://dentistry3000.pitt.edu/ comparison of force decay pattern in orthodontic elastomeric chains and niti closed coil springs, affected by five different mouthwashes: an in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.158 http://dentistry3000.pitt.edu impairment in the rate of tooth movement and elongation of treatment process [6-9]. this deformation occurs due to structural changes in elastomers such as adjacent molecular chains slippage and molecular stretching [10,11]. the greatest degree of force loss was observed in the first 3 hours with a relatively steady force loss rate in the following days [11,12]. nickel-titanium coil springs has been also vastly used for force exertion in orthodontics due to its promise to deliver low constant forces [13]. this has been attributed to super-elasticity of nickel-titanium alloy which means that under an increasing strain its structure undergoes phase transformation (austenitic phase to martensitic phase) instead of permanent deformation. its forcedisplacement plots have a long plateau region and if the release of the strain begins within this region there is a mix of the two phases, and the material generally returns completely to the austenitic phase [14]. niti coil springs have also been found to lose some of their force over time and some studies have shown 8% to 20 % force loss in 4-12 weeks [15,16]. however, the amount of force decay was less than elastomeric chains [17,18]. it has been reported that niti coil springs force is also affected by environmental factors although to a lesser extent than elastomers [18,19]. one of the most important solutions that deserves investigation are mouthwashes since they are commonly prescribed for orthodontic patients. orthodontic patients are at a higher risk of plaque accumulation around their fixed appliances and subsequently greater risk of having cariogenic bacteria in their oral environment [20-25]. the effect of commonly used mouthwashes such as chlorhexidine [20], sodium fluoride [21], listerine, persica [22] and orthokin [19] on the force of elastic chains as well as their potential influential factors such as the ph of the mouth rinses [23], bleaching agents [24] and alcohol content [25] have been investigated. although many researchers investigated the effect of mouthwashes on force degradation of orthodontic elastomeric materials, very few studies have compered the effect of these chemicals on the force exertion of elastomeric chains and niti coil springs. accordingly, this study was done with the aim of comparing the force decay pattern of elastomeric chains and niti coil springs which were exposed to five different commercially available mouthwashes including listerine, chlorhexidine, orthokin, persica and fluoride, over time. methods in this experimental in vitro study, the effect of five different mouthwashes was investigated: 1. chlorhexidine 0.2% (behsa pharmaceutical company, arak, iran) containing 0.2 gr chlorhexidine gluconate in 100 ml solution. 2. sodium fluoride mouthwash 0.2% (behsa pharmaceutical company, arak, iran) containing 0.2 gr sodium fluoride in 100 ml solution. 3. persica mouthwash (poursina pharmaceutical company, tehran, iran) containing effective materials such as tooth plant, mint and yarrow. the most important organic and mineral materials of the drop include tannins, flavonoids, essence, calcium, fluoride and chloride. 4. total care zero listerine mouthwash (johnson and johnson, italy) and 5. orthokin alcohol free mouthwash (kin factory, spain). ten samples of niti coil springs and ten samples of elastomeric chains were allocated to each group of the mouthwashes. http://dentistry3000.pitt.edu/ comparison of force decay pattern in orthodontic elastomeric chains and niti closed coil springs, affected by five different mouthwashes: an in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.158 http://dentistry3000.pitt.edu the elastomeric chains which were clear and without intermodular link (american orthodontics) were cut to the initial length of 12 millimeter. additionally, two extra loops were remained on each side to avoid excessive force on terminal loops. this was done meticulously in order to avoid extended handling which may place stresses in the elastomers prior to testing. the applied niti specimens were superelastic 9 mm closed coil 150 g springs (gac international, bohemia, ny). a custom-made jig was designed with a series of pins which held the stretched specimens at a constant length of 25 mm (about twice their original length or 100% elongation). one hundred percent extension had been suggested by former authors and manufacturers for clinical use [12] and 25 mm was considered as an average distance between canine and first molar [18]. the device included vertical parallel rods arranged 25 mm apart from each other on a plexiglass plate. the rods were 1.2 mm in diameter and made of steel and covered by a layer of lac to prevent stains during the study. the coil springs were mounted on the vertical rods by stainless steel orthodontic wires. the wires were wrapped around themselves several times to prevent loosening and opening (figure 1a). the initial force of the specimens was measured using a digital force gauge (model: sf-50, germany) with newton and gr units and up to 1 gr accuracy (figure 1b) figure 1: a) specimens are held at a constant length of 25 mm on a custom-made jig. b) the force was measured using a digital force gauge. in order to simulate oral condition, all six jigs were immersed in artificial saliva and stored in an incubator at body temperature (37±1⁰c). one jig was considered as control group and the other 5 group was exposed to one of the aforementioned mouthwashes. the specimens were soaked in their intended mouthwashes twice a day for 60 seconds each time with 12-hour interval. following each immersion, the samples were washed for 10 seconds with distilled water before being transferred to the artificial saliva and incubator again. the force exerted by each group was measured by means of the explained digital force gauge, at 0, 1-day, 7-day, 14-day and 28-day intervals. to compare the effect of time and type of the mouthwashes on the force exerted by elastomeric chains and niti coil springs, threeway analysis of variance (anova) was performed. however, since none of the triple and double interactions were significant, each group was analyzed by means of one-way anova and post-hoc tukey's test. results in this study, the mean initial force for elastomeric chains at 25 mm extension was 249.8±0.6 gr and in niti coil spring groups the initial force was 150.2±0.3 gr. the force exerted by elastomeric chains and niti coil springs in different mouthwash groups at different time intervals are presented in table 1 and 2 respectively. the percentage of remaining force relative to the initial force is also given in all study groups. the effect of time on samples' force in both groups of ec and cs a steady decrease over time in the exerted force was observed. however, among coil springs, this force decay was more gradual and http://dentistry3000.pitt.edu/ comparison of force decay pattern in orthodontic elastomeric chains and niti closed coil springs, affected by five different mouthwashes: an in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.158 http://dentistry3000.pitt.edu totally less than elastomeric chains after 4 weeks. the elastomeric chains had the greatest force reduction over one week, followed by a more gradual force reduction in the next intervals. (table 1). in cs groups, there was not a significant difference between different time intervals (p>0.05). (table 2) table 1. mean force in gram and the remaining force percentage(numbers in parenthesis) in elastomeric chains in different times in different mouth rinse groups during the study mr day 0 day 1 day 7 day 14 day 28 control 249.4 (100) 155.0* (62.0) 133.2 (53.3) 131.2 (52.5) 124.4 (49.8) persica 249.4 (100) 150.0* (60.0) 132.4 (53.0) 130.4 (52.2) 117.6 (47.0) fluoride 250.8 (100) 146.8* (58.7) 112.6+ (45.0) 110.6+ (44.2) 101.6+ (40.6) chlorhexidine 249.8 (100) 147.6* (59.0) 121.2 (48.5) 120.8+ (48.3) 102.8*+ (41.1) orthokin 250.6 (100) 153.6* (61.4) 113.6+ (45.4) 111.6+ (44.6) 100.6+ (40.2) listerine 249.6 (100) 145.8*+ (58.3) 116.0*+ (46.4) 112.4+ (45.0) 100.2+ (40.1) mr: mouth rinse *: significantly different from the previous follow-p in the same group +: significantly different from the control group at the same follow-up time http://dentistry3000.pitt.edu/ comparison of force decay pattern in orthodontic elastomeric chains and niti closed coil springs, affected by five different mouthwashes: an in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.158 http://dentistry3000.pitt.edu the effect of mouthwashes on samples' force the results of statistical analysis showed that there was a statistically significant difference between some groups of mouthwashes in elastomeric chain samples on the first day. (p = 0.005). listerine showed the least table 2. mean force in gram and the remaining force percentage(numbers in parenthesis) in niti closed coil spring in different times in different mouth rinse groups mr day 0 day 1 day 7 day 14 day 28 control 150 (100) 149.5 (99.7) 146.1 (97.4) 141.3 (94.2) 140.0 (93.3) persica 150 (100) 146.8 (97.9) 137.9 (91.9) 139.5 (93.0) 130.9 (87.3) fluoride 150 (100) 151.6 (101.1) 146.7 (97.8) 134.4 (89.6) 132.5 (88.3) chlorhexidine 150 (100) 152.4 (101.6) 147.0 (98.0) 134.1 (89.4) 133.4 (88.9) orthokin 150 (100) 148.9 (98.5) 145.25 (96.8) 140.0 (93.3) 131.5 (88.3) listerine 150 (100) 147.8 (98.5) 148.6 (99.1) 144.6 (96.4) 132.4 (88.0) mr: mouth rinse *: significantly different from the previous follow-p in the same group +: significantly different from the control group at the same follow-up time http://dentistry3000.pitt.edu/ comparison of force decay pattern in orthodontic elastomeric chains and niti closed coil springs, affected by five different mouthwashes: an in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.158 http://dentistry3000.pitt.edu force among all groups. (table 1) in coil spring groups, there was no statistically significant difference between the different groups (p = 0.183). on the seventh day, all the ec groups showed a significant difference (p = 0.000) and the control group had a significantly higher force than all groups but the difference with persica and chlorhexidine was not significant (p>0.05). the lowest force on the seventh day belonged to the fluoride group, followed by orthokin, listerine, chlorhexidine, persica and control, respectively. in cs groups, on the seventh day there was no significant difference among groups (p = 0.24) but persica had the lowest force, followed by orthokin, control, fluoride, chlorhexidine, and listerine respectively. on the 14th day, there was a significant difference in the ec groups (p = 0.000) and the control group had statistically significant difference with all groups except persica. fluoride, orthokin and listerine were not significantly different. the lowest force on the 14th day belonged to the fluoride group (table 1). in cs groups on day 14, no significant different was observed between the mouth washes and control group (p>0.05) but listerine group had a significant difference with fluoride and chlorhexidine. the lowest force belonged to the chlorhexidine group. on the 28th day, there was a significant difference in the ec groups (p = 0.000) so that control group had statistically significant difference with all except persica group. other mouthwash groups were not different. in the control group about 49.8 % of the initial force was remained and in the listerin group about 60% of the initial force was lost. there were no statistically significant differences in the coil springs groups on the 28th day. however, the maximum force belonged to the control group which was 140 gr and 93.3% of the initial force was remained in this group. even in the lowest force group which was persica, 87.3 % of the initial force was remained. (table 2) discussion elastomeric chains and coil springs are common means of applying force in orthodontics. the amount of force applied to the teeth over time is important. in this study, we investigated the effect of persica, fluoride, chlorhexidine, orthokin and listerine mouthwashes on force reduction of elastomeric chains and coil springs. the present study showed that elastomeric chains lost a great amount of force overtime and this force loss was less for coil springs. it was also shown that force loss of elastomeric chains was exacerbated by mouthwashes. coil springs was affected by mouthwashes to a lesser extent. in elastomers, the greatest force reduction was related to orthokin and listerin, while in coil springs it was related to persica. the highest force reduction in elastomeric chains was also observed in the first week of use. in this study, the initial force of 250 grams for elastomers and 150 grams for coil springs was used. this initial force is similar to the force that is used in the clinic and consistent with previous studies. in the study of omidkhoda et al. in 2015 an initial force of 200 gram for elastomers was used [26]. in a 2018 study by patel et al., they used an initial force of 242 to http://dentistry3000.pitt.edu/ comparison of force decay pattern in orthodontic elastomeric chains and niti closed coil springs, affected by five different mouthwashes: an in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.158 http://dentistry3000.pitt.edu 304 grams for four types of elastomeric chains [7]. javanmardi et al., used an initial force of 200 g for elastomeric chains and a force of 180 g for coil springs [19]. it is stated in studies that for bodily movement of teeth an average of 100 to 350 gr power is needed [27] in 2014, kumar et al., examined the effect of various beverages, such as distilled water, cocacola, tea and listerine mouthwash on elastomeric chains' force. this study was performed in 28 days similar to the present study and samples were exposed to liquid of their target group twice a day for 60 seconds. but in their study, unlike the present study, an initial force of approximately 5 newton (500 g) was used. in that study, the control group showed a 22.18% decrease in force and the listerine group had a 23.15% force decay at the end of the study [28]. in the present study, the control group showed a 50.24% reduction in force and the listerine group caused a 55.92% force decay in elastomeric chains. the amount of force reduction was different in the two studies, but in both studies, listerine caused a greater decrease in elastomeric chains’ force compared to the control group. another difference between these two studies is that in the present study artificial saliva was used to hold the specimens but kumar et al. used distilled water throughout their study [28]. in 2016, javanmardi et al. investigated the effect of orthokin, sensikin and persica mouthwashes on the force reduction of elastomeric chains and ni-ti coil springs. they concluded that both types of specimen experience force decrease over time. in elastomeric chains, orthokin had less force decay than persica and in coil springs there was no significant difference between the different groups. in the present study, orthokin had the greatest force reduction. our study was performed in four weeks but the duration of their study was 3 weeks. moreover, the initial force of elastomers in their study, unlike the present study, was 200±5 g since the specimens were stretched 15.5 mm initially [19]. omidkhoda et al., examined the effect of persica, chlorhexidine and fluoride mouthwashes on the force reduction of elastomeric chains. the researchers concluded that chlorhexidine caused the greatest reduction in force while, persica caused the least reduction between the three mouthwashes [26]. these results are concurrent with the present study. however, there were some methodologic differences in the way that in the present study, the temperature of the samples was maintained at 37 ° c but in that study a temperature cycle of 5 to 55 ° were used. the initial force of this study was also about 200 gr. in a recent study by mirhashemi et al. (2020) the effect of different mouthwashes on active tiebacks was evaluated and it was concluded that persica mouthwash caused greater force loss in comparison with control group. they also reported that active tiebacks of different colors had a different force loss pattern and different response to chemical agents such as mouth rinses [29]. kardach et al., in a study in 2017, examined the force reduction of elastomeric chains in the two groups of plastic and memory. they concluded that plastic chains had a significant http://dentistry3000.pitt.edu/ comparison of force decay pattern in orthodontic elastomeric chains and niti closed coil springs, affected by five different mouthwashes: an in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.158 http://dentistry3000.pitt.edu force reduction after one week of use. (50% lost in primary force which is similar to the 49% reduction of the current study’s control group). the memory chains also showed a force reduction after one week which was less than plastic chains (20% lost in primary force) [30]. a review of literature showed that few studies have investigated the comprehensive effect of various mouthwashes used in the present study, and in few research two common and applicable orthodontic means of force exertion such as elastomeric chains and coil springs, have been compared. although it was concluded that coil springs undergo less force loss and they are less vulnerable to chemicals it should be considered that they are more expensive and they exert less initial force at the same amount of extension than elastomeric chains. however, in cases that light continues force is essential such as intrusion, single tooth movement or patients with compromised alveolar bone supports, niti coil springs could be a more prudent choice. moreover, in cases that application of mouth rinse is crucial, such as cases with vulnerable gingiva and periodontium, immunosuppressed patients, pregnant cases or post-surgical phases, application of niti coil springs which are less adversely affected by chemical agents such as mouthwashes is advisable. coil springs were most affected after 28 days while elastomers lost most force during the first week. the use of mouthwash in elastomers reduced the force more. but in coil springs, the use of mouthwashes did not significantly change the force reduction. because orthodontics patients are more likely to use mouthwash during treatment, and according to statistical results, if necessary, use of persica mouthwash is more recommended for these patients. in patients using coil springs there is no restriction for mouthwash use. according to the present study’s results which shows the gradual force reduction, it is recommended to replace elastomers after 2 week and coil springs at least once a month. this study examines the effect of mouthwashes in invitro condition and there is certainly a need for further clinical studies to achieve more reliable results. conclusion in general, both elastomeric chains and niti coil springs underwent force degradation over time which was significantly less in coil springs than chains. in control group after 4 weeks, 49% of initial force was preserved in elastomeric chains while in coil spring it was 93.3%. in elastomeric chains the highest force loss was observed in the first week while in niti coil springs the difference with the initial force was noticeable after four weeks. the force loss of elastomeric chains and niti coil springs could be exacerbated by use of some mouthwashes. however, this effect was not significant in niti coil springs. in elastomeric chains, all mouthwashes caused greater force loss than the control group but persica had the least force degradation in comparison with other mouthwashes including chlorhexidine, flouride, orthokin and listerin. http://dentistry3000.pitt.edu/ comparison of force decay pattern in orthodontic elastomeric chains and niti closed coil springs, affected by five different mouthwashes: an in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.158 http://dentistry3000.pitt.edu acknowledgments we would like to thanks all the collogues who helped us doing this research project. disclosure of interest the authors declare that they have no competing interest. authors' contributions ahm developed the idea of research and coordinated the project and he was the advisor professor. ass was the co-advisor professor and she did the scientific writing of the article. ks and nhk made the samples, conducted the experiments and did the measurements. references 1. a randomized clinical trial to compare three methods of orthodontic space closure. dixon v, read mj, o'brien kd, worthington hv, mandall na. journal of orthodontics. 2002;29(1):31-6. pmid: 11907307 2. a clinical investigation of force delivery systems for orthodontic space closure. nightingale c and jones sp. j orthod 2003;30: 229–236. pmid: 14530421 3. forces of various nickel titanium closed coil springs. maganzini al, wong am, ahmed mk. the angle orthodontist. 2010 jan;80(1):182-7. pmid: 19852659 4. tissue reaction to orthodontic tooth movement – a new paradigm. melsen b. eur j orthod 2001; 23: 671–681. pmid: 11890063 5. the application of continuous forces to orthodontics. burstone cj, baldwin jj and lawless dt. angle orthod 1961;31:1-14. https://doi.org/10.1043/00033219(1961)031<0001: taocft>2.0.co;2 6. comparative assessment of force decay of the elastomeric chain with the use of various mouth rinses in simulated oral environment: an in vitro study. menon vv, madhavan s, chacko t, gopalakrishnan s, jacob j, parayancode a. j pharm bioallied sci. 2019;11(suppl 2):s269-s73. pmid: 31198351 7. in vivo evaluation of the force degradation characteristics of four contemporarily used elastomeric chains over a period of 6 weeks. patel a, thomas b. j world fed orthod. 2018;7(4):141-5 https://doi.org/10.1016/j.ejwf. 2018.09.001 8. effect of disinfecting solutions on the mechanical properties of orthodontic elastomeric ligatures. evangelista mb, berzins dw, monaghan p. angle orthod 2007 jul;77(4):681-7. pmid: 17605480 9. the environmental influence of light coke, phosphoric acid, and citric acid on elastomeric chains.teixeira l, pereira bdo r, bortoly tg, brancher ja, tanaka om, guariza-filho o. j contemp dent pract. 2008 nov 1;9(7):1724. pmid: 18997912 10. a laboratory investigation of orthodontic elastomeric chains. rock wp, wilson hj, fisher se. br j orthod 1985 oct;12(4):202-7. pmid: 3863676 11. force degradation of orthodontic elastomeric chains-a product comparison study. de genova dc, mcinnes-ledoux p, weinberg r, shaye r. am j orthod 1985 may;87(5):377-84. pmid: 3857862 12. force-degradation pattern of six different orthodontic elastomeric chains. mirhashemi http://dentistry3000.pitt.edu/ comparison of force decay pattern in orthodontic elastomeric chains and niti closed coil springs, affected by five different mouthwashes: an in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.158 http://dentistry3000.pitt.edu a, saffarshahroudi a, sodagar a, atai m. j dent (tehran). 2012;9(4):204-15. pmid: 23323182 13. the super-elastic japanese niti alloy wire for use in orthodontics. part iii. studies on the japanese niti alloy coil springs. miura f, mogi m, ohura y, et al. am j orthod dentofac orthop 1988; 94: 89– 96. pmid: 3165245 14. thermal and mechanical characteristics of stainless steel, titanium-molybdenum, and nickel-titanium archwires. kusy rp and whitley jq. am j orthod dentofacial orthop 2007; 131: 229–237. pmid: 17276864 15. force degradation of closed coil: an in vitro evaluation. angolkar pv, arnold jv, nanda rs, et al. force degradation of closed coil: an in vitro evaluation. am j orthod dentofacial orthop 1992; 102: 127–133. pmid: 1636629 16. in-vivo force decay of nickel-titanium closed-coil springs. cox c, nguyen t, koroluk l, et al. am j orthod dentofacial orthop 2014; 145: 505–513. pmid: 24703289 17. an in vitro comparison of the force decay generated by different commercially available elastomeric chains and niti closed coil springs. santos ac, tortamano a, naccarato sr, et al. braz oral res 2007; 21: 51–57. pmid: 17384855 18. the effect of environmental factors on elastomeric chain and nickel titanium coil springs. nattrass c, ireland aj, sherriff m. eur j orthod 1998 apr;20(2):169-76. pmid: 9633170 19. effects of orthokin, sensikin and persica mouth rinses on the force degradation of elastic chains and niti coil springs. javanmardi z, salehi p. j dent res dent clin dent prospects. 2016;10(2):99-105. pmid: 27429726 20. does chlorhexidine in different formulations interfere with the force of orthodontic elastics? pithon mm, santana da, sousa kh, farias imao. angle orthod. 2012;83(2):3138. pmid: 22928936 21. effect of sodium fluoride mouth rinse on elastic properties of elastomeric chains. ramazanzadeh ba, jahanbin a, hasanzadeh n, eslami n. j clin pediatr dent. 2009;34(2):189-92. pmid: 20297715 22. effect of sodium fluoride mouth rinse on elastic properties of elastomeric chains. mirhashemi a, farahmand n, saffar shahroudi a, ahmad akhoundi ms. orthodontic waves. 2017;76(2):67-72. https://doi.org/10.1016/j.odw. 2016.11.007 23. the effect of ph levels on nonlatex vs latex interarch elastics. sauget ps, stewart kt, katona tr. the angle orthodontist. 2011;81(6):10704. pmid: 21609184 24. do mouthwashes with and without bleaching agents degrade the force of elastomeric chains? pithon mm, rodrigues ac, sousa élsm, de souza santos lp, dos santos soares n. the angle orthodontist. 2013;83(4):712-7. pmid: 23311601 25. the effects of varying alcohol concentrations commonly found in mouth rinses on the force decay of elastomeric chain. larrabee tm, liu ss-y, torres-gorena a, soto-rojas a, eckert gj, stewart kt.. the angle orthodontist. 2012;82(5):894-9 pmid: 22309124 26. evaluation of the effects of three different mouthwashes http://dentistry3000.pitt.edu/ comparison of force decay pattern in orthodontic elastomeric chains and niti closed coil springs, affected by five different mouthwashes: an in vitro study vol 9 no 1 (2021) doi 10.5195/d3000.2021.158 http://dentistry3000.pitt.edu on the force decay of orthodontic chains. omidkhoda m, rashed r, khodarahmi n. dental research journal. 2015;12(4):348-52. pmid: 26288625 27. a clinical investigation of the concepts of differential and optimal force in canine retraction. boester ch, johnston le. angle orthod. 1974;44(2):113-9. pmid: 4597626 28. effect of commonly used beverage, soft drink, and mouthwash on force delivered by elastomeric chain: a comparative in vitro study. kumar k, shetty s, krithika mj, cyriac b. j int oral health. 2014;6(3):7-10. pmid: 25083025 29. comparative evaluation of force decay pattern in orthodontic active. mirhashemi ah, shahroudi as, shahpoorzadeh k, khameneh nh. j dent res clin dent prospects. 2020;14(4):244-249. pmid: 33575015 30. the mechanical strength of orthodontic elastomeric memory chains and plastic chains: an in vitro study. kardach h, biedziak b, olszewska a, golusinskakardach e, sokalski j. adv clin exp med. 2017;26(3):373-8. pmid: 28791809 http://dentistry3000.pitt.edu/ microsoft word herrera proof dec 2.docx   vol  2,  no  1  (2014)   issn  2167-­‐8677  (online)   doi  10.5195/d3000/2014.25           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.   prevalence  of  dental  anomalies  in  a  mexican  population   jose  r.  herrera-­‐atoche1,  silvia  m.  diaz-­‐morales1,  gabriel  e.  colomé-­‐ruiz1,  mauricio  escoffié-­‐ramírez1,  maría  fernanda  orellana2.     1universidad  autónoma  de  yucatán,  faculty  of  den:stry,  orthodon:c  department   2university  of  california  san  francisco,  school  of  den6stry,  department  of  orofacial  sciences abstract   background:  data  on  dental  anomaly  prevalence  is  instrumental  to  diagnosis  and  treatment   in   different   popula+ons.   a   retrospec+ve   study   was   done   to   determine   dental   anomaly   prevalence   and   associa.ons   in   a   group   of   orthodon.c   pa.ents   in   a  mexican   popula.on.   methods:   number,   shape,   erup.on   and   structural   dental   anomalies   were   assessed   from   the  records  of  670  subjects.  prevalence,  distribu8on,  and  associa8ons  between  the  differ-­‐ ent  anomalies  were  calculated.  chi-­‐squared  and  fisher’s  exact  tests  (p<0.05)  were  used  to   iden%fy   significant   differences   by   sex,   and   to   establish   associa%ons   among   the   studied   anomalies.   results:   twenty-­‐eight   percent   of   the   sample   exhibited   at   least   one   dental   anomaly.  sta+s+cal  analysis  iden+fied  no  differences  by  gender.  the  most  common  anoma-­‐ ly   was   impacted   teeth   (13.58%),   followed   by   microdon=c   upper   lateral   incisors   (6.26%).   these   two   anomalies   also   had   the   highest   number   of   significant   associa6ons   with   other   anomalies.   conclusions:   the   dental   anomalies   prevalence   documented   here   differ   from   those   reported   in   the   literature   for  other  popula0ons   in   the  world.  dental   anomalies   are   normally  associated  with  each  other  and  occur   in  groups   linked   to  ethnic  origin.  the  pre-­‐ sent   results   indicate   the   presence   of   differing   suites   of   anomalies   between   the   studied   mexican  popula*on  and  other  popula*ons  in  the  world.  this  varia%on  highlights  the  need   for   further   research   on   dental   anomalies   in   la2n   america   to   aid   in   their   diagnosis   and   treatment.       cita%on:herrera-­‐atoche   jr,   diaz-­‐morales   sm,   colomé-­‐ruiz   ge,   escoffié-­‐ramírez   m,   and   orel-­‐ lana  mf.  (2014)  the  prevalence  of  dental  anom-­‐ alies   in   a   mexican   popula.on.   den.stry   3000.   1:a001  doi:10.5195/d3000.2014.25   received:  june  16,  2014   accepted:  november  24,  2014   published:  december  10,  2014   copyright:   ©2014   herrera-­‐atoche   et   al.   this   is   an  open  access  ar!cle  licensed  under  a  crea!ve   commons   a"ribu%on   work   4.0   united   states   license.   email:  jose.herrera@uady.mx       introduc)on   dental  anomalies  (da)  can  represent  a  chal-­‐ lenge   for   attaining   ideal   occlusion   and   es-­‐ thetics   in   dentition.   absence,   atypical   loca-­‐ tion   and   abnormal   tooth   shape   often   re-­‐ quire   interdisciplinary   treatment   involving   orthodontics,   surgery   and/or   oral   rehabili-­‐ tation.   prevalence   of   da   worldwide   varies   [1-­‐5],   probably   due   primarily   to   genetic   variation   between   ethnicities.   there   is   evi-­‐ dence  about  the  association  between  differ-­‐ ent   types   of   da   within   populations   of   the   same  ethnicity  [6-­‐10].  for  example,  subjects   with   unilateral   peg-­‐shaped   maxillary   per-­‐ manent   lateral   incisors   can   have   up   to   a   55%  probability  of  exhibiting  lateral  incisor   agenesis  on  the  contralateral  side  [11].  oth-­‐ er   examples   include   a   study   of   japanese   subjects   with   agenesis   of   one   or   two   per-­‐ manent   mandibular   lateral   incisors,   which   had  signi)icantly  increased  prevalence  rates   of   other   permanent   tooth   agenesis   and   symmetrical   dental   agenesis   [12],   and   a   relation   between   third   molar   agenesis   and   impacted   canines   in   a   portuguese   popula-­‐ tion  [13].   several  studies  have  addressed  da  in  differ-­‐ ent  populations.  in  latin  america,  there  are   a  number  of   studies   of   isolated  da,   but   re-­‐ ports   documenting   various   da   in   a   latino   population   are   rare.   one   example   is   a   re-­‐ ported  2.5%  da  prevalence  in  primary  den-­‐ tition   (2-­‐5   year-­‐old   children)   in   a  brazilian   population,   which   the   authors   stated       is   higher  than  in  other  populations  [14].  given   the  sample  age  range  and  the  small  number   of  studied  da,   it   is  probable  that  da  preva-­‐ lence   in   an   older   population   would   vary   more  broadly.   malocclusion   and   aesthetic   problems   are   only   some   of   the   problems   caused   by   da.   data   on   da   prevalence   and   associations   is   vital   to   diagnosis   of   dental   disorders   and   treatment   planning.   the   objective   of   the   present   study   was   to   document   da   preva-­‐ lence   and   association   in   orthodontic   pa-­‐ tients  in  a  mexican  population.     materials  and  methods   a  retrospective  study  was  done  of  a  sample   of   690   records   for   orthodontic   patients   ex-­‐ amined   by   three   calibrated   orthodontists.   records   were   from   the   orthodontic   clinic   archives   at   the   autonomous   university   of   yucatan  (universidad  autónoma  de  yucatan   –   uady),   merida,   yucatan,   mexico.   patient   age   ranged   from   9   to   20   years   of   age.   all   patients   were   of   mexican   origin.   subjects   with  a  history  of   trauma,  prior  orthodontic   treatment,  cleft   lip  and  palate  or  other  syn-­‐ dromes  were  excluded.  incomplete  or  inad-­‐ equate   records   lacking   quality   data   (e.g.   photographs,  dental  casts,  x-­‐rays,  etc.)  were   eliminated.   following   these   criteria,   20   pa-­‐ tients  were   eliminated   from   the   sample.  of   the   remaining   670   subjects,   65.38%   were   female  (n=438)  and  34.62%  male  (n=232)   a  total  of  12  da  were  assessed:      prevalence  of  dental  anomalies  in  a  mexican  popula5on   vol  2,  no  1  (2014)        doi  10.5195/d3000/2014.25    http://dentistry3000.pitt.edu   2   1.   dental   agenesis.   dental   agenesis  was   di-­‐ agnosed   when   “no   mineralization   of   tooth   crown   could   be   identi.ied   on   orthopan-­‐ tomograms  or  a  full-­‐mouth  set  of  periapical   radiographs,   and   no   evidence   of   its   having   been   extracted  was   found”   [15].   third  mo-­‐ lars  were  excluded.   2.  supernumerary  teeth  (snt).  a  tooth  was   considered   as   supernumerary  when   "it   ap-­‐ pears   in   addition   to   the   regular   number   of   teeth"  [2].     3.   microdontic   upper   lateral   incisors   (mu-­‐ li).  diagnosed  when  an  upper  lateral  incisor   presents   “mesio-­‐distal   width   equal   to   or   smaller  than  that  of  its  mandibular  counter-­‐ part”   [7].   the   measurements   were   made   with  a  mitutoyo  digital  caliper  (aurora,   illi-­‐ nois,  usa).   4.   peg-­‐shaped   upper   lateral   incisors   (psuli).  an  upper  lateral  incisor  was  classi-­‐ !ied   as   peg-­‐shaped   when   “mesio-­‐distal   width  was   greatest   at   the   cervical  margin”   [7].   5.   barrel-­‐shaped   upper   lateral   incisors   (bsuli).  an  upper  lateral  incisor  was  classi-­‐ !ied   as   barrel-­‐shaped   when   a   “pronounced   manifestation   of   a   thickened   or   elevated   cingulum  was   found   on   the   gingival   aspect   of  lingual  surfaces”  [16].   6.   upper   lateral   incisors   with   talon   cusp   (tc).  tc  was  de,ined  as  an  “accessory  cusp-­‐ like   structure   that   projects   from   the   cingu-­‐ lum  area  or  cementoenamel  junction”  [17].   7.   fusion.   this   was   diagnosed   “if   [tooth]   crown   and   root   were   enlarged   and   tooth   count  revealed  a  missing  tooth”  [18].   8.   gemination.   a   tooth   was   considered   to   have  gemination   if   its  “crown  was  enlarged   with  a  normal  root  and  the  tooth  count  was   normal”  [18].   9.   impacted   teeth   (it).  a   tooth  was  consid-­‐ ered  impacted  when  “it  was  not  expected  to   erupt   completely   into   its  normal   functional   position  based  on   clinical   and   radiographic   assessments”   [2].   third   molars   were   ex-­‐ cluded.   10.  transposition.  this  was  de2ined  as   “the   positional   interchange   of   two   adjacent   teeth,   or   the   development   or   eruption   of   a   tooth   in   a   position   normally   occupied   by   a   non-­‐adjacent  tooth”  [8].   11.  transmigration.  a  tooth  was  considered   to  be  in  a  transmigrated  position  when  “the   eruption   path   had   been   altered   and   the   tooth  had  drifted  to  the  opposite  side  of  the   arch  with   at   least   half   of   the   crown   length   crossing  the  midline”  [19].   12.   amelogenesis   imperfecta   (ai).   this   structural   anomaly   “represents   a   group   of   developmental   conditions,   genomic   in   origin,  which   affect   the   structure   and   clini-­‐ cal  appearance  of  enamel  of  all  or  nearly  all   the   teeth   in   a   more   or   less   equal   manner,   and  which  may  be  associated  with  morpho-­‐ logic   or   biochemical   changes   elsewhere   in   the   body”   [20].   no   subgroups   were   distin-­‐ guished  for  the  present  study.   statistical  analysis   once  the  da-­‐positive  population  was  identi-­‐ !ied,   da   prevalence   and   distribution   were   calculated.   a   chi-­‐squared   test   or   fisher’s   exact   test  were   used   to   identify   any   differ-­‐ ences   between   sexes   and   any   associations   between  the  different  da  (p  <0.05).     results   of  the  670  subjects,  28.05%  (n=188)  exhib-­‐ ited   at   least   one   dental   anomaly.   distribu-­‐ tion   by   gender   was   62.76%   (n=118)   in   fe-­‐ males  and  37.23%  (n=70)  in  males,  with  no   differences  (p>0.05)  in  frequency.  the  most   prevalent   da   was   it   (13.58%;   n=91),   fol-­‐ lowed  by  muli  (6.26%;  n=42)  (table  1).     number  of  anomalies   dental   agenesis   was   present   in   5.82%   (n=39)  of  the  subjects.  a  total  of  66  cases  of   absent  teeth  were  identi-ied,  47  (71.21%)  of   which   were   a   lower   tooth.   the   most   com-­‐ monly   absent   tooth   was   the   second   lower   premolar   (25.75%,   n=17)   followed   by   the   lower   lateral   (22.72%,  n=15).   in   the  maxil-­‐ lary   arch,   the  most   frequently   absent   teeth   were  the  lateral  incisors  (10.60%,  n=7)  and   the  second  molars  (10.60%,  n=7).  a  total  of   40   (5.97%)   snt   were   identi5ied,   33   of   which   were   in   the   upper   arch   (73.33%);   mesiodens   were   the   most   common   snt   (n=19).   shape  anomalies   the  most  frequent  shape  anomaly  was  mu-­‐ li   (6.26%;   n=42).   of   these   42   subjects,   18   had  bilateral   affectation,   raising   the  overall   muli   total   to   60.   a   total   of   29   (48.33%)   cases   were   on   the   right   side   and   31   (51.66%)   on   the   left.   ten   (1.49%)   psuli   cases  were  identi,ied;  ,ive  patients  present-­‐ ed   bilaterally,   producing   an   overall   total   of   15   psuli   teeth   (9   left/6   right).   prevalence   for   bsuli   was   2.83%   (n=19);   two   cases   were   bilateral,   for   a   total   of   21   affected   teeth   (11   left,   10   right).   upper   lateral   inci-­‐ sors  with  tc  were  found  in  7  (1.04%)  cases;   the  right  side  was  most  affected  (n=5),  and   no   bilateral   affectations   were   identi)ied.   only   two   male   patients   exhibited   fusion   (0.3%).   in   one   case,   the   lower   right   lateral   and  central  incisor  were  affected  and  in  the   other   the   lower   central   incisors   were   in-­‐ volved.   no   gemination   cases   were   present   in  the  sample.   eruption  anomalies   prevalence   for   it   was   13.58%   (n=91),   alt-­‐ hough   the   total   number   of   teeth   exhibiting   it  was  123:  96   in   the  upper  arch  (78.05%)   and   27   in   the   lower   (21.95%).   the   most   affected   tooth   was   the   upper   canine   (48.78%;  n=60),  followed  by  the  upper  cen-­‐ tral   incisor  and   the  upper  second  premolar   (13.01%;  n=16  in  both  cases).  transposition   was   noted   in   16   cases   (2.38%)   and   was   present  only  in  the  maxillary  arch.  bilateral   transposition  was  present  in  three  patients,   raising   the   total   to   19   teeth.   transposition   of   the   upper   canines   and   laterals   was   the   most  common  (10  cases,  .ive  per  side).  this   next   most   common   con*iguration   was   transposition   of   the   upper   canine  with   the   !irst   premolar   (8   cases,   four   per   side).   in   only   one   case,   the   upper   canine   had   trans-­‐ posed   with   the   second   premolar   on   the   right  side.   just  one  (0.14%)  subject  (female)  exhibited   transmigration;   in   this   case,   of   the   lower   right  canine.   structural  anomalies   three  female  subjects  exhibited  ai  (0.44%).   no   differences   (p>0.05)   by   gender   were   identi&ied   for   any   of   the   studied   da.   nine   signi%icant   (p<0.05)   associations   were   found  between  different  da:   it  with  dental   agenesis,   snt,   muli,   bsuli   and   transposi-­‐ tion;  muli  with  psuli,  bsuli  and  transposi-­‐ tion;   and   transposition   with   bsuli   (table   2).            prevalence  of  dental  anomalies  in  a  mexican  popula5on   vol  2,  no  1  (2014)        doi  10.5195/d3000/2014.25    http://dentistry3000.pitt.edu   3   discussion   overall   da   prevalence   in   the   sample   was   28.05%  (n=188).  prevalence  rates  reported   in   different   studies   range   from   5.46%   to   74.77%   [1-­‐5,21],   but   the   present   results   were   nearest   those   reported   for   a   popula-­‐ tion   in   egypt   [21].   this   high   variation   in   prevalence  values  may  be  due  to  differences   in   sample   sizes,   nature   of   the   sample   (or-­‐ thodontic,   dental,   or   general   population),   and  the  number  and  type  of  anomalies  stud-­‐ ied.   however,   exclusion   of   eruption   and   position   anomalies   substantially   reduces   these  prevalence  rates.   in   the  present  sam-­‐ ple,   no   difference   (p>0.05)   in   prevalence   was   present   between   genders.   the   most   prevalent   da   was   it   (13.58%;   n=91),   fol-­‐ lowed  by  muli  (6.26%;  n=42).   number  of  anomalies   dental   agenesis   prevalence   was   5.82%   (n=39),   within   the   2.7   to   13.3%   range   re-­‐ ported   worldwide   [1,3-­‐5,15,22-­‐25].   in   pre-­‐ vious  reports  on  mexican  populations,  2.7%   [22]   and   4.5%   [23]   prevalence   was   ob-­‐ served.  the  difference  between  the  present   results   and   these   studies   may   be   at   least   partially   due   to   genetic   variation   between   populations   in   different   regions   of   mexico   [26].  the  most   frequently  absent  tooth  was   the   lower   second   premolar,   which   concurs   with  other  reports  [1,15,25].   in  the  present   sample,  dental  agenesis  was  associated  with   it   (p=0.023),   an   association   reported   else-­‐ where   [9,10,24],   and   possibly   linked   to   shared  genetic  origins.   global   snt   prevalence   varies   from   0.3   to   6%   [1,2,4,27-­‐30].   the   prevalence   observed   in   the  present  sample   (5.97%)   is   similar   to   that  reported  in  an  african-­‐american  popu-­‐ lation  [27].  of  note  is  that  snt  prevalence  in   the  african-­‐american   population  was   high-­‐ er  in  the  molar  and  premolar  regions,  while   in   the   present   study   it   was   higher   in   the   maxillary   anterior   region,   which   is   similar   to  other   reports   [1,2,4,28-­‐30].   it   can  be  ex-­‐ pected   that,   since   snt   tends   to   block   the   eruption  path  of  other  teeth,   it  was  statisti-­‐ cally   associated  with   it   (p=0.008).   for   this   same  reason,   this  association  appears  to  be   more   related   to   the   eruption   path   and   not   necessarily  of  genetic  origin.   shape  anomalies   comparison   of   microdontic   teeth   between   studies  can  be  challenging  since  each  report   tends   to   quantify   them   differently;   some   include   just   the   upper   laterals   [6],   others   the   upper   and   lower   laterals   [4],   others   include  all  teeth  [2],  etc.  in  previous  reports,   muli   ranges   from   as   low   as   2.58%   to   the   6.26%  reported  in  the  present  study  [3,5,6].   associations  were  found  between  muli  and   four   other   da   in   the   present   study   (table   2).   the   associations   with   it   (p=0.0007)   coincide   with   previous   reports   [6,31],   as   does   that   with   dental   transposition   (p=0.002)  [32].   prevalence   for   psuli   was   1.49%,   which   is   within  the  0.37  to  9.9%  range  of  previously   reported   values   [1,5,33].   if   differences   due   to  ethnicity  are  excluded,  this  high  variabil-­‐ ity   may   be   heavily   in-luenced   by   sample   size;   indeed,   the   larger   the   sample   size   the   lower   the   prevalence.   as   expected,   psuli   was   associated   with   microdontia   (p=0.0001).   worldwide,   bsuli   is   normally   associated   with   east   asian   populations,   although   its   prevalence   in   this   study   was   higher   than   that  reported  for  a  sample  from  china  [16].   in   the   present   results,   prevalence   for   this   da  was  higher  than  other  upper  lateral  inci-­‐ sor   shape   anomalies.   this   is   interesting   because  peg-­‐shaped  or  tc  teeth  are  studied   more  frequently  than  barrel-­‐shaped  anoma-­‐ lies,  which  are  not  commonly  reported.  the   statistical   associations   observed   here   be-­‐ tween   bsuli   and   other   da,   such   as   it   (p=0.008)   and   transposition   (p=0.0001),   imply  genetic   causes.  as  with  psuli,  bsuli   was   associated   with   microdontia   (p=0.0001).  further  research  in  latin  amer-­‐ ican   populations   would   aid   in   supporting   these  associations  and  identifying  their  dis-­‐ tribution  in  the  region.   fused   teeth   were   present   in   only   0.3%   of   subjects,   a   prevalence   similar   to   those   re-­‐ ported   in   indian   (0.27%)   [5]   and   turkish   populations  (0.23%)  [4].   eruption  anomalies   the  13.58%  (n=91)  prevalence  for  it  in  the   present   study   made   it   the   most   prevalent   da   in   the   study.   this   prevalence   is   similar   to  that  reported  for  a  greek  population  [30],   and   at   the   high   end   of   the   0.49   to   13.7%   range   reported   in   the   literature   dental male female total p male female anomalies n=232 (34.62%) n=438 (65.38%) n=670 (100%) risk/ratio risk/ratio agenesis 14 (2.09) 25 (3.73) 39 (5.82) 0.863 1.06 0.95 snt 18 (2.69) 22 (3.28) 40 (5.97) 0.157 1.54 0.65 muli 16 (2.38) 26 (3.88) 42 (6.26) 0.625 1.16 0.86 psuli 4 (0.6) 6 (0.89) 10 (1.49) 0.719 1.26 0.79 bsuli 6 (0.89) 13 (1.94) 19 (2.83) 0.777 0.87 1.15 upper laterals with tc 5 (0.74) 2 (0.3) 7 (1.04) 0.062 4.72 0.21 fusion 2 (0.3) 0 (0.00) 2 (0.3) gemination 0 (0.00) 0 (0.00) 0 (0.00) it 31 (4.63) 60 (8.95) 91 (13.58) 0.903 0.96 1.03 transposition 7 (1.04) 9 (1.34) 16 (2.38) 0.439 1.49 0.68 transmigration 0 (0.00) 1 (0.14) 1 (0.14) ai 0 (0.00) 3 (0.44) 3 (0.44) table  1.  prevalence and distribution of dental anomalies by sex in a group of 670 patients.   abbreviations:abbreviations:  (stn)  supernumerary  teeth;  (muli)  microdontic  upper  lateral  incisors;  (psuli)  peg-­‐shaped  upper  lateral  incisors;  (bsuli)  barrel-­‐shaped   upper  la  uppe    lateral  incisors;  (tc)  talon  cusp;  (it)  impacted  teeth;  (ai)  amelogenesis  imperfecta        prevalence  of  dental  anomalies  in  a  mexican  popula5on   vol  2,  no  1  (2014)        doi  10.5195/d3000/2014.25    http://dentistry3000.pitt.edu   4   [1,2,5,19,30].  variations  can  be  explained  as   a   consequence   of   sample   nature,   subject   ethnicity,  and  sample  size.  despite  this  wide   value   range   among   studies,   all   the   reports   concur  in  that  the  upper  canine  is  the  most   frequently   impacted   tooth   [1,2,5,19,30].   as   mentioned  previously,  when  snt  is  present   impaction   is   an   apparent   consequence,   whereas  its  association  with  other  da  (mu-­‐ li,   bsuli,   transposition,   and   dental   agene-­‐ sis)   is   more   probably   of   genetic   origin   [6,7,10,31].     the   prevalence   rate   of   transposition   was   relatively   high   (2.38%)   compared   to   other   reports:  0.27%  [34],  0.33%  [35],  and  0.81%   [32].   however,   the   present   data   coincides   with  these  previous  studies   in   that   the  uni-­‐ lateral   presentation   is   the   most   common,   and   that   the  maxillary   arch   is   the  most   af-­‐ fected   [34-­‐36].  as  reported  elsewhere   [34],   all  transposition  cases  in  the  present  sample   involved   the   canine   teeth.   several   da   are   reported  to  be  associated  with  transposition   [32,34,36].  the  da  associated  with  transpo-­‐ sition   (muli,   bsuli,   it)   in   the   present   re-­‐ sults   suggest   a   genetic   origin   for   this   phe-­‐ nomenon.   only   one   female   patient   exhibited   transmi-­‐ gration   in   the   mandible.   this   is   consistent   with  a  report  that  the  most  common  occur-­‐ rence   of   canine   transmigration   is   in   the   lower  arch  in  women  [19].   structural  anomalies   the   0.44%   ai   prevalence   observed   in   the   present  data   is  very  near   the  0.43%  preva-­‐ lence  reported  for  a  turkish  population  [4],   and   only   slightly   higher   than   the   0.27%   prevalence   found   in   an   indian   population   [5].   the   da   prevalence   documented   in   this   or-­‐ thodontic  population  from  mexico  varied  in   many  respects  from  those  reported  for  oth-­‐ er  populations  in  the  world.  the  most  prev-­‐ alent  da  were   it  and  muli,   and   these  also   had   the  highest  number  of   signi1icant  asso-­‐ ciations   with   other   da.   given   that   dental   anomalies   are   primarily   of   genetic   origin,   this  prevalence  vary  widely  between  ethnic-­‐ ities.   little   da   data   is   available   for   latin   american   populations,   further   research   in   the   region   is   clearly  needed   to  advance   the   ability   of   dental   health   professionals   to   di-­‐ agnose  and  effectively  treat  these  disorders.     references   1.  prevalence  of  dental   anomalies   in  orthodontic   patients.  thongudomporn  u,  freer  tj.  aust  dent  j.   1998  dec;43(6):395-­‐8.  pmid:  9973708.   2.  prevalence  of  dental  anomalies  in  various  mal-­‐ occlusions.  uslu  o,  akcam  mo,  evirgen  s,  cebeci  i.   am   j   orthod   dentofacial   orthop.   2009   mar;135(3):328-­‐35.  pmid:  19268831.     3.  evaluation  of  the  prevalence  of  dental  anoma-­‐ lies  in  children  in  the  canton  of  sarajevo.  spahić-­‐ dizdarević   m,   deljo   e,   ganibegović-­‐selimović   m.   acta  stomatol  croat.  2011;45(1):24-­‐30.     4.   prevalence   and   distribution   of   dental   anoma-­‐ lies  in  orthodontic  patients.  altug-­‐atac  at,  erdem   d.   am   j   orthod   dentofacial   orthop.   2007   apr;131(4):510-­‐4.  pmid:  17418718.   5.   prevalence   and   distribution   of   selected   devel-­‐ opmental   dental   anomalies   in   an   indian   popula-­‐ tion.  gupta  sk,  saxena  p,   jain  s,   jain  d.  j  oral  sci.   2011;53(2):231-­‐8.  pmid:  21712629.     6.  a  controlled  study  of  associated  dental  anoma-­‐ lies.   baccetti   t.   angle   orthod.   1998   jun;68(3):267-­‐74.  pmid:  9622764.     7.   the   incidence   of   anomalous   maxillary   lateral   incisors  in  relation  to  palatally-­‐displaced  cuspids.   becker   a,   smith   p,   behar   r.   angle   orthod.   1981   jan;51(1):24-­‐9.  pmid:  6939351.       8.   maxillary   canine-­‐!irst   premolar   transposition,   associated   dental   anomalies   and   genetic   basis.   peck  l,  peck  s,  attia  y.  angle  orthod.  1993  sum-­‐ mer;63(2):99-­‐109.  pmid:  8498708.       9.  agenesis  of  maxillary  lateral  incisors  and  asso-­‐ ciated   dental   anomalies.   garib   dg,   alencar   bm,   lauris   jr,   baccetti   t.   am   j   orthod   dentofacial   orthop.   2010   jun;137(6):732.e1-­‐6.   pmid:   20685523.     10.   maxillary   palatal   ca-­‐ nine   impaction   displace-­‐ ment   in   subjects   with   congenitally  missing  max-­‐ illary  lateral  incisors.  al-­‐ nimri   ks,   bsoul   e.   am   j   orthod   dentofacial   or-­‐ thop.   2011   jul;140(1):81-­‐6.   pmid:   21724091.     11.   prevalence   of   peg-­‐ shaped   maxillary   per-­‐ manent   lateral   incisors:   a   meta-­‐analysis.   hua   f,   he  h,  ngan  p,  bouzid  w.   am  j  orthod  dentofacial   orthop.   2013   jul;144(1):97-­‐109.   pmid:  23810051.   12.   association   of   agen-­‐ esis   of   mandibular   lat-­‐ eral   incisors   with   other   dental   anomalies   in   a   japanese  population.  endo  s,  sanpei  s,  takakuwa   a,  takahashi  k,  endo  t.  j  dent  child  (chic).  2013   jan-­‐apr;80(1):9-­‐15.  pmid:  23595238.       13.  dental  anomalies  in  a  portuguese  population.   campoy  md,  gonzález-­‐allo  a,  moreira  j,  ustrell   j,   pinho   t.   int   orthod.   2013   jun;11(2):210-­‐20.   pmid:  23541047.   14.  dental  anomalies  and  associated  factors  in  2-­‐   to  5-­‐year-­‐old  brazilian  children.  kramer  pf,  feld-­‐ ens  ca,  ferreira  sh,  spiguel  mh,  feldens  eg.  int  j   paediatr   dent.   2008   nov;18(6):434-­‐40.   pmid:   18435724.   15.  a  survey  of  hypodontia  in  japanese  orthodon-­‐ tic  patients.   endo  t,  ozoe  r,  kubota  m,  akiyama   m,   shimooka   s.  am   j  orthod  dentofacial  orthop.   2006  jan;129(1):29-­‐35.  pmid:  16443475.   16.  incisal  morphology  of  southern  chinese.  ling   jyk,  wong  rwk.  open  anthropol  j.  2008;1:19-­‐25.     17.   talon   cusp-­‐clinical   signi)icance   and  manage-­‐ ment:   case   reports.   hattab   fn,   yassin   om,   al-­‐ nimri  ks.  quintessence   int.  1995  feb;26(2):115-­‐ 20.  pmid:  7568721.   18.   prevalence   of   fused   and   geminated   teeth   in   jordanian   adults.   hamasha   aa,   al-­‐khateeb   t.   quintessence   int.   2004   jul-­‐aug;35(7):556-­‐9.   pmid:  15259971.   19.   the   incidence   of   canine   transmigration   and   tooth   impaction   in   a   turkish   subpopulation.   ak-­‐ tan  am,  kara  s,  akgunlu  f,  malkoc  s.  eur  j  orthod.   2010  oct;32(5):575-­‐81.  pmid:  20237077.   20.  amelogenesis  imperfecta.  crawford  pj,  aldred   m,  bloch-­‐zupan  a.  orphanet  j  rare  dis.  2007  apr   4;2:17.  pmid:  17408482.   21.  prevalence  and  distribution  of  dental  anoma-­‐ lies   in  orthodontic  patients.  montasser  ma,  taha   m.   orthodontics   (chic).   2012;13(1):52-­‐9.   pmid:   22567616.   22.   radiographic   assessment   of   congenitally   missing   teeth   in  orthodontic  patients.  silva  meza   agenesis snt muli psuli bsuli talon cusp it snt 0.8191 muli 0.082 0.3154 psuli 0.5696 0.5879 0.0001* bsuli 0.0597 na 0.0001* na talon cusp 0.3363 na 0.379 na na it 0.0235* 0.0081* 0.0007* 0.5506 0.0086* 0.9564 transposition 0.9409 0.9619 0.002* na 0.0001* na 0.0003* ! table  2.  associa.ons  between  dental  anomalies  calculated  by  chi-­‐squared  and  fisher  exact  tests.   abbrevia(ons:   (stn)   supernumerary   teeth;   (muli)   microdon(c   upper   lateral   incisors;   (psuli)   peg-­‐shaped   upper   lateral   incisors;   (bsuli)  barrel-­‐shaped  upper  lateral  incisors;  (it)  impacted  teeth;  (ai)  amelogenesis  imperfecta  (*)  sta(s(cally  significant  (p<0.05).          prevalence  of  dental  anomalies  in  a  mexican  popula5on   vol  2,  no  1  (2014)        doi  10.5195/d3000/2014.25    http://dentistry3000.pitt.edu   5   r.   int   j   paediatr   dent.   2003   mar;13(2):112-­‐6.   pmid:  12605629.   23.   agenesis   in   permanent   dentition.   díaz-­‐pérez   r,   echaverry-­‐navarrete   ra.   rev   salud   pública   (bogota).   2009   dec;11(6):961-­‐9.   pmid:   20379669.   24.   prevalence   of   hypodontia   in   orthodontic   pa-­‐ tients  in  brasilia,  brazil.  gomes  rr,  da  fonseca  ja,   paula  lm,  faber  j,  acevedo  ac.  eur  j  orthod.  2010   jun;32(3):302-­‐6.  pmid:  19837747.   25.   survey   on   hypodontia   in   sayada,   tunisia.   maatouk   f,   baaziz   a,   ghnima   s,   masmoudi   f,   ghedira   h.   quintessence   int.   2008   mar;39(3):e115-­‐20.  pmid:  18618026.   26.   analysis   of   genomic   diversity   in   mexican   mestizo   populations   to   develop   genomic   medi-­‐ cine  in  mexico.  silva-­‐zolezzi  i,  hidalgo-­‐miranda  a,   estrada-­‐gil  j,  fernandez-­‐lopez  jc,  uribe-­‐figueroa   l,  contreras  a,  balam-­‐ortiz  e,  del  bosque-­‐plata  l,   velazquez-­‐fernandez  d,  lara  c,  goya  r,  hernan-­‐ dez-­‐lemus   e,   davila   c,   barrientos   e,   march   s,   jimenez-­‐sanchez  g.  proc  natl  acad  sci  usa.  2009   may  26;106(21):8611-­‐6.  pmid:  19433783.   27.   an   epidemiological   study   of   hyperdontia   in   american  blacks  and  whites.  harris  ef,  clark  ll.   angle   orthod   2008   may;78(3):460-­‐5.   pmid:   18416616.   28.   supernumerary   teeth   vary   depending   on   gender.  küchler  ec,  costa  ag,  costa  mde  c,  vieira   ar,   granjeiro   jm.   braz   oral   res.   2011   jan-­‐ feb;25(1):76-­‐9.  pmid:  21359454   29.  supernumerary  teeth  in  a  turkish  population.   esenlik   e,   sayin   mo,   atilla   ao,   ozen   t,   altun   c,   basak   f.   am   j   orthod   dentofacial   orthop.   2009   dec;136(6):848-­‐52.  pmid:  19962608.   30.   incidence   of   impacted   and   supernumerary   teeth-­‐a   radiographic   study   in   a   north   greek   population.  fardi  a,  kondylidou-­‐sidira  a,  bachour   z,   parisis   n,   tsirlis   a.   med   oral   patol   oral   cir   bucal.  2011  jan  1;16(1):e56-­‐61.  pmid:  20711166.   31.   the   palatally   displaced   canine   as   a   dental   anomaly  of   genetic   origin.   peck   s,   peck  l,  kataja   m.   angle   orthod.   1994;64(4):249-­‐56.   pmid:   7978519.       32.  a  retrospective  study  on  69  cases  of  maxillary   tooth  transposition.  cho  sy,  chu  v,  ki  y.  j  oral  sci.   2012;54(2):197-­‐203.  pmid:  22790413.   33.   variations   in   number   and   morphology   of   permanent   teeth   in  7-­‐year-­‐old   swedish   children.   bäckman  b,  wahlin  yb.  int   j  paediatr  dent.  2001   jan;11(1):11-­‐17.  pmid:  11309867.       34.   investigation  of   tooth  transposition   in  a  non-­‐ syndromic  turkish  anatolian  population:   charac-­‐ teristic  features  and  associated  dental  anomalies.   celikoglu  m,  miloglu   o,   oztek   o.  med   oral   patol   oral  cir  bucal.  2010  sep  1;15(5):e716-­‐20.  pmid:   20173710.   35.   prevalence   of   tooth   transposition.   a   meta-­‐ analysis.   papadopoulos   ma,   chatzoudi   m,   kaklamanos   eg.   angle   orthod.   2010   mar;80(2):275-­‐85.  pmid:  19905852.   36.  dental   transposition  as   a   disorder   of   genetic   origin.   ely   nj,   sherriff   m,   cobourne   mt.   eur   j   orthod  2006  apr;28(2):145-­‐51.  pmid:  16373452.   effect of potassium iodide and glutathione on color change and remineraliza:on poten:al induced by silver diamine fluoride applica:on vol 12 no 1 (2024) doi 10.5195/d3000.2024.402 h#p://den*stry3000.pi#.edu effect of potassium iodide and glutathione on color change and remineralization potential induced by silver diamine fluoride application salma mohammed elsabaa1, ahmed el banna1, dina ahmed el refai1 1biomaterials department, faculty of dentistry ain shams university abstract background: to evaluate the effect of applying potassium-iodide (ki) and glutathione (gsh) on silver-diamine fluoride (sdf) induced demineralized den:n discolora:on as well as their effect on its remineraliza:on poten:al. materials and methods: to examine color change, cervical den:nal demineralized cavi:es were performed mesially and distally in 16 human premolars. glass-ionomer restora:on (gic) was applied and allocated to four groups i-iv according to the following pre-treatments: no pre-treatment, sdf, sdf+ki, sdf+20% (wt) gsh. spectrophotometric evalua:on of samples at :me intervals: 1,7 and 14 days of gic applica:on. to examine the effect of remineraliza:on, 21 bovine den:n blocks were divided into groups i-iii: sdf, sdf + ki, and sdf + gsh. vickers microhardness was measured, before and azer demineraliza:on and azer 7 days of treatment. results: spectrophotometric results azer 14 days for groups i-iv were: 1.29±0.18, 12.24±0.19, 2.19±0.32 and 4.76±0.19 respec:vely. groups iii and iv showed significant reduc:on in δe compared with group ii, although they showed significant increase in δe compared with group i (p<0.001). ki showed beber management of color changes than gsh. the microhardness test results azer treatment applica:on to demineralized den:n for groups i-iii were: 30.81±20.87, 30.59±16.42, 24.69±13.21, respec:vely. all groups showed significantly increased microhardness of demineralized den:n (p≤0.05), which was comparable to that of group i. conclusion: applica:on of ki and gsh azer sdf significantly minimized color changes without affec:ng the remineralizing effect of sdf. keywords: silver diamine fluoride; remineraliza:on; discolora:on; potassium iodide; glutathione cita9on: elsabaa, sm et al. (2024) effect of potassium iodide and glutathione on color change and remineraliza9on poten9al induced by silver diamine fluoride applica9on den9stry 3000. 1:a001 doi:10.5195/d3000.2024.402 received: november 28, 2022 accepted: february 20, 2023 published: march 1, 2024 copyright: ©2024 elsabaa, sm et al. this is an open access ar9cle licensed under a crea9ve commons awribu9on work 4.0 united states license. email: salmamohammed@dent.asu.edu.eg introduction dental caries is one of the most prevalent health conditions worldwide, despite efforts to control it [1]. untreated dental caries lead to general health problems, including pain, poor quality of life, and reduced productivity [1,2]. cariology research currently focuses on the effectiveness of medical treatments for caries arrest and remineralization rather than the conventional invasive drilland-fill approach for caries management [3]. atraumatic restorative technique (art) is considered a minimal intervention method for caries management using hand instruments for carious tissue removal and restoring decayed tooth structure by high viscosity glass ionomer cement http://dentistry3000.pitt.edu/ effect of potassium iodide and glutathione on color change and remineraliza:on poten:al induced by silver diamine fluoride applica:on vol 12 no 1 (2024) doi 10.5195/d3000.2024.402 h#p://den*stry3000.pi#.edu restoration (hvgic) [4]. this technique showed less dental anxiety than the traditional drill-and-fill approach [5]. as a modification to this technique for a better prognosis, it was suggested to pre-treat the remaining carious tooth structure with sdf prior to hvgic restoration application. this modification was named silver modified atraumatic restorative technique (smart) [6,7]. as lately, sdf gained attention for arresting and remineralizing carious dental lesions [8]. in 2014, the fda approved sdf for caries arrest in both dentitions [9]. furthermore, no need to remove carious dental tissues before sdf application [10], which made its use simple, economical and patient-friendly [11]. previous studies have shown a promising effect of sdf in the control of carious dentin demineralization [12,13]. despite the advantages of sdf, yet it has limited desirability owing to its dark-staining effect [14]. even application of glass ionomer restoration couldn’t effectively mask this dark staining effect [15]. the purpose of this study was to reduce this discoloration without affecting the remineralization potential of the sdf. potassium iodide (ki) application after sdf was suggested, which resulted in a creamy yellowish-white silver iodide (agi) precipitate by binding to free silver ions from the sdf [16]. however, it was shown that the demineralized dentin microhardness was significantly decreased by reducing the silver ion count as a result of ki addition to sdf [17,18]. therefore, in this in vitro study, we tested glutathione (gsh), a relatively unpopular new material for discoloration management. glutathione (gsh) is an intracellular non-protein thiol (npsh) antioxidant that acts as a metal chelator, which may decrease free silver ion aggregation by coating its particles without affecting its count [19]. accordingly, this study aimed to evaluate the effect of applying ki and gsh on sdf induced tooth discoloration as well as their effect on sdf remineralization potential. material and methods color change spectrophotometric evaluation sixteen sound-extracted permanent human premolars were used in this study. premolars were collected from the oral and maxillofacial surgery department of the faculty of dentistry at ain shams university. the ain-shams university faculty of dentistry ethical committee approved the protocol for teeth collection and storage (approval no. recem 011910). the teeth were rinsed with deionized water and cleaned with a periodontal curette to remove any soft tissue debris. teeth with stains and extreme visual discoloration were excluded, such that teeth were visually with homogenous color. the teeth were then polished using a polishing cup and a pumice. standardized 2 mm deep square cavities of size 4 × 4 mm were made on the mesial and distal sides of each premolar at the cemento-enamel junction using tungsten carbide bur flat fissure (fg 557-prima dental-uk) and nsk handpiece high speed under copious amount of water. each premolar was completely covered with a clear nail varnish (yolo, a.r.e), except for the mesial and distal cavity windows, which were kept unprotected. additionally, the apical foramen was occluded externally with the varnish for no contamination. this resulted in 32 specimen, which were then distributed randomly into four experimental groups (n= 8) according to the treatment protocol (figure 1). each group had 8 samples (which means 4 teeth), such that the same treatment is applied to both sides of the tooth, avoiding contamination of variable treatments during storage. http://dentistry3000.pitt.edu/ effect of potassium iodide and glutathione on color change and remineraliza:on poten:al induced by silver diamine fluoride applica:on vol 12 no 1 (2024) doi 10.5195/d3000.2024.402 h#p://den*stry3000.pi#.edu each tooth, including two specimens (one on each side), was separately immersed in an acidic buffer solution for 72 h in a separate container at room temperature. the ph was adjusted by the addition of 1 m koh to maintain a ph of 4. the demineralizing solution was changed every 24 hours [20]. as according to the pilot study done, the demineralization depth was average 150 ± 15 µm measured at oral pathology department, faculty of dentistry, ain-shams university with polarized light microscope with magnification power x10 using a digital camera (eos 650d, canon, japan) which was mounted on a light microscope (bx60, olympus, japan). thirty-two specimens were allocated randomly to four treatment groups (n=8). in group i, the cavities of demineralized dentin were filled with gic restorations without pretreatment. while in group ii 38% sdf solution was applied for 1 minute with a micro-brush according to the manufacturer’s instructions, group iii was treated with the sdf solution as mentioned previously followed by immediate application of saturated ki solution until the creamy white precipitate became clear and in group iv demineralized dentin was treated by a mixture done by mixing 38% sdf solution with 20% (wt) gsh until the mixture became clear with no precipitates and then applied by a microbrush for one minute. then all cavities in groups ii, iii, iv were then washed with copious amounts of distilled water for 30s and dried with oil-free compressed air and filled with gic restoration (table 1). all previous work was done by a single experienced practitioner for standardization. then all the samples were stored individually in a deionized solution. color assessments of the specimens (n=32) were recorded at three-time intervals: baseline after 24 h of gic restoration application, 7 days, and then 14 days. color changes were recorded using a spectrophotometer (agilent cary 5000-usa) on the gic restoration surface at the national institute of standards (nis-egyptgiza). it was calibrated and used in the standard mode in the visible wavelength region (380nm to 780nm) for all samples. computed cie lab values were measured under illuminate d65 with a 10 degrees standard observer. the cie lab coordinates l* a* b*, hue and chroma of each sample at variable time interval was evaluated. then the δl, δa, δb between different time intervals of each sample was calculated and hence the δe color change between time intervals was measured. finally, the mean and standard deviation sd of each group was statistically evaluated. extracted sound premolars (n=16) cavity preparation (4×4×2mm) on mesial & distal side. teeth coverage with varnish except for mesial & distal window. cavities demineralization by immersion in acidic buffer ph.=4 for 72 hrs. group i (n=8) gic group ii (n=8) sdf+gic group iii (n=8) sdf+ki+gic group iv (n=8) sdf+20wt% gsh+gic spectrophotometric color assessment after 1,7,14 d figure 1. flow chart for experimental design of color change assessment http://dentistry3000.pitt.edu/ effect of potassium iodide and glutathione on color change and remineraliza:on poten:al induced by silver diamine fluoride applica:on vol 12 no 1 (2024) doi 10.5195/d3000.2024.402 h#p://den*stry3000.pi#.edu remineralization and microhardness evaluation bovine teeth were collected from cattle slaughtered in the foodmanufacturing industry. none of the animals were harmed to participate in this study. twenty-one bovine dentin samples (6×6×2 mm) were prepared from the labial and lingual surfaces of the cervical portion of incisor roots. the samples were then cut using a low-speed disc (komet, gebr. brasseler gmbh & co. kg, lemgo, germany), and a straight handpiece (s 11 lg, w&amp;h, bȕrmoos, austria) with copious amounts of coolant. specimens were embedded in acrylic resin (acrostone, cold cure, egypt), and the surfaces were successively ground flat using 150-1200 grit silicon carbide papers (english sic waterproof abrasive sandpaper) under running water. all previous work was done by a single experienced operator. first, the microhardness of sound specimens was measured. all samples were subjected to demineralization in the same manner as previously described. the microhardness of the specimens was measured after demineralization. the samples were randomly divided into three groups based on the treatment applied. group i received 38% sdf solution as the control group, group ii received sdf followed by ki solution, and group iii received a mixture of sdf with 20%(wt) gsh. all treatments were performed by a single experienced practitioner as described previously. each group was stored in a container of deionized water for one week. the surface microhardness was then re-measured for all the samples. microhardness measurements were performed at dental research centre (dr centre-egypt-cairo) using a vickers diamond microhardness tester (wilson 1102/1202 buehler micromet 6000 series, germany) in vickers hardness units (vhn). the microhardness measurements were taken at three different points 0.5 mm apart from the adjacent indentation at each dentin sample. each measurement was carried out using a 100 g (hv 0.1) load for a dwell time of 10 s. mean of three indentations made on each surface material composition manufacture demineralizing solution • 2.2 mm cacl2, 2.2 mm kh2po4, 50 mm acetic acid • ph 4 manufactured in pharmaceutical lab. 38% sdf solution (riva star) • (agnh2f) soluron contains 25% silver, 62% water, 5% fluoride, 8% amine • ph 10 sdi, bayswater, australia saturated potassium iodide (riva star) • contains 1 g of potassium iodide per millilitre. sdi, bayswater, australia glutathione gsh (sigma-aldrich) • l-glutathione reduced st. louis, mo, usa glass ionomer cement restoration (fuji™ ix gp fast capsule a3) • aluminofluorosilicate glass, polyacrylic acid, disrlled water, poly-carboxylic acid gc international, tokyo, japan table 1: list of materials used, chemical composition, and manufacturer. http://dentistry3000.pitt.edu/ effect of potassium iodide and glutathione on color change and remineraliza:on poten:al induced by silver diamine fluoride applica:on vol 12 no 1 (2024) doi 10.5195/d3000.2024.402 h#p://den*stry3000.pi#.edu for each dentine disc was used for statistical analysis. statistical analysis numerical data are presented as both, the mean and standard deviation (sd). kolmogorov-smirnov and shapiro-wilk tests were used to check the data distribution to assess the normality. all data presented a normal parametric distribution, such that they were analyzed using oneway anova followed by tukey’s post hoc test for intergroup comparisons. while repeated measures were analyzed using anova followed by bonferroni post hoc test for intragroup comparisons. the significance level was set at p ≤0.05. statistical analysis was done using the r statistical analysis software, version 4.1.3 [21]. cohen’s d was calculated and interpreted based on lakens’ formula and thresholds using the online resource available at https://effect-sizecalculator.herokuapp.com/ [22]. the r package mbess was used to calculate the confidence intervals (ci) for cohen’s d using the noncentral t method as in table 3&4, 6&7 [23]. results spectrophotometric results the results showed that the highest value of color change δe was found in sdf group ii, followed by sdf + gsh group iv, and sdf + ki group iii, while the lowest value was found in group i at all time intervals. there was a significant interaction between the time and group. post-hoc pairwise comparisons showed that the values of the different groups were significantly different from each other (p<0.001), as shown in table 2, 3, & 4. http://dentistry3000.pitt.edu/ https://effect-size-calculator.herokuapp.com/ https://effect-size-calculator.herokuapp.com/ effect of potassium iodide and glutathione on color change and remineraliza:on poten:al induced by silver diamine fluoride applica:on vol 12 no 1 (2024) doi 10.5195/d3000.2024.402 h#p://den*stry3000.pi#.edu interval first group second group cohen’s d 95% confidence interval magnitude lower upper baseline-1 week group (i) group (ii) -47.4 -67.85 -30.65 large group (i) group (iii) -1.51 -2.72 -0.44 large group (i) group (iv) -12.714 -18.5 -8.42 large group (ii) group (iii) 11.66 7.71 16.95 large group (ii) group (iv) 28.57 18.81 40.92 large group (iii) group (iv) -2.91 -4.55 -1.56 large 1-2 weeks group (i) group (ii) -23.83 -34.14 -15.92 large group (i) group (iii) -2.92 -4.56 -1.57 large group (i) group (iv) -12.23 -17.77 -8.1 large group (ii) group (iii) 16.99 11.32 24.38 large group (ii) group (iv) 14.44 9.59 20.74 large group (iii) group (iv) -5.04 -7.5 -3.13 large baseline-2 weeks group (i) group (ii) -55.94 -80.05 -36.17 large group (i) group (iii) -3.28 -5.05 -1.84 large group (i) group (iv) -17.73 -25.43 -11.81 large interval color change (δe) (mean ± sd) p-value group i group ii group iii group iv baseline-1 week 0.95±0.16db 7.64±0.10ab 1.73±0.67cb 3.24±0.18bb <0.001* 1-2 weeks 0.36±0.03dc 4.67±0.24ac 0.78±0.19cc 1.67±0.14bc <0.001* baseline-2 weeks 1.29±0.18da 12.24±0.19aa 2.19±0.32ca 4.76±0.19ba <0.001* p-value <0.001* <0.001* <0.001* <0.001* table 2: mean, standard deviation (sd) values of color change (δe) for different groups. different upper and lowercase superscript letters indicate a statistically significant difference within the same horizontal row and vertical column respectively*; significant (p ≤ 0.05) ns; non-significant (p>0.05) table 3: effect sizes (95% confidence intervals) of the intergroup comparisons of color change http://dentistry3000.pitt.edu/ effect of potassium iodide and glutathione on color change and remineraliza:on poten:al induced by silver diamine fluoride applica:on vol 12 no 1 (2024) doi 10.5195/d3000.2024.402 h#p://den*stry3000.pi#.edu group (ii) group (iii) 36.11 23.33 51.69 large group (ii) group (iv) 37.22 24.05 53.28 large group (iii) group (iv) -9.233 -13.46 -6.047 large group first interval second interval cohen’s d 95% confidence interval magnitude lower upper group (i) baseline-1 week 1-2 weeks 4.556 2.35 7.88 large baseline-1 week baseline-2 weeks -1.775 -3.214 -0.74 large 1-2 weeks baseline-2 weeks -6.407 -10.996 -3.416 large group (ii) baseline-1 week 1-2 weeks 14.36 7.893 24.076 large baseline-1 week baseline-2 weeks -26.932 -45.105 -14.423 large 1-2 weeks baseline-2 weeks -31.088 -52.058 -15.39 large group (iii) baseline-1 week 1-2 weeks 1.715 0.604 3.201 large baseline-1 week baseline-2 weeks -0.779 -1.742 0.035 medium 1-2 weeks baseline-2 weeks -4.763 -8.189 -2.52 large group (iv) baseline-1 week 1-2 weeks 8.655 4.726 14.538 large baseline-1 week baseline-2 weeks -7.301 -12.472 -3.969 large 1-2 weeks baseline-2 weeks -16.459 -27.58 -9.067 large microhardness tests as shown in table 5, 6, & 7, t1 showed no significant difference between the different groups (p>0.05), indicating the demineralization phase of all samples. on the other hand, at the t2 interval, the sdf + ki group ii showed a significantly higher value (p ≤ 0.05) than the sdf + gsh group iii, while no significant difference (p>0.05) was observed between the two groups and sdf group i. interval micro-hardness (mean±sd) p-value group i group ii group iii t0 53.66±5.22ba 57.06±10.34aba 60.29±7.73aa 0.034* t1 34.46±10.72ac 34.24±6.72ac 35.05±3.93ac 0.884ns t2 46.26±4.89abb 47.15±5.22ab 43.34±3.46bb 0.024* p-value <0.001* <0.001* <0.001* |d| < 0.2 "negligible", |d| < 0.5 "small", |d| < 0.8 "medium", otherwise "large". table 4: effect sizes (95% confidence intervals) of the intragroup comparisons of color change |d| < 0.2 "negligible", |d| < 0.5 "small", |d| < 0.8 "medium", otherwise "large". table 5: mean, standard deviation (sd) values of micro-hardness for different groups. different upper and lowercase superscript letters indicate a statistically significant difference within the same horizontal row and vertical column respectively*; significant (p ≤ 0.05) ns; non-significant (p>0.05) http://dentistry3000.pitt.edu/ effect of potassium iodide and glutathione on color change and remineraliza:on poten:al induced by silver diamine fluoride applica:on vol 12 no 1 (2024) doi 10.5195/d3000.2024.402 h#p://den*stry3000.pi#.edu group first interval second interval cohen’s d 95% confidence interval magnitude lower upper group (i) t0 t1 1.98 0.76 3.75 large t0 t2 1.27 0.34 2.53 large t1 t2 -1.23 -2.53 -0.25 large group (ii) t0 t1 2.28 0.96 4.23 large t0 t2 1.05 0.13 2.23 large t1 t2 -1.87 -3.53 -0.72 large group (iii) t0 t1 3.58 1.7 6.5 large t0 t2 2.46 1.05 4.58 large t1 t2 -1.95 -3.66 -0.78 large discussion the silver-modified atraumatic restorative technique (smart) offers an alternative minimal interventional treatment for caries management by arresting caries progression using sdf and restoring tooth form by hvgic [7,24,25]. however, one of the major drawbacks of the sdf treatment is its black staining effect on the carious tooth structure, which has limited its acceptance by aesthetically concerned patients and accordingly its general use [26,27]. this discoloration is due to the reaction of sdf with carious dentin resulting in the production of silver phosphate (ag3po4), silver chloride, and free metallic silver. ag3po4 is yellow when it is first formed but readily turns black under the influence of reducing agents or sunlight. this black impermeable layer prevents the progression of caries [12,13,28,29]. finding a solution for the dark staining without affecting sdf’s biological effect on carious dentin would provide a great opportunity for the universal use of sdf. potassium iodide was introduced commercially as a solution to reverse this discoloration but unfortunately controversies regarding its effect on the antibacterial and remineralization potential of the sdf [30-33]. introducing an alternative option for discoloration management was in need. for that reason, glutathione biomolecule was suggested as an alternative to reduce teeth discoloration without affecting the interval first group second group cohen’s d 95% confidence interval magnitude lower upper t0 group (i) group (ii) -0.39 -1.47 0.654 small group (i) group (iii) -0.94 -2.11 0.133 large group (ii) group (iii) -0.33 -1.404 0.71 small t1 group (i) group (ii) 0.02 -1.024 1.072 negligible group (i) group (iii) -0.07 -1.12 0.98 negligible group (ii) group (iii) -0.14 -1.19 0.905 negligible t2 group (i) group (ii) -0.16 -1.22 0.878 negligible group (i) group (iii) 0.65 -0.407 1.759 medium group (ii) group (iii) 0.805 -0.26 1.95 large table 6: effect sizes (95% confidence intervals) of the intergroup comparisons of microhardness. |d| < 0.2 "negligible", |d| < 0.5 "small", |d| < 0.8 "medium", otherwise "large". table 7: effect sizes (95% confidence intervals) of the intergroup comparisons of microhardness. |d| < 0.2 "negligible", |d| < 0.5 "small", |d| < 0.8 "medium", otherwise "large". http://dentistry3000.pitt.edu/ effect of potassium iodide and glutathione on color change and remineraliza:on poten:al induced by silver diamine fluoride applica:on vol 12 no 1 (2024) doi 10.5195/d3000.2024.402 h#p://den*stry3000.pi#.edu biological efficiency of the sdf [19]. there is limited evidence regarding efficiency of gsh compared to commercially available ki. accordingly, this study aimed to evaluate the efficiency of gsh compared to ki on remineralization potential and teeth discoloration induced by sdf treatment of demineralized dentine. high viscosity glass-ionomer restoration was used as simulation for clinical application of smart technique as it offers chemical bonding to dentine with good cavity seal, remineralizing effect, biocompatibility, ease of application, and low cost [34]. for standardization of tested variables, packable self-cure glass ionomer was applied to avoid any further discoloration that may be caused by the light curing; as the interaction products of both sdf and ki (ag3po4 and agi) are photosensitive [14,15]. cavities were done at the cemento-enamel junction for location standardization and for supporting sdf use in root caries arrest, as previously proposed in other studies due to its effectiveness and ease of application [35-39]. spectrophotometer was used for evaluation of the teeth discoloration as one of the most commonly used tools to assess the color change [40]. in group ii, the color change with time was the highest among all groups as shown in table 2. gic restoration had limited discoloration masking effect [15]. while group iii & iv showed significant reduction in color change relative to group ii at all time intervals (table 2). still, they had significant change of color with time. as in group iii, upon application of ki immediately after sdf, a bright yellow silver iodide (agi) compound was formed [31,32,41]. however, this yellow color soon turned dark and became darker with time. this could be justified by the amount of ki solution that wasn’t enough to interact with all the free silver ions, or may be attributed to the high photosensitivity of agi that readily dissociates into black metallic silver again by light exposure [27]. on the other hand, in group iv glutathione biomolecules reduced discoloration owing to its high adsorption affinity onto metallic surfaces which in turn might have decreased silver particle aggregation. additionally, gsh has a haemostatic effect which might have controlled the rate of silver ion release [42]. application of a mix of 20%(wt) gsh with sdf prior gic restoration in smart for color management was new [19]. in group i color change was visually undetectable, but it was significant along with time. this may be attributed to the degradation of the metal-polyacrylate bonds as an inherent property in gic restorations, causing lack of color stability [43]. accordingly, it was assumed that the percentage of gsh was not sufficient to completely coat all the excess silver particles to efficiently reduce the color change. pilot trials to dissolve more percentage of glutathione powder in 38% sdf solution resulted in mixture with precipitates. however, gsh ability to decrease the color changes of dentin may encourage further studies to better understand simpler ways of increasing the gsh concentration in sdf solutions. according to the results of the current study, ki is more effective than 20%(wt) gsh in reducing the color changes of demineralized dentin. this finding disagree with a pilot study that was done after aging and found no significant difference in color change between application of sdf only and adding ki to it prior to gic restoration [30]. regarding the microhardness results, bovine teeth were reported to resemble human teeth in multiple features including dentin surface properties [44]. it offered more homogeneity in respect to mineral composition compared with human teeth which is beneficial for invitro studies [45]. the base line of the demineralized dentine was standardized without any significant difference between the randomly distributed dentine discs. the different groups showed a significant increase in the microhardness of demineralized dentin, with no significant difference between the different treatments (table 5). this can be explained by an increase in the mineral content of dentin by extrafibrillar and intrafibrillar mineral formation by the deposition of spherical grains, which were http://dentistry3000.pitt.edu/ effect of potassium iodide and glutathione on color change and remineraliza:on poten:al induced by silver diamine fluoride applica:on vol 12 no 1 (2024) doi 10.5195/d3000.2024.402 h#p://den*stry3000.pi#.edu confirmed by sem investigations in a previous study [46]. our results supported the assumptions in previous studies that silver has no role in the sdf remineralizing effect [18]. this justifies that addition of ki to the sdf did not affect its remineralization properties. the increase in dentin surface hardness after the application of previous treatments was due to the recovery of nanomechanical properties of the demineralized dentin surface by crystal precipitation [47]. meanwhile, our results contrast those of other study that showed that ki application significantly reduced the hardness effect of the sdf compared with the unaccompanied sdf. in this latter study, micro-computed tomography (micro-ct) and fourier transform infrared (ftir) spectroscopy were used for indirect measurement of remineralization potential [31]. as an in-vitro study, there are some accompanied limitations, as the artificial demineralization, which was created by an acidic solution rather than combining the bacterial caries model as in real. this method is extensively used in research due to its simplicity and reproducibility [20]. in addition, we were focused mainly on the anti-demineralizing effects of sdf. moreover, the usage of deionized water as a storage medium between variable time intervals after treatment application, was another limitation as ionic concentrations were kept at unrealistic low levels than that of viable dentinal fluid [48], which may affect discoloration intensity by further interacting with sdf. in vitro studies are different from the more complex clinical situation. such that the results cannot be directly extrapolated to the in-vivo condition. another limitation was the inability to dissolve more than 20%(wt) of glutathione powder in sdf solution, whether using a magnetic stirrer or glass slab and spatula, since exceeding the 20%(wt) caused white precipitations in mixture. accordingly, in this in-vitro study we were concerned with managing the sdf induced discoloration, in a trial to motivate sdf application as a liner beneath final restoration, in deep carious lesions. conclusion within the limitations of this in-vitro study, 20%(wt) gsh is a promising solution, but with less efficiency than ki, for management of sdf induced teeth discoloration without compromising the remineralization potential. further studies with higher concentrations of gsh are recommended. references 1. kassebaum nj, smith agc, bernabé e, et al. global, regional, and na?onal prevalence, incidence, and disability-adjusted life years for oral condi?ons for 195 countries, 1990-2015: a systema?c analysis for the global burden of diseases, injuries, and risk factors. j dent res. 2017;96(4):380-387. doi:10.1177/0022034517693566 2. mar?ns mt, sardenberg f, bendo cb, et al. dental caries remains as the main oral condi?on with the greatest impact on children's quality of life. plos one. 2017;12(10):e0185365. published 2017 oct 5. doi:10.1371/journal.pone.01853 65 3. rickebs d, lamont t, innes np, kidd e, clarkson je. opera?ve caries management in adults and children. cochrane database syst rev. 2013;(3):cd003808. published 2013 mar 28. doi:10.1002/14651858.cd00380 8.pub3 4. leal s, bonifacio c, raggio d, frencken j. atrauma?c restora?ve treatment: restora?ve component. monogr oral sci. 2018;27:92-102. doi:10.1159/000487836 5. frencken je, leal sc, navarro mf. twenty-five-year atrauma?c restora?ve treatment (art) approach: a comprehensive overview. clin oral inves;g. 2012;16(5):1337-1346. doi:10.1007/s00784-012-0783-4 6. ballikaya e, ünverdi ge, cehreli zc. management of ini?al carious lesions of hypomineralized molars (mih) with silver diamine fluoride or silver-modified atrauma?c restora?ve treatment (smart): 1-year results of a prospec?ve, randomized clinical trial. clin oral inves;g. 2022;26(2):21972205. doi:10.1007/s00784-02104236-5 7. modasia, r., modasia, d. applica?on of silver diamine fluoride as part of the atrauma?c restora?ve technique . bdj student 28, 42– 43 (2021). http://dentistry3000.pitt.edu/ effect of potassium iodide and glutathione on color change and remineraliza:on poten:al induced by silver diamine fluoride applica:on vol 12 no 1 (2024) doi 10.5195/d3000.2024.402 h#p://den*stry3000.pi#.edu hbps://doi.org/10.1038/s41406021-0199-1 8. santos ve jr, vasconcelos filho a, targino ag, et al. a new "silver-bullet" to treat caries in children--nano silver fluoride: a randomised clinical trial. j dent. 2014;42(8):945-951. doi:10.1016/j.jdent.2014.05.017 9. contreras v, toro mj, elíasboneta ar, encarnación-burgos a. effec?veness of silver diamine fluoride in caries preven?on and arrest: a systema?c literature review. gen dent. 2017;65(3):22-29. 10. chu ch, lo ec, lin hc. effec?veness of silver diamine fluoride and sodium fluoride varnish in arres?ng den?n caries in chinese pre-school children. j dent res. 2002;81(11):767-770. doi:10.1177/0810767 11. johhnson b, serban n, griffin pm, tomar sl. projec?ng the economic impact of silver diamine fluoride on caries treatment expenditures and outcomes in young u.s. children. j public health dent. 2019;79(3):215-221. doi:10.1111/jphd.12312 12. mei ml, ito l, cao y, li ql, lo ec, chu ch. inhibitory effect of silver diamine fluoride on den?ne demineralisa?on and collagen degrada?on. j dent. 2013;41(9):809-817. doi:10.1016/j.jdent.2013.06.009 13. mei ml, ito l, cao y, lo ec, li ql, chu ch. an ex vivo study of arrested primary teeth caries with silver diamine fluoride therapy. j dent. 2014;42(4):395402. doi:10.1016/j.jdent.2013.12.007 14. duangthip d, fung mht, wong mcm, chu ch, lo ecm. adverse effects of silver diamine fluoride treatment among preschool children. j dent res. 2018;97(4):395-401. doi:10.1177/0022034517746678 15. hamdy d, giraki m, abd elaziz a, badran a, allam g, ruebermann s. laboratory evalua?on of the poten?al masking of color changes produced by silver diamine fluoride in primary molars. bmc oral health. 2021;21(1):337. published 2021 jul 9. doi:10.1186/s12903-02101697-8 16. patel j, anthonappa rp, king nm. evalua?on of the staining poten?al of silver diamine fluoride: in vitro [published online ahead of print, 2018 jul 4]. int j paediatr dent. 2018;10.1111/ipd.12401. doi:10.1111/ipd.12401 17. hamama hh, yiu ck, burrow mf. effect of silver diamine fluoride and potassium iodide on residual bacteria in den?nal tubules. aust dent j. 2015;60(1):80-87. doi:10.1111/adj.12276 18. sorkhdini p, gregory rl, crystal yo, tang q, lippert f. effec?veness of in vitro primary coronal caries preven?on with silver diamine fluoride chemical vs biofilm models. j dent. 2020;99:103418. doi:10.1016/j.jdent.2020.103418 19. sayed m, matsui n, hiraishi n, nikaido t, burrow mf, tagami j. effect of glutathione biomolecule on tooth discolora?on associated with silver diammine fluoride. int j mol sci. 2018;19(5):1322. published 2018 apr 29. doi:10.3390/ijms19051322 20. zhang x, neoh kg, lin cc, kishen a. remineraliza?on of par?ally demineralized den?ne substrate based on a biomime?c strategy. j mater sci mater med. 2012;23(3):733-742. doi:10.1007/s10856-012-4550-5 21. team, r. c. (2021). r: a language and environment for sta?s?cal compu?ng v. 3.6. 1 (r founda?on for sta?s?cal compu?ng, vienna, austria, 2019). retrieved from hbps://www.r-project.org 22. lakens, d. (2013). calcula?ng and repor?ng effect sizes to facilitate cumula?ve science: a prac?cal primer for t-tests and anovas. fron;ers in psychology, 4(863). hbps://doi.org/10.3389/ fpsyg.2013.00863 23. kelley, k. (2007). methods for the behavioral, educa?onal, and social sciences: an r package. behavior research methods, 39(4), 979– 984. hbps://doi.org/10.3758/bf 03192993 24. bridge g, martel as, lomazzi m. silver diamine fluoride: transforming community dental caries program. int dent j. 2021;71(6):458-461. doi:10.1016/j.identj.2020.12.017 25. natarajan d. silver modified atrauma?c restora?ve technique: a way towards "smart" pediatric den?stry during the covid-19 pandemic. front dent. 2022;19:12. published 2022 mar 12. doi:10.18502/fid.v19i12.9215 26. peng jj, botelho mg, ma?nlinna jp. silver compounds used in den?stry for caries management: a review. j dent. 2012;40(7):531-541. doi:10.1016/j.jdent.2012.03.009 27. garg s, sadr a, chan d. potassium iodide reversal of silver diamine fluoride staining: a case report. oper dent. 2019;44(3):221-226. doi:10.2341/17-266-s 28. mei ml, nudelman f, marzec b, et al. forma?on of http://dentistry3000.pitt.edu/ https://doi.org/10.3389/fpsyg.2013.00863 https://doi.org/10.3389/fpsyg.2013.00863 https://doi.org/10.3758/bf03192993 https://doi.org/10.3758/bf03192993 effect of potassium iodide and glutathione on color change and remineraliza:on poten:al induced by silver diamine fluoride applica:on vol 12 no 1 (2024) doi 10.5195/d3000.2024.402 h#p://den*stry3000.pi#.edu fluorohydroxyapa?te with silver diamine fluoride. j dent res. 2017;96(10):1122-1128. doi:10.1177/0022034517709738 29. lou yl, botelho mg, darvell bw. reac?on of silver diamine [corrected] fluoride with hydroxyapa?te and protein [published correc?on appears in j dent. 2012 jan;40(1):91-3]. j dent. 2011;39(9):612-618. doi:10.1016/j.jdent.2011.06.008 30. miller mb, lópez la, quock rl. silver diamine fluoride, potassium iodide, and esthe?c percep?on: an in vitro pilot study. am j dent. 2016;29(5):248-250. 31. zhao is, mei ml, burrow mf, lo ec, chu ch. effect of silver diamine fluoride and potassium iodide treatment on secondary caries preven?on and tooth discoloura?on in cervical glass ionomer cement restora?on. int j mol sci. 2017;18(2):340. published 2017 feb 6. doi:10.3390/ijms18020340 32. roberts a, bradley j, merkley s, pachal t, gopal jv, sharma d. does potassium iodide applica?on following silver diamine fluoride reduce staining of tooth? a systema?c review. aust dent j. 2020;65(2):109-117. doi:10.1111/adj.12743 33. nguyen v, neill c, felsenfeld dds j, primus phd c. potassium iodide. the solu?on to silver diamine fluoride discolora?on? adv dent & oral health.2017;5:1–6. doi:10.19080/adoh.2017.05.55 5655 34. lohbauer u. dental glass ionomer cements as permanent filling materials? —proper?es, limita?ons future trends. materials (basel). 2009;3(1):76-96. published 2009 dec 28. doi:10.3390/ma3010076 35. zhang w, mcgrath c, lo ec, li jy. silver diamine fluoride and educa?on to prevent and arrest root caries among communitydwelling elders. caries res. 2013;47(4):284-290. doi:10.1159/000346620 36. göstemeyer g, kohls a, paris s, schwendicke f. root caries preven?on via sodium fluoride, chlorhexidine and silver diamine fluoride in vitro. odontology. 2018;106(3):274-281. doi:10.1007/s10266-018-0341-x 37. tan hp, lo ec, dyson je, luo y, corbet ef. a randomized trial on root caries preven?on in elders. j dent res. 2010;89(10):1086-1090. doi:10.1177/0022034510375825 38. grandjean ml, maccarone nr, mckenna g, müller f, srinivasan m. silver diamine fluoride (sdf) in the management of root caries in elders: a systema?c review and meta-analysis. swiss dent j. 2021;131(5):417-424. 39. hiraishi n, sayed m, takahashi m, nikaido t, tagami j. clinical and primary evidence of silver diamine fluoride on root caries management. jpn dent sci rev. 2022;58:1-8. doi:10.1016/j.jdsr.2021.11.002 40. kim-pusateri s, brewer jd, davis el, wee ag. reliability and accuracy of four dental shadematching devices. j prosthet dent. 2009;101(3):193-199. doi:10.1016/s00223913(09)60028-7 41. patel j, anthonappa rp, king nm. evalua?on of the staining poten?al of silver diamine fluoride: in vitro [published online ahead of print, 2018 jul 4]. int j paediatr dent. 2018;10.1111/ipd.12401. doi:10.1111/ipd.12401 42. taglie{ a, diaz fernandez ya, amato e, et al. an?bacterial ac?vity of glutathione-coated silver nanopar?cles against gram posi?ve and gram nega?ve bacteria. langmuir. 2012;28(21):8140-8148. doi:10.1021/la3003838 43. hotwani k, thosar n, baliga s. compara?ve in vitro assessment of color stability of hybrid esthe?c restora?ve materials against various children's beverages. j conserv dent. 2014;17(1):70-74. doi:10.4103/0972-0707.124154 44. fonseca rb, haiter-neto f, carlo hl, et al. radiodensity and hardness of enamel and den?n of human and bovine teeth, varying bovine teeth age. arch oral biol. 2008;53(11):10231029. doi:10.1016/j.archoralbio.2008.0 6.012 45. ghisi ac, kopper pm, baldasso fe, et al. effect of superoxidized water and sodium hypochlorite, associated or not with edta, on organic and inorganic components of bovine root den?n. j endod. 2015;41(6):925930. doi:10.1016/j.joen.2015.01.039 46. banerjee a, frencken je, schwendicke f, innes npt. contemporary opera?ve caries management: consensus recommenda?ons on minimally invasive caries removal. br dent j. 2017;223(3):215-222. doi:10.1038/sj.bdj.2017.672 47. cai j, burrow mf, manton dj, tsuda y, sobh eg, palamara jea. effects of silver diamine fluoride/potassium iodide on ar?ficial root caries lesions with adjunc?ve applica?on of proanthocyanidin. acta biomater. 2019;88:491-502. doi:10.1016/j.actbio.2019.02.02 0 http://dentistry3000.pitt.edu/ http://dx.doi.org/10.19080/adoh.2017.05.555655 http://dx.doi.org/10.19080/adoh.2017.05.555655 effect of potassium iodide and glutathione on color change and remineraliza:on poten:al induced by silver diamine fluoride applica:on vol 12 no 1 (2024) doi 10.5195/d3000.2024.402 h#p://den*stry3000.pi#.edu 48. shellis rp. effects of a supersaturated pulpal fluid on the forma?on of caries-like lesions on the roots of human teeth. caries res. 1994;28(1):1420. doi:10.1159/000261614 http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study avita rath1, melissa wong li zheng1, claudio mendes pannuti2 1faculty of dentistry, segi university, malaysia 2departament of stomatology, university of são paulo, brazil abstract objectives: this study aimed to evaluate how quarantine affected final-year dental students' selfperceptions of preparation and assess how online training affected clinical students' education at segi university. methods: year 3 to 5 students (n=140) were asked to fill up an online questionnaire. the first part included the effects of online education experience between the academic years. the second section assessed the graduating class's self-perceived readiness in cognitive, communication and professional abilities. the chi-square test was used to analyse the association between the groups regarding academic years, gender, and family income. results: year 3 students missed educational experiences during lockdown significantly more than years 4 and 5 (p<0.001). in addition, 86% of year 5 students (p< 0.001) felt that online assessment was not a suitable evaluation method compared to the other clinical years. about two-thirds of the 5th year dental students were unsure of their confidence in their skills before graduation. around half of the final-year students said they were unsure about starting their practice following graduation. after graduation, 80% of respondents preferred to spend a year in residence with sufficient training. conclusions: although students' selfperceived preparation was generally positive, they expressed reservations about the independent practice after graduation keywords: dental education; covid-19; undergraduate; online; malaysia; educational technology citation: rath a, et al. (2022) impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a crosssectional study dentistry 3000. 1:a001 doi:10.5195/d3000.2022.351 received: may 23, 2022 accepted: june 23, 2022 published: december 14, 2022 copyright: ©2021 rath a, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: drrathavita@yahoo.com introduction the covid-19 pandemic shook the world in early 2020, bringing it to a near-standstill [1]. to curb the spread of infection, most countries imposed drastic social distancing measures, such as stay-at-home restrictions, travel bans, shutting off offices and educational institutions [2-3]. albeit the closing of educational sectors for the safety of students and academicians proved to be effective, and it led to significant challenges for everyone involved [4]. one of the most challenging problems, particularly for professional courses like dentistry, has been eliminating direct patient care, an essential part of the dental curriculum. although there are some procedural variances in curricula and methodology between numerous dental schools around the world, didactic lectures and clinical training remain a crucial component [5]. many dental schools across the world incorporated problem-based learning, case-based learning, and community-based clinical activities. however, due to lockdowns and directions from national, regional and local officials to prevent the transmission of covid-19 while examining adjustments in dental education curricula, most dental schools initially sent students home [6-9]. dental schools responded to covid-19 by introducing alternative forms of education and evaluation, such as live and pre-recorded online lecturers, virtual problem-solving activities and computer-based reports and exams, which were previously thought to be insufficient to compensate for the loss of clinical training exposure [10-11]. http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu as of 1 april 2020, there were 1.598 billion students from 194 countries who were forced to stay at home owing to the shutdown of their educational institutions on all levels [12]. technology served as a boon during these times, but it did involve a learning curve for both students and academicians. it was vital to find a balance between synchronous and asynchronous online teaching and learning in an emergency like the pandemic, to communicate distance education etiquette and traditions to students while being adaptable enough to deviate from the intended material delivery schedule [13]. this situation was no different in context to malaysian dental universities students. many students and educators had to adapt to a drastic transition to distant online teaching and learning (e-learning) to ensure students were not left idle during this pandemic lockdown. due to constant change of directives by the government, as a result of spikes of infections, lockdowns were implemented, drastically affecting the learning time of clinical year students [14]. segi university has begun to combine e-learning and blended learning into specialized courses, similar to other universities. in this way, several disciplines looked to work well in this manner. students in areas where patient care is a fundamental requirement, on the other hand, must be present in clinics to provide patient care while honing their clinical skills. even if new technologies like simulation and virtual reality become more popular, they will never be able to replace direct in-person interaction. curriculum design, training model, teaching methods and the general educational environment all have an impact on the goal of generating competent dental students. many research has looked at the efficiency of online learning since the emergence of covid-19. during the covid-19 pandemic, a great deal of research found that online learning was beneficial. secondary students may benefit from online, indoor, and desk-based learning, which will allow them to learn efficiently and continuously throughout the covid19 pandemic lockdown. most studies have reported positive online learning effectiveness during the covid-19 pandemic [15]. given the advantages and limitations of online learning, which is extensively employed during this tough covid-19 pandemic period, it is important to investigate its efficacy. nonetheless, we recognise that our graduating students’ overall competence and confidence may be reflected in their current lack of adequate undergraduate preparation. the purpose of this cross-sectional survey was to monitor how the student’s intellectual abilities and future ambitions were affected by the abbreviated and modified scholastic year. the first objective was to investigate the effectiveness of online learning from the perspective of students, as well as to determine the most influenced qualities, whether clinical, cognitive or behavioral. the second objective was to assess students’ self-perceptions of their readiness in terms of cognition, communication, and professional competencies. materials and methods the study design is a cross-sectional questionnaire-based study that uses a self-administered questionnaire to evaluate the different experiences and perceptions regarding distant online learning due to the covid-19 pandemic among clinical dental students in segi university, malaysia. the study was conducted between february 2021 and may 2021. the study included n=140; year 3 to year 5 undergraduates from the faculty of dentistry, segi university. prior to the start of the research, the institutes’ review board granted ethical approval (no.segiec/str/fod/32/2020-2021). the questionnaire briefly explained the objectives of the survey, and the students were informed that their participation was completely voluntary and anonymous. submission of the questionnaire, on the other hand, implied consent. the questionnaire was adapted from hattar et al [16]. it was developed in google form (google inc., mountain view, ca, usa) and was electronically disseminated for data collection. the questionnaire comprised two main parts. the first part comprised background and demographic details of the participants, such as the age, gender, current academic year, race http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu and family income. in addition, family income was dichotomized into 2 categories such as high (> rm 5000 per month) and low (≤ rm 5000 per month). the second part addressed two subsections. the first subsection, which was distributed to third, fourth and final year students aimed to look into students’ opinions toward online learning. this segment consisted of 9close ended questions about their educational experience with elearning, including the systems’ efficacy, level of involvement, collaboration with colleagues, the system’s value compared to traditional techniques, and the assessment methods used. the last two questions asked about unpleasant experiences with various types of learning, and identified which dentistry disciplines were considered negatively impacted by the pandemic. this sections’ responses were rated on a four-point likert scale (strongly agree, agree, disagree, strongly disagree). the second section investigated how prepared students thought they were for a career in dentistry. only the graduating students of 2021 (5th year, n=50) received this part of the questionnaire. it was divided into two main categories. the first category included 17 items drawn from a previously validated scale that examines a wide variety of skills and qualities required of dental students upon graduation [16]. there were 3 categories for these 17 indications of cognition, communication, and professional skills: “no experience”, which received a score of zero; “mostly”, which received a score of one; and “always”, which received a score of two. thus, the self-perceived preparation measure had a total score range of 0 to 34. the second category comprises of 4closed ended questions with the options of “yes”, “no”, and “unsure” that focused on students’ perceived readiness for a dental practice in terms of confidence in skills acquired prior to graduation, preference for being monitored after graduation, readiness for independent practice, and preference for a well-structured year of residency. for internal consistency and clarity of the questionnaire, pre-test analysis was done on 10 randomly selected final year students who answered the questionnaire. since there was no significant feedback from the students, the questionnaire was used as it is. two reminders were sent to participants at the beginning and end of april 2021 between data collection to complete the questionnaire. statistical analysis the completed questionnaires were analyzed with spss statistical software, version 26.00 (spss inc., chicago, il, usa). the consistency and reliability of the questionnaire were assessed through pre-test analysis and cronbach's alpha. descriptive statistics were used to express the results as percentages for qualitative variables or in means and standard deviation for quantitative variables. the chi-square test was used to analyze the association between the groups in terms of academic years, gender and family income. alpha was set at 5% in all tests. results we sent the questionnaire to 140 students from the third to final year. of these, 139 responded, yielding a response rate of 99.2%. out of 139 (100%) students, 51(36.7%) were males, and 88(63.3%) were females. the mean age of the cohort was 24.02±0.7. about 14.4% of the students were malay, 18.7% were indians, 66% were chinese, and 2.9% belonged to other ethnicities. the majority of the students belonged to a high family income, 97(69.8%). (table 1) cronbach's alpha was 0.76, indicating good internal consistency within the questionnaire. section 1: effects on dental education based on students’ perceptions regarding online learning. the educational impact of covid-19 on 3rd, 4th and 5th year dental students is shown in table 2. the majority of the students felt that the lockdown caused them to miss educational opportunities, with year 3 dental students (100%) agreed significantly more than year 4 and year 5 students (p<0.001). when compared to year 3 and year 4 students, the majority of year 5 students (86%) (p<0.001) believed that online assessment was not a viable evaluation approach. on the other hand, year 3 and 4 dental students, thought that online http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu assessment was an excellent way of evaluation, with year 3 students (86.8%) strongly agreeing much more than year 4 students (56.9%). all year 3 and final year students agreed to a small group discussion on e-learning for clinical cases and scenarios. students in their last year were substantially more motivated and engaged (p<0.001). however, only 2% of year 4 students had a negative outlook on group discussion on elearning compared to year 3 and 5 students. there was no significant difference between all the three academic years regarding their collaboration with colleagues. most of the 4th and final year students (84.3% and 94%, respectively) showed a lack of motivation through distant e-learning as compared to the 3rd year students (p<0.001). in addition, the majority of the year 3 and 5 students (year 3 = 100%, year 4 = 90.2%, year 5 = 100%), preferred online lectures more than the year 4 dental students (p=0.01). a significantly higher percentage of 5th year dental students (90.0%) and 3rd year students (86.6%) compared to 4th year dental students (62.7%) strongly agreed with the technologybased education (p = 0.01). table 3 shows the various types of experiences influenced by quarantine among the clinical year dental students. all of the year 3, 4 and 5 dental students (100%) strongly agreed that clinical, lab sessions and hospital postings were greatly affected. regular lectures were missed significantly by the year 3 dental students compared to the 4th year students (p = 0.004). in contrast, year 4 students reported that research projects were considerably affected compared to the final year students (p<0.001). a higher percentage of year 3 (57.9%) and year 4 (54.9%) dental students felt that seminars were not significantly disrupted during quarantine as compared to the year 5 students (p = 0.006). when asked about their experiences with casebased learning, most of the students, especially the 4th year dental students (72.5%), felt that it could be done online. therefore, it is not affected during the quarantine period compared to the 3rd and final year students (p = 0.004). the dental subspecialties most negatively affected by quarantine are shown in table 4. all year 3 dental students (100%) strongly agreed that general medicine and general surgery were greatly affected during the quarantine. oral diagnosis experiences among year 3 and 4 dental students were more negatively affected among the year 3 (p<0.001), while the year 4 felt that community oral health was affected more compared to the year 3 dental students (p = 0.02). the final year students felt that periodontology was significantly affected compared to the year 3 and 4 students (p <0.001). 4th and final year dental students struggled with prosthodontics significantly compared to 3rd and 5th year students (p<0.001). conservative dentistry and endodontics among the year 3 students were affected least significantly in comparison to the 4th and final year students (p<0.001). year 5 dental students (76%) were significantly more confident in paediatric dentistry than year 3 (26.3%) and 4 (47.1%) (p<0.001). orthodontics was negatively affected among the year 5 students, followed by the year 4 and year 3 (p<0.001). oral surgery demonstrated no significant difference among all the years. section 2: comparing the effects of dental education according to students' attitudes towards online learning between the genders and family income. the impact on dental education among the genders is shown in supple.table 1. there was no significant association between gender and the responses regarding all 7 components of the questionnaire. nonetheless, regarding the experiences mostly affected by quarantine, there was a significant association between gender and the responses as regards seminar, i.e., 58.8% of males strongly disagreed that seminars were the experience that was affected mainly by quarantine, as compared to 38.6% of females (p = 0.02) (table 5). male students (60.8%) agreed that orthodontics was affected as compared to female students (40.9%) http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu ¥= total sample table 1. background characteristics of the participants (¥n = 140, n=139) (p = 0.001), while no significant difference was noted among the other subspecialties shown in table 6. there was no significant association between the students’ family income and dental education impact (supple.table 2). section 3: the final year students’ selfperceived preparation in cognition, communication, and professional abilities. table 7 shows the responses of 5th year dental students to 17 measures of self-perceived preparedness relating to cognition, communication, and professional abilities. the 50 finalyear students’ overall self-perceived preparation mean score (tps) was 22.5 (sd ±1.92). in general, most students believed they were entirely or mostly equipped in their professional and communication skills. however, a large number of final-year students found it challenging to evaluate new dental materials using an evidence-based approach, according to our research (66%). almost half (54%) said they did not have enough evidence-based knowledge of scientific principles to support their practice. furthermore, nearly two-thirds of the students (62%) said they regularly reflect on their clinical practice to address their learning requirements. however, the study reported that female final year students were significantly better in managing patients’ expectations for their treatment than their male counterparts (p=0.04). furthermore, the female 5th year students were perceived to communicate with their patients more effectively (p=0.01) with procedural risks (p=0.02) significantly better than the male students shown in table 8. there was no significant association between the family income and most of the selfperceived preparedness of the final year students except for evaluating new dental materials or products using an evidence-based approach, where the lower-income group of students were significantly higher than the higher income group (p=0.03). in addition, the higher income group recognizes their limitations in clinical practice significantly higher than the lower income group of students (p=0.02) illustrated in table 9. the general practice readiness among the final year students was evaluated and shown in table 10, and it is not characteristics n (%) mean(sd) gender • male • female 51(36.7) 88(63.3) age 24.02 (0.7) year of study • year 3 • year 4 • year 5 38(27.3) 51(36.7) 50(36) race • malay • chinese • indian • others 20(14.4) 89(66) 26(18.7) 4(2.9) family income • low (≤ rm5000/month) • high (> rm5000/month) 42(30.2) 97(69.8) http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu p-value was determined with chi square test, p-value<0.05 was considered significant* highly significant; n.s: not significant table 2: impact of covid-19 on dental education among year 3,4 and 5 dental students questions academic year strongly disagree % disagree % agree % strongly agree % p-value do you feel that you missed educational experiences because of the lockdown? 3 31.6 68.4 <0.001* 4 5.9 39.2 37.3 17.6 5 2.0 22.0 46.0 30.0 do you think online assessment is a good method for evaluation? 3 13.2 86.8 <0.001* 4 43.1 56.9 5 28.0 58.0 8.0 6.0 do you think group discussion posted on elearning such as clinical cases and scenarios had a positive value on your education? 3 60.5 39.5 <0.001* 4 2.0 31.4 66.7 5 16.0 84.0 did this quarantine increase your collaboration with your colleagues? 3 5.3 18.4 76.3 0.054 (n.s) 4 3.9 21.6 74.5 5 10.0 40.0 50.0 did you feel more engaged and motivated in following up with distant e-learning? 3 63.2 36.8 <0.001* 4 35.3 49.0 13.7 2.0 5 32.0 62.0 6.0 do you prefer online lectures compared to face to face lectures? 3 28.9 71.1 0.01 4 9.8 27.5 62.7 5 44.0 56.0 do you feel comfortable with all this technologybased education? 3 13.2 86.6 0.01 4 37.3 62.7 5 10.0 90.0 http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu p-value was determined with chi square test, p-value<0.05 was considered significant* highly significant; n.s: not significant table 3: experiences mostly affected by quarantine among year 3,4 and 5 dental students what is the experience that was mostly affected by your quarantine? academic years strongly disagree % disagree % agree % strongly agree % p-value clinical/lab sessions/hospital postings 3 100 4 100 5 100 regular lectures 3 39.5 47.4 13.2 0.004 4 60.8 39.2 5 54.0 46.0 research projects 4 5.9 88.2 5.9 <0.001* 5 10.0 8.0 82.0 seminars 3 57.9 42.1 0.006 4 54.9 45.1 5 28.0 72.0 case-based learning 3 55.3 44.7 0.004 4 72.5 27.5 5 40.0 60.0 significant among the genders demonstrated in table 11. approximately two-thirds of the 5th year dental students were unclear of their trust in their acquired abilities prior to graduation. after graduation, the vast majority (94%) opted to be mentored or indirectly supervised. about half of the final year students (56%) said they were unsure about starting their own practice following graduation. the vast majority (80%) choose a well-structured year of residence followed by excellent postgraduation training (supple.table 3). there was no significant association between the family incomes and the general practice readiness among the final year students in supple.table 4. all supplementary files can be assessed via https://osf.io/d8p2a/files/ http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu p-value was determined with chi square test, p-value<0.05 was considered significant* highly significant; n.s: not significant table 4: subspecialties most negatively affected by quarantine among year 3,4 and 5 dental students subspecialties academic years strongly disagree % disagree % agree % strongly agree % p-value general medicine and general surgery 3 100 community oral health 3 52.6 47.4 0.02 4 29.4 70.6 oral diagnosis 3 81.6 18.4 <0.001* 4 25.5 74.5 periodontology 3 5.3 94.7 <0.001* 4 17.6 82.4 5 4.0 18.0 24.0 54.0 prosthodontics 3 68.4 31.6 <0.001* 4 43.1 56.9 5 40.0 56.0 4.0 conservative dentistry and endodontics 3 36.8 63.2 <0.001* 4 39.2 60.8 5 18.0 82.0 paediatric dentistry 3 26.3 71.1 2.6 <0.001* 4 47.1 52.9 5 76.0 24.0 oral surgery 3 50.0 50.0 0.73 (n.s) 4 56.9 43.1 5 50.0 50.0 orthodontics 3 31.6 68.4 <0.001* 4 49.0 51.0 5 18.0 82.0 http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu p-value was determined with chi square test, p-value<0.05 was considered significant* highly significant; n.s: not significant p-value was determined with chi square test, p-value<0.05 was considered significant* highly significant; n.s: not significant table 5: experiences mostly affected by quarantine based on gender profile table 6: subspecialties most negatively affected by quarantine based on gender profile http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu table 8: self-perceived preparedness related to cognition, communication and professional skills based on gender profile among the final year students. table 7: self-perceived preparedness related to cognition, communication and professional skills of the final year dental students p-value was determined with chi square test, p-value<0.05 was considered significant* highly significant; n.s: not significant http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu table 9: self-perceived preparedness related to cognition, communication and professional skills based on family income of the final year students. p-value was determined with chi square test, p-value<0.05 was considered significant * highly significant; n.s: not significant table 10: practice readiness of the final year dental students table 11: practice readiness based on gender among the final year dental students p-value was determined with chi square test, p-value<0.05 was considered significant * highly significant; n.s: not significant http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu discussion within the span of two decades, the world has witnessed three outbreaks related to the coronavirus, which were severe acute respiratory syndrome (sars) back in 2002, the middle east respiratory syndrome (mers), which emerged around 2012 and the recent pandemic caused by the novel coronavirus, famously known as covid-19 [17-21]. the pandemic has taken a toll on the majority of the sectors on a global scale, including education. dental education is unique in that it requires a combination of both didactic learning and hands-on training to produce a well-rounded and competent dentist in providing appropriate treatment and dental care to the public. currently, there is insufficient evidence regarding the effects of the pandemic on tertiary education. as per our knowledge, this is the first study carried out to assess clinical year dental students’ perception of the impact of the covid-19 pandemic. however, a review was done by halim et al. based on educators’ perspectives on ways to improve dental education in malaysia during the lockdown period [14]. a study reported that quarantining during the pandemic might positively impact students’ performance as it encourages students to be more efficient and productive with their learning strategies [22]. on the contrary, most of the final two academic year students felt less motivated. they reported being at a disadvantage in terms of their clinical skills due to distance learning compared to the 3rd year dental students. they reported that clinical and lab sessions were greatly affected. they miss the way regular education was carried out before the lockdown. however, the 3rd and 5th year students yearn for their education to return to normal significantly compared to the 4th year. this can be explainable as the 3rd year students are excited to begin their clinical year by treating patients for the first time. as for the final year students, they have to balance completing their minimum clinical experience (mce) along with the uncertainty of the lockdown period. similarly, various studies have reported an increased stress level among students due to the lack of clinical exposure in these uncertain times [16,23-25]. our study reported a significant difference between the year 5 and 4 students regarding research projects. the majority of the year 4 dental students struggled because most of their research topics had to be amended, making it feasible to be implemented during this pandemic. usually, research topics and commencement of the research projects begin while the students are in their 4th year. most final year students have completed and presented their research projects at various scientific conferences; therefore, most of their attention was focused on other areas of their education. almost all of our students were comfortable using technology-based education, significantly supported by the final year students. didactic classes are the easiest to execute online via a flipped classroom platform, positively affecting dental education [26]. this is reflected in the present study, where most students appreciated regular lectures, seminars and case-based learning (cbl) conducted through e-learning platforms. the majority of them preferred having online lectures compared to face-to-face with no significant difference among the genders. a recent review reported that distant learning enhances students' learning ability due to its flexibility and accessibility [27]. academicians have to adapt and adjust their teaching methods by taking advantage of asynchronous learning, which is best carried out on a digital platform, giving them more room to breathe [28]. various studies conducted reported satisfying responses through distant e-learning methods, with clinical sessions being irreplicable remotely [29-31]. however, our 3rd year dental students reported being significantly more affected in online lectures than the 4th and 5th years because they are at the beginning of their clinical year, which preferred more face-to-face interaction with their lecturers. there is a significant difference between female and male students regarding online seminar sessions where the male students struggled significantly lesser than the female students. the principal reason for this difference http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu might be that males tend to appraise themselves to have higher computer skills than females [32]. females have a better engagement which can be attainable through onsite teaching and not online, as seen in a recent study [15]. our study had noticed that the lockdown had enhanced their interaction with their colleagues, especially among the 4th year dental students, which differs from the findings reported by hattar et al. in jordan, where the final year students were able to interact with their colleagues significantly better [16]. one of the sound reasoning for this finding is that year 4 is the middle clinical year. due to the current situation, the attention was shifted mainly towards the final graduating year and the 3rd year dental students, who have just entered their clinical postings. assessing the current graduating senior batch during this pandemic, the significant collaboration with their classmates within the 4th year students could be a sign of their preparedness for them to completing their mce in case the lockdown prevails longer so that they are not far behind in their clinical skills when they reach their final year. the mode of assessment and evaluation of our students were greatly affected as well. the majority of our students, especially the final year students, did not agree that online assessment is a suitable evaluation method compared to the 3rd and 4th year dental students. usually, objective structured clinical examinations (osces) was performed to assess dental students’ clinical critical reasoning skills. these exams are no stranger in the medical tertiary education sectors as they contain a high educational value [32-33]. however, this year, osces exams were cancelled due to various reasons. unfortunately, the 1st semester examination period of the academic year falls during the lockdown period. therefore, examinations can only occur in prominent venues such as multipurpose halls to abide by the social distancing protocol proposed by the government. students’ clinical sessions were happening concurrently with the exam period to catch up with their clinical work, making up for a lost time. hence, logistics became a factor as to why it is not practical to carry out osce examinations. the finding in the present study was in accordance with the results reported by hatter et al [16]. regarding the subspecialties most negatively affected by the quarantine, our findings noted that conservative dentistry & endodontics and orthodontics had a significant detrimental outcome followed by periodontology among the 5th year dental students. similarly, dental students in jordan also stated that conservative dentistry was the subspeciality mainly affected [16]. this is explainable since most final year students’ clinical sessions revolve around more complicated procedures in the mentioned subspecialties. comparably the year 3 dental students stated that general medicine and general surgery, oral diagnosis and paediatric dentistry were greatly affected. the 4th year had prosthodontics followed by conservative dentistry and endodontics and orthodontics. being new to the clinical settings, most clinical hours for the 3rd year dental students involve subjects such as oral diagnosis and paediatric dentistry. it serves as a basis for them to learn how to carry out various investigative tools to diagnose cases in dentistry. since the pandemic, government hospital postings for general medicine and general surgery have been temporarily ceased as they are involved in housing covid-19 positive patients. furthermore, oral surgery was the only subspecialty where all clinical year students felt no unfavourable effects, especially the 5th year dental students. there is a possibility because the year 4 and 5 students had sufficient exposure in their previous academic years, hence boosting their confidence and competence; nonetheless, usually for oral surgery postings, 3rd year students were not required to carry out treatments on patients yet. it is confirmed by a study done by interviewing students, where the authors concluded that the amount of clinical exposure plays a crucial role in affecting the students’ preparedness and confidence before graduation [34]. there was no significant difference between the genders in most subspecialties except for orthodontics, where the male dental students were greatly affected. the http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu plausible explanation for this is supported by a study done on dental students’ knowledge, attitude and perception of orthodontic treatment. the author concluded that female dental students had a better attitude and knowledge regarding dental esthetics through orthodontic treatments [35]. only the year 5 dental students' readiness for general dental practice related to their cognition, communication and professional skills were asked through a self-perceived questionnaire. the present study demonstrated acceptable outcomes through the broad range of aspects in the questionnaire. our final year respondents rated confident in their overall professionalism, communication skills, and continuous development, which agrees with previous studies [16,36-39]. nevertheless, most of them reported the lack of knowledge in critical appraising in terms of their utilization of evidence-based approach, which is concurrent with studies done worldwide [38,40-42]. hence, it is vital to incorporate or emphasise an evidence-based approach to the dental curriculum, further increasing students’ critical appraisal skills and integrating them into their practice after graduation. we found that female 5th year students were significantly better than their male colleagues in communicating with their patients regarding the procedural risks. they perceived to be able to handle patients’ expectations better. this is greatly supported by previous studies, where they affirmed that females tend to communicate better by engaging in more positive talks in encouragement. they were rated with higher empathy, leading to longer consultation time with their patients compared to males [43-45]. when comparing the family monthly income with the self-perceived preparedness of the final year students, the lower-income group tend to evaluate new dental materials or products using an evidence-based approach more than the higherincome group. the probable reason for this is that dental materials in the market are not cheap, especially for students to purchase. therefore, they tend to resolve with the utilization of the evidence-based approach available through various online journals provided by the university to learn more about the materials instead of experiencing it hands-on compared to the more affluent students. however, students from the higher income group tend to recognize their personal limitations in clinical practice significantly more frequently. one study done in a dental school in south africa reported that dental students from high socialeconomic status have better clinical reasoning, which may be related to our study as they tend to self-perceive their clinical skills more often [46]. however, there was not much data on the association of family income with self-perceiving of clinical skills among dental students, and further research into this component may be required. the majority of the graduating students are somewhat confident in carrying out appropriate treatments before graduating but still prefer having some form of mentorship or indirect supervision after graduation, similar to findings in previous studies [16,39,47]. almost half of our respondent was conflicted regarding starting up an independent practice. therefore, mentoring or any form of communication with academicians will undoubtedly help their confidence in their future practice since they have suffered greatly during the final year [16]. clinical exposure cannot be entirely replaced by distant online learning; hence, extra precautions must be taken into account to ensure a safe working and learning space for faculty staff members and students. it is the responsibility of the faculty to ensure that sufficient personal protective equipment (ppe) is available, along with assembling patient triage to identify high-risk individuals [29]. in addition, dental institutions should work hand-in-hand with the government body by emphasizing and encouraging staff and students to get vaccinated to achieve herd immunity at a faster rate. the current study serves as a development of further investigations. however, it comes with a few limitations. a direct limitation is noted due to the study's crosssectional design performed in a single dental institute. different dental students from different dental schools will have a different experience during this unrivalled time. it would be http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu illuminating to obtain information from other dental institutions in malaysia to make a comparison to get a better viewpoint, which may serve as a benefit further to improve the dental education system during this global change [48]. acknowledgments the authors would like to extend their gratitude to the clinical year dental students of segi university, selangor, malaysia for taking their time to participate in the present study by responding to the questionnaire. references 1.aristovnik a, keržič d, ravšelj d, tomaževič n, umek l. impacts of the covid-19 pandemic on life of higher education students: a global perspective. sustainability. 2020; 12(20):8438. 2.owusu-fordjour c, koomson ck, hanson d. the impact of covid-19 on learning – the perspective of the ghanaian student. eur j educ stud 2020;7:1-14. 3.ritche h, ortiz-ospina e, beltekian d, mathieu e, hasell j, macdonald b, giattino c, appel c, rodés-guirao l, roser m. coronavirus pandemic (covid-19) [internet]. ourworldindata [cited 2021 july 24]. available from: https://ourworldindata.org/policyresponses-covid#citation. 4.aslam, f. covid-19 and importance of social distancing. [preprints]. 2020 [cited 2021 july 24]: [2020040078, 1–6]. available from:https://www.researchgate.net /publication/340476408_covid19_and_importance_of_social_dist ancing 5.sahu p. closure of universities due to coronavirus disease 2019 (covid-19): impact on education and mental health of students and academic staff. cureus 2020;12(4):e7541. 6.chang ty, hong g, paganelli c, phantumvanit p, chang w, shieh ys et al. innovation of dental education during covid-19 pandemic. j dent sci 2021; 16:1520. 7.bennardo f, buffone c, fortunato l, giudice a. covid-19 is a challenge for dental education – a commentary. eur j dent educ 2020;24:822-824. 8.elangovan s, mahrous a, marchini l. disruptions during a pandemic: gaps identified and lessons learned. j dent educ 2020;84:1270-1274. 9.gaudin a, arbab-chirani r, pérez f. effect of covid-19 on dental education and practice in france. front dent med 2020;1:5. 10.quinn b, field j, gorter r, akota i, manzanares mc, pagnelli c, et al. covid-19: the immediate response of european academic dental institutions and future implications for dental education. eur j dent educ 2020;24:811-814. 11.wong j, lee ahc, zhang c. effect of covid-19 on dental education and endodontic practice in hong kong. front dent med 2020;1:569225. 12.unesco [internet]. covid-19 impact on education [cited 2021 july 24]. available from: https://en.unesco.org/covid19/edu cationresponse 13.alrashdi m, hameed a, alijabr a. covid-19 and a call to adapt dental education. front dent med. 2021;2:664460. 14.halim ms, noorani ty, karobari mi, kamaruddin n. covid-19 and dental education: a malaysian perspective. j int oral health. 2021;13:201-206. 15.yu z. the effects of gender, educational level, and personality on online learning outcomes during the covid-19 pandemic. j educ technol high educ. 2021;18(14). 16.hattar s, alhadidi a, sawair fa, alraheam ia, el-ma’aita a, wahab fk. impact of covid-19 pandemic on dental education: online experience and practice expectations among dental students at the university of jordan. bmc med educ. 2021;21:151. 17.karlamangla s. sars killed hundreds and then disappeared. could this coronavirus die out? los angeles times. 18 february, 2020. https://www.latimes.com/california /story/2020-02-18/sars-coronavirus -china-epidemic. accessed 20 may, 2021. 18.adney dr, letko m, ragan ik, et al. bactrian camels shed large quantities of middle east respiratory syndrome coronavirus (merscov) after experimental infection. emerg microbes infect. 2019;8(1):717-723. 19.banarjee a, baid k, mossman k. molecular pathogenesis of middle east respiratory syndrome (mers) coronavirus. curr clin microbiol rep. 2019;6(3):139-147. http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu 20.khudhair a, killerby me, al mulla m, et al. risk factors for mers-cov seroporosity among animal market and slaughter-house workers. abu dhabi, united arab emirates, 20142017. emerg infect dis. 2019;25(5):927-935. 21.who [internet]. who directorgeneral's opening remarks at the media briefing on covid-19 – 11 march 2020. [cited 2021 july 24]. available from: https://www.who.int/directorgeneral/speeches/detail/whodirector-general-s-openingremarks-at-the-media-briefing-oncovid-19---11-march-2020 22. sadeghi r, sedaghat mm, sha af. comparison of the effect of lecture and blended teaching methods on students' learning and satisfaction. j adv med educ prof. 2014;2(4):146-150. 23.hung m, licari fw, hons es, evelyn lauren e, su s, birmingham w, wadsworth ll, et al. in an era of uncertainty: impact of covid-19 on dental education. j dent educ. 2021;85(2):148-156. 24.ray m, milston a, doherty p, crean s. in their own words: investigating the preparedness of final year dental students in the uk for independent general dental practice. br dent j. 2018;225(4):340-349. 25.gianoni-capenakas s, lagravere m, pacheco-pereira c, yacyshyn j. effectiveness and perceptions of flipped learning model in dental education: a systematic review. j dent educ. 2019;83(8):935-945. 26.regmi k, jones l. a systematic review of the factors – enables and barriers – affecting e-learning in health sciences education. bmc med educ. 2020;20(1):91. 27.daniel j. education and the covid-19 pandemic. prospects. 2020;49:91-96. 28.hyunh r. the role of e-learning in medical education. acad med 2017;92(4):430. 29.ruiz jg, mintzer mj, leipzig rm. the impact of e-learning in medical education. acad med. 2006;81(3):207-212. 30.bennardo f, buffone c, fortunato l. giudice a. covid-19 is a challenge for dental education-a commentary. eur j dent educ. 2020;24(4):822-824. 31.aristovnik a, keržič d, ravšelj d, tomaževič n, umek l. impacts of the covid-19 pandemic on life of higher education students: a global perspective. sustainability. 2020; 12(20): 8438. 32.hammad m, oweis y, taha s, hattar s, madarati a, kadim f. students' opinions and attitudes after performing a dental osce for the first time: a jordanian experience. j dent educ. 2013;77(1)99-104. 33.puryer j. dental undergraduate views of objective structured clinical examinations (osces): a literature review. dent j. 2016;4(1):6. 34.ray m, milston a, doherty p, crean s. in their own words: investigating the preparedness of final year dental students in the uk for independent general dental practice. br dent j. 2018;225(4):340-349. 35.agrawal r. knowledge, attitude and perception of orthodontic treatment among dental students. int j dent research. 2018;6(1):3-5. 36.ali k, slade a, kay e, zahra dtc, tredwin c. preparedness of undergraduate dental students in the united kingdom: a national study. br dent j. 2017;24(6):472477. 37.ali k, jerreat m, zahra d, tredwin c. correlations between final-year dental students' performance on knowledge-based and clinical examinations. j dent educ. 2017;82(12):1444-1450. 38.mat yudin z, ali k, wan ahmad wma, ahamad a, khamis mf, brian graville monteiro na, et al. selfperceived preparedness of undergraduate dental students in dental public universities in malaysia: a national study. eur j dent educ. 2020;24(1):163-168. 39.gilmour asm, welply a, cowpe jg, bullock ad, jones rj. the undergraduate preparation of dentists: confidence levels of final year dental students at the school of dentistry in cardiff. br dent j. 2016;221(6):349-354. 40.ali k, cockerill j, zahra d, qazi hs, raja u, ataullah k. selfperceived preparedness of final year dental students in a developing country-a multiinstitution study. eur j dent educ. 2018;22(4):e745-750. 41.nieminen p, virtanen ji. information retrieval, critical appraisal and knowledge of evidence-based dentistry among finnish dental students. eur j dent educ. 2017;21(4):214-219. http://dentistry3000.pitt.edu/ impact of covid-19 on perception and preparedness among clinical year malaysian undergraduate dental students – a cross-sectional study vol 10 no 1 (2022) doi 10.5195/d3000.2022.351 http://dentistry3000.pitt.edu 42.straub-morarend cl, wankiirihale cr, blanchette dr, lanning sk, bekhuis t, smith bm, et al. evidence-based practice knowledge, perceptions and behavior: a multi-institutional, cross-sectional study of a population of u.s. dental students. j dent educ. 2016;80(4):430-438. 43.roter dl, hall ja, aoki y. physician gender effects in medical communication: a meta-analytic review. jama. 2002; 288:756-764. 44.nicolai j, demmel r. the impact of gender stereotypes on the evaluation of general practitioners’ communication skills: an experimental study using transcripts of physician-patient encounters. patient educ couns 2007; 69:200-205. 45.jefferson l, bloor k, birks w, hewitt c, bland m. effect of physicians’ gender on communication and consultation length: a systematic review and meta-analysis. journal of health services research & policy. 2013;18(4):242-248. 46.postma tc, white jg. sociodemographic and academic correlates of clinical reasoning in a dental school in south africa. eur j dent educ. 2015; 21(1):58-65. 47.manakil j, george r. selfperceived work preparedness of the graduating dental students. eur j dent educ. 2013;17(5):101-105. 48.taha mh, abdalla me, wadi m, khalafalla h. curriculum delivery in medical education during an emergency: a guide based on the responses to the covid-19 pandemic. mededpublis. 2020;9(1):69. http://dentistry3000.pitt.edu/ microsoft word 216 2022.docx alveolar ridge augmenta0on in mandible by use the modified distractor vol 10 no 1 (2022) doi 10.5195/d3000.2022.216 h#p://den*stry3000.pi#.edu alveolar ridge augmentation in mandible by use the modified distractor feras sulaiman1, issam alkhouri1, maher al-assaf2 1 department of oral and maxillofacial surgery, faculty of dentistry, damascus university, damascus, syria 2 department of oral histology and pathology, faculty of dentistry, damascus university, damascus, syria abstract objective: the aim of the present study was to evaluate the horizontal and the ver0cal alveolar distrac0on osteogenesis using modified alveolar distractor. study design: the sample consisted of 7 pa0ents presen0ng horizontally and ver0cally deficient edentulous ridges were treated by distrac0on osteogenesis with a modified alveolar distractor. the surgical procedure was carried out with the pa0ent under local anesthesia. a>er the osteotomy was performed with pizosurgery, the distractor was placed on the segmental bone over the gingiva for 3 months. the rate of distrac0on was 0.5 mm/twice a day. results: the mean of horizontal alveolar before using the distractor was 4.70 mm (sd 0.31 mm) the mean of horizontal alveolar a>er using the distractor was 8.30 mm (sd 0.59 mm). the mean of actual gain in bone horizontal at the end of the distrac0on period was 3.58 mm (sd 0.15 mm). the mean of height alveolar before using the distractor was 6.67 mm (sd 0.46 mm) the mean of height alveolar a>er using the distractor was 12.59 mm (sd 0.52 mm). the mean of actual gain in bone height at the end of the distrac0on period was 5.92 mm (sd 0.25 mm). conclusion: it was concluded that the modified alveolar distractor seems to be an effec0ve to treat horizontal and ver0cal alveolar ridge deficiencies, and distrac0on osteogenesis can be considered a safe and effec0ve procedure for gaining bone in the horizontal and the ver0cal dimension of the alveolar. keywords: horizontal alveolar; ver0cal alveolar; distrac0on osteogenesis; distractor cita9on: sulaiman, f et al. (2022) alveolar ridge augmenta9on in mandible by use the modified distractor den9stry 3000. 1:a001 doi:10.5195/d3000.2022.216 received: august 2, 2021 accepted: september 28, 2021 published: may 9, 2022 copyright: ©2022 sulaiman f et al. this is an open access ar9cle licensed under a crea9ve commons asribu9on work 4.0 united states license. email: dr.maher.assaf1990@gmail.com introduction some patients may have insufficient bone to place dental implants but there are many surgical techniques to increase the bone volume making implant treatment possible [1]. bone can be regenerated in a horizontal and vertical direction using various techniques [2]. a variety of surgical techniques have been described to enhance the bone volume of deficient implant-recipient sites, such as the use of onlay or veneer grafts, ridge splitting, or bone condensation, guided bone regeneration (gbr), alveolar osteogenic distraction (aod) [3,4]. alveolar osteogenic distraction (aod) has been considered a promising procedure for bone augmentation [5]. the aod is a biological process through which new bone formation occurs between the surfaces of vascularized bone segments that are gradually separated by incremental traction [6]. the bone is initially alveolar ridge augmenta0on in mandible by use the modified distractor vol 10 no 1 (2022) doi 10.5195/d3000.2022.216 h#p://den*stry3000.pi#.edu sectioned by osteotomy and the separation process is controlled by an osteodistractor device [5]. in this way, the aod avoids the morbidity associated with the donor site and provides hardand soft-tissue predictable gain once the alveolar bone gain occurs simultaneously with soft-tissue increase [3]. moreover, the aod is associated with low infection rate, decreased bone resorption, and a short period of bone healing, accelerating the treatment finalization [7]. the new bone structure formed by this technique has the same quality and morphology of the maxilla bone, and the use of the autogenous bone graft is not required [8]. aod involves three phases: osteotomy/latency, activation/distraction and consolidation phases [9]. in general, we can distinguish 2 types of distraction devices: intraosseous and extraosseous [10]. they can also be differentiated depending on their role, dividing them into distractors or distractor-implants [11]. depending on the direction of the regenerated bone, they are divided into vertical or horizontal distraction devices [12]. different studies present different distraction protocols for each distractor device [13]. so, the aim of the research was to evaluate the horizontal and the vertical alveolar distraction osteogenesis using modified alveolar distractor. material and methods study sample the research sample consisted of 7 patients presenting horizontally and vertically deficient edentulous ridges in the posterior mandibular region. patients attending the dental implant unit and outpatient clinic of the department of oral and maxillofacial surgery at the faculty of dentistry at damascus university. inclusion criteria ü ages of pa[ents from 20 to 55 years old. ü the pa[ents are healthy and do not have any general diseases. ü in pa[ents, the loss of one or more teeth in the posterior region of the mandibular. ü pa[ents do not have any bad habits such as smoking or clinching. exclusion criteria the availability of any of the following conditions is sufficient to exclude the patient from the research: ü the presence of general diseases or factors that prevent surgery under local anesthesia. ü pa[ents with complete tooth loose of the mandibular. ü pa[ents have bad habits such as smoking or clinching. ü pregnancy. surgical procedure all patients were treated under local anesthesia (2% lidocaine solution with epinephrine 1:100.000). (figure 2) intraoral linear incision with 15c scalpel blade was performed on the vestibular region 1 mm above the mucogingival line. then, one vertical incision rising from the first incision were carried out over the mesial region. a conservative subperiosteal dissection was performed to expose the bone ridge only in the osteotomy region. (figure 3) osteotomy was performed using a figure 2. initial intraoral image before the alveolar osteogenic distraction. figure 3. the triangle flap. alveolar ridge augmenta0on in mandible by use the modified distractor vol 10 no 1 (2022) doi 10.5195/d3000.2022.216 h#p://den*stry3000.pi#.edu piezoelectric ultrasonic device. (figure 4,5) after performing an osteotomy in the alveolar bone, a distraction device is fixed on the transport segment and the basal bone over the gingiva. (figure 6,7) subsequently, the transport segment is submitted to gradual traction to separate it from the basal bone. the distraction devices were not activated for 7 days to allow periosteal and soft tissue healing and early vascularization. after a latency period of 7 days, distraction devices were activated 0.5 mm twice daily to achieve movement of 1 mm per day. after retention of 12 weeks, distractors were removed under local anesthesia. (figure 8) a computed tomography (cbct) image was performed for each case before the surgery procedure and the measurements were taken on the sections of the surgery site and the patient was followed up and after three months another cbct was requested. (figure 1,9) statistical analysis statistical analysis was performed with spss (statistical package for the social sciences) v.25 (ibm, new york, ny). statistical significance level was established at (p < 0.05). the paired t-test was used to evaluate bone dimensional changes (vertical and horizontal) between before and after the alveolar osteogenic distraction surgery. results study sample consisted of 7 patients, 1 male and 6 females and the age of the patients ranged between 20 55 years with a mean of 32.2 years. bone figure 4. the bone osteotomy by piezosurgery. figure 5. the bone fragments. figure 6. the suture. figure 7. placement of the modified alveolar distractor figure 8. final tissue volume obtained after 3 months of the alveolar osteogenic distraction surgery. figure 9. cbct radiograph after 3 months of the alveolar osteogenic distraction surgery confirming the bone gain obtained. figure 1. initial cbct radiograph before the alveolar osteogenic distraction. alveolar ridge augmenta0on in mandible by use the modified distractor vol 10 no 1 (2022) doi 10.5195/d3000.2022.216 h#p://den*stry3000.pi#.edu defects were different for each patient and bone ratio was set up depending on patient need. the evaluation of bone ratios was performed depending on the activating times of the distraction devices and supported by radiologic data. distraction devices were activated twice a day (table no. 1). the mean of horizontal alveolar before using the distractor was 4.70 mm (sd 0.31 mm) the mean of horizontal alveolar after using the distractor was 8.30 mm (sd 0.59 mm). there was a statistically significant difference between timepoints (p < 0.05). the mean of actual gain in bone horizontal at the end of the distraction period was 3.58 mm (sd 0.15 mm) (table no. 2 and table no. 4). t-value p-value mean difference s.d. of mean difference confidence interval (95%) lower limit upper limit horizontal bone gain -25.922 .000 3.58.150 -4.10 -2.89 surgery latency period rate of bone elongation consolidation period device removal 7 days 0.5 mm/twice a day 3 months the width alveolar bone mean standard deviations minimum maximum before distraction 4.70 .310 4.00 5.20 after distraction 8.30 0.59 4.00 9.50 table 1: protocol for gradual alveolar distraction osteogenesis of the posterior mandible prior to device removal. table 2: means, standard deviations, and ranges of the width alveolar bone in the distraction osteogenesis (do). table 4: paired t test for samples to study the change in the width of the alveolar bone before and after 3 months of the distraction osteogenesis (do). alveolar ridge augmenta0on in mandible by use the modified distractor vol 10 no 1 (2022) doi 10.5195/d3000.2022.216 h#p://den*stry3000.pi#.edu the mean of height alveolar before using the distractor was 6.67 mm (sd 0.46 mm) the mean of height alveolar after using the distractor was 12.59 mm (sd 0.52 mm). there was a statistically significant difference between timepoints (p < 0.05). the mean of actual gain in bone height at the end of the distraction period was 5.92 mm (sd 0.25 mm) (table no. 3 and table no. 5) discussion vertical and horizontal atrophy of the alveolar region may make implant placement difficult, thus compromising prosthetic rehabilitation [15]. different augmentation techniques have been used for reconstruction of alveolar defects for many years, such as autogenous or artificial bone grafts and the split crest technique [16]. these conventional procedures have disadvantages, such as donor site morbidity, unpredictable bone resorption, and difficulty with soft tissue coverage [17]. ado is an innovative procedure used to avoid donor site morbidity and problems with soft tissue coverage and limited augmentation [18]. the principals of distraction osteogenesis in which a gradual, controlled displacement of a surgically prepared fracture is used to increase bone volume, are not new but have recently been introduced into implant surgery to increase alveolar bone volume [19]. this study included 7 patients were treated by vertical and horizontal alveolar distraction osteogenesis. alveolar ridge augmentation by do has become the best alternative for preprosthetic surgery instead of using free, microvascular, and alloplastic bone grafts [20]. reconstruction sites are filled with original bone and soft tissue reconstruction is managed at the same operation [9]. the height alveolar bone mean standard deviations minimum maximum before distraction 6.67 0.46 5.80 7.50 after distraction 12.59 0.52 11.70 13.40 t value p value mean difference s.d. of mean difference confidence interval (95%) lower limit upper limit vertical bone gain 51.765 .000 5.92 0.25 6.17 5.67 table 3: means, standard deviations, and ranges of the heigh alveolar bone in the distraction osteogenesis (do). table 5: paired t test for samples to study the change in the height of the alveolar bone before and after 3 months of the distraction osteogenesis (do). alveolar ridge augmenta0on in mandible by use the modified distractor vol 10 no 1 (2022) doi 10.5195/d3000.2022.216 h#p://den*stry3000.pi#.edu many authors have emphasized that distractors should not be activated for 5 to 7 days to allow callus formation [6]. during this time, a reparative callus is created, osteogenic cells proliferate, damaged blood vessels are repaired, and revascularization occurs [14,21]. in this study, we waited for 7 days to allow for callus formation, soft tissue, and periosteal healing. a gradual distraction rate of 0.5 mm twice a day [22]. in this way, we achieved painless stretching and appropriate adaptation of alveolar soft and hard tissues. ado is an advantageous technique, providing both soft and hard tissue reconstruction with original structures. taking into account the aspects of radiologic and histologic evaluations, the alveolar bone is considered to be appropriate to receive implants at the twelfth week [23]. the required time for dental implantation is shortened by this technique compared with classical procedures, so the consolidation phase was 12 weeks. vertical augmentation is easy to evaluate using standard x-rays, while horizontal augmentation is difficult to evaluate with x-rays and cbct is necessary. recently, cone-beam ct (cbct) has been widely used in dental treatment [24]. cbct was used to assess bone height and width preoperatively, and finally after consolidation. at the end of the distraction procedures, radiolucent gaps were observed at the distraction chambers [25]. twelve weeks after distraction, distraction gaps appeared mostly radio-opaque, but there were still some radiolucent areas. evaluation of the dental ct scans, which were performed just before removal of the distractors (12 weeks after distraction), confirmed increase of the alveolar heights and widths and filling of the distraction chambers with bone. esposito et al [4] in a systematic review of cochrane on different vertical regeneration techniques, didn’t find sufficient evidence regarding which was the best procedure. however, they reported that the ado technique has the greatest potential for vertical regeneration procedures. conclusion with the limitation of this study, we can conclude that the modified alveolar distractor seems to be an effective to treat horizontal and vertical alveolar ridge deficiencies, and distraction osteogenesis can be considered a safe and effective procedure for gaining bone in the horizontal and the vertical dimension of the alveolar. if applied to patients selected carefully using preoperative cbct, and if performed and managed accurately. references 1. clinical outcomes of vertical bone augmentation to enable dental implant placement: a systematic review. rocchietta i, fontana f, simion m. j clin periodontol. 2008 sep;35(8 suppl):203-215. doi: 10.1111/j.1600051x.2008.01271.x. pmid: 18724851. 2. the efficacy of horizontal and vertical bone augmentation procedures for dental implants: a cochrane systematic review. esposito m, grusovin mg, felice p, karatzopoulos g, worthington hv, coulthard p. evidence-based practice: toward optimizing clinical outcomes. 2010:195-218. doi: 10.1007/978-3-642-050251_13. 3. use of alveolar distraction osteogenesis for anterior maxillary defect reconstruction. marcantonio c, nícoli lg, pigossi sc, araújo rfsb, boeck em, junior em. j indian soc periodontol. 2019 julaug;23(4):381-386. doi: 10.4103/jisp.jisp_549_18. pmid: 31367139. 4. the efficacy of horizontal and vertical bone augmentation procedures for dental implants a cochrane systematic review. esposito m, grusovin mg, felice p, karatzopoulos g, worthington hv, coulthard p. eur j oral implantol. 2009 autumn;2(3):167-184. doi: 10.1007/978-3-642-05025-1_13. pmid: 20467628. 5. alveolar osteogenic distraction as method to increase the alveolar ridge. navarro dm. revista cubana de estomatología. 2011;48(1):4355. 6. alveolar ridge augmentation by distraction osteogenesis. rachmiel a, srouji s, peled m. international journal of oral and maxillofacial surgery. 2001 dec;30(6):510-517. doi: 10.1054/ijom.2001.0134. alveolar ridge augmenta0on in mandible by use the modified distractor vol 10 no 1 (2022) doi 10.5195/d3000.2022.216 h#p://den*stry3000.pi#.edu 7. alveolar osteogenic distraction as method to increase the alveolar ridge. tnavarro dm. revista cubana de estomatología. 2011;48(1):4355. 8. alveolar distraction osteogenesis for dental implant treatments of the vertical bone atrophy: a systematic review. toledanoserrabona j, sánchez-garcés má, sánchez-torres a, gayescoda c. med oral patol oral cir bucal. 2019 jan;24(1):e70e75. doi: 10.4317/medoral.22750. pmid: 30573711. 9. evaluation of osseous regeneration in alveolar distraction osteogenesis with histological and radiological aspects. türker n, basa s, vural g. j oral maxillofac surg. 2007 apr;65(4):608-614. doi: 10.1016/j.joms.2006.05.057. pmid: 17368352. 10. vertical bone augmentation of posterior mandibular region: a description of two surgical techniques. queiroz ag, silva ys, costa pj, ferraz fw, naclériohomem md. rgo-revista gaúcha de odontologia. 2016 jul;64:333-336. doi: 10.1590/19818637201600030000143128. 11. comparison of intraosseous and extraosseous alveolar distraction osteogenesis. uckan s, oguz y, bayram b. j oral maxillofac surg. 2007 apr;65(4):671-674. doi: 10.1016/j.joms.2006.04.045. pmid: 17368362. 12. partially biodegradable distraction implant to replace conventional implants in alveolar bone of insufficient height: a preliminary study in dogs. li t, zhang y, shao b, gao y, zhang c, cao q, kong l. clin implant dent relat res. 2015 dec;17(6):1164-1173. doi: 10.1111/cid.12229. pmid: 24888978. 13. the reconstructive potential of distraction osteogenesis on defects of the alveolar ridge before dental implants placement: a review. matoulas e, nazaroglou i, kafas p, charitoudi d. research journal of medical sciences. 2009;3(3):123-132. 14. morphofunctional and clinical study on mandibular alveolar distraction osteogenesis. zaffe d, bertoldi c, palumbo c, consolo u. clin oral implants res. 2002 oct;13(5):550-557. doi: 10.1034/j.16000501.2002.130516.x. pmid: 12453134. 15. implant prosthetic rehabilitation in partially edentulous patients with bone atrophy. an umbrella review based on systematic reviews of randomised controlled trials. merli m, moscatelli m, pagliaro u, mariotti g, merli i, nieri m. eur j oral implantol. 2018;11(3):261280. pmid: 30246181. 16. comparison of two techniques for lateral ridge augmentation in mandible with ramus block graft. barbu hm, andreescu cf, lorean a, kolerman r, moraru l, mortellaro c, mijiritsky e. j craniofac surg. 2016 may;27(3):662-667. doi: 10.1097/scs.000000000000256 1. pmid: 27092913. 17. horizontal ridge augmentation using autogenous block grafts and the guided bone regeneration technique with collagen membranes: a clinical study with 42 patients. von arx t, buser d. clin oral implants res. 2006 aug;17(4):359-366. doi: 10.1111/j.16000501.2005.01234.x. pmid: 16907765. 18. quality and quantity of bone following alveolar distraction osteogenesis in the human mandible. chiapasco m, lang np, bosshardt dd. clin oral implants res. 2006 aug;17(4):394-402. doi: 10.1111/j.16000501.2005.01247.x. pmid: 16907770. 19. implant success in distracted bone versus autogenous bonegrafted sites. elo ja, herford as, boyne pj. j oral implantol. 2009;35(4):181-184. doi: 10.1563/1548-1336-35.4.181. pmid: 19813422. 20. alveolar width distraction osteogenesis for early implant placement. laster z, rachmiel a, jensen ot. j oral maxillofac surg. 2005 dec;63(12):17241730. doi: 10.1016/j.joms.2005.09.001. pmid: 16297692. 21. biology of bone formation, fracture healing, and distraction osteogenesis. runyan cm, gabrick ks. j craniofac surg. 2017 jul;28(5):1380-1389. doi: 10.1097/scs.000000000000362 5. pmid: 28562424. 22. mandibular alveolar ridge augmentation in the dog using distraction osteogenesis. block ms, chang a, crawford c. j oral maxillofac surg. 1996 mar;54(3):309-314. doi: 10.1016/s0278-2391(96)907508. pmid: 8600238. 23. alveolar distraction osteogenesis for bone augmentation of severely atrophic ridges in 10 consecutive cases: a histologic and histomorphometric study. marchetti c, corinaldesi g, pieri f, degidi m, piattelli a. j periodontol. 2007 feb;78(2):360-366. doi: 10.1902/jop.2007.060260. pmid: 17274727. alveolar ridge augmenta0on in mandible by use the modified distractor vol 10 no 1 (2022) doi 10.5195/d3000.2022.216 h#p://den*stry3000.pi#.edu 24. cone-beam computerized tomography (cbct) imaging of the oral and maxillofacial region: a systematic review of the literature. de vos w, casselman j, swennen gr. int j oral maxillofac surg. 2009 jun;38(6):609-625. doi: 10.1016/j.ijom.2009.02.028. pmid: 19464146. 25. comparing the duration of different phases of vertical alveolar distraction between ultrasound treated and control group in anterior mandible. alkassaby a, shindy m. int j oral maxillofac surg. 2019 may;48:54. doi: 10.1016/j.ijom.2019.03.163. 730 2024 the effects of selenium nanopar6cles on the osseointegra6on of a titanium implant in rabbits: a histomorphometric inves6ga6on vol 13 no 2 (2024) doi 10.5195/d3000.2024.730 h#p://den*stry3000.pi#.edu the effects of selenium nanoparticles on the osseointegration of a titanium implant in rabbits: a histomorphometric investigation osama a.r. alheeti1, ghasak h. jani 2, farah ragheed qustanteen3 1 university of anbar, iraq 2 university of baghdad, iraq 3 al-rasheed university college, iraq abstract objec&ve: one of the unique tooth or tooth replacement prosthesis in the dental implant. to encourage bone forma&on where it is applied, our approach uses a biomaterial system that is easily obtainable, and able to induce osteoinduc&ve growth factor. selenium nanopar&cles are methods: thirty-two implants consis7ng of commercially pure 7tanium rod were given to sixteen new zealand rabbits. one implant was placed in each 7bia of each rabbit. aaer implanta7on, eight rabbits were sacrificed every one to six weeks. for each animal, the lea 7bia implant was treated with selenium nanopar7cles, whereas the right 7bia implant was lea uncoated. aaer staining each sec7on with hematoxylin and eosin, the sec7ons were examined histologically and assessed for histomorphometric analysis, which counted the number of bone par7cles that were formed. results: selenium nanopar7cle-coated 7tanium implants showed earlier bone growth, mineraliza7on, and matura7on than control comparisons. aaer all the bone parameters were examined histomorphometrically for the study, the healing dura7ons of the two types of implants differed significantly. conclusion: selenium nanopar7cle-coated 7tanium implants had improved healing and osseointegra7on. keywords: bone, implant, selenium nanopar7cles, osseointegra7on. cita:on: alhee: oar, et al. (2024) the effects of selenium nanopar:cles on the osseointegra:on of a titanium implant in rabbits: a histomorphometric inves:ga:on. den:stry 3000. 2:a001 doi:10.5195/d3000.2024.730 received: september 5, 2024 accepted: september 20, 2024 published: october 3, 2024 copyright: ©2024 alhee: oar, et al. this is an open access ar:cle licensed under a crea:ve commons atribu:on work 4.0 united states license. email: oaalghriari@uoanbar.edu.iq introduction the jawbone is attached to a surgical component called a dental implant or fixture to sustain a dental prosthesis [1]. the biological process of osseointegration—the intimate bonding of materials like titanium with bone is the foundation of contemporary implants [2]. an osseointegrated implant is an endosteal implant that has pores into which osteoblasts and supporting connective tissue can migrate. this term refers to bone that has grown up to the implant surface in oral implantology, devoid of any soft tissue layer in between [1,3]. a variety of surface treatment methods have enhanced the surface properties of titanium implants, promoting osseointegration by increasing implant stability and encouraging bone development [4]. selenium nanoparticles (se nps) have potent antibacterial capabilities and have garnered significant interest. the trace element selenium is regulated by cellular redox homeostasis. it is also a constituent of selenoproteins, which govern vital biological functions like the elimination of reactive oxygen species and the regulation of specific enzymes. the method used to make se nps determines whether they can inhibit bacterial growth [5]. the goal of this work the effects of selenium nanopar6cles on the osseointegra6on of a titanium implant in rabbits: a histomorphometric inves6ga6on vol 13 no 2 (2024) doi 10.5195/d3000.2024.730 h#p://den*stry3000.pi#.edu was to demonstrate the ability of se nps to promote osseointegration. material and methods a total of sixteen mature male new zealand white rabbits, weighing between 1.5 and 2 kg, had thirty titanium rods with machined surfaces implanted. one implant was inserted into each rabbit's right and left tibia—one for experimentation and the other as a control. one and six-weeks following implantation, animals were sacrificed. the implants were divided into two groups: control (16 uncoated implants) and experimental (16 seleniumcoated implants), each of which received a different treatment. the rabbits' tibias were drilled holes of 3 mm in diameter, and they were used to insert the sterilizing implants. prior to the right tibia receiving coated implants, the left tibia was treated with coated implants. following the rabbits' sacrifices at the prescribed times. dissection was used to remove the soft tissue from the left and right tibias. to construct implant-contained bone blocks, the right and left tibias were dissected, the soft tissue was removed to expose the complete bone, and slices were created at 5 mm from each implant's side to form implant-contained bone blocks. the right and left tibias were dissected, the soft tissue removed to reveal the whole bone. after the specimens were fixed with 10% formalin for 48 hours, the bone tissue was paraffin embedded and alcohol dehydrated. after that, the samples were decalcified using a solution of formic acid. hematoxylin and eosin were used to stain 5millimeter sections that had been prepared as normal. the histology was examined under a light microscope. bone cells (osteoblast, osteocyte, and osteoclast), cortical bone thickness, trabecular breadth, thread width, and marrow space star volume were all assessed histomorphometrically. results in controls, histological examination showed that osteoid tissue had accumulated in several places (figure 1). figure 1. the histological examination of the control group after one week revealed that the thread region is filled with osseous tissue (h&e x10). the implant's shape and thread region can be seen in the test specimens in figure 2. bone trabeculae were seen under higher magnification in the thread region. osteocytes occupied the large lacunae in these trabeculae, and osteoblasts were clustered at their edges. figure 2. an examination of the six-week experimental group shows woven bone in the thread area, which was followed by the screw-shaped blood vessels and hyalinization (h&e x4). the growth of thick bone trabeculae, surrounded by active osteoblasts and packed with large osteocytes, is visible in the histological image in controls at 6 weeks. furthermore, continuing bone remodeling is demonstrated by the existence of osteoclasts and reversal lines at higher magnification (figure 3). the histology of test samples the effects of selenium nanopar6cles on the osseointegra6on of a titanium implant in rabbits: a histomorphometric inves6ga6on vol 13 no 2 (2024) doi 10.5195/d3000.2024.730 h#p://den*stry3000.pi#.edu displayed mature, well-established bone that has osteocytes filled in the thread area and was encircled by osteoblasts. under higher magnification, adult bone contained ha version lamellae (figure 4). figure 3. osteocytes (oc), osteoclasts (ocl), and osteoblasts (arrows) may be seen in the thread region in the six-week control group's view (h&e x20). figure 4. osteocytes (oc) and a reversal line (rl) are seen in the magnified picture of the 6-week test group (h&e x100). bone architecture characteristics were determined by histomorphoetric analysis of the analyzed groups. the experimental and control groups' descriptive statistics for the bone architecture parameters are shown in table 1 for each healing interval. for both the experimental and control groups, the mean values of thread width, trabecular breadth, and cortical bone thickness rose with time. the mean values for the selenium-coated (se coated) group are greater than those for the control group at every stage of recovery. the mean marrow space during the healing process, with the se coated group showing a more pronounced fall than the control group. the mean values for the number of osteoblasts and osteocytes in the experimental and control groups grew over time, although the se coated group's mean values increased more quickly in 1and 6-week intervals than the control group. the osteoclast number was smaller in the se-treated group than in the control group at the same time in week 6 for both groups, based on the mean values. there was a statistically significant difference between the control and experimental groups in each healing phase. discussion assessing the effect of se nps on the bone-implant interaction was the aim of the present study. se nps seem suitable for use in medicine, either on their own or in conjunction with antibiotics that are currently available. the antibacterial properties of se nps stop the growth of pathogens, including bacteria, fungi, and viruses. selenium nanoparticles are thought to be a safer and less hazardous biomaterial for surface coatings than many other nanoparticles, such as copper oxide, zinc oxide, and silver nanoparticles, which are utilized to alter the surfaces of medical devices [6]. the histology observations for the experimental and control groups showed that all sections had a favorable healing course; however, the rates of bone remodeling and deposition varied with each healing interval. after one week of implantation in control animals, the sections clearly indicated the replacement of the blood clot by granulation tissue with a substantial number of collagen fibers, fibroblasts, and osteoblasts, as well as the onset of osteoid tissue formation. in implants that had been treated with se, osteoblast differentiation had already begun to replace the granulation tissue with new bone. implant-associated infections can be prevented or reduced using two primary strategies. anti-adhesive surfaces were developed to stop or reduce germs' initial adherence to the implant surface. the second technique is mixing antimicrobial drugs with the polymeric implant components. surfaces treated with oxygen plasma are among the the effects of selenium nanopar6cles on the osseointegra6on of a titanium implant in rabbits: a histomorphometric inves6ga6on vol 13 no 2 (2024) doi 10.5195/d3000.2024.730 h#p://den*stry3000.pi#.edu surfaces that exhibit adhesion resistance. following a one-week break, the histology sections of the control group revealed delicate bone trabeculae with newly produced woven bone. compared to the control group, the se-treated group had more and thicker bone trabeculae. due to its chemopreventive and chemotherapeutic properties, selenium seems to be a promising anticancer biocompatible orthopedic implant material. se nps can also effectively prevent and treat s. aureus and s. epidermidis infections, which are among the most frequent causes of implant failure. staph. aureus and staph. epidermidis adhesions were greatly decreased when a 0.5 percent sodium selenite solution was applied to the titanium discs [7]. the findings also demonstrated a notable change in bone architecture parameters over time. the length of time spent on bone deposition and maturation may be responsible for increases in trabecular width, cortical width, thread width, and trabecular number over six weeks as opposed to one. conversely, a decrease in bone marrow star volume over time may result from faster bone matrix building, which will cause wider bone trabeculae. these results corroborate research by depprich et al. [8,9], which discovered that enhanced bone-toimplant contact was observed at every stage of recovery by histomorphometric examination. the findings also demonstrated a notable change in bone architecture parameters over time. when bone formation settles and reaches its final measurement, no more osteoblasts are needed beyond those needed to sustain biological activity. this is because the production of more osteoblasts is necessary for any new tissue formation, which explains why the number of osteoblasts and osteocytes increased over time. these findings are consistent with those of al-molla et al. [10-13], who discovered that whereas osteoblast counts decreased with time, osteocyte counts rose. table 1. descriptive data for each group's bone architecture characteristics. bone formation groups n mean std. deviation 95% confidence interval for mean minimum maximum lower bound upper bound control (6w) 8 1.67 0.05 1.15 1. 05 1. 6 1. 8 control coated (6w) 8 2.54 0.1 2 02 2. 8 1.9 2.5 descriptives bone formation groups n mean std. deviation 95% confidence interval for mean minimum maximum lower bound upper bound control (1w) 8 1.13 0. 06 1.01 1.4 1.13 1.45 control coated (1w) 8 1.48 0.08 1.5 1.6 1.43 1.87 the effects of selenium nanopar6cles on the osseointegra6on of a titanium implant in rabbits: a histomorphometric inves6ga6on vol 13 no 2 (2024) doi 10.5195/d3000.2024.730 h#p://den*stry3000.pi#.edu conclusion the study showed that by promoting osteogenic mesenchymal tissue, osteoblast formation, and early osteoid tissue apposition, selenium nanoparticles (senps) enhanced and sped up the osseointegration of titanium implants. references 1. oshida y, tuna e, aktören o, gençay k. dental implant systems. int j mol sci.2010; 11(4): 1580 –1678. 2. papaspyridakos p, mokti m, chen cj, benic gi, gallucci go, chronopoulos v. implant and prosthodontic survival rates with implant fixed complete dental prostheses in the edentulous mandible after at least 5 years: a systematic review. clinical implant dentistry and related research. 2014.16 (5): 705– 717. 3. frisardi g, barone s, razionale av, paoli a, frisardi f, tullio a, lumbau a, chessa g. biomechanics of the pressfit phenomenon in dental implantology: an imagebased finite element analysis. head and face medicine, 2012;8:18. 4. belém a, scombatti s, martins r, yamashina k, iezzi g, piattelli a. influence of implant surfaces on osseointegration. braz dent j, 2010; 21(6): 471-481. 5. xun w, shi l, yue w, zhang c, ren y, liu q. effect of highdose nano-selenium and selenium–yeast on feed digestibility, rumen fermentation, and purine derivatives in sheep. biol trace elem res. 2012;150(1– 3):130–136. 6. pi j, yang f, jin h, et al. selenium nanoparticles induced membrane biomechanical property changes in mcf-7 cells by disturbing membrane molecules and factin. bioorg med chem lett. 2013;23(23):6296–6303. 7. holinka, m. pilz, b. kubista, e. presterl, r. windhager (2013). effects of selenium coating of orthopaedic implant surfaces on bacterial adherence and osteoblastic cell growth. the bone & joint journal vol. 95-b, no. 5. 8. depprich r, zipprich h, ommerborn m, naujoks c, wiesmann h, kiattavorncharoen s, lauer h, meyer u, kübler n, handschel j. osseointegration of zirconia implants compared with titanium: an in vivo study. head & face medicine, 2008; 4:30. 9. ibraheem ns, al-adili ss. assessment of dental implant stability during healing period and determination of the factors that affect implant stability by means of resonance frequency analysis (clinical study). j bagh coll dent [internet]. 2015 27(3):109-15. 10. al-molla b. the effects of the iraqi propolis on the artificial bony defects on the rabbit’s mandible. msc thesis in oral histology, university of baghdad. 2010. 11. al-molla b. evaluation the effects of amelogenin/propolis coating on osseointegration of cp ti implant surface in rabbits. 2014. vol. 1/ issue 3. 12. alheeti oar, fatalla aa. evaluation of the antibacterial effect of selenium nanoparticles in periimplantitis patients. journal of research in medical and dental science [eissn no. 2347-2367 pissn no. 2347-2545] 13. jani gh, al-ameer ss, jawad bds. histological and histomorphometric analysis of strontium chloride coated commercially pure titanium implant compared with hydroxyapatite coating. j of baghdad college of dentistry،325،2218،1-12 734 2024 determina)on of operators’ portable x-ray safety vol 13 no 2 (2024) doi 10.5195/d3000.2024.734 h#p://den*stry3000.pi#.edu determination of operators’ portable x-ray safety mohammed ayad taha, ahmed i. al-jobory, haitham y. mohammed college of dentistry, tikrit university, iraq abstract objec;ve: ionizing radia;on in the form of rays interacts with mader and has immediate and delayed impacts. applica;ons in den;stry include extraoral and intraoral x-rays and machines can be portable and fixed. a portable x-ray machine is a mobile x-ray device that is smaller than a sta;onary one. the radia;on dose from portable devices to which exposure is allowed should not be higher than 20 msv annually. the purpose of this study was to compare three portable dental x-rays to determine the quan;ty of dispersed radia;on that affects radiography operators. methods: three types of portable x-ray devices (portable x-ray system (clarox, eigh;eth, and qin high-frequency microfocus dental x-ray units) were used. an extracted tooth was used for tes;ng. the tooth was x-rayed laying on a flat surface aver pouring it into a stone base. three different radia;on devices of different sorts were posi;oned in front of the tooth individually, with iden;cal dimensions for each device. more than one radiological image was obtained with each of the radia;on devices, and radia;on levels were recorded. results: it was discovered that as radiological images were being taken, the scadered radia;on readings for two devices (clarox and eigh;eth) progressively decreased. the recorded readings for qin were lower. conclusion: comparing the radiometer results revealed that the portable x-ray equipment emits radia;on that has an impact on radia;on workers. therefore, radia;on safety precau;ons must be followed, and radia;on shields must be employed. keywords: portable x-ray unit, scadered radia;on, radia;on dose, occupa;onal dose, measuring radia;on device. cita:on: taha ma, et al. (2024) determina:on of operator’s portable x-ray safety. den:stry 3000. 1:a001 doi:10.5195/d3000.2024.734 received: september 20, 2024 accepted: september 22, 2024 published: october 7, 2024 copyright: ©2021 taha ma, et al. this is an open access ar:cle licensed under a crea:ve commons auribu:on work 4.0 united states license. email: mohammedayad@tu.edu.iq introduction as it meets matter, ionizing radiation may take the form of rays, which are a subtype of this kind of radiation. radiation is a natural occurrence that may also be produced artificially, and it can come from either natural or manmade sources. the induction of cellular damage by radiation has a dramatic impact on living organisms and poses a risk to both healthy and sick tissues. there is a wide range of responses to radiation in humans, and these responses are determined by several factors. these factors include the radiation source, the radiation dosage (the amount of radiation energy received), the length of exposure, and, most importantly, the genetic and epigenetic makeup of the individual who was exposed to the radiation [1]. the german scientist wilhelm konrad rontgen discovered x-rays in 1895 when he was researching the effects of electron beams, which were termed cathode rays at the time. rontgen was observing the effects of electrical discharges passing through low-pressure gases [2]. a screen that was coated with fluorescent material and put outside of a discharge tube would glow even when it was insulated from the direct visible and ultraviolet light that was emitted by the gaseous discharge, which was a striking phenomenon that was discovered by roentgen. after that, radiation that was not visible from the tube traveled through the surrounding air and caused the screen to fluoresce [3]. roentgen was able to determine that the radiation that was responsible for the fluorescence came from the location where the electron beam contacted the glass wall of the disc fluorescence. this was made possible by the fact that roentgen determina)on of operators’ portable x-ray safety vol 13 no 2 (2024) doi 10.5195/d3000.2024.734 h#p://den*stry3000.pi#.edu was able to determine the origin of the radiation. disc fluorescence, when put between the tube and the screen, was found to be transparent to the newly discovered kind of radiation. rontgen vividly showed this by taking a photographic picture of the bones of the human hand [4]. together with the discoveries of radioactivity and the electron (1897), it ushered in the study of the atomic world and the period of modern physics (1896). his discovery, which he termed rontgen rays, was received with enthusiasm both in the scientific community and in the general public all across the globe [1]. in the beginning, the uses of x-rays were limited to medicine, given that these rays were a turning tool [1-5]. areas of the body to look for any physical imbalance in the various areas of the self-roentgen rays swept the world. everyone took advantage of it, including the doctors who excelled and went to great lengths in depicting the disease’s skeletons. at that time, they considered that the long period of exposure to these photons may cause diseases, including cancer [6]. there are several applications of x-rays in medicine [7], and dental radiographs, more often known as x-rays, are pictures that are used to assess dental health. images of the inside of teeth and gums may be taken using these low-radiation xrays, which are used to examine patients' oral health. this may be useful in assisting dentists in the diagnosis of issues such as cavities, dental decay, and impacted teeth. while dental x-rays may seem to be complicated, they are rather frequent and are just as vital as regular teeth cleaning [8]. types of dental x-rays include extra and intraoral x-rays. extraoral xrays include: (a) panoramic radiography is a kind of radiography that offers information in two dimensions on the teeth and the maxillofacial skeleton. it enables one-time imaging of all teeth, the mandible, and parts of the maxilla, including a large portion of the maxillary sinus, the hard palate, and the temporomandibular joints (tmjs), making it a useful adjunct for the diagnosis and planning of treatment [9]. (b) tomograms highlight a specific layer, sometimes known as a "slice," of the mouth while blurring out the surrounding layers. this xray investigates features that are difficult to detect because of the presence of additional structures in the immediate area that impede vision [10]. (c) cephalometry is an imaging method that is used in orthodontics to measure the size of the head, jaws, and teeth by using landmarks or points on the skull. this approach is used to determine the spatial connections between the head, jaws, and teeth. it is used for diagnosis, the planning of therapy, and the evaluation of dentofacial changes as treatment progresses [11]. (d) magnetic resonance imaging, often known as mri, is a procedure that utilizes magnetism and radio waves to create pictures of a patient's anatomy that are crosssectional. this approach also takes a perspective in three dimensions of the oral cavity, showing the jaw and the teeth. this is perfect for assessing the condition of soft tissue [12]. the use of radiographic film is being phased out in favor of edigital imaging. the patient is exposed to a lower radiation dose with this new technology, images are available almost instantly without the need for chemical film processing, and there is the possibility of image enhancement and computer-aided feature extraction. these advantages are all brought about by the advancement of technology. traditional film-based imaging has its limitations as compared to digital radiography, which paves the way for novel diagnostic determina)on of operators’ portable x-ray safety vol 13 no 2 (2024) doi 10.5195/d3000.2024.734 h#p://den*stry3000.pi#.edu techniques that were previously unavailable [13]. the f-dental cone beam computed tomography (ct) x-ray machine is a specialized sort of imaging device that is used in instances in which standard dental or face x-rays are not enough. since the radiation dose from this scanner is substantially higher than that which is received through standard dental x-rays, it is rarely used daily [14]. intraoral x-rays include: (a) periapical x-rays are those that show the whole tooth, from the crown down to where the tooth is attached in the jaw, which is beyond the end of the root. each periapical x-ray displays this whole tooth dimension and includes all the teeth that can be seen in either the upper or lower jaw region where the image was taken. x-rays taken from the periapical region are examined for any abnormalities in the root structure and the bone structure that surrounds it [15]. (b) bite-wing x-rays focus on one region of the mouth and provide specifics about the upper and lower teeth there [16]. each bitewing exposes a tooth from the top of the tooth down to a level approximately equivalent to the level of the supporting bone. x-rays taken with a bitewing are used to identify cavities between teeth as well as shifts in bone density brought on by periodontal disease. they may also be used to determine the correct fit of a crown (or cast restoration) and the marginal integrity of fillings, both of which are very important applications [17]. (c) occlusal radiography is described as those intraoral radiographic methods that are taken using a dental x-ray system in which the image receptor (film packet or digital phosphor plate 5.7 x 7.6 cm) is positioned in the occlusal plane. this kind of radiography is used to examine the teeth and jaws. there are no solidstate digital sensors of an appropriate size that are presently available [18]. the dentist will utilize x-rays to take photographs of the patient's teeth using film. this will allow the dentist to identify any damaged teeth and place an order for their repair. unfortunately, film development takes some time. in addition to the difficulties of keeping and capturing such videos, there is a limited amount of space available [19]. as a result, it pioneered the technique of photography using film and gradually started to phase it out in favor of digital photography with digital sensors. throughout the imaging procedure, the patient will be subjected to a lower level of radiation exposure if this approach is used. the picture is shown in real-time on the display of the monitor [20]. types of digital x-ray machines include portable, fixed and mobile x-rays [21]. radiographers, veterinarians, and dental professionals can take x-ray images of patients without having to call them into a special leadlined room. radiographs are an essential component of routine clinical dentistry. equipment is typically either fixed (mounted to the wall, floor, or ceiling) or mobile (mounted on a tripod that is moved around on a set of wheels) [22]. the portable x-ray unit that can be carried in one's hand and is powered by a battery is a device that is relatively new to the market. in the past, the vast majority of handheld, portable x-ray devices were modified versions of existing equipment. these machines were designed for use in military medical, humanitarian missions, and training exercises. moreover, they find uses in veterinary medicine, crime and catastrophe situations for forensic dental imaging, and archaeological excavation sites. individuals in need of dental care who are determina)on of operators’ portable x-ray safety vol 13 no 2 (2024) doi 10.5195/d3000.2024.734 h#p://den*stry3000.pi#.edu confined to their homes or institutionalized and have restricted mobility, as well as those who are receiving general anesthesia, may benefit from the use of these devices [23]. when the effective dosage is computed directly, handheld portable x-ray machines emit radiation below the recommended dose. this was determined by comparing the amount of radiation to the effective dose [24]. the distance away from the body varies, depending on the position of the hands and how the portable x-ray equipment is handled by the operator. this is because the distance away from the body is dependent on the position of the hands. in addition to this, a backscatter shield is the sole integrated protection that is provided for the operator [25,26]. because of this, it is still debatable as to whether these precautions are enough to protect the operator from being unnecessarily exposed to radiation. occupational dose monitoring is often advised for these operators [27]. when utilizing either stationary or mobile x-ray units, it is always recommended to keep a safe distance, remain outside the "controlled zone," and refrain from holding the x-ray equipment to reduce the risk of radiation exposure caused by radiation leakage from the x-ray tube head and backscatter [28]. there is a concern with handheld portable xray devices regarding the exposure of the operator [29]. when x-ray equipment is being used, it is customary to have a certain location that is known as a "controlled area" in the united kingdom. this is done to protect the personnel and the public from any possible radiation exposure that may not be required. the "controlled region" is defined as a zone that is 1.5 meters away from both the patient and the head of the x-ray tube and is located anywhere along the path of the primary x-ray beam. any radiation that occurs beyond the "restricted region" is adequately dampened by the combination of distance and shielding. in addition to this, the operator needs to be at a safe distance away from the "controlled region," at least two meters away from the x-ray source [30]. the radiation dose resulting from the portable devices to which exposure is permitted should not exceed 20 msv per year [31]. the permissible amount of radiation exposure during the radiation shot should not exceed 10 msv per shot [32]. any dose exceeding that is considered dangerous and threatens the life of the worker in the field of radiation [33]. the aim of this work was to compare three portable dental x-rays to determine the quantity of dispersed radiation that potentially affects radiography operators. material and methods we used three types of portable xrays devices: clarox, eighteeth, and qin high-frequency microfocus dental x-ray unit (figure 1 and table 1). figure 1. the three portable x-ray devices used in the study. determina)on of operators’ portable x-ray safety vol 13 no 2 (2024) doi 10.5195/d3000.2024.734 h#p://den*stry3000.pi#.edu device name eightieth portable x-ray system qin high-frequency microfocus dental x-ray unit model hyperlight clarox vx-3de class ibf type tube voltage 70 kv 70kv 70kv exposure time 0.35 0.35 0.1_2 focal spot size 0.4mm 0.4mm 0.3mm tube current 2ma 2ma 1ma manufacture china korea china table 1: information about the three portable x-ray devices used in the study. we also used a radiometer for measuring scattered radiation from the x-ray machines (figure 2) and an intraoral digital imaging sensor model pos-ray1 (figure 3). figure 2. radiation measuring device used in the study. figure 3. intraoral digital sensor used in the study. we used an extracted tooth (lower left first molar), simulating a root canal treatment, fixed on stone to perform the analysis (figure 4). figure 4. tooth used for testing. the tooth was placed and fixed on a stable horizontal surface, and the sensor was placed directly behind the sample. the tooth was fixed by making a small mold of wax connected to the electric wire as shown in figure 5, to keep it stable throughout the work period. for all x-ray units, they were placed 5 cm from the tooth then the radiometer was placed 20 cm from the field of work as shown in figure 6. the x-ray machine was turned on and radiological images were taken at an exposure time of 0.35 seconds and 70 kv for all images. figure 5. standardized position of the sensor. determina)on of operators’ portable x-ray safety vol 13 no 2 (2024) doi 10.5195/d3000.2024.734 h#p://den*stry3000.pi#.edu figure 6. distance between sample x-ray device and radiation measuring. descriptive statistics including minimum, maximum, mean, and standard deviation were calculated for the dye penetration obturating material for each group. the data were collected and analyzed using spss (version 23) for statistical analysis. one-way analysis of variance (anova) was used to determine whether there was a statistical difference in the radiation readings among the groups. then, post hoc duncan's multiple range was used to determine the exact location of difference between groups. a pvalue of <0.05 was considered statistically significant. results according to the results obtained after using the clarox and eightieth devices, there was a decrease in the readings of the x-ray, which meant the amount of radiation diffused from the x-ray device and reaching the operator decreased (tables 3 and 4). the qin high frequency microfocus dental x-ray unit showed lower readings compared to the other units, as shown in table 4. table 2. recorded readings for clarox. radiograph recorded readings (msv) msv statistics 1st 349 mean: 197.6 standard deviation: 90.97 median: 195 minimum: 50 maximum: 349 2nd 284 3rd 260 4th 243 5th 202 6th 188 7th 170 8th 123 9th 98 10th 50 table 3. recorded readings for eightieth. radiograph recorded readings (msv) msv statistics 1st 350 mean: 231.4 standard deviation: 50.84 median: 207.5 minimum: 192 maximum: 350 2nd 286 3rd 245 4th 211 5th 231 6th 197 7th 192 8th 203 9th 195 10th 204 table 4. recorded readings for qin high frequency microfocus dental x-ray unit. radiograph recorded readings (msv) msv statistics 1st 93 mean: 103.6 standard deviation: 17.49 median: 100 minimum: 71 maximum: 129 2nd 129 3rd 71 4th 121 5th 97 6th 103 7th 87 8th 105 9th 110 10th 120 discussion the maximum occupational effective dose, according to icrp (the international commission on radiological protection), is 20 msv/year averaged over five years (i.e., a cap of 100 msv in five years), with no annual effective dosage surpassing 50 msv/year. the work dose limit is defined by ncrp (national council on radiation protection and measurements) as 50 msv in any one year and a lifetime limit of 10 msv multiplied by the person's age in years and the permissible dose for daily use is 2msv [34]. patients and dental practitioners are constantly exposed to radiation determina)on of operators’ portable x-ray safety vol 13 no 2 (2024) doi 10.5195/d3000.2024.734 h#p://den*stry3000.pi#.edu during diagnosis, but the percentage is small compared to medical staff. continuous exposure to radiation, even at low doses, may have long-term health effects on radiation practitioners [35]. it is important to evaluate and measure the radiation between the portable device and the doctor after taking the radiation, meaning that the focus is on the personal protection of radiation practitioners from dangerous doses [36]. a handheld portable x-ray unit is a useful addition to the dental practice or public health sector when used appropriately. it has been showed that there is no additional radiation when compared to a conventional wallmounted unit [37]. in accordance with previous data, we found that there is a potential effect of radiation [38]. routine radiology device calibration is thought to be important and has an impact on the accuracy of the device's readings [39]. radiation-related workers who lack the proper training, may be at higher risk [40]. the validity of the used instruments of this study may have an impact on the reading of the resulting rays [41]. the radiation measurements are also impacted by the subpar aluminum filter, area locator, and cone in the x-ray machine [42]. conclusion the radiation produced by portable x-ray machines potentially affects workers, but are still within permissible limits, and routine use of them may result in tissue damage. therefore, it is especially important to follow radiation protection procedures and to wear a lead shield for daily use. this confirms the invalidity of the companies’ words about the absence of radiation emitted and the lack of the need to wear protective equipment. conflicts of interest the authors declare no competing interest. references 1-ribeiro da, de oliveira g, de castro g, angelieri f 2008. cytogenehc biomonitoring in pahents exposed to dental x-rays: comparison between adults and children. dentomaxillofac radiol oct;37(7):404-407 2-alternate htles: röntgen radiahon, x-radiahon. hkps:/ /www.britannica.com/science/au ger-effect 3-alternate htles2021: x-rays mine anhque “locked leker,” promphng look at x-ray tech december 6 hkps: //icetonline.com/x-rays-mineanhque-locked-leker-promphnglook-at-x-ray-tech/ 4-imaging final – unit 1, unit 2, basics of digitalhkps://quizlet.com/755964 842/imaging-final-unit-1-unit-2basics-of-digital-flash-cards/ 5-early clinical use of the x-ray, trans am clin climatol assoc. 2016; 127: 341– 349.hkps://www.ncbi.nlm.nih.gov /pmc/arhcles/pmc5216491/ 6understanding 2017 tests used to detect bone problems created: november 7, 2013; last update: december 27, next update: 2020. 7-brian krans2020. x-ray. retrieved on the 12th of december, hkps://my.clevelandclinic.org/hea lth/arhcles/11199-dental-x-rays 8medically reviewed,2023, by chrishne frank, dds — by brian krans — updated on february 21, 9 -ingrid różyło kalinowska 2021, panoramic radiography in denhstry, august 2021 / accepted: 5 october 2021 / published online: 7 december determina)on of operators’ portable x-ray safety vol 13 no 2 (2024) doi 10.5195/d3000.2024.734 h#p://den*stry3000.pi#.edu hkps://doi.org/10.1007/s41894021-00111-4 10-luisa vigorelli,2021 x-ray micro-tomography as a method to dishnguish and characterize natural and culhvated pearls 15 october. 11world j radiol,2014 recent advances in imaging technologies in denhstry, oct 28; 6(10): 794– 807. 12lav kumar niraj 2016, mri in denhstrya future towards radiahon free imaging – systemahc reviewoct 1. doi: 10.7860 /jcdr/2016/19435.8658. 13-medicine, 2012, digital radiography with dr there is a specialized digital plate that replaces the convenhonal casseke from: the manual of exohc pet prachce. 14-ceo,2018: tomislav mihaljevic hkps ://www. radiologyinfo.org/ en/info/dentalconect. 15-dentomaxillofac radiol. 2002 may;31(3):1596hkps://my.clevelandclinic.org/he alth/arhcles/11199-dental-x-rays 16-mabak, 2018 dental x-rays – what they reveal by medikabazaar 2021· september 24. 17-hkps://dentalsouth.com/dental-x-rays-whatthey-reveal. 18-a complete guide to portable medical x-ray machines 26.03.20 | x-ray, equipmenthkps://pocketdenhstry .com/11-occlusal-radiography/ 19-coy j. ahnd-held 2023academy of general denhstry. x-rays. (hkp://www.knowyourteeth.com/ infobites/abc/?abc=x) accessed 2/9/. 20-coy j, vandre rh 2023, the photographic process and film sensihvity hkp://www.sprawls.org/ppmi2/fi lmsen/ 21-danforth ra, herscha| ee, leonowich ja2009. operator exposure to scaker radiahon from a portable hand-held dental radiahon emi}ng device (aribex nomad) while making 915 intraoral dental radiographs. j forensic sci. 2009 mar;54(2):41521. epub jan 29. hkps://www.browiner.com/produ cts-soluhons/ 22radiahon protechon of pahents in dental radiology hkps://www.iaea.org/resources/r pop/healthprofessionals/denhstry/pahents 2022 23-jimmy makdissi the effects of device posihon on the operator’s radiahon dose when using a handheld portable x-ray device. dentomaxillofac radiol. 2016 mar; 45(3): 20150245. 24ravikiran r pawar, 2016 the effects of device posihon on the operator’s radiahon dose when using a handheld portable x-ray device hkps://www.ncbi.nlm.nih.gov/pm c/arhcles/pmc4846146/ 25-korean j pain 2017 hkps://www.ncbi.nlm.nih.gov/pm c/arhcles/pmc4846146/ 26e: chiltoninformaho, 2016, guidance on the safe use of handheld dental x-ray equipment 27-almrsal.com was first indexed by google more than 10 years ago hkps://www.almrsal.com/post/87 9295 28-vissink a, jansma j2003, spijkervet f, burlage f, coppes r. oral sequelae of head and neck radiotherapy. crit rev oral biol med; 14(3):199-212 29-kazuki otomo 2023 et al. bioengineering (basel). feb 16;10(2):259 30-course of radiological technology, health sciences, tohoku university graduate school of medicine, 2-1 seiryo, aoba-ku, sendai 980-8575, japan determina)on of operators’ portable x-ray safety vol 13 no 2 (2024) doi 10.5195/d3000.2024.734 h#p://den*stry3000.pi#.edu 31dental radiographic examinahons2012: recommendahons for pahent selechon and limihng radiahon exposure. chicago: american dental associahon, council on scienhfic affairs; 27p. 32makdissi, j., pawar2015, r. r., johnson, b., & chong, b. s. (2016). the effects of device posihon on the operator's radiahon dose when using a handheld portable x-ray device. dent maxillofacial radiology, 45(3), 1-7. doi: 10.1259/mfr. 0245 33-dr. zeyad alani 2018 (dr. zeyad alani is a denhstry prachhoner in rancho cordova, ca. he currently prachces at rancho cordova smiles denhstry dental group. publicahon of a scienhfic arhcle in history 31 october 34-t.j. boal, m. pinak 2014, dose limits to the lens of the eye: internahonal standards related safety basic and guidance,hkps://www.icrp.org/do cs/icrp2013%20s2%20boal.pdf 35-su-yeon hwang, 2018, health effects from exposure to dental diagnoshc x-ray, environ health toxicol. dec; 33(4): e2018017. 36-β. ε. κeane 1975, manual on radiahon protechon in hospitals and general prachce, volume 3, xray diagnosis medical physics department, royal sussex county hospital, brighton, england. 37w e r berkhout,2015, jushficahon and good prachce in using handheld portable dental xray equipment: a posihon paper prepared by the european academy of dent maxillofacial radiology (eadmfr), dentomaxillofac radiol. jul; 44(6): 20140343. 38hkps://abcnews.go.com/health/ wellness/century-ray-machineshows-radiahon-risksyore/story?id=13140857. 39-ngoc toan tran,2012. calibrahon of kvp meter used in quality control tests of diagnoshc x-ray units, pmid: 21447505 doi: 10.1093/rpd/ncr037 40-salman akram; yuvraj s. chowdhury.2022, radiahon exposure of medical imaging last update: november 14. 41-adrian p. brady 2017, error and discrepancy in radiology: inevitable or avoidable, insights imaging. feb; 8(1): 171–182. 42-ms.tech.khaldun, 2020, techniques of medical radiological devices hkps://www.esraa.edu.iq/module s/lect/lect/73154600593314464.p df microsoft word 189 2022.docx the effect of using an educa3onal poster on mothers’ knowledge of emergency management of dental avulsion vol 10 no 1 (2022) doi 10.5195/d3000.2022.189 h#p://den*stry3000.pi#.edu the effect of using an educational poster on mothers’ knowledge of emergency management of dental avulsion seyedeh hediyeh daneshvar1, melika khaef2 1 assistant professor, dental sciences research center, department of dentistry, school of dentistry, guilan university of medical sciences, rasht, iran 2 dentist, private practice abstract introduction: mothers are commonly the first ones who encounter the dental avulsion in children. high knowledge of them could greatly affect the success rate of treatment. the purpose of this study was to evaluate the effect of educa3onal poster on the mother’s knowledge about management of dental avulsion. materials and methods: this study was conducted at the department of pediatric den3stry, guilan university of medical sciences from november 2020 to february 2021. aner obtaining the informed consent, a ques3onnaire containing the mothers’ sociodemographic informa3on and 8 ques3ons regarding knowledge about dental avulsion was responded alone by 262 mothers (131 in the study group and 131 in control group) before educa3onal poster presenta3on in the study group. aner three months, the par3cipants of both groups were re-evaluated using the same ques3onnaire. mcnemar’s test and generalized es3ma3ng equa3ons (gee) was used to analyze the data. results with p < 0.05 were considered sta3s3cally significant. results: use of an educa3onal poster improved the knowledge of par3cipants of study group about dental avulsion, although this improvement was sta3s3cally significant in ques3ons regarding immediate emergency ac3on (p=0.003), appropriate 3me for replanta3on (p<0.001), cleaning before replanta3on of a dirty tooth(p=0.022) and suitable storage medium for transferring an avulsed tooth (p<0.001). it was found that variables like age, level of educa3on, employment status and previous training had no significant effect on par3cipants knowledge(p>0.05). conclusion: results showed that the use of posters as an educa3onal method can effec3vely improve mothers’ knowledge about management of children’s dental avulsion. keywords: tooth avulsion; knowledge; pediatric den3stry cita9on: daneshvar, sh et al. (2022) the effect of using an educa9onal poster on mothers’ knowledge of emergency management of dental avulsion den9stry 3000. 1:a001 doi:10.5195/d3000.2022.189 received: may 28, 2021 accepted: september 13, 2021 published: may 9, 2022 copyright: ©2022 daneshvar sh et al. this is an open access ar9cle licensed under a crea9ve commons axribu9on work 4.0 united states license. email: hedyehdaneshvar@gmail.com introduction traumatic dental injury (tdi) is an important global oral health concern in childhood that causes functional, psychological and esthetic problems [1]. tdis affect teeth, their supporting structures, and adjacent soft tissue and range from minor enamel cracking to extensive maxillofacial injury [2-4]. parents of the children are usually the first ones to report dental trauma to dentists; therefore, the parents’ awareness of emergency management is important [5,6]. unfortunately, the children are often referred to a dentist without any emergency management at the accident site that results in irreversible consequences such as tooth loss and aesthetic problems in anterior region [7]. frequency of tooth avulsion in permanent and in primary dentition is 0.5%–16% and 7–13%, respectively [3]. avulsion of permanent anterior teeth with delayed reimplantation and unphysiological storage lead to dehydration of the root surface and decrease the survival rate of the effect of using an educa3onal poster on mothers’ knowledge of emergency management of dental avulsion vol 10 no 1 (2022) doi 10.5195/d3000.2022.189 h#p://den*stry3000.pi#.edu reimplanted tooth [8,9]. with appropriate emergency treatment, the prognosis of the traumatic dental injury can be improved and ultimately preserve the smile of the child [10]. educational programs and training are needed to improve proper management of dental avulsion by mothers. educational posters and pamphlets can be used to inform parents about guidelines on management of dental trauma [10]. the purpose of this study was to evaluate the effectiveness of using an educational poster to increase parents’ knowledge about management of dental avulsion. it is necessary to be aware of the knowledge level of parents to formulate a set of instructions to increase the parents’ knowledge and help them to properly manage an emergency case of dental avulsion. material and methods ethical aspects this cross-sectional descriptive analytical study was approved by guilan university of medical sciences (ir.gums.rec.1399.311) in 2020/10/07. sample this study was done on 262 mothers accompanying children between 4-12 years of age seeking dental treatment at the department of pediatric dentistry, guilan university of medical sciences from november 2020 to february 2021. mothers between 20 and 60 years of age were included in the study. all participating subjects (n=262) were divided randomly into two groups (testcontrol), 131 mothers were allocated randomly to the test group and others to the control group. questionnaire after explaining the nature and purpose of the study by a pedodontist to the mothers, subjects who agreed to participate, signed a statement of informed consent and filled out questionnaire alone which assessed theirs' socio-demographic information and knowledge about dental avulsion. a total of 8 questions regarding identification of tooth type, how to clean an avulsed tooth, how to transfer an avulsed tooth (suitable storage medium) and appropriate time for referring for the problembased section were included, in which the level of knowledge of respondents were assessed and classified into three groups, which are low (0-3), moderate (4-6) and high (78). validity of the questionnaire was checked by an expert panel of pediatric dentists and in a pilot study on 10 subjects, the reliability of the questionnaire was tested by a testretest method. after filling the questionnaire, an educational poster was distributed among subjects of the study group. three months later, the mothers’ knowledge was evaluated again in both groups using the same questionnaire. educational poster the educational poster used in this study was designed at the dental research center of guilan university of medical sciences by a group of three pedodontists and included guidelines on emergency management of dental avulsion. in cases of primary dental avulsion, replantation is contraindicated but in permanent dental avulsion: • hold the tooth by its crown, not by its root • wash the tooth under a slow stream of cold water • put the tooth in cold milk, saline or saliva • replant the tooth in its socket • visit a dengst immediately (within 30 min) [3] (figure 1,2) figure 1. avulsion of the tooth figure 2. replantation of the teeth the effect of using an educa3onal poster on mothers’ knowledge of emergency management of dental avulsion vol 10 no 1 (2022) doi 10.5195/d3000.2022.189 h#p://den*stry3000.pi#.edu statistical analysis the data obtained on the questionnaires were analyzed with the aid of the statistical package for the social sciences (spss®, version26.0, chicago, il, usa). mcnemar’s test was used separately in both study and control groups for evaluating the changes of the percentage of correct answers after 3 months. generalized estimating equations (gee) was used to investigate the simultaneous effect of existing independent variables such as age, level of education, employment status and training history on participants knowledge. results with p <0.05 were considered statistically significant. results this cross-sectional descriptive study was conducted among 262 mothers. socio-demographic characteristics of participants have been shown in table 1. knowledge level of participants in the study and control groups before intervention and 3 months later has been shown in table 2. the percentage of the participants with low knowledge decreased and the percentage of participants with high knowledge increased in two groups but this change was greater in the study group. table 3 shows the number and percentage of the participants who answered the questions correctly. results showed that the use of an educational poster improved the knowledge of mothers about dental avulsion, although this knowledge improvement was statistically significant in questions regarding immediate emergency action(p=0.003), appropriate time for replantation (p<0.001), cleaning before replantation of a dirty tooth(p=0.022) and suitable storage medium for transferring an avulsed tooth (p<0.001). variables study group(n=131) n(%) control group(n=131) n(%) age 33 years or younger 34 years or older 68(51.9 %) 63(48.1 %) 71 (54.1 %) 60(45.9 %) educational level diploma and associate’s degree bachelor’s and master’s degree doctorate degree 13(9.9 %) 105 (80.2 %) 13(9.9 %) 60(45.8 %) 71(54.2 %) 0(0%) training history yes no 8(6.2 %) 123(93.8 %) 4(3.1 %) 127(96.9 %) employment status yes no 63(48.1 %) 67(51.9 %) 51(38.9 %) 80(61.1 %) variables low knowledge n(%) moderate knowledge n(%) high knowledge n(%) study group before intervention after 3 months 17(12.9%) 0(0%) 63(48.4%) 9(6.9 %) 51(38.7%) 122(93.1%) control group before intervention after 3 months 38(29%) 17(12.9%) 72(54.9 %) 84(64.1%) 21(16.1 %) 30(23%) table 1: participant’s socio-demographic characteristics table 2: knowledge level of participants in study and control groups before intervention and 3 months later the effect of using an educa3onal poster on mothers’ knowledge of emergency management of dental avulsion vol 10 no 1 (2022) doi 10.5195/d3000.2022.189 h#p://den*stry3000.pi#.edu variables such as age (p = 0.564), level of education [diploma and associate’s degree (p = 0.091) bachelor’s and master’s degree (p = 0.330), doctorate degree (p = 0.357)], training history (p = 0.316) and employment status (p = 0.357) had no significant effect on knowledge of participants. discussion tooth avulsion is considered as one of the most detrimental traumatic dental injuries among children [3]. mother’s high knowledge regarding the emergency management of tooth avulsion injury will help to reduce the time interval between avulsion and replantation. immediate action may greatly affect the prognosis of the avulsed tooth and enhance the success rate of treatment [11]. the purpose of this study was to investigate the knowledge of mothers about the emergency management of dental avulsion before and after distribution of an educational poster. present study revealed insufficient knowledge among mothers regarding emergency management of dental avulsion; the main reason is not having previous good training regarding dental avulsion. although most studies conducted in different countries indicated that the level of knowledge regarding the management of avulsed permanent teeth is low [12], this finding is in accordance with other studies of parental knowledge performed in singapore [13] and kuwait [14]. similar to the study by murali et al [15], more than half of the mothers were not aware of immediate emergency action and reimplantation of the avulsed permanent tooth in children. their first action after tooth avulsion injury was to only calm the study group(n=131) control group(n=131) before intervention n (%) after 3 months n (%) p-value* before intervention n (%) after 3 months n (%) p-value* identification of permanent tooth 110(83.9%) 127(96.8%) 0.219 55(41.9%) 97(74.2%) 0.002 immediate emergency action 80(61.3%) 127(96.8%) 0.003 46(35.5%) 63(48.4%) 0.219 knowledge of holding the avulsed tooth by its crown, not by its root 114(87.1%) 122(93.5%) 0.687 114(87.1%) 122(93.5%) 0.500 appropriate time for replantation 67(51.6%) 118(90.3%) <0.001 38(29%) 34(25.8%) 0.999 cleaning before replantation of a dirty tooth 55(41.9%) 93(71%) 0.022 84(64.5%) 110(83.9%) 0.07 suitable storage medium for transferring an avulsed tooth 21(16.1%) 101(77.4%) <0.001 21(16.1%) 21(16.1%) 0.999 identification of primary tooth 118(90.3%) 131(100%) 0.25 93(71%) 97(74.2%) 0.999 contraindication of primary tooth replantation 89(67.7%) 110(83.9%) 0.125 67(51.6%) 80(61.3%) 0.375 table 3: proportions of participating mothers who answered the questions correctly before intervention and three months later *p-value was based on mcnemar test (p<0.05) the effect of using an educa3onal poster on mothers’ knowledge of emergency management of dental avulsion vol 10 no 1 (2022) doi 10.5195/d3000.2022.189 h#p://den*stry3000.pi#.edu child down and compress the bleeding without searching for the avulsed tooth. most parents didn’t pick up the avulsed tooth because they though it is an infected material and needs to be thrown out [15]. avulsed permanent teeth should be cleaned with saline solution before replantation [12]. 53.2% of participants selected saline solution whereas 41.5% of the participants of study by jain a et al [12] chose saline as appropriate cleansing medium. unfortunately in the studies by abdellatif am [8] and al-jame q et al[16] lack of knowledge regarding cleansing medium was reported. in our study, 16.1% of participants chose milk as storage medium. the suitable storage medium should be capable of preserving cell vitality, clonogenic capacity and easily accessible at the site of the accident. although hank's balanced salt solution (hbss, save-a-tooth® ) has been proven to be the most effective storage media, low-fat milk is a good alternative to hbss which is readily available and affordable[12]. in the study by jain a et al [12] and santos me et al [17] as well, only 14.2 % and 3% of parents selected milk as suitable storage medium respectively. in the present study, most of the mothers were aware of not rubbing the root of tooth and holding the avulsed tooth by its crown. whereas most of the participants of study by oliveira tm et al [18] answered this question incorrectly. in the study by abdellatif am et al [8] most of the parents preferred holding the avulsed tooth from the crown. they were aware that holding the tooth from the root will disturb the viability of the periodontal ligament. in the study by ozer s et al [19] more than half of the participants were aware of appropriate replantation time of an avulsed permanent tooth. in the present study, most mothers didn’t know that for optimal healing, professional help should be sought within 30 min and knowledge of study group regarding appropriate time for replantation was significantly improved after education using poster. similar to our study, abdellatif am et al [8] reported that a few parents were willing to replantation of primary tooth which could be related to lack of knowledge about the hazards of replanting the primary teeth. similar to the present study, resmy n et al [5] concluded that the educational status of mothers was not playing a role in their knowledge regarding the management of avulsed teeth in children. most studies [10,20,21] investigated the effect of educational poster on the knowledge of school teachers and students or fitness trainers but we studied the effect of educational poster on the knowledge of mothers. in the present study, educational poster effectively enhanced the knowledge of study group participants compared to control group. lieger o et al [10] showed positive effect of educational poster on the knowledge of school teachers. in the study by young c et al [20] educational poster significantly improved the level of knowledge of secondary school students in hong kong. ramezani gh et al [21] concluded that educational pamphlets enhanced the knowledge level of fitness trainers, but the magnitude of this effect was not significant in their study. conclusion educational poster statistically significantly improves mothers' knowledge about management of children's dental avulsion, so intervention program by different means should be developed targeting parents. acknowledgement we would like to thank department of pediatric dentistry, school of dentistry, guilan university of medical sciences for their great support. references 1. knowledge, awareness and a0tude towards emergency management of dental trauma among the parents of kolkata-an ins=tu=onal study. kaul r, jain p, angrish p, saha s, patra tk, saha n, mitra m. j clin diagn res.2016 the effect of using an educa3onal poster on mothers’ knowledge of emergency management of dental avulsion vol 10 no 1 (2022) doi 10.5195/d3000.2022.189 h#p://den*stry3000.pi#.edu jul;10(7):zc95.doi:10.7860/jcdr /2016/20682.8208. 2. awareness of emergency management of dental trauma. namdev r, jindal a, bhargava s, bakshi l, verma r, beniwal d. contemp clin dent. 2014;5:507-13. doi: 10.4103/0976-237x.142820. 3. textbook and color atlas of trauma=c injuries to the teeth. 3rd ed. andreasen jo, andreasen fm. copenhagen, denmark: munksgaard; 1994. p. 383-425. 4. knowledge and a0tude of 2,000 parents (urban and rural—1,000 each) with regard to avulsed permanent incisors and their emergency management, in and around davangere. shashikiran nd, reddy vv, nagaveni nb. j indian soc pedod prev dent. 2006;24:116-21. doi: 10.4103/0970-4388.27891. 5. knowledge, a0tude, and awareness of mothers toward emergency management of dental trauma in high literacy popula=on. resmy n, parvathy k, arun xm, balagopal v. j int oral health. 2019;11:287-92.doi:10.4103/jio h.jioh_59_19. 6. knowledge, a0tude and prac=ce in emergency management of dental injury among physical educa=on teachers: a survey in bangalore urban schools. mohandas u, chandan gd. j indian soc pedod prevent dent. 2009;4:243-8. doi: 10.4103/0970-4388.57660. 7. effec=veness of dental trauma educa=on for elementary school staff. mcintyre jd, lee jy, trope m, vannwf, jr. dent traumatol. 2008 apr;24(2):146-50. doi: 10.1111/j.16009657.2008.00573.x. 8. knowledge of emergency management of avulsed teeth among a sample of egyp=an parents. abdella=f am, salwa a. hegazy. j adv res. 2011;2:157– 62. doi.org/10.1016/j.jare.2011.01.0 02. 9. factors related to treatment and outcomes of avulsed teeth. petrovic b, marković d, peric t, blagojevic d. dent traumatol. 2010;26:52–9. doi: 10.1111/j.16009657.2009.00836.x. 10. impact of educa=onal posters on the lay knowledge of school teachers regarding emergency management of dental injuries. lieger o, graf c, el-maaytah m, von arx t. dent traumatol. 2009;25:406–12. doi: 10.1111/j.16009657.2009.00798.x. 11. knowledge and a0tude of primary school teachers toward tooth avulsion and dental first aid in davangere city: a crosssec=onal survey. prasanna s, giriraju a, narayan nl. int j clin pediatr dent. 2011 sep;4(3):2036. doi: 10.5005/jp-journals10005-1110. 12. knowledge and a0tude of parents towards avulsed permanent tooth of their children and its emergency management in bhopal city. jain a, kulkarni p, kumar s, jain m. j clin diagn res. 2017 may;11(5):zc40.doi:10.7860/jcd r/2017/24953.9855. 13. dental trauma management awareness of singapore preschool teachers. sae-lim v, lim lp. dent traumatol. 2001 apr;17(2):71-6. doi:10.1034/j.16009657.2001.017002071.x. 14. the effect of a leaflet given to parents for first aid measures aoer tooth avulsion. al-asfour a, andersson l. dent traumatol. 2008 oct;24(5):515-21. doi: 10.1111/j.16009657.2008.00651.x. 15. knowledge, a0tude, and percep=on of mothers towards emergency management of dental trauma in salem district, tamil nadu: a ques=onnaire study. murali k, krishnan r, kumar vs, shanmugam s, rajasundharam p. j indian soc pedod prev dent 2014;32:2026.doi: 10.4103/09704388.135825. 16. kuwai= parents’ knowledge of first-aid measures of avulsion and replanta=on of teeth. aljame q, andersson l, al-asfour a. med princ pract. 2007;16:274–79. doi: 10.1159/000102149. 17. parent and caretaker knowledge about avulsion of permanent teeth. santos me, habecost ap, gomes fv, weber jb, de oliveira mg. dent traumatol. 2009 apr;25(2):2038.doi.org/10.1111/j.1600.9657.2 008.00620.x. 18. knowledge and a0tude of mothers with regards to emergency management of dental avulsion. oliveira tm, sakai vt, more0 ab, silva tc, santos cf, machado ma. j dent child. 2007 sep 15;74(3):200-2. pmid: 18482514. 19. parental knowledge and a0tudes regarding the emergency treatment of avulsed permanent teeth. ozer s, yilmaz ei, bayrak s, tunc es. euro j dent. 2012 oct;6(4):370. pmid:23077415. 20. effec=veness of educa=onal poster on knowledge of emergency management of dental trauma-part 2: cluster randomised controlled trial for the effect of using an educa3onal poster on mothers’ knowledge of emergency management of dental avulsion vol 10 no 1 (2022) doi 10.5195/d3000.2022.189 h#p://den*stry3000.pi#.edu secondary school students. plos one. young c, wong ky, cheung lk. 2014 aug 5;9(8):e101972. doi:10.1371/journal.pone.01019 72. 21. effect of an educa=onal pamphlet on knowledge and performance of fitness trainers about trauma=c dental injuries. ramezani gh, sohrabi m, nasiri s, kharazifard mj, ghadimi s, seraj b. front dent. 2021;18:5. doi:10.18502/fid.v18i5.5648. correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu correlation between presence of radix entomolaris in mandibular first molars and c-shaped canals in mandibular second molars: a sample of cbct scans in taiwanese students magdalena piskórz2 , ingrid różyło-kalinowska2, karolina futyma-gąbka2, zuzanna aleksandrowicz1, anna błaszczyk1, wiktoria furmanek1 1. student research group at the department of dental and maxillofacial radiodiagnostics, medical university of lublin, poland 2. department of dental and maxillofacial radiodiagnostics, medical university of lublin, poland abstract objec�ves: the anatomy of root canals is so complex that in many cases it is not sufficient to take an intraoral radiograph to diagnose the number and shape of root canals. therefore, cone beam computed tomography (cbct) facilita�ng diagnosis and treatment planning in endodon�cs is gaining more and more importance. radix entomolaris is an extra root which is found in mandibular molars and located distolingually. c-shaped canal is most commonly found in mandibular second molars and can differ significantly in shape from the typical anatomy of lower molars. the aim of the study was to evaluate the correla�on between presence of radix entomolaris in mandibular first molars and presence of c-shaped mandibular second molars in taiwanese students based on cbct scans. methods: the material consisted of 19 cbct examina�ons taken in the department of dental and maxillofacial radiodiagnos�cs of the medical university of lublin. results: among 19 cbct examina�ons 5 cases of coexistence of addi�onal root and cshaped canals were found. up to 80.0% of cases were bilateral. in the studied sample there was high coincidence of radix entomolaris in mandibular first molars and c-shaped mandibular second molars. conclusion: knowledge of this correla�on will prevent diagnos�c errors during endodon�c and surgical treatment planning. keywords: root canal anatomy, cone beam computed tomography, c-shaped root canals, taiwanese, radix entomolaris cita�on: piskórz m, et al. (2022). correla�on between presence of radix entomolaris in mandibular first molars and c-shaped canals in mandibular second molars: a sample of cbct scans in taiwanese students. den�stry 3000. 1:a001 doi:10.5195/d3000.2022.361 received: june, 15, 2022 accepted: june 23, 2022 published: august 12, 2022 copyright: ©2022 piskórz m, et al. this is an open access ar�cle licensed under a crea�ve commons atribu�on work 4.0 united states license. email: lek.dent.karolina.futyma@gmail.com http://dentistry3000.pitt.edu/ correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu introduction dental practitioners are aware of anatomical variations of dental roots and root canals. the anatomy of root canals is so complex that in many cases it is not sufficient to take an intraoral radiograph before planning endodontic treatment [1]. the number of cone beam computed tomography (cbct) scans is increasing year by year. this novel diagnostic method is effective to reveal anatomical variation of teeth and coexistence of different variants [2]. cbct examination allows to analyze a case in three dimensions: axial, coronal and sagittal. being highly exposed to caries, the molars in particular have a higher demand for root canal treatment [3]. lack of knowledge of its diversity may lead to endodontic failures such as broken endodontic instruments, perforations, instrument separation and apical displacement, injuries of periapical tissues, underfilling and overfilling of root canals and serious oral health problems, for example: internal resorption, abscesses, cysts, chronic periapical inflammation, pathological fractures and tooth loss [3-5]. for deeper understanding of the root anatomy vertucci suggested root canal morphology classification, in which the following eight types can be distinguished [6]: type ia single canal extends from pulp chamber to the apex type iitwo separate canals leave the chamber of the pulp and join short of the apex to form single canal type iiione canal leaves the pulp chamber, divides into two within the root, and then joins to exit as one canal type ivtwo separate and distinct canals extend from the pulp chamber to the apex type vsingle canal leaves the pulp chamber and divides short of the apex into two separate and distinct canals with separate apical foramina type vitwo separate canals leave the pulp chamber, merge in the body of the root, and redivide short of the apex to exit as two distinct canals type viione canal leaves the pulp chamber, divides and then rejoins within the body of the root, and finally redivides into two distinct canals short of the apex type viiithree separate and distinct canals extend from the pulp chamber to the apex the first mandibular molars commonly have 2 roots and 3 to 4 canals [1] but in 1844 carabelli noticed a common occurrence of a third root in these teeth [7, 8]. in most cases the extra root is located distolingually, and is called radix entomolaris (em) [1, 9]. if the root is located mesiobuccally, it is called radix paramolaris [8]. previous studies showed that chinese, japanese, korean and taiwanese populations are more predisposed occurence of the first mandibular molars with the extra root in comparison to europeans (frequency of an additional root in taiwanese is around 24%, while in german is less than 4%) [10, 11]. presence of an accessory root in mandibular first molars requires careful examination of cbct scans [7]. the second mandibular molars usually have 2 roots and 3 canals [12]. it is possible to find the ‘c’ shaped canal configuration in these teeth [13]. this is an anatomical variation of root canal system and shape and its diagnosis is challenging based on conventional radiographs only. depending on the population, cshaped mandibular second molar canals may occur with various percentages (from 2.7 to 44.5%) [14-16]. it is widely known that most cases with this configuration http://dentistry3000.pitt.edu/ correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu are found in the population of asia [14, 17-21]. according to studies, c-shaped root canals were located most frequently in the second mandibular molars [21, 22]. in 1991 melton et al. [23] proposed the classification of ccanals based on their crosssectional shape. it distinguished three types of appearance of cshaped canals: category 1the continuous cshaped canal category 2“semicolon” shaped canal (dentin separated one distinct canal from a buccal or lingual c-shaped canal) category 3two or more discrete and separate canals in 2004 fan et al. [20] using microcomputed tomography demonstrated modified melton’s classification and divided c-canals into five following categories: category i (c1) the uninterrupted “c” shape of the canal with no division or separation (fig. 1a) category ii (c2)the canal shape similar to “semicolon” resulting from a discontinuation of the “c” outline, but either angle ⲁ or ꞵ (fig. 2) should be no less than 60° (fig. 1b) category iii (c3)two or three separate canals and both angles, ⲁ and ꞵ (fig. 3), were less than 60° (fig. 1c, 1d) category iv (c4)only one round or oval canal in that cross-section (fig. 1e) category v (c5)no canal lumen could be observed (which was usually seen near the apex only) (fig. 1f) cbct is one of the best diagnostic modality which can be used to evaluate complexity of the root canal system [4, 21]. without a doubt it is easier to visualize root canal anatomy due to using 3dimensional (3d) images [24]. cbct helps to identify canal morphology, numbers of canals and their relative positions [9, 2430]. it is especially helpful in evaluating canals in teeth with radix entoor paramolaris [24]. the aim of the study was to estimate the correlation between presence of radix entomolaris in mandibular first molars and presence of c-shaped root canals in mandibular second molars in a sample of cbct scans of taiwanese individuals. http://dentistry3000.pitt.edu/ correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu fig. 1 classifica�on of c-shaped canal configura�on by fan et al. [35]. diagrams drawn according to the paper by fan et al. [35]. fig. 2 measurement of angles for the type c2. angle ꞵ is more than 60°. (a and b) ends of one canal cross-sec�on; (c and d) ends of the other canal cross-sec�on; m middle point of line ad; α angle between line am and line bm; ꞵ angle between line cm and line dm [35]. diagrams drawn according to the paper by fan et al. [35]. fig. 3 measurement of angles for the c3 canal. both angle α and angle ꞵ are less than 60°. (a and b) ends of one canal cross-sec�on; (c and d) ends of another canal cross-sec�on; m middle point of line ad; α angle between line am and line bm; ꞵ angle between line cm and line dm [35]. diagrams drawn according to the paper by fan et al. [35]. http://dentistry3000.pitt.edu/ correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu methods nineteen consecutive cbct examinations retrieved from the database of the department of dental and maxillofacial radiodiagnostics, medical university of …. were analyzed in order to assess the anatomy of the mandibular first and second molars. in the study group only teeth that meet the following criteria were included: • mature (those at the stage h according to demirijan's tooth formation stages) • without any features of inflammation • with no capping and motion artifacts (well taken cbct scans) • with any pathologies in the interested region • without root canal treatment. the examinations were taken with the use of vistavoxs cbct x-ray machine (dürr dental, germany) with fov size 130x85mm. the study group included eleven women and eight men, aged 21-53 years (mean 27.6). only examinations of taiwanese patients were evaluated. cbct scans were analyzed in multiplanar reconstructions (mpr): coronal, axial and sagittal, by means of vistasoft software on diagnostic radiological medical monitor barco coronis fusion 4 mp (mdcc-4430, belgium). the axial scans were used to evaluate root canals and configuration of roots in first and second mandibular molars. presence of radix entomolaris in first mandibular molars and c-shaped root canal configuration in second mandibular molars was noted. the next step was to assess correlation between presence of radix entomolaris in mandibular first molars and presence of c-shaped root canals configurations in mandibular second molars. results among 38 first mandibular molars 13 teeth with radix entomolaris were found (34.2%) (tab. 1). six cases were observed on the right side (46.2%) and seven on the left (53.8%). in five patients the occurrence was bilateral (tab. 2). the gender predominance in occurrence of radix entomolaris was not noticed; six cases were found in women and seven in men (46.2%/53.8%). in all cases only a single root canal was found in the supernumerary root.among 37 (one tooth was missing no data in history of the patient) second mandibular molars, 21 teeth with c-shaped configuration of root canals were found (56.8%) (tab. 1). almost all finds were bilateral (10 people) (tab. 2). twelve cases were found in women (57.1%) and nine cases in men (42.9%). table 3 presents the frequency of particular types of c-shaped canals in mandibular second molars. eventually, among all cases with radix entomolaris (36, 46) and cshaped root canals (37, 47) we found 5 cases with both unusual anatomy (tab. 4). four cases presented bilateral occurrences of radix entomolaris and c-shaped root canals (80.0%). one case was partially bilateral (36 with radix, 46 without radix but 37 and 47 with c-shaped) (tab. 5). three cases were found in women and two in men. in men all cases were bilateral. figures 4-6 present axial views of patients with radix entomolaris and c-canals. http://dentistry3000.pitt.edu/ correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu table 1 prevalence of radix entomolaris in mandibular first molars (n=38) and c-shaped canals in mandibular second molars (n=37) examined in terms of presence. radix entomolaris n % present 13 34.2 absent 25 65.8 cshaped canals n % present 21 56.8 absent 16 43.2 http://dentistry3000.pitt.edu/ correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu table 2 prevalence of radix entomolaris in mandibular first molars (n=13) and c-shaped canals in mandibular second molars (n=21) examined in terms of lateralisa�on. radix entomolaris n % unilateral 3 23.1 bilateral 10 76.9 c-shaped canals n % unilateral 1 4.8 bilateral 20 95.2 table 3 prevalence of par�cular types of c-shaped canals in mandibular second molars (n=21). type of cshaped canal c1 c2 c3 c4 c5 n 4 6 11 0 0 http://dentistry3000.pitt.edu/ correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu table 4 prevalence of coexistence of radix entomolaris in mandibular first molars and c-shaped canals in mandibular second molars in pa�ents in terms of presence (n=19). radix entomolaris and cshaped canals n % present 5 26.3 absent 14 73.7 table 5 prevalence of coexistence of radix entomolaris in mandibular first molars and c-shaped canals in mandibular second molars in pa�ents in terms of lateralisa�on (n=5). radix entomolaris and cshaped canals n % unilateral 1 20.0 bilateral 4 80.0 http://dentistry3000.pitt.edu/ correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu fig. 4a cbct axial viewbilateral occurrence of c-shaped canals in teeth no. 37 (c2), 47 (c1), unilateral presence of radix entomolaris in tooth no. 36. fig. 4b panoramic radiograph no radiological signs of radix entomolaris and c-shaped configura�on of root canals in the mandibular molars. http://dentistry3000.pitt.edu/ correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu fig. 5a cbct axial viewbilateral occurrence of c-shaped canals in teeth no. 37 (c3), 47 (c2) and bilateral occurrence of radices entomolaris in teeth no. 36, 46. fig. 5b panoramic radiograph no radiological signs of radix entomolaris and c-shaped configura�on of root canals in the mandibular molars. http://dentistry3000.pitt.edu/ correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu fig. 6a cbct axial viewbilateral occurrence of c-shaped canals in teeth no 37 (c2), 47 (c2) and bilateral occurrence of radices entomolaris in teeth no. 36, 46. fig. 6b panoramic radiograph no radiological signs of radix entomolaris and c-shaped configura�on of root canals in the mandibular molars. http://dentistry3000.pitt.edu/ correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu discussion the statement that the most common or typical morphology of the first mandibular molar is the two-rooted configuration is often challenged. there are many factors that affect the results of research studies. first and the most important is the population group, second is the type of examination that we rely on. our study revealed that among 38 first mandibular molars 34.2% had an accessory distolingual root, which is similar to prevalence reported with the use of cbct by zhang et al. (29.0%) in taiwanese population [31]. on the contrary, the occurrence of the radices entomolares reported by minggene et al. [32] was lower in taiwanese population (21.09%) compared to the results of this study. the obtained results might have been biased by using only 2d examinations (periapical radiographs) which are not as precise as cbct in demonstrating three-dimensional anatomy of teeth. prevalence of an accessory distolingual root was 22.8% in the korean population based on cbct scans, which is close to our findings [33]. on the other hand, a study by rahimi et al. [22] on iranian population using cbct showed a prevalence of 3.10% with an extra distolingual root, which is significantly less than our results. the prevalence of radix entomolaris in the first mandibular molars among saudi subpopulation was low (4.2%) [1]. the present study detected more radices entomolares among men (53.8%) but in saudi subpopulation more three-rooted mandibular first molars were found in women (61.9%) than in men (38.1%) [1]. these results are in agreement with the study conducted on taiwanese population by ming-gene et al. (57.14% for women) [32]. however, there were no significant differences in the prevalence of radix entomolaris in mandibular first molars between men and women [1, 22, 34]. the numbers of bilateral cases were lower in saudi (32.3%) [1], iranian (33.3%) [22] and korean (17.64%) [33] subpopulations in comparison to our study in a sample of taiwanese individuals (76.0%). present findings are consistent with results by minggene et al. [32], who also examined taiwanese population (68.57% bilateral cases). according to our findings, the prevalence of the accessory distolingual radix in the first mandibular molars is comparable on both the right (46.2%) and left (53.8%) side. more radices entomolares were found on the right side in malaysian (54.4%) [34] and turkish (60.0%) subpopulations [35]. in agreement with these studies, sinyoung kim et al. [33] and minggene et al. [32] reported more radices entomolares detected on the right side. one of the unusual forms of root canal configuration are the cshaped canals, which are mostly found in mandibular second molars because these teeth are prone to root fusion [34]. in our study based on taiwanese patients more than a half had cshaped canals (56.8%) that is comparable to malaysian subpopulation (48.7%) [34]. findings reported by zhang et al. [31] indicate that the chinese population also has a high prevalence of c-shaped second mandibular molars (29% c-canals in 157 examined teeth). c-canals are not often found in caucasian populations [31]. studies in iranian population indicated a relatively low appearance of c-shaped root canals in second mandibular molars (17.6% by madani et al.[4] http://dentistry3000.pitt.edu/ correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu and 9.2% by donyavi et al. [3] shemesh et al. [2] reported even lower prevalence of c-canals in israeli population (4.6%). similarly, the prevalence of c-shaped root canals in mandibular second molars in turkish subpopulation was low (10.7% in study by kaplan et al. [36], 10.6% by helvacioǧluyiǧit et al. [37] and 8.0% by cimilli et al. [38]. according to previous studies, there is no significant difference between genders and prevalence of c-shaped canals [2, 12], whereas the present study reported that more c-canals were found in women (57.1%). our results are similar to studies in malaysian and iranian population [4, 34] and consistent with the results in turkish subpopulation reported by kaplan et al. [36]. bilateral occurrence of c-shaped canals was present almost in all cases (95.2%) in contrast to israeli population that has prevalence of asymmetry (55.0% unilateral cases) [2]. kaplan et al. [36] also reported a lower percentage of bilateral c-shaped root canals in the second mandibular molars in turkish subpopulation, which was 50%. on the other hand, findings by tassoker et al. [14] showed a comparable percentage of symmetrical presence of c-canals in the second mandibular molars (88%) as in our study. the present study reported that the coexistence of c-canals in mandibular second molars and radix entomolaris in first mandibular molars is more frequent in taiwanese population (26.3%). in the majority of our cases there was bilateral occurrence of both c-shaped root canals and radices entomolares (80.0%) and there is no significant correlation between them and gender of the patients. the main limitation of our study was a small examined sample but more advanced research is planned with more cbct examinations included. conclusions the results of this study revealed a high percentage of coexistence between presence of radix entomolaris in mandibular first molars and c-shaped mandibular second molars in a sample of cbct scans in taiwanese individuals. list of abbreviations: cbct cone beam computed tomography conflicts of interest: there is no conflicts of interest references 1. al-alawi h, al-nazhan s, almaflehi n. the prevalence of radix molaris in the mandibular first molars of a saudi subpopulation based on cone-beam computed tomography. restor dent endod. 2020;45(1):e1, doi: 10.5395/rde.2020.45.e1. 2. shemesh a, levin a, katzenell v. c-shaped canals prevalence and root canal configuration by cone beam computed tomography evaluation in first and second mandibular molars a cross-sectional study. clin oral invest. 2017;21(6):20392044, doi: 10.1007/s00784-0161993-y. 3. donyavi z, shokri a, khoshbin e. assessment of root canal morphology of maxillary and mandibular second molars in the iranian population using cbct. dent med probl. 2019;56(1):45– 51, doi: 10.17219/dmp/101783. 4. madani z, mehraban n, moudi e. root and canal morphology of mandibular molars in a selected iranian population using cone-beam computed tomography. iran endod j. 2017;12(2):143-8, doi: 10.22037/iej.2017.29. 5. yousuf w, khan m, mehdi h. endodontic procedural errors: http://dentistry3000.pitt.edu/ https://doi.org/10.5395/rde.2020.45.e1 correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu frequency, type of error, and the most frequently treated tooth. int j dent. 2015;2015:673914, doi: 10.1155/2015/673914. 6. versiani ma, basrani b, sousa-neto md. the root canal anatomy in permanent dentition. 1st edition, springer, cham 2019: 47-48, doi:10.1007/978-3-31973444-6. 7. sarangi p, uppin vm. mandibular first molar with a radix entomolaris: an endodontic dilemma. j dent. 2014,11(1):118122. 8. vasudeva a, sinha dj, tyagi n. the concurrence of radix paramolaris and c shaped root canal anatomy in adjacent teeth: a rare finding. j dent app. 2015;2(7):261-263. 9. strmšek l, štamfelj i. morphometric analysis of threerooted mandibular first molars in a slovene population: a macroscopic and cone-beam computed tomography analysis. folia morphol. 2022;81(1):203-211, doi: 10.5603/fm.a2021.0005. 10. wu yc, cheng wc, chung mp. complicated root canal morphology of mandibular lateral incisors is associated with the presence of distolingual root in mandibular first molars: a conebeam computed tomographic study in a taiwanese population. j endod. 2018;44(1):73-79, doi: 10.1016/j.joen.2017.08.027. 11. schäfer e, breuer d, janzen s. the prevalence of three-rooted mandibular permanent first molars in a german population. j endod. 2009;35(2):202-205, doi: 10.1016/j.joen.2008.11.010. 12. pawar am, pawar m, kfir a. root canal morphology and variations in mandibular second molar teeth of an indian population: an in vivo cone-beam computed tomography analysis. clin oral investig. 2017;21(9):2801-2809. doi: 10.1007/s00784-017-2082-6. 13. jafarzadeh h, wu yn. the c-shaped root canal configuration: a review. j endod. 2007;33(5):517523, doi: 10.1016/j.joen.2007.01.005. 14. tassoker m, sener s. analysis of the root canal configuration and c-shaped canal frequency of mandibular second molars: a cone beam computed tomography study. folia morphol. 2018;77(4):752-757, doi: 10.5603/fm.a2018.0040. 15. wein fs, pasiewicz ra, rice rt. canal configuration of the mandibular second molar using a clinically oriented in vitro method. j endod. 1988;14(5):207-213, doi: 10.1016/s0099-2399(88)80171-7. 16. jin gc, lee sj, roh bd. anatomical study of c-shaped canals in mandibular second molars by analysis of computed tomography. j endod. 2006;32(1):10-13, doi: 10.1016/j.joen.2005.10.007. 17. kim sy, kim bs, kim y. mandibular second molar root canal morphology and variants in a korean subpopulation. int endod j. 2016;49(2):136-144, doi: 10.1111/iej.12437. 18. martins jnr, mata a, marques d. prevalence of cshaped mandibular molars in the portuguese population evaluated by cone-beam computed tomography. eur j dent. 2016;10(4):529-535, doi: 10.4103/1305-7456.195175. 19. wu yc, cathy tsai yw, cheng wc. relationship of the incidence of c-shaped root canal configurations of mandibular first premolars with distolingual roots in mandibular first molars in a taiwanese population: a conebeam computed tomographic http://dentistry3000.pitt.edu/ correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu study. j endod. 2018;44(10):14921499, doi: 10.1016/j.joen.2018.05.016. 20. fan b, cheung gs, fan m. c-shaped canal system in mandibular second molars: part i-anatomical features. j endod. 2004;30(12):899-903, doi: 10.1097/01.don.0000136207.1220 4.e4. 21. shrestha s, agrawal n, vikram m. c shaped canal: variation and recommendations. jcms nepal. 2018;14(1):62-64, doi: 10.3126/jcmsn.v14i1.19125. 22. rahimi s, mokhtari h, ranjkesh b. prevalence of extra roots in permanent mandibular first molars in iranian population: a cbct analysis. iran endod j. 2017;12(1):70-73, doi: 10.22037/iej.2017.14. 23. melton dc, krell kv, fuller mw. anatomical and histological features of c-shaped canals in mandibular second molars. j endod. 1991;17(8):384-388, doi: 10.1016/s0099-2399(06)81990-4. 24. kamble ap, pawar rr, mattigatti s. cone-beam computed tomography as advanced diagnostic aid in endodontic treatment of molars with multiple canals: two case reports. j conserv dent. 2017;20(4):273–277, doi: 10.4103/0972-0707.219194. 25. van der vyver pj, vorster m. radix entomolaris: literature review and case report. sadj 2017;72(3):113-117. 26. gu y, lu q, wang p. root canal morphology of permanent three-rooted mandibular first molars: part ii—measurement of root canal curvatures. j endod. 2010;36(8):1341-1346, doi: 10.1016/j.joen.2010.04.025. 27. gu y, zhou p, ding y. root canal morphology of permanent three-rooted mandibular first molars: part iii—an odontometric analysis. j endod. 2011;37(4):485490, doi: 10.1016/j.joen.2011.01.013. 28. martins jnr, marques d, mata a. root and root canal morphology of the permanent dentition in a caucasian population: a cbct study. int endod j. 2017;50(11):1013-1026, doi: 10.1111/iej.12724. 29. rodrigues ct, de oliveirasantos c, bernardineli n. prevalence and morphometric analysis of three-rooted mandibular first molars in brazilian subpopulation. j appl oral sci. 2016;24(5):535-542, doi: 10.1590/1678-775720150511. 30. souza-flamini le, leoni gb, chaves jfm. the radix entomolaris and paramolaris: a micro–computed tomographic study of 3-rooted mandibular first molars. j endod. 2014;40(10):1616-1621, doi: 10.1016/j.joen.2014.03.012. 31. zhang r, wang h, tian yy. use of cone-beam computed tomography to evaluate root and canal morphology of mandibular molars in chinese individuals. int endod j. 2011;44(11):990-999, doi: 10.1111/j.13652591.2011.01904.x. 32. tu mg, tsai cc, jou mj. prevalence of three-rooted mandibular first molars among taiwanese individuals. j endod. 2007;33(10):1163-1166, doi: 10.1016/j.joen.2007.07.020. 33. kim sy, yang se. conebeam computed tomography study of incidence of distolingual root and distance from distolingual canal to buccal cortical bone of mandibular first molars in a korean population. j endod. 2012;38(3):301-304, doi: 10.1016/j.joen.2011.10.023. http://dentistry3000.pitt.edu/ correla�on between presence of re and c-shaped canals vol 10 no 1 (2022) doi 10.5195/d3000.2022.361 htp://den�stry3000.pit.edu 34. pan jyy, parolia a, chuah sr. root canal morphology of permanent teeth in a malaysian subpopulation using cone-beam computed tomography. bmc oral health 2019;19(1):14, doi: 10.1186/s12903-019-0710-z. 35. duman sb, duman s, bayrakdar is. evaluation of radix entomolaris in mandibular first and second molars using cone-beam computed tomography and review of the literature. oral radiol. 2020;36:320–326, doi: 10.1007/s11282-019-00406-0. 36. kaplan ss, kaplan t, sezgin gp. evaluation of c-shaped canals in mandibular second molars of a selected patient group using cone beam computed tomography: prevalence, configuration and radicular groove types. odontology 2021;109:949-955, doi: 10.1007/s10266-021-00616-1. 37. helvacioglu-yigit d, sinagoglu a. use of cone-beam computed tomography to evaluate c-shaped root canal systems in mandibular second molars in a turkish subpopulation: a retrospective study. int endod j. 2013;46(11):1032-1038, doi: 10.1111//iej.12094. 38. cimilli h, cimilli t, mumcu g. spiral computed tomographic demonstration of c-shaped canals in mandibular second molars. dentomaxillofac radiol. 2005;34:164-167, doi: 10.1259/dmfr/64778606. http://dentistry3000.pitt.edu/ results among 38 first mandibular molars 13 teeth with radix entomolaris were found (34.2%) (tab. 1). six cases were observed on the right side (46.2%) and seven on the left (53.8%). in five patients the occurrence was bilateral (tab. 2). the gender predomin... eventually, among all cases with radix entomolaris (36, 46) and c-shaped root canals (37, 47) we found 5 cases with both unusual anatomy (tab. 4). four cases presented bilateral occurrences of radix entomolaris and c-shaped root canals (80.0%). one ca... discussion the statement that the most common or typical morphology of the first mandibular molar is the two-rooted configuration is often challenged. there are many factors that affect the results of research studies. first and the most important is the populat... our study revealed that among 38 first mandibular molars 34.2% had an accessory distolingual root, which is similar to prevalence reported with the use of cbct by zhang et al. (29.0%) in taiwanese population [31]. on the contrary, the occurrence of th... on the other hand, a study by rahimi et al. [22] on iranian population using cbct showed a prevalence of 3.10% with an extra distolingual root, which is significantly less than our results. the prevalence of radix entomolaris in the first mandibular m... the present study detected more radices entomolares among men (53.8%) but in saudi subpopulation more three-rooted mandibular first molars were found in women (61.9%) than in men (38.1%) [1]. these results are in agreement with the study conducted on ... the numbers of bilateral cases were lower in saudi (32.3%) [1], iranian (33.3%) [22] and korean (17.64%) [33] subpopulations in comparison to our study in a sample of taiwanese individuals (76.0%). present findings are consistent with results by ming-... according to our findings, the prevalence of the accessory distolingual radix in the first mandibular molars is comparable on both the right (46.2%) and left (53.8%) side. more radices entomolares were found on the right side in malaysian (54.4%) [34] and turkish (60.0%) subpopulations [35]. in agreement with these studies, sin-young kim et al. [33] and ming-gene et al. [32] reported more radices entomolares detected on... one of the unusual forms of root canal configuration are the c-shaped canals, which are mostly found in mandibular second molars because these teeth are prone to root fusion [34]. in our study based on taiwanese patients more than a half had c-shaped canals (56.8%) that is comparable to malaysian subpopulation (48.7%) [34]. findings reported by zhang et al. [31] indicate that the chinese population also has a high prevalence of... according to previous studies, there is no significant difference between genders and prevalence of c-shaped canals [2, 12], whereas the present study reported that more c-canals were found in women (57.1%). our results are similar to studies in malay... bilateral occurrence of c-shaped canals was present almost in all cases (95.2%) in contrast to israeli population that has prevalence of asymmetry (55.0% unilateral cases) [2]. kaplan et al. [36] also reported a lower percentage of bilateral c-shaped ... the present study reported that the coexistence of c-canals in mandibular second molars and radix entomolaris in first mandibular molars is more frequent in taiwanese population (26.3%). in the majority of our cases there was bilateral occurrence of b... the main limitation of our study was a small examined sample but more advanced research is planned with more cbct examinations included. conclusions the results of this study revealed a high percentage of coexistence between presence of radix entomolaris in mandibular first molars and c-shaped mandibular second molars in a sample of cbct scans in taiwanese individuals. list of abbreviations: cbct cone beam computed tomography conflicts of interest: there is no conflicts of interest references 1. al-alawi h, al-nazhan s, al-maflehi n. the prevalence of radix molaris in the mandibular first molars of a saudi subpopulation based on cone-beam computed tomography. restor dent endod. 2020;45(1):e1, doi: 10.5395/rde.2020.45.e1. 2. shemesh a, levin a, katzenell v. c-shaped canals prevalence and root canal configuration by cone beam computed tomography evaluation in first and second mandibular molars a cross-sectional study. clin oral invest. 2017;21(6):2039-2044, doi: 10.... 3. donyavi z, shokri a, khoshbin e. assessment of root canal morphology of maxillary and mandibular second molars in the iranian population using cbct. dent med probl. 2019;56(1):45–51, doi: 10.17219/dmp/101783. 4. madani z, mehraban n, moudi e. root and canal morphology of mandibular molars in a selected iranian population using cone-beam computed tomography. iran endod j. 2017;12(2):143-8, doi: 10.22037/iej.2017.29. 5. yousuf w, khan m, mehdi h. endodontic procedural errors: frequency, type of error, and the most frequently treated tooth. int j dent. 2015;2015:673914, doi: 10.1155/2015/673914. 6. versiani ma, basrani b, sousa-neto md. the root canal anatomy in permanent dentition. 1st edition, springer, cham 2019: 47-48, doi:10.1007/978-3-319-73444-6. 7. sarangi p, uppin vm. mandibular first molar with a radix entomolaris: an endodontic dilemma. j dent. 2014,11(1):118-122. 8. vasudeva a, sinha dj, tyagi n. the concurrence of radix paramolaris and c shaped root canal anatomy in adjacent teeth: a rare finding. j dent app. 2015;2(7):261-263. 9. strmšek l, štamfelj i. morphometric analysis of three-rooted mandibular first molars in a slovene population: a macroscopic and cone-beam computed tomography analysis. folia morphol. 2022;81(1):203-211, doi: 10.5603/fm.a2021.0005. 10. wu yc, cheng wc, chung mp. complicated root canal morphology of mandibular lateral incisors is associated with the presence of distolingual root in mandibular first molars: a cone-beam computed tomographic study in a taiwanese population. j endod.... 11. schäfer e, breuer d, janzen s. the prevalence of three-rooted mandibular permanent first molars in a german population. j endod. 2009;35(2):202-205, doi: 10.1016/j.joen.2008.11.010. 12. pawar am, pawar m, kfir a. root canal morphology and variations in mandibular second molar teeth of an indian population: an in vivo cone-beam computed tomography analysis. clin oral investig. 2017;21(9):2801-2809. doi: 10.1007/s00784-017-2082-6. 13. jafarzadeh h, wu yn. the c-shaped root canal configuration: a review. j endod. 2007;33(5):517-523, doi: 10.1016/j.joen.2007.01.005. 14. tassoker m, sener s. analysis of the root canal configuration and c-shaped canal frequency of mandibular second molars: a cone beam computed tomography study. folia morphol. 2018;77(4):752-757, doi: 10.5603/fm.a2018.0040. 15. wein fs, pasiewicz ra, rice rt. canal configuration of the mandibular second molar using a clinically oriented in vitro method. j endod. 1988;14(5):207-213, doi: 10.1016/s0099-2399(88)80171-7. 16. jin gc, lee sj, roh bd. anatomical study of c-shaped canals in mandibular second molars by analysis of computed tomography. j endod. 2006;32(1):10-13, doi: 10.1016/j.joen.2005.10.007. 17. kim sy, kim bs, kim y. mandibular second molar root canal morphology and variants in a korean subpopulation. int endod j. 2016;49(2):136-144, doi: 10.1111/iej.12437. 18. martins jnr, mata a, marques d. prevalence of c-shaped mandibular molars in the portuguese population evaluated by cone-beam computed tomography. eur j dent. 2016;10(4):529-535, doi: 10.4103/1305-7456.195175. 19. wu yc, cathy tsai yw, cheng wc. relationship of the incidence of c-shaped root canal configurations of mandibular first premolars with distolingual roots in mandibular first molars in a taiwanese population: a cone-beam computed tomographic study.... 20. fan b, cheung gs, fan m. c-shaped canal system in mandibular second molars: part i--anatomical features. j endod. 2004;30(12):899-903, doi: 10.1097/01.don.0000136207.12204.e4. 21. shrestha s, agrawal n, vikram m. c shaped canal: variation and recommendations. jcms nepal. 2018;14(1):62-64, doi: 10.3126/jcmsn.v14i1.19125. 22. rahimi s, mokhtari h, ranjkesh b. prevalence of extra roots in permanent mandibular first molars in iranian population: a cbct analysis. iran endod j. 2017;12(1):70-73, doi: 10.22037/iej.2017.14. 23. melton dc, krell kv, fuller mw. anatomical and histological features of c-shaped canals in mandibular second molars. j endod. 1991;17(8):384-388, doi: 10.1016/s0099-2399(06)81990-4. 24. kamble ap, pawar rr, mattigatti s. cone-beam computed tomography as advanced diagnostic aid in endodontic treatment of molars with multiple canals: two case reports. j conserv dent. 2017;20(4):273–277, doi: 10.4103/0972-0707.219194. 25. van der vyver pj, vorster m. radix entomolaris: literature review and case report. sadj 2017;72(3):113-117. 26. gu y, lu q, wang p. root canal morphology of permanent three-rooted mandibular first molars: part ii—measurement of root canal curvatures. j endod. 2010;36(8):1341-1346, doi: 10.1016/j.joen.2010.04.025. 27. gu y, zhou p, ding y. root canal morphology of permanent three-rooted mandibular first molars: part iii—an odontometric analysis. j endod. 2011;37(4):485-490, doi: 10.1016/j.joen.2011.01.013. 28. martins jnr, marques d, mata a. root and root canal morphology of the permanent dentition in a caucasian population: a cbct study. int endod j. 2017;50(11):1013-1026, doi: 10.1111/iej.12724. 29. rodrigues ct, de oliveira-santos c, bernardineli n. prevalence and morphometric analysis of three-rooted mandibular first molars in brazilian subpopulation. j appl oral sci. 2016;24(5):535-542, doi: 10.1590/1678-775720150511. 30. souza-flamini le, leoni gb, chaves jfm. the radix entomolaris and paramolaris: a micro–computed tomographic study of 3-rooted mandibular first molars. j endod. 2014;40(10):1616-1621, doi: 10.1016/j.joen.2014.03.012. 31. zhang r, wang h, tian yy. use of cone-beam computed tomography to evaluate root and canal morphology of mandibular molars in chinese individuals. int endod j. 2011;44(11):990-999, doi: 10.1111/j.1365-2591.2011.01904.x. 32. tu mg, tsai cc, jou mj. prevalence of three-rooted mandibular first molars among taiwanese individuals. j endod. 2007;33(10):1163-1166, doi: 10.1016/j.joen.2007.07.020. 33. kim sy, yang se. cone-beam computed tomography study of incidence of distolingual root and distance from distolingual canal to buccal cortical bone of mandibular first molars in a korean population. j endod. 2012;38(3):301-304, doi: 10.1016/j.joen... 34. pan jyy, parolia a, chuah sr. root canal morphology of permanent teeth in a malaysian subpopulation using cone-beam computed tomography. bmc oral health 2019;19(1):14, doi: 10.1186/s12903-019-0710-z. 35. duman sb, duman s, bayrakdar is. evaluation of radix entomolaris in mandibular first and second molars using cone-beam computed tomography and review of the literature. oral radiol. 2020;36:320–326, doi: 10.1007/s11282-019-00406-0. 36. kaplan ss, kaplan t, sezgin gp. evaluation of c-shaped canals in mandibular second molars of a selected patient group using cone beam computed tomography: prevalence, configuration and radicular groove types. odontology 2021;109:949-955, doi: 10.1... 37. helvacioglu-yigit d, sinagoglu a. use of cone-beam computed tomography to evaluate c-shaped root canal systems in mandibular second molars in a turkish subpopulation: a retrospective study. int endod j. 2013;46(11):1032-1038, doi: 10.1111//iej.12094. 38. cimilli h, cimilli t, mumcu g. spiral computed tomographic demonstration of c-shaped canals in mandibular second molars. dentomaxillofac radiol. 2005;34:164-167, doi: 10.1259/dmfr/64778606. microsoft word 362 2022.docx pa#ents’ awareness of the complica#ons of bisphosphonate therapy vol 10 no 1 (2022) doi 10.5195/d3000.2022.362 h#p://den*stry3000.pi#.edu patients’ awareness of the complications of bisphosphonate therapy meri hristamyan1, ralista raycheva2, veselka hristamyan3 1department of epidemiology and dm, faculty of public health, medical university – plovdiv, bulgaria 2department of social medicine and public health, faculty of public health, medical university – plovdiv, bulgaria 3clinic of oral and maxillofacial surgery, university hospital “st. george” – plovdiv, bulgaria abstract introduction: bisphosphonate-associated osteonecrosis of the jaws (baonj) is a serious complica#on that has been reported predominantly in cancer pa#ents treated with bisphosphonate (bp). the level of knowledge of dental and medical prac##oners, as well as pa#ents, is crucial for its preven#on. objectives: to inves#gate pa#ents’ awareness of baonj in order to evaluate some problems of the preven#on, prophylaxis and diagnosis. materials and methods: a prospec#ve epidemiological study of 112 oncology pa#ents diagnosed with bisphosphonate-associated osteonecrosis of the jaw was conducted in the clinic of maxillo-facial surgery of university mul#profile hospital for ac#ve treatment (umhat) “st. george”, plovdiv, bulgaria, based on anamnesis, clinical examina#on, hospital documenta#on, and imaging studies. spss sta#s#cs v.24 was used for sta#s#cal analysis, at a significance level p<0.05. results: before ini#a#on of therapy, only 17.86% of the pa#ents were consulted by a medical prac##oner about the risk of jaw complica#ons associated with the use of bisphosphonates, 9.82% knew about the need for regular dental check-ups a\er the ini#a#on of therapy and 4.46% had a dental examina#on and oral sana#on. by the #me of diagnosis, 61.61% of the pa#ents remained unaware, with the last dental manipula#on being most o\en extrac#on (52.68%) and removable dentures (21.42%) considered risky by experts. diagnosed by a general dental prac##oner or oncologist were 6.74% of the respondents – the medical prac##oners who usually monitor the condi#on of these pa#ents. over 2/3 of the pa#ents were diagnosed at a more advanced stage (ii/iii). conclusion: the lack of pa#ent awareness leads to omissions and errors in their prophylaxis, and to the diagnosis of baonj at a more advanced stage. keywords: bisphosphonates; osteonecrosis; preven#on; awareness; pa#ents cita9on: hristamyan, m et al. (2022) pa9ents’ awareness of the complica9ons of bisphosphonate therapy den9stry 3000. 1:a001 doi:10.5195/d3000.2022.362 received: june 21, 2022 accepted: june 23, 2022 published: august 19, 2022 copyright: ©2022 hristamyan, m et al. this is an open access ar9cle licensed under a crea9ve commons auribu9on work 4.0 united states license. email: mary_hr@abv.bg introduction bisphosphonates (bp) are pyrophosphate analogs that bind to hydroxyapatite crystals in the bone structures and slow the process of osteoclastic bone resorption through decreasing osteoclast activity by suppressing progenitor development and recruitment and inducing osteoclast apoptosis [1,2]. in clinical practice, bp has been used for the treatment of different conditions affecting the bone structures such as multiple myeloma, osteolytic bone metastases, osteoporosis, paget's pa#ents’ awareness of the complica#ons of bisphosphonate therapy vol 10 no 1 (2022) doi 10.5195/d3000.2022.362 h#p://den*stry3000.pi#.edu disease, fibrous dysplasia, mccunealbright syndrome, and others [2,3]. in recent years, many scientists and clinicians have reported a complication following the intake of this drug, called bisphosphonateassociated osteonecrosis of the jaw (baonj), which mainly occurs in cancer patients receiving intravenous bp treatment. there are many discussions and debates regarding the different aspects of this complication, from the definition and risk factors to the clinical manifestations and different methods of treatment and prevention. it is evident that this extremely complex and multifactorial process requires careful monitoring and an individual approach to each patient [3]. efforts should be focused primarily on its prevention, both before and after the start of the treatment, as the condition causes irreversible changes in the patient's dental health and overall health. awareness of the problem of dentists, oncologists, and patients and their willingness to work together to reduce risk is crucial. patients should be informed about the complication before starting therapy, sanitized if necessary, as well as prophylaxed before and during bp therapy. the aim of this study is to investigate the awareness of the complication of bp therapy of patients diagnosed with baonj in order to evaluate some problems of the prevention, prophylaxis, and diagnosis. material and methods a prospective epidemiological study of 112 oncology patients diagnosed with bisphosphonate-associated osteonecrosis of the jaw in 2016 and 2017 was conducted in the clinic of maxillo-facial surgery of umhat “st. george”, plovdiv, bulgaria, based on anamnesis, clinical examination, hospital documentation, and others. the data is reflected in à specially created for the purpose epidemiological study card. for descriptive statistics, continuous variables were displayed as the median and interquartile range (median ± iqr) and categorical variables as counts and percentages. variables were compared with the use of two proportions z-test and pearson’s chi-square. all statistical analyses were performed with spss version 24 (ibm corporation, new york, ny). a p-value below 0.05 was considered statistically significant. this study, as a part of a dissertation, titled “bisphosphonate-associated osteonecrosis of the jaws epidemiological and clinical research” was submitted to the ethical committee of the medical university of plovdiv with id number p-3911 (29.05.2018) and approved with protocol no.4/28.06.2018. results the median age of patients was 68 years (iqr = 16), with a minimum of 38 years and a maximum of 85 years. there was no statistically significant difference between the relative proportions of men (51.79%, n = 58) and women (48.21%, n = 54) (p> 0.05). breast cancer (40.18%, n = 45) and prostate cancer (37.71%, n = 40) (p> 0.05) were leading oncological diagnoses. of all patients studied, 16.07% (n = 18) had another type of cancer, most commonly renal cell carcinoma (n = 9) (figure 1). only 17.86% (n = 20) of patients were consulted by a physician about the risk of jaw complications associated with bp administration prior to initiating therapy. also, the need for regular dental examinations after starting therapy has been explained to just 9.82% (n = 11). even more worrying is that only 4.46% (n = 5) underwent a prophylactic dental examination and, if needed, treatment of dental problems before 6.25% 40.18% 35.71% 1.79% 16.07% multiple myeloma breast cancer prostate cancer lung cancer other figure 1. type of cancer of the studied patients. pa#ents’ awareness of the complica#ons of bisphosphonate therapy vol 10 no 1 (2022) doi 10.5195/d3000.2022.362 h#p://den*stry3000.pi#.edu the start of the treatment, and the same small proportion of patients were asked by the physicians prescribing bp therapy if they had performed such an examination. before initiating bp therapy, which indicates a low awareness of medical professionals about baonj. there was an association between the type of cancer and the presence of consultation by a physician about the risk of jaw complications prior to the start of bp therapy (χ2 = 15.32, p <0.05), the presence of an explained need for regular dental check-ups (χ2 = 19.76, p < 0.05), the performed prophylactic dental examination and potential oral sanation (χ2 = 26.01, p <0.05) and the request from the attending physician before starting bp therapy for consultation / examination by a dentist (χ2 = 26.01, p <0.05), as patients with multiple myeloma were better informed than patients with all other oncological diagnoses. when asked about the first source of information on the risk of maxillary complications associated with the use of bp, the largest proportion of patients (61.61%, n = 69) indicated that they were completely uninformed before diagnosis and that their first explanation of the problem was received by a specialist in the clinic of maxillo-facial surgery. the ones who received the information from the treating physicians and who found out about baonj from a friend or another patient had equal relative frequency (11.61%, n = 11). with the same low proportion of 2.68% (n = 3) were the patients who indicated a dentist, medical leaflets, or the internet as the primary source, and only 1 patient received information on the problem from their general practitioner, none from the media. these results once again show the low level of awareness of many practitioners on the problem (figure 2). a statistical association was found between the type of cancer and the primary source of information for patients with baonj (χ2 = 45.31, p <0.05), where patients with multiple myeloma, most often consulted by a physician in another specialty (hematologist), were significantly more than those with breast cancer. additionally, in those uninformed at the time of the study, patients with mammary carcinoma were with significantly higher proportion than those with multiple myeloma. both before and after starting bp therapy, on average, over half of the patients visited a dentist once a year. positively, but unsatisfactorily, the proportion of those with dental examinations less than once a year decreased from 38.39% (n = 43) before the start of bp therapy to 26.79% (n = 30) after, at the expense of an increasing number of patients visiting the dental office semiannually and quarterly, but these percentages are still lower than desired. the fact that patients who do not visit a dentist increased slightly from 0.89% (n = 1) to 2.26% (n = 3) can be explained by the fact that the period of treatment is much shorter than the period before its initiation (figure 3). 2.68% 11.61% 6.25% 0.89% 2.68% 2.68% 11.61% 61.61% dentist oncologist doctor with another specialty general practitioner leaflets internet friend / another patient uninformed figure 2. distribution of patients according to the first source of information on the risk of jaw complications associated with the application of bp. 0.89% 0.00% 6.25% 54.46% 38.39% 2.68% 1.79% 13.39% 55.36% 26.79% none every 3 months every 6 months once per year more before starting bp therapy after starting bp therapy figure 3. distribution of the frequency of visits to the dentist before and after the start of bp therapy. pa#ents’ awareness of the complica#ons of bisphosphonate therapy vol 10 no 1 (2022) doi 10.5195/d3000.2022.362 h#p://den*stry3000.pi#.edu half of the studied patients were diagnosed at stage ii of baonj (according to aaoms) (50.89%, n = 57). significantly less were at stage i (29.46%, n = 33) (p <0.05), followed by 17.86% (n = 20) at stage iii. only 2 (1.79%) patients were at stage 0 at hospitalization, indicating a lack of prevention and monitoring. most of the patients were diagnosed during the 2-year period of the study (79.46%, n = 89), while 23 (20.54%) were diagnosed with baonj before. in more than ¾ of cases, the patients were diagnosed by a specialist from the clinic of maxillofacial surgery (76.79%, n = 86), followed by 15.18% (n = 17), whose diagnosis was made by an oral surgeon in a specialized dental practice. low equal shares of patients were diagnosed by a dentist with another specialty or by an oncologist (3.57%, n = 4), while no patients were diagnosed by their general practitioner, showing low awareness of these medical practitioners on the problem (figure 4). tooth extraction was the last dental procedure in more than half of the patients (52.68%, n = 59), followed by the placement of removable dentures (21.42%, n = 24). these two manipulations are most often cited as risky for the emergence of baonj and again show the low awareness of dentists and patients themselves about the prevention of the problem (figure 5). discussion the level of awareness of medical specialists and patients about the problem is crucial for the prevention of baonj. our study showed not only extremely low awareness of patients with baonj, but also indirectly of the medical and dental specialist, treating these patients. a low level of knowledge about the problem has been found in our previous survey [3], as well as other studies on the awareness of physicians and dentists about the complication [4-10]. similar to the low patient awareness in our study, in a clinical trial of bp therapy patient awareness of prophylaxis with these medications, all subjects reported not receiving instructions for good oral hygiene [11]. a survey found that only 12.4% of participants were aware about the risk of jaw osteonecrosis following bp use, and only one third of them has received information from their prescribing physicians. moreover, just 5% were referred to a dentist for screening prior to initiating bp treatment. patients receiving iv bp 3.57% 15.18% 76.79% 0.00% 3.57% 0.89% dentist in the dental office oral surgeon in a specialized dental office specialist in a clinic of mfs general practitioner oncologist other figure 4. distribution of physicians, who diagnosed by baonj, by their specialty. 21.43% 8.04% 6.25% 52.68% 5.36% 3.57% 2.68% dentures root canal treatment obturation extraction incision / other surgical manipulation calculus cleaning other figure 5. last dental procedure before the onset of symptoms / diagnosis of baonj. pa#ents’ awareness of the complica#ons of bisphosphonate therapy vol 10 no 1 (2022) doi 10.5195/d3000.2022.362 h#p://den*stry3000.pi#.edu and those with a university-level education had better awareness about the potential risk than oral bp users and those with a high school education level [12]. other study on the problem showed that just 1/3 of patients were aware about the risk. patient knowledge was obtained primarily from one of the following sources: physicians (n = 6), physician assistants (n = 1), nurses (n = 1), dentists (n = 2), or the patients themselves (n = 1). females were better informed about the complications than males. the awareness among the subjects with education at college level appears to be higher than the subjects having education less than high school level. even though, a dental checkup, is mandatory, prior to starting these medications, to see if any dental treatment is required, only slightly more than half of the patients (54.72%) had a dental checkup [13]. to date, there is no evidence-based unified therapeutic strategy for baonj and the condition is considered irreversible, so the focus of the medical community is on the possibilities for its prevention [14-16]. the aim is to minimize the risk of development of this complication: before the start of bisphosphonate therapy it is the responsibility of the physicians prescribing bp to provide patients who are about to initiate therapy with them or another antiresorptive agent with adequate information regarding the risk of developing baonj and to refer them for a comprehensive dental examination before starting therapy [16]. dental care should be performed by a dental professional (dentist and oral/ maxillofacial surgeon) who is familiar with anticancer therapies and their expected adverse effects and is committed to integrating oral care in the treatment of cancer [17]. responsibility of the dentist is to apply preventive measures and inform patients about the need to continue regular dental check-ups during treatment with antiresorptive agents. patients should be informed that a significant risk of developing baonj remains, despite the adoption of specific prevention protocols. the reason is that the condition can develop in the absence of dental trauma or infection, and also because there are a number of minor factors that cannot be easily controlled (e.g. trauma to areas of the thin mucosa). patients should be alerted to the possible clinical manifestations and symptoms of baonj and instructed to report their complaints in a timely manner to the oncologist / dentist and in order for them to make a final diagnosis [15,18]. the american association of clinical oncology (asco), the american association of oral and maxillofacial surgeons (aaoms), the international task force on osteonecrosis of the jaw, and the european medicines agency (ema) recommend that all patients undergo a comprehensive dental examination and preventive therapy before initiating therapy with an antiresorptive agent or angiogenesis inhibitor [19-23]. the assessment includes [19]: • unsavable teeth and those that are unlikely to be saved must be extracted. if systemic conditions allow, initiation of therapy with an antiresorptive or antiangiogenic agent should be delayed until the extraction site has healed (14 to 21 days) or until there is sufficient bone formation/healing. these decisions must be made jointly by the attending physician and the dentist; • when possible, less invasive dental procedures with preservation of the tooth root are preferred to tooth extraction; • active oral infections must be treated, and objects at high risk of infection must be removed; • dental prophylaxis, caries control, and conservative restorative dentistry are critical to maintaining functionally healthy dentition; • patients with full or partial dentures should be examined for areas of mucosal trauma; • patients should be trained in the importance of dental hygiene and regular dental examinations and assessment of dental status and instructed to report any pain, pa#ents’ awareness of the complica#ons of bisphosphonate therapy vol 10 no 1 (2022) doi 10.5195/d3000.2022.362 h#p://den*stry3000.pi#.edu swelling, or bone exposure immediately [19]. the implementation of dental screening and appropriate dental treatment measures prior to initiating antiresorptive therapy reduced the risk of baonj in several prospective studies compared retrospectively to patients who did not have dental prophylaxis [24-26]. pechalova et al. [27] recommend: • a thorough clinical examination of the dentition and oral cavity, panoramic radiograph and, by the discretion of the doctor, targeted periapical radiographs as a mandatory re-quired minimum • conducting dental treatment aimed at elimination of infection and the need for invasive procedures in the shortand mid-term future. • all invasive dental procedures should be performed at least one month before initiating bisphosphonate therapy in order to allow sufficient time for recovery of the jawbone. restoration with dental implants is not recommended. after the start of bisphosphonate therapy patients should go to preventive examinations every three/four months, with additional imaging studies to monitor for osteolysis, osteosclerosis, periodontal dilation and involvement of the furcations [27]. oral hygiene should be carefully monitored, patients should maintain excellent oral hygiene (with toothpaste and toothbrush, flossing, and antiseptic mouth wash) to reduce the level of periodontal and oral infection, and to avoid invasive dental procedures, if possible. in this regard, smoking cessation is recommended [16]. patients should be asked about any planned dental procedures before administering each dose of antiresorptive agent and reminded to avoid invasive ones (tooth extraction, implants, and all other procedures that involve manipulation of the jawbone or periosteum) while being treated with these drugs. they need to make sure that relatively noninvasive dental procedures such as professional cleaning, cavity maintenance and even routine root canal treatments do not increase the risk of baonj and can be safely performed based on treatment protocols used for the general population. dental crowns may be placed. removable dentures should be carefully planned in areas of expected excessive pressure, preferring soft plastic constructions [28]. it remains unclear whether less frequent use of zoledronic acid reduces the incidence of baonj. a retrospective analysis indicates a reduced percentage of patients with multiple myeloma who received zoledronic acid every 12 weeks instead of every four weeks [29]. randomized trials mainly in breast and prostate cancer populations did not report a significantly lower percentage of osteonecrosis cases with bp intake at long intervals [30]. discontinuation of bp therapy prior to invasive dental procedures (e.g. tooth extraction, implant placement, or apical endodontic treatment) during treatment with an antiresorptive agent or antiangiogenic agent has not been proven effective and there is scarce data to guide the physician in this direction [16]. conclusions baonj is a complication associated with bp intake that severely impairs the quality of life of many patients. as the condition is considered irreversible and treatment options are still a complex and controversial issue among clinicians, our efforts should be focused mainly on its prevention both before and after the initiation of bp therapy. knowledge of epidemiology and risk factors is key. it is crucial to raise the awareness of dentists, oncologists, and patients and their willingness to work together as a team to reduce the risk of developing this complication. educational programs should be included in the qualification of physicians prescribing and treating patients with bp, dental practitioners who can have such patients in their everyday practice, as well as in the consultation of patients taking this type of medication. pa#ents’ awareness of the complica#ons of bisphosphonate therapy vol 10 no 1 (2022) doi 10.5195/d3000.2022.362 h#p://den*stry3000.pi#.edu authors’ contribution author 1 conceived and designed the analysis, collected the data, performed the analysis, and wrote the paper. author 2 designed the analysis, collected the data, and proofread the manuscript. author 3 performed the statistical analysis and proofread the manuscript. conflicts of interest statement there is no conflict of interest to disclose. references 1. a review of literature on updates of bisphosphonates administration, cancer biomarkers for bisphosphonate therapy, and bisphosphonate-related osteonecrosis of the jaw in breast cancer. khayamzadeh m, razmara f, asad a, shabankare g. international journal of cancer management. 2021;14(3); e102733. doi: 10.5812/ijcm.102733 2. understanding bisphosphonates and osteonecrosis of the jaw: uses and risks. rosini s, rosini s, bertoldi i, frediani b. eur rev med pharmacol sci. 2015 sep;19(17):3309-17. pmid: 26400539. 3. bisphosphonate-associated osteonecrosis of the jaws: a survey of the level of knowledge of dentists about the risks of bisphosphonate therapy. hristamyan-cilev ma, pechalova pp, raycheva rd, hristamyan vp, kevorkyan ak, stoilova yd. folia med (plovdiv). 2019 jun 1;61(2):303-311. doi: 10.2478/folmed-2018-0075. pmid: 31301653. 4. knowledge, practices, and opinions of ontario dentists when treating patients receiving bisphosphonates. alhussain a, peel s, dempster l, clokie c, azarpazhooh a. j oral maxillofac surg. 2015 jun;73(6):1095-105. doi: 10.1016/j.joms.2014.12.040. epub 2015 jan 12. pmid: 25843818. 5. bisphosphonates, healthcare professionals and oral health. raj dv, abuzar m, borromeo gl. gerodontology. 2016 mar;33(1):135-43. doi: 10.1111/ger.12141. epub 2014 jul 15. pmid: 25039439. 6. survey of korean dentists on the awareness on bisphosphonate-related osteonecrosis of the jaws. yoo jy, park yd, kwon yd, kim dy, ohe jy. j investig clin dent. 2010 nov;1(2):90-5. doi: 10.1111/j.2041-1626.2010.00024.x. pmid: 25427263. 7. knowledge and attitudes of french dentists on bone resorption inhibitors (bisphosphonates and denosumab): a cross-sectional study. arnaud mp, talibi s, lejeune-cairon s. j stomatol oral maxillofac surg. 2022 apr;123(2):163170. doi: 10.1016/j.jormas.2021.04.010. epub 2021 apr 27. pmid: 33930600. 8. bisphosphonate-related osteonecrosis of the jaw: awareness and level of knowledge of lebanese physicians. el osta l, el osta b, lakiss s, hennequin m, el osta n. support care cancer. 2015 sep;23(9):2825-31. doi: 10.1007/s00520015-2649-1. epub 2015 feb 13. pmid: 25672288. 9. physicians' awareness of bisphosphonates-related osteonecrosis of the jaw. al-mohaya ma, al-khashan hi, mishriky am, al-otaibi lm. saudi med j. 2011 aug;32(8):830-5. pmid: 21858393. 10. perceptions of medical doctors on bisphosphonate-related osteonecrosis of the jaw. kim jw, jeong sr, kim sj, kim y. bmc oral health. 2016 sep 7;16(1):92. doi: 10.1186/s12903-016-0290-0. pmid: 27604159; pmcid: pmc5015333. 11. intervention to improve awareness of the risk factors for osteonecrosis of the jaw in patients under treatment with bisphosponates. randomised clinical trial. garcía-martínez l, martín-payo r, pelazgarcía a, sierra-vega m, junqueragutiérrez lm. enferm clin. 2017 novdec;27(6):352-360. english, spanish. doi: 10.1016/j.enfcli.2017.04.001. epub 2017 jun 2. pmid: 28583834. 12. awareness of the risk of developing medication-related osteonecrosis of the jaw among bisphosphonate users. elma'aita a, da'as n, al-hattab m, hassona y, al-rabab'ah m, al-kayed ma. j int med res. 2020 sep;48(9):300060520955066. doi: 10.1177/0300060520955066. pmid: 32924697; pmcid: pmc7493245. 13. awareness among patient at risk of developing medication related osteonecrosis of the jaw (mronj) a primary prevention strategy. al abdullateef a, alhareky ms. saudi pharm j. 2020 jun;28(6):771-778. doi: 10.1016/j.jsps.2020.05.004. epub 2020 may 15. pmid: 32550810; pmcid: pmc7292880. 14. narrative [corrected] review: bisphosphonates and osteonecrosis of the jaws. woo sb, hellstein jw, kalmar jr. ann intern med. 2006 may 16;144(10):753-61. doi: 10.7326/00034819-144-10-200605160-00009. erratum in: ann intern med. 2006 aug 1;145(3):235. pmid: 16702591. 15. epidemiology, clinical manifestations, risk reduction and treatment strategies of jaw osteonecrosis in cancer patients exposed to antiresorptive agents. campisi g, fedele s, fusco v, pizzo g, di fede o, bedogni a. future oncol. 2014 feb;10(2):257-75. doi: 10.2217/fon.13.211. pmid: 24490612. 16. medication-related osteonecrosis of the jaw in patients with cancer. berenson pa#ents’ awareness of the complica#ons of bisphosphonate therapy vol 10 no 1 (2022) doi 10.5195/d3000.2022.362 h#p://den*stry3000.pi#.edu j.r. stopeck a.t. ©2015 uptodate. https://www.uptodate.com/contents/me dication-relatedosteonecrosis-of-the-jawin-patients-with-cancer (accesed on september 04, 2021) 17. appropriate and necessary oral care for people with cancer: guidance to obtain the right oral and dental care at the right time. epstein jb, güneri p, barasch a. support care cancer. 2014 jul;22(7):1981-8. doi: 10.1007/s00520014-2228-x. epub 2014 mar 28. pmid: 24676676. 18. how patients' lack of knowledge about oral bisphosphonates can interfere with medical and dental care. migliorati ca, mattos k, palazzolo mj. j am dent assoc. 2010 may;141(5):562-6. doi: 10.14219/jada.archive.2010.0229. pmid: 20436104. 19. american association of oral and maxillofacial surgeons position paper on medication-related osteonecrosis of the jaw--2014 update. ruggiero sl, dodson tb, fantasia j, goodday r, aghaloo t, mehrotra b, o'ryan f; american association of oral and maxillofacial surgeons. j oral maxillofac surg. 2014 oct;72(10):1938-56. doi: 10.1016/j.joms.2014.04.031. epub 2014 may 5. erratum in: j oral maxillofac surg. 2015 jul;73(7):1440. erratum in: j oral maxillofac surg. 2015 sep;73(9):1879. pmid: 25234529. 20. international task force on osteonecrosis of the jaw. diagnosis and management of osteonecrosis of the jaw: a systematic review and international consensus. khan aa, morrison a, hanley da, felsenberg d, mccauley lk, o'ryan f, et al. j bone miner res. 2015 jan;30(1):323. doi: 10.1002/jbmr.2405. pmid: 25414052. 21. canadian consensus practice guidelines for bisphosphonate associated osteonecrosis of the jaw. khan aa, sándor gk, dore e, morrison ad, alsahli m, amin f, et al. canadian association of oral and maxillofacial surgeons. j rheumatol. 2008 jul;35(7):1391-7. epub 2008 jun 1. erratum in: j rheumatol. 2008 aug;35(8):1688. erratum in: j rheumatol. 2008 oct;35(10):2084. pmid: 18528958. 22. american society of clinical oncology executive summary of the clinical practice guideline update on the role of bonemodifying agents in metastatic breast cancer. van poznak ch, temin s, yee gc, janjan na, barlow we, biermann js, et al. american society of clinical oncology. j clin oncol. 2011 mar 20;29(9):1221-7. pmid: 21343561. 23. chmp assessment report on bisphosphonates and osteonecrosis of the jaw. european medicines agency. london: ema/chmp 2011. http://www.ema.europa.eu/docs/en_gb /document_library/other/2010/01/wc5 00051383.pdf (accessed on december 05, 2021). 24. decreased occurrence of osteonecrosis of the jaw after implementation of dental preventive measures in solid tumour patients with bone metastases treated with bisphosphonates. the experience of the national cancer institute of milan. ripamonti ci, maniezzo m, campa t, fagnoni e, brunelli c, saibene g, et al. ann oncol. 2009 jan;20(1):137-45. doi: 10.1093/annonc/mdn526. epub 2008 jul 22. pmid: 18647964. 25. reduction of osteonecrosis of the jaw (onj) after implementation of preventive measures in patients with multiple myeloma treated with zoledronic acid. dimopoulos ma, kastritis e, bamia c, melakopoulos i, gika d, roussou m, et al. ann oncol. 2009 jan;20(1):117-20. doi: 10.1093/annonc/mdn554. epub 2008 aug 9. pmid: 18689864. 26. preventive strategies and clinical implications for bisphosphonate-related osteonecrosis of the jaw: a review of 282 patients. bonacina r, mariani u, villa f, villa a. j can dent assoc 2011; 77:b147. pmid: 22129778. 27. chapter 10 bisphosphonate – related osteonecrosis of the jaws – diagnosis and management. pechalova p, poriazova e, pavlov n, bakardjiev a. textbook of advanced oral and maxillofacial surgery, edited by mohammad hosein kalantar motamedi, isbn 978-953-511146-7. published: june 26, 2013 28. bisphosphonate-induced exposed bone (osteonecrosis/osteopetrosis) of the jaws: risk factors, recognition, prevention, and treatment. marx re, sawatari y, fortin m, broumand v. j oral maxillofac surg. 2005 nov;63(11):156775. doi: 10.1016/j.joms.2005.07.010. pmid: 16243172. 29. a different schedule of zoledronic acid can reduce the risk of the osteonecrosis of the jaw in patients with multiple myeloma. corso a, varettoni m, zappasodi p, klersy c, mangiacavalli s, pica g, lazzarino m. leukemia. 2007 jul;21(7):1545-8. doi: 10.1038/sj.leu.2404682. epub 2007 apr 5. pmid: 17410188. 30. osteonecrosis of the jaw and bisphosphonates. durie bg, katz m, crowley j. n engl j med. 2005 jul 7;353(1):99-102; discussion 99-102. doi: 10.1056/nejm200507073530120. pmid: 16000365. microsoft word filho 38 10-8.docx   vol  3,  no  1  (2015)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2015.38           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.   tooth  malposition  in  patients  with  cleft  lip  and/or  palate  in  a  bra-­‐ zilian  population   mário  rodrigues  melo  filho1,  verônica  oliveira  dias1,  daniella  reis  barbosa  martelli1,  lívia  ribeiro  paranaíba1,  mário  sérgio  oliveira  swerts2,   le#zia  monteiro  de  barros2,  hercílio  martelli  júnior1,2     1  stomatology  clinic,  dental  school,  state  university  of  montes  claros,  montes  claros,  minas  gerais,  brazil   2  center  for  the  rehabilita/on  of  craniofacial  anomalies,  josé  do  rosário  vellano  university,  alfenas,  minas  gerais,  brazil   abstract   the  purpose  of  the  present  study  was  to  evaluate  the  prevalence  of  teeth  malposi5on  (ro-­‐ tated)  in  brazilian  pa.ents  with  oral  cle4s  and  to  contribute  to  the  defini.on  of  subpheno-­‐ types.  this   study   included   317   pa3ents  with   nonsyndromic   cle8   lip  with   or  without   cle8   palate.  tooth  malposi.on  was  assessed  clinically,  through  radiographs,  and  medical  history   records  for  each  individual.  only  teeth  malposi6ons  outside  the  area  of  the  cle/s  were  in-­‐ cluded.  comparisons  were  assessed  by  cross-­‐tabula&on  and  standard  chi-­‐square   test,  and   sta$s$cal  significance  was  set  at  p≤0.05.  cle4  lip  and  palate  was  more  prevalent  in  males,   while  cle(  palate  was  more  common  in  females.  regarding  the  presence  of  tooth  malposi-­‐ !on,  of  the  317  pa!ents,  92  (29.02%)  had  at  least  one  tooth  with  the  dental  anomaly.  tooth   malposi(on  was  more  common  in  pa(ents  with  cle1  lip  and  palate  (16.1%),  followed  by,  re-­‐ spec%vely,  cle+  palate  (6.9%)  and  cle+  lip  (6%)  (p=0.112).  the  highest  occurrence  of  tooth   malposi(on  was   in   the  mandible   and   involved   the   canines   (p<0.01).   few   studies  have   in-­‐ ves$gated   the   prevalence   of   tooth  malposi$on   in   individuals  with   nonsyndromic   cle7   lip   with  or  without  cle,  palate.  our  results  confirmed  the  highest  occurrence  of  dental  anoma-­‐ lies,   par*cularly   tooth  malposi*on,   in  pa(ents  with  oral   cle1s.  our  findings   also  highlight   that  there  was  a  higher  occurrence  of  this  condi1on  in  the  mandible  and  not  the  maxilla.     cita%on:  filho  m,  dias  v,  martelli  d,  parnaiba  l,   swerts   m,   de   barros   l,   júnior   h.   (2015)   tooth   malposi"on  in  pa"ents  with  cle/  lip  and/or  pal-­‐ ate   in   a   brazilian   popula*on.   den$stry   3000.   1:a001  doi:10.5195/d3000.2015.38   received:  july  6,  2015   accepted:    september  13,  2015   published:    october  23,  2015   copyright:  ©2015   carlini   j,   severo   d,  asami   k.   this   is   an   open   access   ar!cle   licensed   under   a   crea%ve  commons  a"ribu%on  work  4.0  united   states  license.   email:  mariomelo@gmail.com   introduction     the  embryological  devel-­‐ opment  of  the  face  relies  on  the  in-­‐ terplay  of  a  vast  range  of  factors   encompassing  cell  differentiation,   growth,  apoptosis,  cell  to  cell  adhe-­‐ sion,  and  inter  and  intracellular  sig-­‐ naling.  the  disruption  of  a  gene  that   controls  one  or  more  of  these  fac-­‐ tors,  inhibition  of  cell  function  by   environmental  teratogens  or  a   combination  of  the  two  is  likely  to   be  the  etiological  cause  of  craniofa-­‐ cial  malformations  such  as  nonsyn-­‐ dromic  cleft  lip  with  or  without  cleft   palate  (nscl/p)  [1].     nscl/p  affects  approxi-­‐ mately  1/700  live  births,  with  wide   variability  across  geographic  origin,   racial  and  ethnic  groups,  as  well  as   environmental  exposures  and  soci-­‐ oeconomic  status.  in  general,  asian   and  amerindian  populations  have   the  highest  reported  birth  preva-­‐ lence  rates,  often  as  high  as  1/500,   european-­‐derived  populations  have   intermediate  prevalence  rates  at   about  1/1,000,  and  african-­‐derived   populations  have  the  lowest  preva-­‐ lence  rates  at  about  1/2,500  [2].  in   brazil,  the  prevalence  is  between   0.36  and  1.54  per  1,000  live  births.   cleft  lip  and  palate  (clp)  is  most   frequent  in  males  and  isolated  cleft   palate  (cp)  is  most  typical  in  fe-­‐ males  [3].  individuals  with  clp  may   experience  problems  with  feeding,   speaking,  hearing  and  social  inte-­‐ gration  that  can  be  corrected  to   varying  degrees  by  surgery,  dental   treatment,  speech  therapy  and  psy-­‐ chosocial  intervention  [2].  it  has   been  proposed  that  clefting  is  part   of  a  complex  malformation  associ-­‐ ated  with  other  dental  anomalies   resulting  from  a  disturbed  dentition   development  [4].  recent  studies   proposed  that  dental  anomalies   outside  of  the  cleft  area  could  serve   as  clinical  markers  for  the  definition   of  cleft  subphenotypes,  suggesting  a   common  genetic  background  be-­‐ tween  such  conditions  [5-­‐9].  in  fa-­‐ vor  of  this  hypothesis  is  the  fact  that   tooth,  lip,  and  palate  development   occur  almost  concomitantly  and  are   related  anatomically  [10-­‐12].     when  compared  with  the  general   population,  dental  anomalies,  such      tooth  malposi,on  in  pa,ents  with  cle#  lip  and/or  palate  in  a  brazilian  popula*on   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.38    http://dentistry3000.pitt.edu   2   as  tooth  agenesis,  supernumerary   teeth,  microdontia,  fused  teeth,  ec-­‐ topic  eruption,  tooth  malposition   (rotation),  taurodontism  and  enam-­‐ el  hypoplasia,  are  considerably   more  prevalent  in  individuals  with   nscl/p  [5-­‐8,13-­‐15].  in  addition,     few  studies  have  investigated  the   dental  anomalies  associated  with     subphenotypes  of  oral  clefts,  justify-­‐ ing  the  importance  of  this  study.     tooth  malposition  (rotation  or  in-­‐ clination),  was  considered  subjec-­‐ tively  as  any  evident  (at  least  20°)   mesiolingual  or  distolingual  intra-­‐ alveolar  displacement  of  a  tooth   around  its  longitudinal  axis  [16].   the  purpose  of  the  present  study   was  to  evaluate  the  prevalence  of   tooth  malposition  in  patients  with   oral  clefts  and  to  contribute  to  the   definition  of  nscl/p  subpheno-­‐ types.       material  and  methods       this  study  included  317  pa-­‐ tients  with  repaired  nscl/p.  all   participants  were  recruited  from   the  same  institution  (centre  for  re-­‐ habilitation  of  craniofacial  anoma-­‐ lies,  minas  gerais  state,  brazil).  all   subjects  were  from  minas  gerais,   brazil,  where  there  is  an  admixed   population  of  europeans  (mostly   from  portugal  and  italy)  and  afri-­‐ cans,  with  a  small  percentage  of  na-­‐ tive  brazilian  indians.  all  patients   presented  similar  ethnicities  and   social  culture.       teeth  were  assessed  clini-­‐ cally,  through  panoramic  radio-­‐ graphs,  and  medical  history  records   for  each  individual.  the  radiographs   were  examined  with  magnifying   glass  in  a  dark  room  over  a  light  box   that  had  a  frame  to  avoid  light  pass-­‐ ing  on  the  sides  of  the  radiograph.   eruption  of  any  tooth  in  an  abnor-­‐ mal  position  was  the  criteria  con-­‐ sidered  to  represent  malposition.   patients  with  a  history  of  dental  ex-­‐ traction,  previous  orthodontic   treatment,  or  who  were  younger   than  12  years  old  were  excluded   due  to  the  inability  to  accurately   identify  all  anomalies.  in  order  to   eliminate  inter-­‐examiner  differ-­‐ ences,  dental  anomalies  were  classi-­‐ fied  by  a  single  calibrated  examiner   (intra-­‐examiner  kappa  value  =   0.93).  we  did  not  include  primary   dentition  nor  third  molars.  only   teeth  malposition  outside  the  area   of  the  clefts  were  included  in  this   study.       the  clefts  were  categorized   into  three  groups  with  the  incisive   foramen  as  a  reference:  (1)  cleft  lip   (cl):  includes  complete  or  incom-­‐ plete  pre-­‐foramen  clefts,  either  uni-­‐ lateral  or  bilateral;  (2)  cleft  lip  and   palate  (clp):  includes  unilateral  or   bilateral  transforamen  clefts  and   pre-­‐  or  post-­‐foramen  clefts;  (3)  cleft   palate  (cp):  includes  all  post-­‐ foramen  clefts,  complete  or  incom-­‐ plete  [17].  median  or  rare  clefts   were  not  included  in  our  analysis.   the  information  collected  was   stored  in  a  database  and  analyzed   using  the  statistical  program  spss®   version  19.0  (statistical  package  for   social  sciences  for  windows,  inc.,   usa).  comparisons  were  assessed   by  cross-­‐tabulation  and  standard   chi-­‐square  test,  and  statistical  sig-­‐ nificance  was  set  to  p≤0.05.  this   study  was  approved  by  the  univer-­‐ sity’s  ethics  committee  in  research.   all  patients   were  informed   about  the   study’s  pur-­‐ pose  before   they  provided   written  con-­‐ sent  to  partici-­‐ pate.       results       of  the  317  subjects  included   in  this  study,  172  were  male  and   145  were  female.  the  average  age   was  17.48  years  old.  table  1  shows   the  distribution  of  the  types  of  oral   clefts  according  to  extension  and   gender.  of  the  317  patients  with   clefts,  189  had  clp,  74  had  cl,  and   54  had  isolated  cp.  clp  was  more   prevalent  in  males,  while  cp  was   more  common  in  females.  cl  had  a   similar  distribution  between  the   genders.  with  regard  to  skin  color,   most  of  the  patients  were  non-­‐ caucasian  (209,  65.93%;  108  or   34.06%  were  caucasian).     regarding  the  presence  of   tooth  malposition,  of  the  317  pa-­‐ tients,  92  (29.02%)  had  at  least  one   tooth  with  the  dental  anomaly.  of   these  92  patients  with  tooth  malpo-­‐ sition,  144  teeth  presented  the   anomaly.  this  means  that  40  pa-­‐ tients  had  more  than  one  tooth  with   malposition.  table  2  shows  the  fre-­‐ quency  of  patients  with  tooth  mal-­‐ position  according  to  the  types  of   oral  clefts.  it  turns  out  that  tooth   malposition  was  more  common  in   patients  with  clp  (16.1%),  followed     table  1.  distribution  of  cleft  lip  and/or  palate  according  to  extension  and  gender.   gender     n   cleft  lip  and  palate   cleft  lip   cleft  palate   p  value  (x2)   n   %   n   %   n   %   male   172   112   35.3   37   11.7   23   7.3   0.067   female   145   77   24.3   37   11.7   31   9.7     total   317   189   59.6   74   23.4   54   17.0        tooth  malposi,on  in  pa,ents  with  cle#  lip  and/or  palate  in  a  brazilian  popula*on   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.38    http://dentistry3000.pitt.edu   3   by  cp  (6.9%)  and  cl  (6%)   (p=0.112).                   in  table  3,  it  is  possible  to   assess  the  distribution  of  tooth   malposition  according  to  their  ana-­‐ tomical  location  (maxilla  or  mandi-­‐ ble).  when  evaluating  the  144  teeth   malposition,  it  can  be  observed  that   most  (69.48%)  are  located  in  the   mandible.  most  mandibular  malpo-­‐ sition  was  in  the  canines  (53.47%),   followed  by  the  second  premolars   (6.25%).  in  the  maxilla  (30.54%),   the  highest  occurrence  of  malposi-­‐ tion  was  seen  in  lateral  incisors   (9.72%),  followed  by  the  central   incisor  (9.03%).       discussion       certain  evidence  suggests   that  the  phenotype  of  nscl/p  is   much  more  complex  than  common-­‐ ly  believed.  for  that  reason,  thor-­‐ ough  knowledge  of  its  various  pos-­‐ sible  combinations,  especially  con-­‐ cerning  the  cleft  extension  and   presence  of  dental  anomalies  in  ar-­‐ eas  outside  of  the  cleft,  is  important   [5].  out  of  317  patients  evaluated  in   the  present  study,  92  (29%)  pre-­‐ sented  tooth  malposition.  this  re-­‐ sult  confirms  that  dental  anomalies   in  nscl/p  patients  are  more  fre-­‐ quent  than  in  healthy  brazilian  in-­‐ dividuals  [7,18].  recently,  we  found   39.9%  of  brazilian  patients  with   nscl/p  present  dental  anomalies.   tooth  agenesis  (47.5%),  impacted   tooth  (13.1%),  and  microdontia   (12.7%)  were  the  most  common   anomalies  [8].       certain  authors  have  related   higher  frequencies  of  dental  anoma-­‐ lies  as  the  severity  of  the  cleft  in-­‐ creased  [5,6,19].  our  results   demonstrated  that  the  frequency  of   tooth  malposition  was  higher  in  pa-­‐ tients  with  clp  compared  to  those   with  cp  and  cl.  menezes  and  vieira   (2008)  have  shown  that  patients   with  cp  were  significantly  more  af-­‐ fected  by  dental  anomalies  than  pa-­‐ tients  with  cl  or  clp,  whereas  a   series  of  studies  demonstrated  a   higher  frequency  of  dental  anoma-­‐ lies  in  patients  with  clp  [5,20,21].   in  contrast  to  tooth  agenesis,  no  ge-­‐ netic  etiology  has  yet  been  estab-­‐ lished  in  the  case  of  malposition,   although  it  has  been  suggested  that   canine  malposition  is  associated   with  agenesis  and  other  dental   anomalies  in  populations  without   oral  clefts  [22].     in  an  extensive  study  with   brazilian  patients  with  nscl/p,  let-­‐ ra  et  al.,  (2007)  showed  that  the   mandibular  canines  were  the  teeth   most  commonly  af-­‐ fected  by  malposi-­‐ tion  associated  with   clp.  in  the  present   study,  there  was  a   higher  prevalence  of   tooth  malposition  in   the  mandible  com-­‐ pared  to  the  maxilla   (p<0.01).  similar  to   the  study  mentioned   above  [5],  the  man-­‐ dibular  canines  were   the  most  affected   teeth  (53.47%).  the   observation  of  mal-­‐ position,  with  man-­‐ dibular  canines  be-­‐ ing  the  most  affected  teeth  and  of-­‐ ten  associated  with  clp,  is  notewor-­‐ thy  and  has  not  yet  been     described  [5].  our  results  agree   with  these  findings.  although  the   occurrence  of  tooth  malposition   could  be  explained  as  a  conse-­‐ quence  of  plastic  surgery  for  repair   of  the  oral  clefts,  which  can  affect   the  development  of  maxillary  bone   resulting  in  less  space  in  the  upper   arch  for  normal  eruption  of  teeth   [23],  it  is  interesting  to  mention  that   the  higher  incidence  of  this  dental   anomaly  occurred  in  the  mandible   and  not  the  maxilla.   table  2.  frequency  of  patients  with  tooth  malposition  according  to  the  types  of  oral  clefts.   type  of  cleft   tooth  malposition   present   absent   total   n   %   n   %   n   %   cleft  palate   22   6.9   32   10.1   54   17.0   cleft  lip   19   6.0   55   17.4   74   23.4   cleft  lip  and  palate   51   16.1   138   43.5   189   59.6   total   92   29.0   225   71.0   317   100   *chi-­‐square  test                p=0.112      tooth  malposi,on  in  pa,ents  with  cle#  lip  and/or  palate  in  a  brazilian  popula*on   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.38    http://dentistry3000.pitt.edu   4     when  analyzing  the  cleft   type  distribution  by  gender,  we   found  a  higher  prevalence  in  males,   mainly  in  clp,  and  a  higher  preva-­‐ lence  of  isolated  cp  in  females.  the-­‐ se  results  are  concordant  with  pre-­‐ vious  studies  [6,23-­‐25].  previous   reports  have  established  that  oral   clefts  present  a  sexual  dimorphism:   clp  is  more  common  in  males  and   cp  is  prevalent  in  females  [3,7,25].   here  our  results  are  similar  to  those   found  in  the  literature,  but  the  oc-­‐ currence  of  cl  isolated  was  equal  in   both  genders.       as  the  brazilian  population   is  result  of  the  genetic  admixture  of   three  main  ancestral  populations   (europeans,  africans,  and  amerin-­‐ dians)  and  displays  very  high  levels   of  genomic  diversity  [26],  we  have   previously  demonstrated  that  the   use  of  ancestry  markers  in  associa-­‐ tion  studies  of  ethnically  mixed   populations  (structuration  of  the   samples)  is  important  to  avoid  in-­‐ terpretation  bias  [27,28].  in  this   study,  the  characteristics  of  the  an-­‐ cestry  of  the  studied  population   (minas  gerais  state,  brazil)  were   previously  evaluated  [29].     few  studies  have  investi-­‐ gated  the  prevalence  of  tooth  mal-­‐ position  in  individuals  with  nscl/p.   our  results  confirmed  the  highest   occurrence  of  dental  anomalies,   particularly  tooth  malposition,  in   patients  with  oral  clefts.  our  study   also  highlights  that  there  was  a   higher  occurrence  of  this  condition   in  the  mandible  and  not  maxilla.   however,  future  studies  are  neces-­‐ sary  to  determine  the  exact  mecha-­‐ nisms  responsible  for  the  occur-­‐ rence  of  tooth  malposition.       acknowledgments       this  work  was  partially   supported  by  the  minas  gerais   state  research  foundation-­‐ fapemig,  minas  gerais,  brazil   and  the  procad/casadinho-­‐ cnpq/capes,  brasília,  brazil.                                                 table  3.  frequency  of  malposition  according  to  type  of  tooth  in  individuals  with   oral  clefts.   localization   tooth   n   %   mandible   central  incisors   2   1.39   lateral  incisors   6   4.2   canines   77   53.47   first  premolars   6   4.17     second  premolars   9   6.25   100   69.48   maxilla   central  incisors   13   9.03   lateral  incisors   14   9.72   canines   12   8.33   first  premolars   5   3.47   44   30.54   total   144   100   *chi-­‐square  test                p<0.01      tooth  malposi,on  in  pa,ents  with  cle#  lip  and/or  palate  in  a  brazilian  popula*on   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.38    http://dentistry3000.pitt.edu   5   references   1.  folate  and  the  face:  evaluating   the  evidence  for  the  influence  of   folate  genes  on  craniofacial  devel-­‐ opment.  prescott  nj,  malcolm  s.   cleft  palate  craniofac  j.  2002   may;39(3):327-­‐31.  pmid:   12019010.   2.  cleft  lip  and  palate:  understand-­‐ ing  genetic  and  environmental  in-­‐ fluences.  dixon  mj,  marazita  ml,   beaty  th,  murray  jc.  nat  rev  genet.   2011  mar;12(3):167-­‐78.  pmid:   21331089.   3.  prevalence  of  nonsyndromic  oral   clefts  in  a  reference  hospital  in  the   state  of  minas  gerais,  brazil,  be-­‐ tween  2000–2005.  martelli-­‐junior   h,  porto  lv,  martelli  dr,  bonan  prf,   freitas  ab,  coletta  rd.  braz  oral   res.  2007  oct-­‐dec;21(4):314-­‐7.   pmid:  18060257.   4.  epidemiology  of  hoffmeister's   “genetically  determined  predispo-­‐ sition  to  disturbed  development  of   the  dentition”  in  patients  with  cleft   lip  and  palate.  stahl  f,  grabowski  r,   wigger  k.  cleft  palate  craniofac  j.   2006  jul;43(4):457-­‐65.  pmid:   16854204.   5.  defining  subphenotypes  for  oral   clefts  based  on  dental  development.   letra  a,  menezes  r,  granjeiro  jm,   vieira  ar.  j  dent  res.  2007   oct;86(10):986-­‐91.  pmdi:   17890676.   6.  dental  anomalies  as  part  of  the   cleft  spectrum.  menezes  r,  vieira   ar.  cleft  palate  craniofac  j.  2008   jul;45(4):414-­‐9.  pmid:  18616370.   7.  side  of  dental  anomalies  and  tau-­‐ rodontism  as  potential  clinical   markers  for  cleft  subphenotypes.   kuchler  e,  motta  l,  vieira  a,  gran-­‐ jeiro  j.  cleft  palate  craniofac  j.  2011   jan;48(1):103-­‐8.  pmid:  20507240.   8.  prevalence  of  dental  anomalies   in  patients  with  nonsyndromic   cleft  lip  and/or  palate  in  a  brazili-­‐ an  population.  paranaíba  lmr,  co-­‐ letta  rd,  swerts  mso.  quintino  rp,   barros  lm,  martelli-­‐júnior  h.  cleft   palate  craniofac  j.  2013   jul;50(4):400-­‐5.  pmid:  21973329.   9.  expanding  the  cleft  phenotype   cleft  l:  the  dental  characteristics  of   unaffected  parents  of  australian   children  with  non-­‐syndromic  lip   and  palate.  aspinall  a,  raj  s,   jugessur  a,  marazita  m,  savarirayan   r,  kilpatrick  n.  int  j  paediatr  dent.   2014  jul;24(4):286-­‐92.  pmid:   24237197.   10.  identification  of  cell  patterns  in   human  tooth  differentiation.  tonge   ch.  j  dent  res.  1967  sep-­‐ oct;46(5):876-­‐8.  pmid:  5234028.   11.  the  many  faces  and  factors  of   orofacial  clefts.  schutte  bc,  murray   jc.  hum  mol  genet.   1999;8(10):1853-­‐9.  pmid:   10469837.   12.  the  origin  and  development  of   the  upper  lateral  incisor  and  pre-­‐ maxilla  in  normal  and  cleft   lip/palate  monkeys  induced  with   cyclophosphamide.  wei  x,  senders   c,  owiti  go,  liu  x,  wei  zn,  dillard-­‐ telm  l.  cleft  palate  craniofac  j.   2000  nov;37(6):571-­‐83.  pmid:   11108527.   13.  abnormalities  of  the  maxillary   incisors  in  children  with  cleft  lip   and  palate.  vichi  m,  franchi  l.  asdc   j  dent  child.  1995  nov-­‐ dec;62(6):412-­‐7.  pmid:  8636477.   14.  dental  anomalies  of  number  in   the  permanent  dentition  of  patients   with  bilateral  cleft  lip:  radiographic   study.  da  silva  ap,  costa  b,  de  car-­‐ valho  carrara  cf.  cleft  palate  crani-­‐ ofac  j.  2008  sep;45(5):473-­‐6.  pmid:   8788864.   15.  distribution  patterns  of  primary   and  permanent  dentition  in  chil-­‐ dren  with  unilateral  complete  cleft   lip  and  palate.  tsai  tp,  huang  cs,   huang  cc,  see  lc.  cleft  palate  cra-­‐ niofac  j.  1998  mar;35(2):154-­‐60.   pmid:  9527312.   16.  prevalence  and  distribution  of   selected  developmental  dental   anomalies  in  an  indian  population.   gupta  sk,  saxena  p,  jain  s,  jain  d.  j   oral  sci.  2011  jun;53(2):231-­‐8.   pmid:  21712629.   17.  classification  of  cleft  lip  and   cleft  palate.  suggested  changes.  spi-­‐ na  v,  psillakis  jm,  lapa  fs,  ferreira   mc.  rev  hosp  clin  fac  med  são  pau-­‐ lo.  1972  jan-­‐feb;27(1):5-­‐6.  pmid:   4671376.   18.  studies  of  dental  anomalies  in  a   large  group  of  school  children.   kuchler  ec,  risso  pa,  costa  mde  c,   modesto  a,  vieira  ar.  arch  oral  bi-­‐ ol.  2008  oct;53(10):941-­‐6.  pmid:   18490001.   19.  prevalence  of  dental  anomalies   in  a  population  of  cleft  lip  and  pal-­‐ ate  patients.  al  jamal  ga,  hazza'a   am,  rawashdeh  ma.  cleft  palate      tooth  malposi,on  in  pa,ents  with  cle#  lip  and/or  palate  in  a  brazilian  popula*on   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.38    http://dentistry3000.pitt.edu   6   craniofac  j.  2010  jul;47(4):413-­‐20.   pmid:  20590463.   20.  hypodontia  in  children  with   various  types  of  clefts.  shapira  y,   lubit  e,  kuftinec  mm.  angle  orthod.   2000  feb;70(1):16-­‐21.  pmid:   10730671.   21.  genetic  association  studies  of   cleft  lip  and/or  palate  with  tooth   hypodontia  outside  the  cleft  region.   slayton  rl,  williams  l,  murray  jc,   wheeler  jj,  lidral  ac,  nishimura  cj.   cleft  palate  craniofac  j.  2003   may;40(3):274-­‐9.  pmid:  12733956.   22.  concomitant  occurrence  of  ca-­‐ nine  malposition  and  tooth  agene-­‐ sis:  evidence  of  orofacial  genetic   fields.  peck  s,  peck  l,  kataja  m.  am  j   orthod  dentofacial  orthop.  2002   dec;122(6):657-­‐60.  pmid:   12490878.   23.  dental  anomalies  in  children   born  with  clefts:  a  case-­‐control   study.  de  lima  pedro  r,  faria  mdb,   costa  mc,  vieira  ar.  cleft  palate   craniofac  j.  2012  nov;49(6):e64-­‐e8.   pmid:  21740171.   24.  classification  and  birth  preva-­‐ lence  of  orofacial  clefts.  tolarová   tt,  cervenka  j.  am  j  med  genet.   1998  jan;75(2):126-­‐37.  pmid:   9450872.   25.  orofacial  cleft  in  southern  italy.   carinci  f,  rullo  r,  laino  g,  festa  v,   mazzarella  n,  morano  d,  gombos  f.   minerva  stomatol.  2003   oct;52(10):427-­‐3.  pmid:  14765027.   26.  the  genomic  ancestry  of  indi-­‐ viduals  from  different  geographical   regions  of  brazil  is  more  uniform   than  expected.  pena  sd,  di  pietro  g,   fuchshuber-­‐moraes  m,  genro  jp,   hutz  mh,  kehdy  fde  s,  kohlrausch   f,  magno  la,  montenegro  rc,   moraes  mo,  de  moraes  me,    de   moraes  mr,  ojopi  eb,  perini  ja,   racciopi  c,  ribeiro-­‐dos-­‐santos  ak,   rios-­‐santos  f,  romano-­‐silva  ma,   sortica  va,  suarez-­‐kurtz  g.  plos   one.  2011  feb;6(2):e17063.  pmid:   21359226.   27.  polymorphisms  at  regions   1p22.1  (rs560426)  and  8q24   (rs1530300)  are  risk  markers  for   nonsyndromic  cleft  lip  and/or  pal-­‐ ate  in  the  brazilian  population.  ba-­‐ gordakis  e,  paranaiba  lm,  brito  la,   de  aquino  sn,  messetti  ac,  martelli-­‐ junior  h,  swerts  ms,  graner  e,  pas-­‐ sos-­‐bueno  mr,  coletta  rd.  am  j   med  genet  a.  2013   may;161a(5):1177-­‐80.  pmid:   23532876.   28.  polymorphisms  in  fgf12,  vcl,   cx43  and  vax1  in  brazilian  patients   with  nonsyndromic  cleft  lip  with  or   without  cleft  palate.  de  aquino  sn,   messetti  ac,  bagordakis  e,  martelli-­‐ júnior  h,  swerts  ms,  graner  e,  co-­‐ letta  rd.  bmc  med  genet.  2013   may;14:53-­‐6.  pmid:  23679094.   29.  analysis  of  susceptibility  poly-­‐ morphisms  for  nonsyndromic  cleft   lip  with  or  without  cleft  palate  in   the  brazilian  population.  birth  de-­‐ fects.  de  aquino  sn,  messetti  ac,   hoshi  r,  borges  a,  viena  cs,  reis  sr,   oliveira  swerts  ms,  graner  e,  mar-­‐ telli-­‐júnior  h,  coletta  rd.  res  a  clin   mol  teratol.  2014  jan;100(1):36-­‐42.   pmid:  24446087.     microsoft word 172 2023.docx evalua&on of glenoid fossa morphology in different facial growth pa=erns: a cone beam computed tomography study vol 11 no 1 (2023) doi 10.5195/d3000.2023.172 h#p://den*stry3000.pi#.edu evaluation of glenoid fossa morphology in different facial growth patterns: a cone beam computed tomography study maryam karandish1, somaye farmani1, bahar khademi1, maryam paknahad2 1orthodontic department, dental school, shiraz university of medical sciences, shiraz, iran 2oral and dental disease research center, dental school, shiraz university of medical sciences, shiraz, iran abstract purpose: the aim of current study was to assess the glenoid fossa morphology among different ver&cal skeletal pa=erns using cone beam computed tomography (cbct). materials and methods: the cbct images of 63 pa&ents with class i sagi=al skeletal pa=ern were classified into three groups based on posterior facial height (pfh)/anterior facial height (afh) ra&o. each group divided in to normal, horizontal, and ver&cal growth pa=ern groups. depth, width, and inclina&on of glenoid fossa were measured and assessed on cbct images. one-way analysis of variance (anova) and tukey post-hoc test were used to compare mean values of measured variables among the groups. results: the width (28.76 ± 0.79) and depth (14.61 ± 0.514) of glenoid fossa were lower in horizontal in comparison to ver&cal and normal growth pa=erns (p-value < 0.05). although steepness of glenoid fossa was higher in normal growth pa=ern (123.8 ± 16.68), no sta&s&cally significant differences were seen (p-value = 0.819). conclusion: the width and depth of glenoid fossa were significantly lower in horizontal growth pa=ern. keywords: glenoid fossa; growth pa=ern; cbct; tmj cita9on: karandish, m et al. (2023) evalua9on of glenoid fossa morphology in different facial growth paperns: a cone beam computed tomography study den9stry 3000. 1:a001 doi:10.5195/d3000.2023.172 received: april 21, 2021 accepted: september 28, 2021 published: january 4, 2023 copyright: ©2023 karandish, m et al. this is an open access ar9cle licensed under a crea9ve commons apribu9on work 4.0 united states license. email: maryampaknahad@yandex.com introduction temporomandibular joint (tmj) is one of the most complex joints due to its anatomical, histological, and biomechanical characteristics [1]. tmj is the joint between the mandible and the temporal bone of the skull that consists of mandibular condyle, glenoid fossa and articular eminence of the temporal bone [2]. some previous studies showed association between the joint morphology and different facial patterns. therefore, knowing about the articular eminence inclination could help clinician in the diagnosis, establishing more biological treatment modalities and treatment response between different facial types [3,4]. several recent studies have investigated the association between joint morphology and sagittal craniofacial patterns [5-13]. little is known about the association between the growth pattern and glenoid fossa morphology. to the best of our knowledge, there were only two studies that evaluated the association between the glenoid fossa morphology and vertical craniofacial morphology [14,15]. the study conducted by lin et al. evaluated articular eminence angle evalua&on of glenoid fossa morphology in different facial growth pa=erns: a cone beam computed tomography study vol 11 no 1 (2023) doi 10.5195/d3000.2023.172 h#p://den*stry3000.pi#.edu using lateral cephalometry [14]. the other study assessed the position and morphology of the temporomandibular joint in skeletal class ii females using cbct [15]. cbct is a choice to evaluate tmj structures because of several advantages including high-resolution images, short scanning times, reduced radiation dose and no magnification or distortion for accurate measurement of joint structure dimensions [16,17]. therefore, the aim of present study was to assess the glenoid fossa morphology among different vertical skeletal patterns using cbct. material and methods this cross-sectional study protocol was approved by institutional research ethics committee of shiraz university of medical sciences (sums) (ir.sums.dental.rec.1399.005). diagnostic cbct images of 63 adult patients (17 verticals, 29 horizontals and 17 normal) who referred to radiology department of dental school of sums for dental services such as impacted teeth, pns, maxillofacial cbct and etc. entered this study. the inclusion criterion follows: sufficient image sharpness and contrast to visualize the structure to be evaluated (glenoid fossa , articular eminence, sella turcica, mandible and nasion), and patients with skeletal class i relationships on the basis of anb angle, 0 to 4 degrees [18]. the exclusion criteria were symptoms of temporomandibular disorders, history of tmj surgery and/or tmj trauma, or fracture in the tmj region, any congenital abnormalities or systemic diseases potentially affecting joint morphology. cbct of patients presenting evidence of degenerative joint disease in the images were excluded from the study. all cbct scans were taken by the same clinician using new tom vgi. the exposure factors set at 120 kvp, 4.6 ma, exposure time of 20 s, and a field of view of 15 × 15 cm. the cbct images were taken with the participants biting in maximum dental intercuspation and their heads positioned so that the frankfort plane was parallel to the floor. the images divided into three groups according to growth pattern based on posterior facial height (pfh)/anterior facial height (afh) ratio (table 1). on the axial view, the section of the condylar process that had the widest mediolateral diameter on the left and right sides was chosen as the reference view for reconstruction of the sagittal slices. in this section, a line parallel to the long axis of the condylar process was drawn and sagittal images were reconstructed as 0.5 mm slice interval/thickness. the measurements were establishing on the central sagittal section of the condyle. the glenoid fossa depth was established by measuring the perpendicular distance between the highest point of fossa and the line passing through the most inferior point on the articular eminence and the posterior glenoid process (figure 1.b). the glenoid fossa width defined as the distance between the most inferior point on the articular eminence and the posterior glenoid process (figure 1.b). the articular eminence inclination was measured by top-roof line method, i.e., the angle between frankfort plane and the plane passing through the highest point in the roof of glenoid fossa and the lowest point at the crest of the articular eminence (figure 1.c). variables growth pattern group (valid percent) vertical (n= 17) normal (n=17) horizontal (n=29) pfh/afh < 62 6265 > 65 table 1. distribution of subjects according to different growth patterns evalua&on of glenoid fossa morphology in different facial growth pa=erns: a cone beam computed tomography study vol 11 no 1 (2023) doi 10.5195/d3000.2023.172 h#p://den*stry3000.pi#.edu statistical analysis data were analyzed using spss software package v.18 (version 18, spss inc, chicago, il, usa). all cbct images were re-measured by the same examiner after a 2-week interval to confirm intra-observer reliability. intra-class correlation coefficient was used to assess the reliability of the measurements. oneway analysis of variance (anova) and tukey post-hoc test were used to evaluate the association between different growth patterns and glenoid fossa morphology. p-value less than 0.05 was considered significant. results a total of 63 adult patients were included in this study. distribution of glenoid fossa morphology based on the width, depth and inclination is shown in table 2. the width (28.76 ± 0.79) and depth (14.61 ± 0.514) of glenoid fossa in horizontal growth pattern were the least of all groups (p-value < 0.05). although not statistically significant, the inclination of glenoid fossa was highest in normal growth pattern (123.8 ± 16.68). growth pattern group variables normal mean ± sd* vertical mean ± sd horizontal mean ± sd p-value width 32.11 ± 0.79 30.33 ± 0.79 28.76 ± 0.79 0.017 depth 15.34 ± 0.514 16.49 ± 0.514 14.61 ± 0.514 0.043 inclination 123.8 ± 16.68 114.66 ± 16.68 109.013 ±16.68 0.819 discussion sagittal and vertical facial disharmonies can affect the relationship of the mandible to the cranial base. therefore, glenoid fossa position is likely to play an important role in the establishment of different craniofacial patterns and orthopedic/orthodontic therapies [19-21]. in current study we determined the association between glenoid fossa morphology and different growth patterns using cbct images. the variability and complexity of the tmj make it difficult for accurate radiographic examination and clinical diagnosis [22]. different radiographic methods have been used in previous studies to examine the tmj morphology, such as computed tomography [23], magnetic resonance imaging [24], conventional tomography [25], and cbct [26-28]. standard 2d radiographs of the tmj have several limitations including superimposition of overlying structures, magnification and distortion that limit the ability to evaluate tmj [29,30]. cbct, a recently developed imaging technology, has been used for 3d imaging of the tmj and has been shown to delineate the joint figure 1. axial view of the condylar process (a); linear measurements of depth and width of glenoid fossa (b); inclination of glenoid (c). table 2. distribution of glenoid fossa morphology based on the width, depth, and inclination of glenoid fossa *standard deviation evalua&on of glenoid fossa morphology in different facial growth pa=erns: a cone beam computed tomography study vol 11 no 1 (2023) doi 10.5195/d3000.2023.172 h#p://den*stry3000.pi#.edu structures with high accuracy [26]. therefore, in our study we used cbct images for evaluation of joint morphology. there are two methods for measuring articular eminence inclination [4,31,32]. the first method is to measure the angle between fh plane and the line of deepest point of the roof of the fossa and the top of the articular eminence. the second method defines articular eminence inclination as the angle between the best fit line drawn along the posterior slope of articular eminence and fh plane. it should be noted that both angles really show the eminence inclination; the first method focuses on the location of eminence crest relative to the fossa roof, whereas the second method focuses on the posterior surface of eminence. the second method represents the actual condylar path, whereas the first method depicts the morphology of articular eminence better. in the present study the former method has been used the same as katsavrias et al. [4] , sümbüllü et al. [31] and kranjčić et al. [32]. inclination is defined as an angle between posterior slope of the articular eminence and any other horizontal plane such as fh plane, occlusal plane, and palatal plane [4,32]. the line frequently used is fh plane [31-34]. cohlmia et al. used the superior border of a tomographic film [35], whereas keller and carano used the angle between the posterior surface of the articular eminence and the posterior occlusal plane [34]. since some other previous studies selected the fh plane as the reference plane. we also used fh plane as the reference plane in the present study like sümbüllü et al. [31], kranjčić et al. [32] , wu et al. [33] and gökalp et al. [34]. there were different concepts about the depth and width of the glenoid fossa. our results showed that the depth and width of glenoid fossa in horizontal growth pattern was significantly smaller than other groups in skeletal cl i. lower depth seems to be in accordance with clockwise rotation of anterior and posterior cranial bases [36]. lin et al. [15] and katsavrias et al. [37] expressed that the depth of the glenoid fossa in high-angle was significantly smaller than control and the low-angle groups but there were no statistical differences between three groups in regard to width of glenoid fossa. the differences might be due to different case selections; skeletal cl i in current study vs skeletal class ii female cases and different sagittal skeletal patterns in lin's and katsavrias' respectively. the inclination of the glenoid fossa was lower although not statistically significant in the horizontal compared with vertical and normal growth pattern groups. on the other hand, lin et al. [15] and widman et al. [14] demonstrated that inclination of articular eminence and mandibular plane angle were inversely related. the variation in the results from these studies might be due to different radiographic methods and the index for growth pattern classification with varying accuracy. current study analyzed cbct images of class i adult patients and adopted jaraback's as a reliable index to represent the growth pattern. widman et al. [14] and lin et al. [15] examined the angle of articular eminence with lateral cephalometry and adopted mandibular plane angle to classify patients. we assumed that unlike sn-mp and fma, representing the relation of the mandibular base to cranial base, jaraback's is a special index to classify patients according to their growth patterns. conclusion due to the importance of muscular function on the tmj it is recommended further studies focusing on the relationship of muscle function and glenoid fossa morphology. the width and depth of glenoid fossa were significantly lower in horizontal growth pattern. no statistically significant differences were found between inclinations of glenoid fossa among three groups. references 1. temporomandibular joint anatomy assessed by cbct images. caruso s, storti e, nota a, ehsani s, gatto r. biomed res int. 2017;2017. 2. skin doses on the lens for temporomandibular joint exam in cone beam computed evalua&on of glenoid fossa morphology in different facial growth pa=erns: a cone beam computed tomography study vol 11 no 1 (2023) doi 10.5195/d3000.2023.172 h#p://den*stry3000.pi#.edu tomography. oliveira mvld, andrade mea, batista wo, campos psf. brazil arch biol technol. 2015;58(6):886-90. 3. comparison of condylar position in hyperdivergent and hypodivergent facial skeletal types. girardot jr ra. angle orthodontist. 2001;71(4):240-6. 4. changes in articular eminence inclination during the craniofacial growth period. katsavrias eg. angle orthodontist. 2002;72(3):25864. 5. condyle and fossa shape in class ii and class iii skeletal patterns: a morphometric tomographic study. katsavrias eg, halazonetis dj. ame j orthodont dentofac orthoped. 2005;128(3):337-46. 6. three-dimensional evaluation of tmj parameters in class ii and class iii patients. krisjane z, urtane i, krumina g, zepa k. stomatologija. 2009;11(1):32-6. 7. condylar volume and condylar area in class i, class ii and class iii young adult subjects. saccucci m, d’attilio m, rodolfino d, festa f, polimeni a, tecco s. head face med. 2012;8(1):34. 8. correlation between condylar position and different sagittal skeletal facial types. paknahad m, shahidi s, abbaszade h. j orofacial orthoped. 2016;77(5):350-6. 9. computed tomography evaluation of the temporomandibular joint in class ii division 1 and class iii malocclusion patients: condylar symmetry and condyle-fossa relationship. rodrigues af, fraga mr, vitral rwf. am j orthodont dentofac orthoped. 2009;136(2):199-206. 10. computed tomography evaluation of temporomandibular joint alterations in patients with class ii division 1 subdivision malocclusions: condyle-fossa relationship. vitral rwf, de souza telles c, fraga mr, de oliveira rsmf, tanaka om. am j orthodont dentofac orthopedic. 2004;126(1):48-52. 11. three-dimensional cone-beam computed tomography based comparison of condylar position and morphology according to the vertical skeletal pattern. park iy, kim jh, park yh. korean j orthodont. 2015;45(2):66-73. 12. association between condylar position and vertical skeletal craniofacial morphology: a cone beam computed tomography study. paknahad m, shahidi s. int orthodont. 2017;15(4):74051. 13. comparison of glenoid fossa morphology between different sagittal skeletal pattern using cone beam computed tomography. khademi b, karandish m, paknahad m, farmani s. j craniofac surg. 2020;31(8):e789-e92. 14. functional and morphologic considerations of the articular eminence. widman dj. angle orthodont. 1988;58(3):221-36. 15. comparahve cone-beam computed tomography evaluahon of temporomandibular joint posihon and morphology in skeletal class ii females. lin yxm, wu h, zhang h, wang s, qi k. j int med res 2020;48(2):300060519892388. doi: 10.1177/0300060519892388. 16. clinical applications of conebeam computed tomography in dental practice. scarfe wc, farman ag, sukovic p. j canada dent associat. 2006;72(1):75. 17. cone beam computed tomography 3d reconstruction of the mandibular condyle. schlueter b, kim kb, oliver d, sortiropoulos g. angle orthodont. 2008;78(5):880-8. 18. cephalometrics for you and me. steiner c. am j orthodontic. 1953;39(10):729-55. http://dx.doi.org/10.1016/00029416(53)90082-7 19. the cranial base as an aetiological factor in malocclusion. hopkin g, houston w, james g. angle orthodontist. 1968;38(3):250-5. 20. some relationships between the glenoid fossa position and various skeletal discrepancies. droel r, isaacson rj. am j orthodontic. 1972;61(1):64-78. 21. tissue reactions in the temporomandibular joint resulting from anterior displacement of the mandible in the monkey. stöckli pw, willert hg. am j orthodontic dentofacial orthopedic. 1971;60(2):142-55. 22. cone-beam ct evaluation of temporomandibular joint in permanent dentition according to angle's classification. song j, cheng m, qian y, chu f. oral radiol. 2020;36(3):261-6. 23. characteristics of articular fossa and condyle in patients with temporomandibular joint complaint. okur a, ozkiris m, kapusuz z, karaçavus s, saydam l. eur rev med pharmacol sci. 2012;16(15):2131-5. 24. condylar position assessed by magnetic resonance imaging after various bite position registrations. kandasamy s, boeddinghaus r, kruger e. am j orthodontic dentofacial orthopedic. 2013;144(4):512-7. 25. tomographic assessment of temporomandibular joint osseous articular surface contour and spatial relationships associated with disc displacement and disc length. major pw, kinniburgh rd, nebbe b, prasad ng, glover ke. evalua&on of glenoid fossa morphology in different facial growth pa=erns: a cone beam computed tomography study vol 11 no 1 (2023) doi 10.5195/d3000.2023.172 h#p://den*stry3000.pi#.edu am j orthodontic dentofacial orthopedic. 2002;121(2):152-61. 26. assessment of optimal condylar position with limited cone-beam computed tomography. ikeda k, kawamura a. am j orthodontic dentofacial orthopedic. 2009;135(4):495-501. 27. cone beam computed tomography imaging in the evaluation of the temporomandibular joint. barghan s, merrill r, tetradis s. j california dent associat. 2010;38(1):33-9. 28. assessment of condyle and glenoid fossa morphology using cbct in south-east asians. alkoshab m, nambiar p, john j. plos one. 2015;10(3):e0121682. 29. spatial analysis of condyle position according to sagittal skeletal relationship, assessed by cone beam computed tomography. arieta-miranda jm, silva-valencia m, flores-mir c, paredes-sampen na, arriolaguillen le. progress orthodontic. 2013;14(1):36. 30. imaging of the temporomandibular joint: a position paper of the american academy of oral and maxillofacial radiology. brooks sl, brand jw, gibbs sj, hollender l, lurie ag, omnell kå, et al. oral surg oral med oral pathol oral radiol endodontol. 1997;83(5):609-18. 31. radiological examination of the articular eminence morphology using cone beam ct. sümbüllü m, cağlayan f, akgül h, yilmaz a. dentomaxillofacial radiol. 2012;41(3):234-40. 32. differences in articulareminence inclination between medieval and contemporary human populations. kranjčić j, vojvodić d, žabarović d, vodanović m, komar d, mehulić k. arch oral biol. 2012;57(8):1147-52. 33. assessments of inclinations of the mandibular fossa by computed tomography in an asian population. wu c-k, hsu jt, shen yw, chen jh, shen wc, fuh lj. clin oral invest. 2012;16(2):443-50. 34. correlation between eminence steepness and condyle disc movements in temporomandibular joints with internal derangements on magnetic resonance imaging. gökalp h, türkkahraman h, bzeizi n. europ j orthodontic. 2001;23(5):579-84. 35. tomographic assessment of temporomandibular joints in patients with malocclusion. cohlmia jt, ghosh j, sinha pk, nanda rs, currier gf. angle orthodontist. 1996;66(1):27-36. 36. graber t, vanarsdall r, vig k. current principles and techniques1994. 685 p. 37. condyle and fossa shape in class ii and class iii skeletal patterns: a morphometric tomographic study. katsavrias eg, halazonetis dj. am j orthod dentofacial orthop. 2005;128(3):337-46. http://dx.doi.org/10.1016/j.ajod o.2004.05.024 www.ncbi.nlm.nih.gov/pubmed/ 16168330 785 2025 final comparison of adhesion of so1-liner between conven8onal pmma and 3d printed resin denture bases vol 13 no 1 (2025) doi 10.5195/d3000.2025.785 h#p://den*stry3000.pi#.edu comparison of adhesion of soft-liner between conventional pmma and 3d printed resin denture bases *dawood jadaan1,2, juzailah roffie2, husniyati roslan2, nahas p3 ¹ college of dentistry, tikrit university, tikrit, salahddin , iraq 2 advanced medical and dental institute, university sains malaysia, kepala batas, pulau pinang, malaysia ³ faculty of dental medicine, lebanese university, hadat, beirut, lebanon abstract aims: the purpose of this study was to evaluate the tensile strength of the so6-liner (molloplast-b®) between the denture founda>on made of 3d printed resin and tradi>onal acrylic (pmma). materials and methods: in this experiment, 60 acrylic specimens in the shape of reverse dumbbells were employed. the first group consisted of 24 specimens made from tradi>onal acrylic polymethyl methacrylate (pmma) (vertex, veracril, and duradent) while the second group consisted of 24 specimens made from 3d printed resin (dentona). the third group consisted of 12 samples that were packed at the dough stage of acrylic and so6 liner at the same >me. a6er that, the samples from the first and second groups were split in two, with one half receiving no surface treatment and the other half undergoing sandblas>ng. every sample is separated into two halves with precise measurements. the thickest and thinnest sec>ons were 80 mm in length (10 mm x 25 mm) and 8 mm x 15 mm, respec>vely, and were joined in the middle by the so6-liner material. the experiment's tensile strength was examined in two separate direc>ons to ascertain the so6-liner material's strength of adhesion to the sample's chosen materials. results: the data analysis of the tensile strength test revealed a sta>s>cally significant difference in the so6-liner adhesion strength between the 3d-printed acrylic resin with sandblast treatment (0.2133 ± 0.03939 kn/mm²) and the 3d-printed acrylic resin without surface treatment (0.1567 ± 0.04677 kn/mm²). similar results were observed for conven>onal acrylic pmma, where the sandblasted acrylic pmma (0.0950 ± 0.03606 kn/mm²) exhibited significantly be^er bond strength compared to the untreated acrylic pmma (0.0875 ± 0.02491 kn/mm²). dough stage samples had be^er values complared to tradi>onal acrylic (0.1483± 0.03689 kn/mm²). conclusion: the results demonstrate that sandblast treatment significantly improves the so6-liner adhesion strength of both 3dprinted acrylic resin and conven>onal acrylic pmma. surface treatment enhances bond strength compared to untreated materials. these findings suggest that sandblas>ng is an effec>ve method for improving adhesion in acrylic-based materials. keywords: heat polymerized acrylic, sandblast, so6-liner, tensile strength. cita:on: jadaan d, et al. (2025) comparison of adhesion of soh-liner between conven:onal pmma and 3d printed resin denture bases. den:stry 3000. 2.a001 doi:10.5195/d3000.2025.785 received: november 29, 2024 accepted: february 1, 2025 published: february 5. 2025 copyright: ©2025 jadaan d, et al. this is an open access ar:cle licensed under a crea:ve commons azribu:on work 4.0 united states license. email: dawoodsalmantech@gmail.com introduction long-term use of dentures can lead to significant changes in the supporting structures, often resulting in chronic pain and discomfort, particularly in the mandible [1]. accelerated resorption of the alveolar ridges leads to the formation of a sharp, narrow crest, which generates excessive pressure, causing severe complications for the patient. as a result, it becomes essential to adopt denture design that provides adequate ridge protection [2]. relining procedures play a critical role in addressing denture related problems [3]. this procedure is non-invasive, cost-effective, and offers greater patient comfort compared to fabricating new dentures [4,5]. there are two types of denture liners: stiff (usually made of polymethyl methacrylate) and resilient [6]. resilient liners fall into two categories: short-term and long-term liners. in contrast to comparison of adhesion of so1-liner between conven8onal pmma and 3d printed resin denture bases vol 13 no 1 (2025) doi 10.5195/d3000.2025.785 h#p://den*stry3000.pi#.edu short-term liners, which are intended to be worn for a maximum of 30 days, long-term liners can be worn for up to a year and maintain their durability for more than 30 days [7–9]. resilient denture lining materials are preferred over hard denture liners because they engage soft tissue undercuts to increase prosthesis retention, relieve constrained pressure, and cushion the fitting surface of dental prostheses to enable more uniform force delivery [4]. however, surface imperfections and porosity, residual palatable after use, odor and water absorption, color instability, difficulties maintaining hygiene, and early thickening due to plasticizer solubility are some of the issues with resilient denture liners [7]. one major issue with denture liners is the liner's inability to adhere to the acrylic resins [10,11]. to prevent interfacial separation at the borders of the denture, it is recommended that the acrylic resins and denture liners retain a robust and durable binding. additionally, the disintegration of the adhesive between the soft liners and the acrylic resins encourages the growth of bacteria and speeds up the deterioration of the soft lining material. [12,13]. previous studies have suggested several techniques to increase the adhesion of liners to the denture base. these include airborne particle abrasion, bur roughening acrylic, chemical etching, and lasing to give the denture base a rougher surface [8, 14–18]. denture processing techniques have changed dramatically in recent years due to the application of computer-aided design and manufacturing (cad/cam) technology [19–21]. although dentures have been made using cad/cam technology since the 1990s, the lack of scientific evidence still makes them appear to be a relatively new technique [22]. cad-cam-fabricated dentures have better material properties and require fewer treatment visits, which saves money and time for both patients and physicians. every day, more and more professional dentistry practices are utilizing cad/cam technology. nevertheless, there is little scientific data on the bonding properties of denture liners to denture resins made using cad/cam technology [3]. research on the use of 3d-printed denture resins is limited, with existing studies indicating that the adhesion strength of 3d-printed denture resins is generally lower than that of traditional acrylic. while many studies have focused on mixing powder and liquid softliner materials, this study utilizes a pre-made silicone-based soft liner (molloplast b®), which is commonly used in maxillofacial prosthetics. the aim of this study is to evaluate the adhesion properties between resilient denture liners and various denture base materials, including those fabricated using traditional methods and advanced cad/cam or 3d printing technologies. material and methods materials the materials used are listed in the following table (table 1). table 1. materials used in the study. item brand country soft liner pestsilicon based molloplast ® b (detax) germany heat cure acrylic polymer and monomer duradant turkey 3d printer resin denture base dentona® germany dental stone type 4 efes dental turkey wax disc milling for vericom /mazic wax korea comparison of adhesion of so1-liner between conven8onal pmma and 3d printed resin denture bases vol 13 no 1 (2025) doi 10.5195/d3000.2025.785 h#p://den*stry3000.pi#.edu cad/ca m separating medium duradent turkey water tap water iraq sand renfert ® germany alcohol clear logo iraq modeling wax dura dent turkey fabrication of the samples three types of samples were prepared: pmma samples, 3d printed resin samples, and dough stage samples. samples were prepared by joining two rectangular blocks of acrylic (pmma) with a soft-liner material, while another group consisted of two rectangular blocks fabricated from 3d printed resin denture material connected by the soft liner (figure 1). figure 1. sample measurements. the preparation of the samples involved two distinct groups: acrylic (pmma) and 3d printed resin. for acrylic samples, a traditional technique was employed. a wax pattern was first designed and cut to the desired measurements. the wax pattern was placed inside a flask and fixed in position using dental stone (figure 2). after the wax was eliminated, a mold space was created and was ready to be packed with acrylic material. after that, the acrylic was cured inside a water bath for one hour after reaching boiling state. once curing was completed, the specimens were removed from the mold and finished. the acrylic samples were divided into two groups: one group underwent sandblasting with air pressure to treat the surface, while the other group was left untreated. figure 2. wax patterns inside the dental stone. for the 3d printed resin samples, the required dimensions for the tensile testing were designed using autodesk (figure 3). the design was then sent as an stl file to a 3d printer, which used dentona® resin for removable dentures. after printing, the samples were removed from the machine and cleaned using an ultrasonic device with alcohol to ensure thorough cleaning. the samples were then light cured for two minutes and divided into two groups: one group was sandblasted to treat the surface, and the other remained untreated. figure 3. autodesk software. the next steps were similar for both groups. acrylic samples (those made by traditional method and 3d printed samples) were joined in the middle by wax and place inside a dental flask filled with dental stone (figure 4). after wax elimination, a mold space was created to be packed with the softliner material. the soft-liner material (molloplast-b®) was then packed into the mold space, with the flask placed under pressure for five minutes. it was transferred to a clamp to maintain pressure and submerged in a water bath at room temperature. the curing process involved dipping the flask into a water bath heated to 70°c for 90 minutes, followed by raising the temperature to 100°c for 30 minutes. after curing, the flask was removed and allowed to cool gradually to ambient temperature for 30 minutes, followed by an additional 15 minutes of cooling under running water. comparison of adhesion of so1-liner between conven8onal pmma and 3d printed resin denture bases vol 13 no 1 (2025) doi 10.5195/d3000.2025.785 h#p://den*stry3000.pi#.edu figure 4. samples are connected by wax that was replaced by soft-liner. for dough stage samples, both the pmma acrylic and the soft liner were packed together inside the dental mold at dough stage. this was followed by curing as mentioned earlier. in all cases, the preparation process involved careful handling, curing, and cooling to ensure the proper bonding of the soft-liner material with the acrylic or 3d printed resin base. study design the experiment employed 60 specimens in the form of reverse rectangular blocks. 12 specimens were dough stage packed specimens which received no treatment. 24 of the specimens were made from 3d printed resin (dentona® optiprint® laviva), while the other 24 specimens were made from traditional heat-cured acrylic pmma resin (veracril®, duradent®). in line with chladek et al. 2014 (figure 1), the specimens were designed as follows: the entire length of 80 mm of resin (acrylic pmma or 3d printed) was split into two sections of 40 mm each, separated by 3 mm. the surface was sanded and airtreated, with 25 mm having a thickness of 10 mm. on the other hand, the borders of 15mm were 8mm thick. surface treatment half of the sample underwent the surface treatment before the bonding of the soft reline material (molloplast b®). the surface treatment was done using airborne-particle abrasion to the bonded surface by sandblast size 90–150-micron alumina with air pressure of 34 bar. this was to increase the surface roughness to increase the mechanical boned between the materials. tensile bond strength test the testing procedure was carried out at the university of tikrit college of engineering using a hoytom d1922fi testing machine (figure 5) with a load cell capacity of 170 kn and a crosshead speed of 0.5mm/min. the readings obtained from the device's digital display represented the maximum load of failure, and the value bond strength was calculated by dividing the maximum load of failure by the cross-sectional area of each sample figure 5. hoytom testing machine. statistical analysis the statistical analysis involved the use of one-way anova at p ≤0.05 to reveal if there were any differences between the groups. results tensile strength test the adhesion strength of soft liners was found to be significantly higher with 3d printed resin denture bases compared to conventional pmma. there were highly significant differences between the five groups as revealed by one-way anova test. surface treated 3d printed resin samples had the highest value of tensile bond strength (0.2133) followed by untreated 3d printed resin (0.1567), dough stage samples (0.1483), surface treated pmma samples (0.0950), while the lowest value was recorded for the untreated pmma samples (0.0875) as indicated in (tables 2 and 3). comparison of adhesion of so1-liner between conven8onal pmma and 3d printed resin denture bases vol 13 no 1 (2025) doi 10.5195/d3000.2025.785 h#p://den*stry3000.pi#.edu evaluation sample under the scanning electron microscope (sem). all samples were sent for sem evaluation to determine the morphology of the denture bases before and after treatment by the sand airborne-particle abrasion surface treatment (figure 6). figure 6. scanner electron microscopic (sem), right are the untreated samples, left are the treated samples. discussion this study compares the tensile strength of soft liners between conventional pmma and 3dprinted resin denture bases. the results show that the soft liner, molloplast-b®, adheres better to 3d-printed samples compared to conventional acrylic specimens. the tensile bond strength of 3dprinted samples was significantly higher than that of acrylic pmma samples (traditional and dough stage), particularly when the 3dprinted surfaces were treated with sandblasting and air pressure (0.2133 ± 0.03939 kn/mm²), compared to untreated 3d-printed surfaces (0.1567 ± 0.04677 kn/mm²). this could be related to the printing process which can results in a micro-roughness and irregularities that can improve mechanical interlocking between the soft liner and the 3d printed sample. this roughness increases the surface area available for bonding, promoting stronger adhesion through mechanical bonding (micromechanical interlocking) between the liner and the resin [23]. in contrast, conventional pmma samples, tends to have a smoother, more homogeneous surface. while sandblasting can induce surface roughness by creating microabrasions, the overall roughness of pmma samples remains lower than that of 3d-printed resin surfaces, which may explain the lower bond strength observed in the untreated acrylic samples [2429]. in some cases, the sandblasted 3d-printed resin samples showed failure of the soft liner without separating from the bond surface, indicating strong adhesion. for conventional pmma, sandblasting improved the bond strength of molloplast-b® soft liner, with a bond strength of 0.0950 ± 0.03606 kn/mm², compared to untreated pmma (0.0875 ± 0.02491 kn/mm²). in the case of the 3d-printed resins, surface treatments like sandblasting and air pressure can enhance the bonding sites by increasing surface energy [29, 30]. sandblasting causes the surface to become microscopically rougher, which can expose more reactive functional groups, increasing the surface's affinity for chemical bonding with the liner [31-33]. polymerization and the degree of crosslinking in the resin also play a role in determining the bond strength. 3d-printed resins generally exhibit better control over polymerization, allowing for a more uniform and complete curing process [34]. this results in a more stable, tightly packed polymer matrix that can provide superior adhesion. in contrast, pmma, while also polymerized, may not have as consistent or efficient a curing process as 3d-printed resins, leading to slight variations in surface hardness and cohesion [36]. traditional pmma denture bases can also suffer from degradation over time due to the migration of plasticizers, which are used to modify the material's flexibility. this can weaken the surface and reduce its ability to maintain a strong bond with soft liners like molloplast-b® [37]. 3d-printed resins, however, often have a more comparison of adhesion of so1-liner between conven8onal pmma and 3d printed resin denture bases vol 13 no 1 (2025) doi 10.5195/d3000.2025.785 h#p://den*stry3000.pi#.edu stable polymer matrix and less plasticizer migration, which can result in better long-term adhesion [38]. in addition, the surface energy of a material is a key factor in determining how well it bonds to another material. 3d-printed resins typically have higher surface energy compared to pmma, especially after surface treatments like sandblasting. this increased surface energy improves wettability, which enhances the ability of the soft liner to spread evenly over the denture base and form a stronger bond [39]. in comparison, untreated pmma has lower surface energy, leading to poorer wetting and a weaker bond [40]. as mentioned above, dough stage samples had better tensile strength values compared to pmma samples packed by the traditional methods. this is because packing both acrylic and soft liner at the dough stage allows the materials to intermingle or bond more effectively during the molding process. when packed with acrylic at the dough stage, the two materials have a better chance of distributing the applied tensile stress more evenly across the surface. this means that the soft liner can absorb some of the forces, reducing localized stress points. at the dough stage, both acrylic and soft liners are in a form that allows for better molecular interaction [41]. acrylic resins are typically polymerized during curing, and when mixed with soft liners, the soft liner's material may undergo some crosslinking or slight interaction with the acrylic. this can result in a composite material with enhanced mechanical properties which explains the high tensile strength compared to pmma [42]. conclusions this study provides valuable insights into the adhesive properties of soft-liners when used with different denture base materials. the superior performance of 3d printed resin denture bases in terms of adhesion strength suggests that these materials could offer improved outcomes for patients requiring dentures with soft liners. however, the need for surface treatment, such as sandblasting, remains critical to optimizing the bond strength. these findings contribute to the growing body of evidence supporting the use of advanced manufacturing techniques in prosthodontics and underscore the importance of continued research in this evolving field. conflicts of interest the authors declare no competing interest. references 1. boucher, c.o. complete denture prosthodonjcs—the state of the art. j. prosthet. dent. 2004,92, 309–315. 2. mahboub, f.; salehsaber, f.; parnia, f.; gharekhani, v.; kananizadeh, y.; taghizadeh, m. effect of denture cleansing agents on tensile and shear bond strengths of sop liners to acrylic denture base. j. dent. res. dent. clin. dent. prospect. 2017, 11, 183–188. 3. choi, j.e.; ng, t.e.; leong, c.k.; kim, h.; li, p.; waddell, j.n. adhesive evaluajon of three types of resilient denture liners bonded to heat-polymerized, autopolymerized, or cadcam acrylic resin denture bases. j. prosthet. dent. 2018,120, 699– 705. 4. nowakowska-toporowska, a.; raszewski, z.; wieckiewicz, w. color change of sop silicone relining materials aper storage inarjficial saliva. j. prosthet. dent. 2015,115, 377–380. 5. kutlu, i.u.; yaniko˘glu, n.d.; kul, e.; duymu¸s, z.y.; sa˘gsöz, n.p. effect of sealer coajng and storage methods on the surface roughness of sop comparison of adhesion of so1-liner between conven8onal pmma and 3d printed resin denture bases vol 13 no 1 (2025) doi 10.5195/d3000.2025.785 h#p://den*stry3000.pi#.edu liners. j. prosthet. dent. 2015, 115, 371–376. 6. bail, m.; jorge, j.; urban, v.m.; campanha, n.h. surface roughness of acrylic and siliconebased sop liners: in vivo study in a rat model. j. prosthodont. 2013,23, 146–151. 7. kreve, s.; dos reis, a.c. denture liners: a systemajc review relajve to adhesion and mechanical properjes. sci. world j.2019, 2019, 1–11. 8. atsu, s.; keskin, y. effect of silica coajng and silane surface treatment on the bond strength of sop denture liner to denture base material. j. appl. oral sci. 2013, 21, 300–306. 9. valenjni, f.; luz, m.s.; boscato, n.; pereira-cenci, t. biofilm formajon on denture liners in a randomised controlled in situ trial. j. dent. 2013, 41, 420– 427. 10. elias, c.; henriques, f.q. effect of thermocycling on the tensile and shear bond strengths of three sop liners to a denture base resin. j. appl. oral sci. 2007,15, 18–23. 11. emmer, t.j.; emmer, t.j.; vaidynathan, j.; vaidynathan, t.k. bond strength of permanent sop denture liners bonded to the denture base. j. prosthet. dent. 1995, 74, 595–601. 12. al-athel, m.; jagger, r.; jagger, d. effect of ageing on the bond strength of a permanent denture sop lining material. j. oral rehabil. 2002, 29. 13. 13. pinto, j.r.r.; mesquita, m.f.; nóbilo, m.a.d.a.; henriques, g.e.p. evaluajon of varying amounts of thermal cycling on bond strength and permanent deformajon of two resilient denture liners. j. prosthet. dent. 2004, 92, 288–293. 14. jacobsen, n.l.; mitchell, d.l.; johnson, d.l.; holt, r.a. lased and sandblasted denture base surface preparajons affecjng resilient liner bonding. j. prosthet. dent. 1997, 78, 153–158. 15. bulad, k.; taylor, r.l.; verran, j.; mccord, j.f. colonizajon and penetrajon of denture sop lining materials by candida albicans. dent. mater. 2004, 20, 167–175. [google scholar] 16. mack, p.j. denture sop linings: clinical indicajons. aust. dent. j. 1989, 34, 454–458. 17. el-hadary, a.; drummond, j.l. comparajve study of water sorpjon, solubility, and tensile bond strength of two sop lining materials. j. prosthet. dent. 2000, 83, 356–361. 18. usumez, a.; inan, o.; aykent, f. bond strength of a silicone lining material to aluminaabraded and lased denture resin. j. biomed. mater. res. 2004, 71b, 196–200. 19. al-fouzan, a.f.; al-mejrad, l.a.; albarrag, a.m. adherence of candida to complete denture surfaces in vitro: a comparison of convenjonal and cad/cam complete dentures. j. adv. prosthodont. 2017, 9, 402–408. 20. saber-sheikh, k.; clarke, r.l.; braden, m. viscoelasjc properjes of some sop lining materials. ii-ageing characterisjcs. biomaterials 1999, 20, 2055–2062. 21. atalay, s.; çakmak, g.; fonseca, m.; schimmel, m.; yilmaz, b. effect of thermocycling on the surface properjes of cadcam denture base materials aper different surface treatments. j. mech. behav. biomed. mater. 2021, 121, 104646. 22. gruber, s.; kamnoedboon, p.; özcan, m.; srinivasan, m. cad/cam complete denture resins: an in vitro evaluajon of color stability. j. prosthodont. 2020, 31, 13246. 23. goodacre, b.j.; goodacre, c.j.; baba, n.z.; ka{adiyil, m.t. comparison of adhesion of so1-liner between conven8onal pmma and 3d printed resin denture bases vol 13 no 1 (2025) doi 10.5195/d3000.2025.785 h#p://den*stry3000.pi#.edu comparison of denture base adaptajon between cad-cam and convenjonal fabricajon techniques. j. prosthet. dent. 2016, 116, 249–256. 24. wimmer, t.; gallus, k.; eichberger, m.; stawarczyk, b. complete denture fabricajon supported by cad/cam. j. prosthet. dent. 2016, 115, 541– 546. 25. botega, d.m.; sanchez, j.l.l.; mesquita, m.f.; henriques, g.e.p.; consani, r.l.x. effects of thermocycling on the tensile bond strength of three permanent sop denture liners. j. prosthodont. 2008, 17, 550–554 26. chladek, g.; ˙zmudzki, j.; kasperski, j. long-term sop denture lining materials. materials2014,7, 5816–5842. 27. parvizi, a., lindquist, t., schneider, r., williamson, d., boyer, d., & dawson, d. v. (2004). comparison of the dimensional accuracy of injecjon-molded denture base materials to that of convenjonal pressure-pack acrylic resin. journal of prosthodonjcs: implant, esthejc and reconstrucjve denjstry, 13(2), 83-89. 28. dohiem, m. m. (2022). comparison of bond strength between acrylic based sop liner and two different denture bases (3dprinted and convenjonal denture base). egypjan dental journal, 68(1), 659-663. 29. muddugangadhar, b.c.; mawani, d.p.; das, a.; mukhopadhyay, a. bond strength of sop liners to denture base resins and the influence of different surface treatments and thermocycling: a systemajc review. j. prosthet. dent. 2020, 123, 800–806. 30. material arjcle the sop denture liner adhesion to convenjonal and cad/cam processed poly (methyl methacrylate)acrylic denture resins –an in – vitro study 31. dawood a, marj marj b, sauret-jackson v, darwood a. 3d prinjng in denjstry. br dent j. 2015 dec;219(11):521–9. 32. 3d printed dental model using this technologysim j-y, jang y, kim w-c, kim h-y, lee d-h, kim j-h. comparing the accuracy (trueness and precision) of models of fixed dental prostheses fabricated by digital and convenjonal workflows. j prosthodont res. 2019 jan;63(1):25–30. 33. van noort r. the future of dental devices is digital. dent mater off publ acad dent mater. 2012 jan;28(1):3–12. 34. barazanchi a, li kc, alamleh b, lyons k, waddell jn. addijve technology: update on current materials and applicajons in denjstry: addijve technology. j prosthodont. 2017 feb;26(2):156–63. 35. us – turkey workshop on rapid technologies, september 24 25, 2009 36. gozalo-diaz, d. j., et al. (2012). "impact of 3d prinjng on the performance of denture bases." internajonal journal of prosthodonjcs, 25(3), 237-244 37. mutluay, m. m., et al. (2008). "surface treatments of pmma denture base resin to improve adhesion of sop denture liners." journal of prosthejc denjstry, 99(5), 393-398. 38. (anadioj et al., 2018). anadioj, e., kane, b. and soulas, e. (2018). current and emerging applicajons of 3d prinjng in restorajve denjstry. current oral health reports, 5(2), pp.133-139 39. awad, a.n., cho, s.h., kesterke, m.j. and chen, j.h. (2021). comparison of tensile bond strength of denture reline materials on denture bases fabricated with cad-cam comparison of adhesion of so1-liner between conven8onal pmma and 3d printed resin denture bases vol 13 no 1 (2025) doi 10.5195/d3000.2025.785 h#p://den*stry3000.pi#.edu technology. the journal of prosthejc denjstry 40. braden, m., wright, p. s., & parker, s. (1995). sop lining materials--a review. european journal of prosthodonjcs and restorajve denjstry, 3(4), 163174. 41. saeed f, muhammad n, khan as, sharif f, rahim a, ahmad p, irfan m. prosthodonjcs dental materials: from convenjonal to unconvenjonal. materials science and engineering: c. 2020 jan 1;106:110167. 42. haghi hr, shiehzadeh m, gharechahi j, nodehi d, karazhian a. comparison of tensile bond strength of sop liners to an acrylic resin denture base with various curing methods and surface treatments. int j prosthodont. 2020 jan 1;33(1):56-62. table 2. statistical analysis for tensile bond strength. vickers microhardness group mean ±sd acrylic without sandblasting a 0.0875 0.02491 acrylic with sandblasting b 0.0950 0.03606 3d printed resin without sandblasting c 0.1567 0.04677 3d printed resin with sandblasting d 0.2133 0.03939 dough stage 0.1483 0.03689 anova: f=22.448; p=0.000 levene statistics=0.080, p-value=0.923 comparison of adhesion of so1-liner between conven8onal pmma and 3d printed resin denture bases vol 13 no 1 (2025) doi 10.5195/d3000.2025.785 h#p://den*stry3000.pi#.edu table 3. post-hoc tukey’s test results. i j mean difference (i-j) standard error p-value acrylic without sandblasting acrylic with -0.00750 0.01530 0.988 3d without -0.06917 0.01530 0.000 3d with -0.12583 0.01530 0.000 dough stage -0.06083 0.01530 0.002 acrylic with sandblasting acrylic without 0.00750 0.01530 0.988 3d without -0.06167 0.01530 0.002 3d with -0.11833 0.01530 0.000 dough stage -0.05333 0.01530 0.008 3d printed resin without sandblasting acrylic without\ 0.06917 0.01530 0.000 acrylic with 0.06167 0.01530 0.002 3d with -0.05667 0.01530 0.004 dough stage 0.00833 0.01530 0.982 3d with sandblasting acrylic without\ 0.12583 0.01530 0.000 acrylic with 0.11833 0.01530 0.000 3d without 0.05667 0.01530 0.004 dough stage 0.06500 0.01530 0.001 use of fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders among university students a cross sectional study. vol 10 no 1 (2022) doi 10.5195/d3000.2022.178 http://dentistry3000.pitt.edu use of fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders among university students a cross sectional study. prashantha g shivamurthy1, nutan kumari1, ashfiya sadaf1, meghana mb1, heba azhar1, sharanya sabrish1, 1 faculty of dental sciences, ramaiah university of applied sciences, bengaluru, india abstract aim: temporomandibular disorders (tmd) are one of the main causes of non-dental orofacial pain. it has been reported that tmd occurred more frequently among individuals in stressful situations and that university students are more likely to have a high risk of anxiety related disorders. this study aims to assess the prevalence and severity of tmd in undiagnosed population consisting of students of different faculties in a university campus in bengaluru using the fonseca’s questionnaire. material and methods: descriptive cross-sectional study conducted at a university campus, bengaluru. completed forms of fonseca’s questionnaire were collected from the sample consisted of 300 students. the questionnaire had two main parts. first part collected demographic information and past relevant histories. second part had fonseca’s 10 questions. the tmd severity was categorized as no tmd (0-15), mild tmd (20-40), moderate tmd (45-65) and severe tmd (70-100). results: 33.3% on the sample showed varying signs of tmd. there was no statistically significant differences found between the tmd and non tmd groups with respect to age and sex. but when comparing varying grades of tmd there was a statistically highly significant difference seen in relation to age of the subjects. conclusions: a high prevalence of tmd was found in the university students with majority of the cases having milder signs of tmd. keywords: temporomandibular joint dysfunction; fonseca’s questionnaire; stress. citation: shivamurthy pg, et al. (2022) use of fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders among university students a cross sectional study. dentistry 3000. 1:a001 doi:10.5195/d3000.2022.178 received: april 30, 2021 accepted: september 30, 2021 published: february 21, 2022 copyright: ©2022 shivamurthy pg gs, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: drsharanyaortho@gmail.com introduction temporomandibular disorders (tmd) are one of the main causes of non-dental orofacial pain. the pain is generally located on the masticatory muscles, in the preauricular area and the temporomandibular joint (tmjs).[1] the term temporomandibular disorders is a collective term which includes all the problems involving painful orofacial disorders, complaints of pain in tmj region and fatigue of the craniocervicofacial muscles, especially masticatory muscles, limitation of mandible movement, and presence of articular clicking.[2] many factors such as oral habits, fatigued masticatory muscles and premature loss of natural teeth have been linked to the disorder.[3] other important aetiological factors include emotional stress and high anxiety levels.[4] previous studies of various populations have reported a high prevalence of tmd.[5] the presence of tmd signs has been reported in more than 50% of nonpatients, with the presence of at least one symptom being reported among more than 30% of subjects.[6] the variability in prevalence may be attributed to differences in the race of the population, in the sampling design and criteria, and in the methods used for collecting information.[7] due to the high prevalence and variability of the complaints, tmd http://dentistry3000.pitt.edu/ use of fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders among university students a cross sectional study. vol 10 no 1 (2022) doi 10.5195/d3000.2022.178 http://dentistry3000.pitt.edu is diagnosed by associating signs and symptoms, as some characteristics may be frequent even in a non-patient population.[8] it has been reported that tmd occurred more frequently among individuals in stressful situations.[9] it has been reported that university students are more likely to have a high risk of anxietyand depression-related clinical disorders.[10] in a study on an indian population, among all the participants examined sixty participants had no tmd (30%), 110 participants had mild tmd (55%); 28 patients had moderate tmd (14%) and two participants had severe tmd (1%).[11] in another study among adolescents in haryana the prevalence of tmd signs and symptoms was 51%. females had higher prevalence (57.1%) of tmd symptoms when compared to males (43.2%). tmd signs has shown a statistical significant relationship with overbite (p = 0.007) and bruxism (p < 0.0001) only.[12] the prevalence of tmd has been studied in a brazilian university population, a saudi arabian university, etc.[13],[5] since the factors associated with tmd are of a diverse nature, there are expected to be variations between different populations and across different demographics. currently, there are no published articles related to prevalence of tmd in students of different faculties in a given university in bengaluru. several instruments for tmd diagnosis have been proposed, but there is no single appropriate diagnostic criterion. to address this issue, schiffman and colleagues in 2014 proposed a new comprehensive tool known as the diagnostic criteria for temporomandibular disorders (dc/tmd).[14] this diagnostic instrument is validated and considered a good method for diagnosing tmd in clinical studies. despite the advantages, the dc/tmd is quite a cumbersome assessment tools in that it requires the individual to be present in order to render a tmd diagnosis, and it is difficult to use on large samples. due to the need of simpler assessment procedures that can be widely applicable and to standardize research samples involving tmd patients, questionnaires have been created to address the main clinical tmd findings and assign clinical indexes for patient classification in terms of severity levels.[15] according to oliveira, a simple selfadministered questionnaire would offer the advantage of faster application and, thus, low cost.[16] an additional advantage is that a self-applied questionnaire would provide a severity index with less influence from the examiner and less variability in the measures. the anamnestic index (ai) presented by campos ja, et al. is one of the available instruments in portuguese language for characterization of tmd symptoms, and it was developed to classify patients according to severity of those symptoms.[17] anamnesis questionnaires have the limitation of providing data only on tmd symptoms and it cannot provide tmd diagnosis. in 1992 fonseca developed his anamnestic questionnaire based on many earlier indices. the fonseca’s anamnestic index (fai) is a self-administered questionnaire that has been proposed as a low-cost, easily applied alternative tmd assessment tool for the nonpatient population.[18] hence in this study we have evaluated, using the fonseca’s questionnaire (table 1), the prevalence and severity of tmd in undiagnosed indian population consisting of students of different faculties in a university campus. the assessment of volunteers with undiagnosed tmd would help understanding its prevalence in the indian university population. the aim of this study was to assess the prevalence and the severity of http://dentistry3000.pitt.edu/ use of fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders among university students a cross sectional study. vol 10 no 1 (2022) doi 10.5195/d3000.2022.178 http://dentistry3000.pitt.edu tmds in the students of different faculties in a university in bengaluru using fonseca's questionnaire. material and methods this was a descriptive crosssectional study. the study was carried out between september 2019 and february 2020. the study subjects were students from various disciplines of professional and degree courses which included medical, dental, business management, commerce & finance and allied sciences. initially 340 undergraduate students from the university campus were randomly selected and were requested to answer the fonseca’s questionnaire. prior to that, the volunteers received proper instructions about the goals of the research and experiment procedure, risk and benefits and signed an informed consent form. questionnaires and a cover letter stating the instructions, rationale, and purpose of the survey were distributed to a conveniently selected sample of university students from the university campus. subjects who were willing to participate in the study filled the questionnaire by hand and returned it in person. participants were not given a time limit for completing the questionnaire. informed consent was signed and collected from the sample. since it was questionnaire-based study and no intervention was done, the clearance was given after assessment of the study protocol and the study was approved by the university’s ethical committee (ramaiah university of applied sciences, bengaluru). the research project protocol number was ec2020/ug/076. participants having a history of tmj trauma, receiving orthodontic treatment or treatment for tmd, and/or suffering from any immunocompromised disease were excluded from the study. the sample size estimated at 300 based on previous studies.[13],[19],[18] sample size was calculated based on the previous studies using g* power 3.1.2 software.[13] the minimum sample size was calculated, following these input conditions: power of 95% , alpha error of 0.05 and the effect size was 0.35. initially the questionnaire was given to 340 students and based on the exclusion criteria and after removing incompletely filled questionnaires, we reached the sample size of 300. the study tool used was the fonseca’s questionnaire (table 1) because it ensures collection of large quantity of information in a relatively short period of time. it evaluates the degree of tmd in the participant. it does not diagnose the presence of tmd. the questionnaire is framed in such a way that it assesses for the presence of pain in tmj and head while chewing, parafunctional habits, limitations of movement, joint clicking, perception of malocclusion and emotional stress. the fonseca questionnaire is an already validated and standardized questionnaire for the evaluation of tmd in relation to various orofacial movements and exercises, with each question having a scale of calibration to answer ranging from yes, no and sometimes in order to assess the degree of severity. the questionnaire comprised two main parts. the first part collected demographic information and past medical, dental, tmj, and facial trauma histories. the second part asked fonseca’s 10 questions. participants were requested to select one answer: yes, no, or sometimes. each ‘‘yes’’ answer was assigned a value of 10, each ‘‘sometimes’’ answer a value of 5, and each ‘‘no’’ answer a value of 0. the sum of the values for all 10 answers was used to classify each subject according to the criteria shown in table 1. hence, the collective score of each questionnaire ranged from 0 to 100. incomplete and partially completed questionnaires were excluded. a clinical index was described on the basis of collective score of key questions (table 1). tmd severity was categorized as no tmd (0-15), mild tmd (20-40), moderate tmd (45-65) and severe tmd (70-100). the frequency and severity of tmd were compared in terms of students’ gender, age and college. after collecting the data all the subjects who were diagnosed as having any stomatognathic system impairment either http://dentistry3000.pitt.edu/ use of fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders among university students a cross sectional study. vol 10 no 1 (2022) doi 10.5195/d3000.2022.178 http://dentistry3000.pitt.edu tmd or orofacial pain were subjected to appropriate treatment. sl. no fonseca’s key question yes sometimes no 1. do you feel that it is difficult to open your mouth? 2. do you feel that right and left movement of your jaw is difficult? 3. do you feel muscular pain and feel tired while chewing? 4. do you have headaches frequently? 5. do you feel stiffness in your neck? 6. do you feel pain in your ears or tmj? table 1 fonseca’s questionnaire key questions in fonseca’s questionnaire and classification of tmd severity http://dentistry3000.pitt.edu/ use of fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders among university students a cross sectional study. vol 10 no 1 (2022) doi 10.5195/d3000.2022.178 http://dentistry3000.pitt.edu 7. do you hear any sounds of the tmj clicking while chewing or opening your mouth? 8. do you grind or clench your teeth? 9. do you feel that your teeth do not articulate properly? 10. do you feel that you are a tense or nervous person? classification of tmd severity based on the key questions in fonseca’s questionnaire score category total between 0 and 15 points no tmd total between 20 and 40 points mild tmd total between 45 and 65 points moderate tmd total between 70 and 100 points severe tmd http://dentistry3000.pitt.edu/ use of fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders among university students a cross sectional study. vol 10 no 1 (2022) doi 10.5195/d3000.2022.178 http://dentistry3000.pitt.edu the data was collected and saved in excel format and then subjected to statistical analysis. descriptive statistics and frequency analyses of the collected data were performed in the statistical package for social sciences (spss) software (version 17 spss, chicago, illinois, usa). age of the participants was expressed as the mean± standard deviation (sd), and 95% confidence intervals were reported. chi-square tests for statistical significance were applied to the frequencies within each fonseca’s severity category and to the frequencies of responses to each fonseca’s question, with p < 0.05 regarded as statistically significant. the effect size was calculated using cramers v table. results table 2 shows the distribution of socio demographic characteristics of the study participants. table 3 shows the distribution of varying grades of tmd among study participants based on fonseca questionnaire total score. 66.7% of the subjects showed no signs of tmd while 33.3% showed varying signs of tmd. table 4 includes the age and gender wise comparison of prevalence of tmd among study participants using chi square test. there was no statistically significant differences found between the groups. table 5 includes the age and gender wise comparison of distribution of varying grades of tmd among study participants using chi square test. there was a statistically highly significant difference seen in relation to age of the subjects with p value of 0.001.(figure 1). the association between the individual parameters of fonseca’s questionnaire and the subjects with and without temporomandibular disorders according to fonseca criteria using chi square test is presented in table 6. in all the parameters there was a statistically significant difference seen. the responses which were positive for temporomandibular disorders according to fonseca criteria are shown in figure 2. figure 1: age and gender wise comparison of distribution of varying grades of tmd among study participants figure 2: responses positive for temporomandibular disorders according to fonseca criteria http://dentistry3000.pitt.edu/ use of fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders among university students a cross sectional study. vol 10 no 1 (2022) doi 10.5195/d3000.2022.178 http://dentistry3000.pitt.edu discussion the distribution of socio demographic characteristics of the study participants is shown in table 2. majority of the subjects in this study were between 17-25 years and 66.3% were females. among the subjects, 33.7% were medical students and 33% were dental students. the distribution of varying grades of tmd among study participants based on fonseca questionnaire total score is presented in table 3. 66.7% of the subjects showed no signs of tmd while 33.3% showed varying signs of tmd. in such a small sample of 300 subjects this prevalence of tmd can be considered to be high. possible reasons could be high stress levels among students’ especially medical and dental undergraduates ass supported by literature.[5] also of the 33.3% showing signs of tmd, 29.3% showed only mild signs which indicates that majority of the tmd cases in this age range were in the initial stages of this disorder. in another study done on an indian population on the signs and symptoms of tmds in vidarbha population[11] using the fonseca’s questionnaire it was found that among all the participants examined sixty participants had no tmd (30%), 110 participants had mild tmd (55%); 28 patients had moderate tmd (14%) and two participants had severe tmd (1%). these values were for a general population and was not focused on student population. thus, in these studies a higher prevalence of tmd was found compared to the results of this study. the possible reasons for the lower prevalence of tmd in this study could be the younger sample age in this study compared to studies on general population and hence the tmd could be in initial stages. in a similar study on brazilian undergraduates it was found that 53.21% of the participants had tmd.[13] garcia et al, found that 61% of the 200 undergraduates evaluated had some sign of tmd.[13] pedroni et al. found a prevalence of 68% of the evaluated volunteers, which is again higher than the present study.[8] the probable reasons could be different grading variables category n % age 17-20 yrs 152 50.7% 21-25 yrs 145 48.3% 26-30 yrs 3 1.0% sex males 101 33.7% females 199 66.3% faculty medical 101 33.7% dental 99 33.0% allied science 32 10.7% others 68 22.7% table 2distribution of socio demographic characteristics of study participants. (others include business management, commerce & finance students)severity http://dentistry3000.pitt.edu/ use of fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders among university students a cross sectional study. vol 10 no 1 (2022) doi 10.5195/d3000.2022.178 http://dentistry3000.pitt.edu methods and diagnostic tests used and population variations. table 4 includes the age and gender wise comparison of prevalence of tmd among study participants using chi square test. there was no statistically significant differences found between the groups. also, among those who did show signs of tmd between the ages of 17-20 and 2125 years, there was almost a similar prevalence of tmd (50%), hence both age ranges had a similar risk of tmd. there was a higher prevalence of tmd in females (table 4) but this was not statistically significant. similar gender based findings were found in other studies.[13] women (63.11%) were the more affected than men (40.62%).[13] the high prevalence of tmd in women may be related to their different physiological characteristics, such as regular hormonal variations, muscle structures and different characteristics of the connective tissue.[15],[20] table 5 includes the age and gender wise comparison of distribution of varying grades of tmd among study participants using chi square test. as observed in table 5 there was no difference seen with respect to sex. interestingly, there was a statistically highly significant difference seen in relation to age of the subjects with p value of 0.001.(figure 1) this can be interpreted as, the subjects in the age range of 1725 years if suffering from tmd, were more likely to have mild tmd rather than moderate or severe tmd. the sample size in the age range of 2630 years was very low to extrapolate the results to include them. (figure1) the association between the individual parameters of fonseca’s questionnaire and the subjects with and without temporomandibular disorders according to fonseca criteria using chi square test is presented in table 6. in all the parameters there was a statistically significant difference seen. hence the individual parameters of this questionnaire can be considered to be valid. the responses which were positive for temporomandibular disorders according to fonseca criteria are shown in figure 2. in a study on tmd, nearly onethird of participants had a history of psychological stress.[18] in our study 79.6% of the people who had tmd reported that they felt they were tensed. these outcomes are in agreement with those of pesqueira and colleagues[4] and bonjardim and colleagues[10] who asserted that stress and anxiety play important roles in tmds by acting as predisposing or aggravating factors. the limitations of this study are that of limited sample size, nonhomogeneous ethnicity of the sample and possible errors in recording information in questionnaire such as underreporting, overreporting etc. also the emotional factors were not considered which play a role in symptoms of tmd. the fonseca’s questionnaire allows collecting a large quantity of information in a relatively short period and at low cost, it is easy to understand and has almost no influence from the examiner.[15] the potential uses could be regular public health and screening services, using this questionnaire, for a wide population coverage. with proper diagnosis and treatment, this could manage orofacial pain at early stages in a large contingent of people. http://dentistry3000.pitt.edu/ use of fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders among university students a cross sectional study. vol 10 no 1 (2022) doi 10.5195/d3000.2022.178 http://dentistry3000.pitt.edu grades of tmd fqts range n % tmd absent 0-15 200 66.7% mild tmd 20-45 88 29.3% mod. tmd 50-65 10 3.3% severe tmd 70-100 2 0.7% variables category without tmd with tmd effect size rr 95% ci value pvalue n % n % age 17-20 yrs 103 51.5% 49 49.0% 0.03 0.402 0.82 21-25 yrs 96 48.0% 49 50.0% 0.97 0.74 to 1.28 26-30 yrs 1 0.5% 2 1.0% 0.98 0.77 to 1.25 sex males 72 36.0% 29 28.6% 0.07 0.85 0.58to 1.25 1.628 0.20 females 128 64.0% 71 71.4% table 4age and gender wise comparison of prevalence of tmd among study participants using chi square testmanagement, commerce & finance students)severity table 3distribution of varying grades of tmd among study participants based on fonseca questionnaire. fqts fonseca questionnaire total scoretestmanagement, commerce & finance students)severity http://dentistry3000.pitt.edu/ use of fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders among university students a cross sectional study. vol 10 no 1 (2022) doi 10.5195/d3000.2022.178 http://dentistry3000.pitt.edu variables categories mild tmd mod. tmd severe tmd effect size value p-value n % n % n % age 17-20 yrs 43 87.8% 5 10.2% 1 2.0% 0.35 24.571 <0.001* 21-25 yrs 44 89.8% 5 10.2% 0 0.0% 26-30 yrs 1 50.0% 0 0.0% 1 50.0% sex males 24 82.8% 4 13.8% 1 3.4% 0.10 1.144 0.57 females 64 90.1% 6 8.5% 1 1.4% fonseca items without tmd with tmd value effect size rr 95% ci p-value n % n % q1 3 1.5% 26 26.5% 46.910 0.41 13.96 4.3 to 45.18 <0.001* q2 7 3.5% 26 26.5% 35.426 0.36 6.10 2.72 to 13.6 <0.001* q3 23 11.5% 48 49.0% 40.907 0.44 3.14 2.00 to 4.9 <0.001* q4 48 24.0% 64 65.3% 47.838 0.50 2.01 1.46 to 2.77 <0.001* q5 16 8.0% 35 35.7% 35.613 0.37 3.5 2.01 to 6.07 <0.001* q6 8 4.0% 28 28.9% 37.916 0.37 5.68 2.67 to 12.01 <0.001* q7 27 13.5% 38 38.8% 24.638 0.30 2.31 1.48 to 3.61 <0.001* q8 33 16.5% 37 38.1% 16.987 0.32 1.90 1.25 to 2.89 <0.001* q9 23 11.5% 46 46.9% 46.424 0.45 3.05 1.93 to 4.81 <0.001* q10 55 27.5% 78 79.6% 72.223 0.57 2.03 1.52 to 2.70 <0.001* table 5 age and gender wise comparison of distribution of varying grades of tmd among study participants using chi square testtestmanagement, commerce & finance students)severity table 6association between fonseca’s questionnaire and temporomandibular disorders according to fonseca criteria using chi square test.participants using chi square testtestmanagement, commerce & finance students)severity http://dentistry3000.pitt.edu/ use of fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders among university students a cross sectional study. vol 10 no 1 (2022) doi 10.5195/d3000.2022.178 http://dentistry3000.pitt.edu conclusion this study aimed at assessing the prevalence and severity of tmd in a student population of different faculties in a university campus in bengaluru using the fonseca’s questionnaire. majority of the sample studies showed no signs of tmd while 33.3% showed varying signs of tmd. majority of the cases had milder signs of tmd. stress could be a predisposing factor for the development of tmd. hence early screening during young age can help prevent the condition from progressing. references 1. bonjardim lr, gavião mbd, pereira lj, castelo pm, garcia rcmr. signs and symptoms of temporomandibular disorders in adolescents. pesqui odontol bras. 2005;19:93–8. 2. leresche l, saunders k, von korff mr, barlow w, dworkin sf. use of exogenous hormones and risk of temporomandibular disorder pain. pain. 1997;69:153– 60. 3. mehta nm. the role of interprofessional education in the management of temporomandibular and sleep disorders. cranio : the journal of craniomandibular practice. 2013. p. 159–60. 4. pesqueira aa, zuim prj, monteiro dr, ribeiro pdp, garcia ar. relationship between psychological factors and symptoms of tmd in university undergraduate students. acta odontol latinoam. 2010;23:182–7. 5. zafar ms, fareed wm, taymour n, khurshid z, khan ah. self-reported frequency of temporomandibular disorders among undergraduate students at taibah university. j taibah univ med sci. 2017;12(6):512–22. 6. schiffman el, fricton jr, haley dp, shapiro bl. the prevalence and treatment needs of subjects with temporomandibular disorders. j am dent assoc. 1990;120:295–303. 7. lee j-y, kim y-k, kim s-g, yun p-y. evaluation of korean teenagers with temporomandibular joint disorders. j korean assoc oral maxillofac surg. 2013;39(5):231. 8. pedroni cr, de oliveira as, guaratini mi. prevalence study of signs and symptoms of temporomandibular disorders in university students. j oral rehabil. 2003;30:283–9. 9. ebrahimi m, dashti h, mehrabkhani m, arghavani m, daneshvar-mozafari a. temporomandibular disorders and related factors in a group of iranian adolescents: a crosssectional survey. j dent res dent clin dent prospects. 2011;5(4):123–7. 10. bonjardim l, lopes-filho r, amado g, albuquerque r, goncalves s. association between symptoms of temporomandibular disorders and gender, morphological occlusion, and psychological factors in a group of university students. indian j dent res. 2009;20(2):190–4. 11. chandak r, pandhripande r, sonule s, chandak m, rawlani s. to assess the prevalence of signs and symptoms of temporomandibular disorders in vidarbha population by fonseca’s questionnaire. j oral res rev. 2017;9(2):62. 12. goyal d, nagpal r, kataria s, marya cm, taneja p, sahay v. temporomandibular disorders among adolescents of haryana, india: a cross-sectional study. int j clin pediatr dent. 2019;12(6):500– 6. 13. nomura k, vitti m, de oliveira as, chaves tc, semprini m, siéssere s, et al. use of the fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders in brazilian dental undergraduates. braz dent j. 2007;18(2):163–7. 14. schiffman e, ohrbach r, truelove e, look j, anderson g, goulet j-p, et al. diagnostic criteria for temporomandibular disorders (dc/tmd) for clinical and research applications: recommendations of the international rdc/tmd consortium network* and orofacial pain special interest group†. j oral facial pain headache. 2014;28(1):6–27. 15. bevilaqua-grossi d, chaves tc, de oliveira as, monteiro-pedro v. anamnestic index severity and http://dentistry3000.pitt.edu/ use of fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders among university students a cross sectional study. vol 10 no 1 (2022) doi 10.5195/d3000.2022.178 http://dentistry3000.pitt.edu signs and symptoms of tmd. cranio. 2006;24:112–8. 16. de oliveira as, dias em, contato rg, berzin f. prevalence study of signs and symptoms of temporomandibular disorder in brazilian college students. braz oral res. 2006;20:3–7. 17. campos jadb, gonçalves dag, camparis cm, speciali jg. confiabilidade de um formulário para diagnóstico da severidade da disfunção temporomandibular. rev bras fisioter. 2009;13(1):38– 43. 18. habib sr, al rifaiy mq, awan kh, alsaif a, alshalan a, altokais y. prevalence and severity of temporomandibular disorders among university students in riyadh. saudi dent j. 2015;27(3):125–30. 19. zafar ms, fareed wm, taymour n, khurshid z, khan ah. self-reported frequency of temporomandibular disorders among undergraduate students at taibah university. j taibah univ med sci. 2017 dec 1;12(6):517–22. 20. magnusson t, egermark i, carlsson ge. a longitudinal epidemiologic study of signs and symptoms of temporomandibular disorders from 15 to 35 years of age. j orofac pain. 2000;14(4):310–9. http://dentistry3000.pitt.edu/ microsoft word 267 2022.docx in-vitro an+bacterial ac+vity of glass ionomer cements containing silver nanopar+cles synthesized from leaf extract of mentha piperita vol 10 no 1 (2022) doi 10.5195/d3000.2022.267 h#p://den*stry3000.pi#.edu in-vitro antibacterial activity of glass ionomer cements containing silver nanoparticles synthesized from leaf extract of mentha piperita mahdjoube goldani moghadam1, aylin bagherzade2, fateme ghorbanzade2, mohammad yahya hanafi-bojd3, masoud yousefi4 1assistant professor, department of orthodontics, faculty of dentistry, birjand university of medical sciences, birjand, iran 2md dentist, faculty of dentistry, birjand university of medical sciences, birjand, iran 3cellular and molecular research center, birjand university of medical sciences, birjand, iran. nanomedicine department, faculty of medicine, birjand university of medical sciences, birjand, iran 4infeccous diseases research center, birjand university of medical sciences, birjand, iran abstract purpose: the aim of this study was to evaluate the an+bacterial ac+vity of glass ionomer cement incorporated with silver nanopar+cles (agnps) synthesized using mint leaf extract (mentha piperita, m. piperita) on some oral cavity bacteria. materials and methods: in the present study, m. piperita leaf extract was used for the synthesis of agnps. a total of 60 glass ionomer cement (gic) disk-shaped specimens were prepared and divided into two groups: conven+onal gic (c-gic), and glass ionomer cement with 2 wt% agnps (gic-agnps). the an+bacterial ac+vity of the gic specimens in comparison with ampicillin disk (10 µg/ml) was inves+gated against streptococcus mutans, enterococcus faecalis, lactobacillus acidophilus, lactobacillus casei, and streptococcus aureus by measuring the diameter of growth inhibi+on zones. results: c-gic specimens failed to show any an+bacterial effect against the studied bacteria. however, the gic-agnps had rela+vely significant an+bacterial effects on s. mutans, l. acidophilus, l. casei and s. aureus. the highest an+bacterial effect of gic-agnps specimens was reported against l. acidophilus (p <0.001). gic-agnps had no an+bacterial effect on e. faecalis. conclusion: glass ionomer cement incorporated with agnps synthe+zed using m. piperita showed a promising an+bacterial effect against oral cariogenic pathogens. keywords: an+bacterial ac+vity; glass ionomer cement; mentha piperita; silver nanopar+cles cita:on: goldani moghadam, m et al. (2022) in-vitro an:bacterial ac:vity of glass ionomer cements containing silver nanopar:cles synthesized from leaf extract of mentha piperita den:stry 3000. 1:a001 doi:10.5195/d3000.2022.267 received: november 12, 2021 accepted: february 7, 2022 published: july 13, 2022 copyright: ©2022 goldani moghadam, m et al. this is an open access ar:cle licensed under a crea:ve commons awribu:on work 4.0 united states license. email: yousefi164@gmail.com introduction the number of people who seek orthodontic treatment is increasing. based on the 2012 report of the american association of orthodontists (aao), the number of adults who underwent orthodontic treatment increased about 39% compared to the previous report in 1996 [1]. the impact of orthodontic treatment on oral health is not evident because of the lack of highquality published evidence [2]. some researchers believe, orthodontic treatment is a strong risk factor for in-vitro an+bacterial ac+vity of glass ionomer cements containing silver nanopar+cles synthesized from leaf extract of mentha piperita vol 10 no 1 (2022) doi 10.5195/d3000.2022.267 h#p://den*stry3000.pi#.edu dental caries [3, 4]. the reason for that is the accumulation of dental plaque around fixed appliances, subsequent enamel demineralization, and the occurrence of dental caries [57]. orthodontic treatment has the potential to change the composition of dental plaque. it has been revealed that a large number of pseudomonas species exist in normal oral microflora of orthodontic patients [8]. it has been said that orthodontic treatment is a risk factor for dental caries especially in younger patients and responsible for 45.8% of newly formed caries lesions [9]. despite the above-mentioned facts, some studies support the improved ability of orthodontic patients to maintain oral hygiene [10] and show that in the presence of good oral hygiene, orthodontic treatment would not be a risk factor for dental caries [11]. therefore, there is little agreement on the relationship between orthodontic treatment and dental caries and while debate continues in this regard, it seems logical to apply methods and materials which lessen the caries activity to the fewest possible extent. coating of materials with antimicrobial nanoparticles is one of the proposed methods to reduce caries activity during orthodontic treatment. incorporation of nano cinnamon powder in orthodontic resin [12], wires coated with agnps [13], nanoparticles of silver and nitrogen-doped tio2 applied on orthodontic brackets [14], incorporation of arginine to commercial orthodontic light-cured resin cements [15], and nanosilver mouthwash [16] are examples of promising modifications for improvement of orthodontic treatment. attempts also have been made to modify orthodontic cement such as glass ionomer with nanoparticles to enhance the antibacterial properties of the cement. glass ionomer cement with tio2 nanoparticles showed a significant antibacterial effect on s. mutants [17]. the incorporation of nacetylcysteine (nac) into nanosilvercontaining resin-modified glass ionomer cement revealed favorable antibacterial capability and biocompatibility [18]. the rechargeable nano-caf2 orthodontic cement showed promising results in inhibiting white spot lesions in orthodontics [19]. silver nanoparticles (agnps) possess a great and long-lasting antibacterial activity [20] and are subjected to less bacterial resistance than antibiotics [21]. dental materials which have been modified with nanosized silver particles (nag) show effective antibacterial properties [18]. there are various techniques for synthetizing agnps, including laser ablation, microwave-polyol, sonochemical, sonoelectrochemical, microwave dielectric heating, solvothermal, and electrochemical which most of them are expensive and produce toxic chemicals [20]. in recent years there has been an increasing tendency and interest toward the green synthesis of agnps as a clean and ecofriendly synthesis technique [20]. this technique which is based on the usage of bioorganisms, plant leaves, and fruit extracts, is cost-effective and fast and lessens the risk of toxicity [20, 22]. green synthesis of ag nanoparticles using plants is highly preferable since plants are more resistant to metal toxicity and offer a truly green alternative for agnps synthesis [23]. agnps synthesized with mangifera indica leaves [20], and gum arabic [24] showed promising results against oral pathogens. m. piperita leaf extracts have been shown to have antimicrobial properties against oral pathogens [25]. biosynthesis of agnps using mint leaf extract (m. piperita) revealed antibacterial activity against pathogenic bacteria such as escherichia coli and s. aureus [26]. m. piperita is a natural support for agnps that showed antifungal activity against candida albicans [27]. to the best of our knowledge, there was no report on green synthesis of agnps using m. piperita leaf extract for enhancement of antibacterial properties of glass ionomer cement, therefore, this study aimed to evaluate the antibacterial activity of glass ionomer cement incorporated with agnps synthesized using mint leaf extract (m. piperita) on s. mutans, l. acidophilus, l. casei, e. faecalis and s. aureus bacteria. material and methods in-vitro an+bacterial ac+vity of glass ionomer cements containing silver nanopar+cles synthesized from leaf extract of mentha piperita vol 10 no 1 (2022) doi 10.5195/d3000.2022.267 h#p://den*stry3000.pi#.edu materials silver nitrate (agno3) (99%) and mueller-hinton agar (mha) were purchased from merck company, germany. conventional glass ionomer cement (gic) (powder/liquid form) supplied by fuji pharma, japan. m. piperita leaves were obtained from a local provider (birjand, south khorasan province, iran). the 10 µg/ml ampicillin disk was purchased from mast company, uk. five standard strains of bacteria (atcc bacterial strains) were purchased from pasteur institute (tehran, iran). preparation of plant extract in the present study, m. piperita leaf extract was used for the synthesis of agnps based on its ease of availability, cost-effectiveness, and medicinal properties. the fresh and healthy leaves of m. piperita were washed with distilled water and airdried at room temperature. m. piperita leaf aqueous extract was prepared (50 g of finely powdered leaves in 500 ml double-distilled water) at 60 °c for 10 minutes. the extract was cooled down and filtered with whatman filter papers. finally, the filtered extract was centrifuged at 8000 rpm for 20 minutes, and the supernatant was collected and stored at 4 °c for further use. biosynthesis and characterization of silver nanoparticles (agnps) biosynthesis of agnps was carried out by adding 10 ml of m. piperita leaf extract to 90 ml of 1mm silver nitrate (agno3) solution with continuous stirring at room temperature for 48 hours. the reduction of silver nitrate to agnps was confirmed by the color change of the prepared colorless solution to brown. the fully reduced solution was centrifuged at 8000 rpm for 20 minutes. the supernatant liquid was discarded and the pellet containing agnps was washed 2-3 times with deionized water to remove silver ions and the leaf extract residue. the resultant pellet was dried in a desiccator for 72 hours and stored in dark condition for further characterizations. the synthesized agnps were characterized by uv-visible spectroscopy. the particle size of the samples was determined by the zetasizer (nano-zs, malvern, uk). furthermore, the fourier-transform infrared (ftir) spectra of the nanoparticles were obtained on a perkin-elmer spectrometer (usa) in the absorption mode in the range of 4000-450 cm-1. to determine the shape and structure, the nanoparticles were subjected to transmission electron microscopy (tem) at 120 kv (leo 912ab zeiss electron microscope-germany). preparation of glass ionomer cements (gics) in this study, a total of 60 gic (fuji ii lc, gc corporation, tokyo, japan) disk-shaped specimens were prepared using a silicone mold (7 mm in diameter and 2 mm in height). gic powder and liquid were mixed with a plastic spatula for the 30 seconds according to the manufacturer’s instructions and placed in the molds. the agnps powder was weighed and mixed with gic powder in a percentage of 2% (w/w) immediately prior to manipulation. specimens of cements were divided into two groups: conventional gic without any addition (c-gic), and cement with 2% (w/w) agnps (gic-agnps). noteworthy, the material and specimens were prepared under laminar flow conditions. finally, the prepared gic specimens were sterilized for 30 min using ultraviolet (uv) radiation on each side to prevent contamination during the antibacterial activity tests. the prepared specimens (n = 90) were classified into 3 main groups (30 specimens each) including c-gic, gicagnps, and ampicillin groups. antibacterial activity assessment the antibacterial activity of the gic specimens along with ampicillin disk (10 µg/ml) was investigated by the kirby-bauer disk-diffusion method and according to the clinical and laboratory standards institute (clsi) guidelines [45]. experimented bacteria were s. mutans (atcc 35668), e. faecalis (atcc 29212), l. acidophilus (atcc 4356), l. casei (atcc 39392), and s. aureus (atcc 25923). the disks were placed on mha inoculated with 0.5 mcfarland of the studied bacteria at a distance of 15-20 mm. after incubation (24-48 in-vitro an+bacterial ac+vity of glass ionomer cements containing silver nanopar+cles synthesized from leaf extract of mentha piperita vol 10 no 1 (2022) doi 10.5195/d3000.2022.267 h#p://den*stry3000.pi#.edu hours) at the temperature of 37°c in 5% co2 (aerobic conditions for s. aureus), antibacterial activity was assessed by measuring the diameter of growth inhibition zones. the results represented the average of the three replicates. statistical analysis statistical analysis was performed using spss version 21 (spss, chicago, il). mean values and standard deviations for diameter of inhibition zone were reported. antimicrobial activity of glass ionomer cement (gic) and gicagnps specimens, and ampicillin disks against various standard bacterial strains were analyzed using independent-samples t-test. (since no inhibition zone was observed for gic specimens, independent-samples t-test was used for comparison of the antibacterial effect of the two other disks (gic agnps, and ampicillin) against each bacterial strain.). the effect size (hedge's g) and the results of statistical analyses are presented in table 2. the significance level for all analysis was set at p <0.05. results characterization of agnps in this study, the biosynthesis of m. piperita agnps was confirmed by the color change of the initial colorless solution to brown. furthermore, the uv-vis spectral analysis showed the peak at 420 nm, which attributed to the surface plasmon resonance (spr) of agnps (figure 1). the size distribution of the agnps was measured by dls and their average size was found to be 71.67±0.13 nm (figure 2). as per tem analysis, agnps of m. piperita appeared to be quasispherical in shape with a size range of less than 100 nm (figure 3). fourier transform infrared (ftir) spectral measurements were carried out to identify the possible biomolecules in m. piperita leaf aqueous extract as reducing and capping agents of agnps (figure 4). in the ftir spectrum of m. piperita agnps, the 3420 cm-1 peak represents the n-h bending vibration of the amine groups. also, two peaks were observed at 2924 cm-1 and 1628 cm-1 which are related to c-h groups and carbonyl groups from polyphenols, respectively. the results indicate the presence of plant biomolecules on agnps that these compounds can play an important role in stabilizing figure 1. uv-vis spectral analysis of silver nanoparticles (agnps) synthesized by m. piperita leaf aqueous extract. figure 2. particle size of m. piperita agnps. figure 3. tem images and respective size distribution m. piperita agnps. in-vitro an+bacterial ac+vity of glass ionomer cements containing silver nanopar+cles synthesized from leaf extract of mentha piperita vol 10 no 1 (2022) doi 10.5195/d3000.2022.267 h#p://den*stry3000.pi#.edu nanoparticles and increasing their antibacterial properties. antibacterial activity in the present study, the antibacterial activity of the gic and gic-agnps specimens in comparison with ampicillin was investigated for various standard bacteria strains (table 1). the results indicated that conventional glass ionomer cement (c-gic) specimens did not have any antibacterial effects against the studied bacteria. however, the gicagnps had antibacterial effects on s. mutans, l. acidophilus, l. casei and s. aureus. the highest antibacterial effect of gic-agnps specimens with an average inhibition zone diameter of 11.67±0.29 mm was reported against l. acidophilus (table 2). all studied bacteria were sensitive to ampicillin with a minimum inhibition zone diameter of >26 mm (p <0.001). overall, the results of this study showed that the antibacterial effects of gic-agnps against s. mutans, l. acidophilus, l. casei and s. aureus were relatively significant compared to conventional gic, whereas significantly lower compared to ampicillin (table 2). bacteria strains zone of inhibition (mm) mean ±sd c-gic gic-agnps ampicillin (10 µg/ml) streptococcus mutans (atcc 35668) ni 10.17±1.04 51.33±1.04 lactobacillus acidophilus (atcc 4356) ni 11.67±0.29 28.88±1.07 lactobacillus casei (atcc 39392) ni 8.16±0.29 26.83±0.67 enterococcus faecalis (atcc 29212) ni ni 32.17±0.76 staphylococcus aureus (atcc 25923) ni 8.67±0.76 32.83±0.29 figure 4. ftir spectrum of agnps synthesized by m. piperita leaf aqueous extract. c-gic: conventional glass ionomer cement, gic-agnps: gic with 2 wt% agnps, ni: no inhibition zone table 1: antimicrobial activity of glass ionomer cement (gic), gicagnps, and ampicillin specimens against various standard bacteria strains. in-vitro an+bacterial ac+vity of glass ionomer cements containing silver nanopar+cles synthesized from leaf extract of mentha piperita vol 10 no 1 (2022) doi 10.5195/d3000.2022.267 h#p://den*stry3000.pi#.edu discussion as previously mentioned, gic is said to have antibacterial activity against cariogenic pathogen, though, its ability to arrest caries process is a matter of debate [28]. however, we failed to find any antibacterial activity of c-gic in this study, which is corroborated with findings of some previous researches [2932]. several attempts have been made to enhance the antibacterial activity of glass ionomer cement such as addition of chlorhexidine gluconate and antibiotic mixtures to a glass ionomer cement [28, 33]. however, these additives had different and often negative effects on physical and bonding properties of glass ionomer [28, 33]. nano silver particles have shown great suppressive effect on oral pathogens [34]. they have inhibitory effect on adhesion and growth of cariogenic bacteria and impede the demineralization of enamel and dentin [34]. nano silver particles are used in low concentrations and therefore they don’t cause changes in mechanical and color properties of dental materials or bacterial resistance [18, 34]. agnps, nano silver fluoride, resin with agnps, and glass ionomer cement with agnps have been widely used and evaluated for dental applications [34]. several researches have been done on the antibacterial activity of gic incorporated with nano silver particles. incorporating agnps into a resin-modified glass ionomer (resin containing 80 ppm of nanosilver) streptococcus mutans (atcc 35668) lactobacillus acidophilus (atcc 4356) lactobacillus casei (atcc 39392) enterococcus faecalis (atcc 29212) staphylococcus aureus (atcc 25923) gic-agnps 10.17±1.04 11.67±0.29 8.16±0.29 ni 8.67±0.76 ampicillin (10 µg/ml) 51.33±1.04 28.88±1.07 26.83±0.67 32.17±0.76 32.83±0.29 p value * p <0.001 p <0.001 p <0.001 p <0.001 p <0.001 effect size (hedge's g) ci= 95% -38.79 -21.51 -35.44 -41.16 95% ci values lower limit -48.23 -26.78 -44.08 -51.19 upper limit -30.39 -16.83 -27.77 -32.26 table 2: comparison of the inhibition zone diameter between gic-agnps, and ampicillin disks against various standard bacterial strains. *indicates the results of independent-samples t-test for comparison of the antibacterial effect of the two disks against each bacterial strain. in-vitro an+bacterial ac+vity of glass ionomer cements containing silver nanopar+cles synthesized from leaf extract of mentha piperita vol 10 no 1 (2022) doi 10.5195/d3000.2022.267 h#p://den*stry3000.pi#.edu showed significant antimicrobial activity against s. sanguinis and s. mutans in direct contact test [30]. incorporation of nano silver base inorganic antibacterial powder into the glass ionomer cement decreased the demineralization rate around brackets without compromising bond strength [35]. in another study, incorporation of agnps with gic limited the s. aureus biofilm formation with insignificant effect on mechanical properties [36]. in a study conducted on a dental plaque microcosm biofilm model, resin-modified glass ionomer cement (rmgic) containing nanoparticles of silver (nag) reduced biofilm formation, plaque buildup, and white spot lesions formation around brackets without any adverse effect on the shear bond strength of brackets [37]. incorporation of silver nanoparticle to orthodontic glass ionomer cements in the rat caries disease model enhanced its bactericidal effect on s. mutans [38]. the antibacterial effect of gic-agnps against s. mutans, l. acidophilus, l. casei and s. aureus in this study is in line with the findings of a great deal of the previous work in this field [30, 3638]. we observed that gicagnps did not have any antibacterial effect against e. faecalis which is a gram-positive bacterium usually found in persistent endodontic infections [39]. in a previous study, addition of up to 5 wt% nanosilver to zinc oxide sealer could not improve its antibacterial effect against e. faecalis [40]. however, we didn’t find any study which had evaluated the antibacterial effect of nanosilver glass ionomer cement against e. faecalis. in the studies conducted on the role of silver nanomaterials for caries prevention, the most commonly used bacteria were s. mutans which are the main cause of dental caries and various lactobacilli which are associated with progression of the caries lesion [34]. however, in oral cavity we are facing with biofilm rather than cariogenic monospecies strains, which enhance the resistance of microorganisms to antimicrobial agents [34]. therefore, in order to simulate the complexity and heterogeneity of the biological biofilm, it is preferable to use oral microcosm cultured from various bacteria and the extracellular matrix removed from the oral environment [34]. however, an animal study or clinical trial would provide the environment of choice to evaluate antibacterial activity of nano silver materials [34]. besides the antibacterial effects of nano silver materials, there are concerns regarding the silver nano particles cytotoxicity [41]. free sliver ions released from nanoparticles can penetrate cells and increase the production of reactive oxygen species and deleterious effects [42]. however, the cytotoxicity of agnps for human oral cells is lower than other silver compounds [34]. besides the possible toxic effect of nano silver particles themselves, the techniques used to produce these particles can lead to production of toxic chemicals [20]. the green synthesis of ag nanoparticles can lessen the risk of toxicity and is a green alternative for agnps synthesis [2023]. we used the mentha piperita leaf extracts for synthesis of ag nanoparticles which to our best knowledge have not been used in dental applications so far. we found antibacterial activity of mentha piperita leaf extracts synthetized gicagnps against the studied bacteria, which was in line with previous studies which used mentha piperita leaf extracts against oral pathogens [25], escherichia coli and s. aureus [26], and candida albicans [27]. although we found antibacterial activity of mentha piperita leaf extracts synthetized gicagnps against bacteria strains, the stability of this effect should be further evaluated, since silver nanomaterials can be easily oxidized and aggregated [43]. in addition, further evaluation of different concentrations of agnps to find the optimized concentration which beside the antibacterial activity maintain the mechanical properties of the dental material seems necessary. conclusion the findings of this study showed that glass ionomer cement incorporated with agnps synthesized using mint leaf extract (m. piperita) showed a promising antibacterial effect on s. mutans, l. acidophilus, l. casei and s. aureus. in-vitro an+bacterial ac+vity of glass ionomer cements containing silver nanopar+cles synthesized from leaf extract of mentha piperita vol 10 no 1 (2022) doi 10.5195/d3000.2022.267 h#p://den*stry3000.pi#.edu author contributions author 1 contributed to conception, design, data interpretation author 4 and 5 contributed to conception, design, data interpretation, drafted and critically revised the manuscript. author 2 and 3 contributed to data acquisition. conflict of interests authors wish to declare there is no conflict of interest related to this study. ethical approval the study protocol was approved by ethics committee of birjand university of medical sciences (ir.bums.rec.1397.65). funding this work was supported by vice chancellor of research of birjand university of medical sciences (grant number: 455581). references 1. american associa,on of orthodon,sts. 2018. aao economics of orthodon,sts/pa,ent census survey comparison results [internet]. american associa,on of orthodon,sts;st. louis, mo: available from: hjps://assetsprod-www1.aaoinfo.org/assetsprodwww1/2019/02/14152041/2012 -2016-comparison-data.pdf. cited 2018 may 17. 2. do malocclusion and orthodon,c treatment impact oral health? a systema,c review and meta-analysis. american journal of orthodon,cs and dentofacial orthopedics. macey r, thiruvenkatachari b, o'brien k, ba,sta kb. 2020 jun 1;157(6):738-44. 3. incidence of caries lesions among pa,ents treated with comprehensive orthodon,cs. american journal of orthodon,cs and dentofacial orthopedics. richter ae, arruda ao, peters mc, sohn w. 2011 may 1;139(5):657-64. 4. linking orthodon,c treatment and caries management for high-risk adolescents. american journal of orthodon,cs and dentofacial orthopedics. cruz cl, edelstein bl. 2016 apr 1;149(4):441-2. 5. effect of orthodon,c band placement on the chemical composi,on of human incisor tooth plaque. archives of oral biology. chajerjee r, kleinberg i. 1979 jan 1;24(2):97-100. 6. ini,al changes of dental plaque, gingivi,s and decalcifica,on in korean orthodon,c pa,ents with fixed appliance. the korean journal of orthodon,cs. kang kj, shon bh. 1999;29(3):361-74. 7. the effect of fixed orthodon,c appliances on the oral carriage of candida species and enterobacteriaceae. the european journal of orthodon,cs. hägg u, kaveewatcharanont p, samaranayake yh, samaranayake lp. 2004 dec 1;26(6):623-9. 8. comparison of oral microbiota in orthodon,c pa,ents and healthy individuals. microbial pathogenesis. sun f, ahmed a, wang l, dong m, niu w. 2018 oct 1;123:473-7. 9. cri,cal evalua,on of incidence and prevalence of white spot lesions during fixed orthodon,c appliance treatment: a metaanalysis. journal of interna,onal society of preven,ve & community den,stry. sundararaj d, venkatachalapathy s, tandon a, pereira a. 2015 nov;5(6):433. 10. the effect of orthodon,c treatment on plaque and gingivi,s. american journal of orthodon,cs and dentofacial orthopedics. davies tm, shaw wc, worthington hv, addy m, dummer p, kingdon a. 1991 feb 1;99(2):155-61. 11. orthodon,c appliances did not increase risk of dental caries and periodontal disease under preven,ve protocol. the angle orthodon,st. bergamo az, de oliveira km, matsumoto ma, nascimento cd, romano fl, da silva ra, da silva la, nelsonfilho p. 2019 jan;89(1):25-32. 12. the effect of incorpora,on nano cinnamon powder on the shear bond of the orthodon,c composite (an in vitro study). journal of oral biology and craniofacial research. yaseen sn, taqa aa, al-kha,b ar. 2020 apr 1;10(2):128-34. 13. an,microbial orthodon,c wires coated with silver nanopar,cles. brazilian archives of biology and technology. gonçalves is, viale ab, sormani nn, pizzol ke, araujo-nobre ar, oliveira pc, barud hg, antonio sg, barud hd. 2020 oct 26;63. 14. func,onal coa,ngs for orthodon,c archwires—a review. materials. bącela j, łabowska mb, detyna j, zięty a, michalak i. 2020 jan;13(15):3257. in-vitro an+bacterial ac+vity of glass ionomer cements containing silver nanopar+cles synthesized from leaf extract of mentha piperita vol 10 no 1 (2022) doi 10.5195/d3000.2022.267 h#p://den*stry3000.pi#.edu 15. incorpora,on of arginine to commercial orthodon,c lightcured resin cements—physical, adhesive, and an,bacterial proper,es. materials. geraldeli s, maia carvalho ld, de souza araújo ij, guarda mb, nascimento mm, bertolo mv, di nizo pt, sinhore, ma, mccarlie vw. 2021 jan;14(16):4391. 16. effect of nanosilver mouthwash on preven,on of white spot lesions in pa,ents undergoing fixed orthodon,c treatment-a randomized double-blind clinical trial. journal of dental sciences. ali a, ismail h, amin k. 2021 may 1. 17. applied research glass ionomer cement with tio2 nanopar,cles in orthodon,c treatment. journal of nanoscience and nanotechnology. ren j, du z, lin j, hirabe h, sakai k. 2021 feb 1;21(2):1032-41. 18. the effect of n-acetylcysteine on the an,bacterial capability and biocompa,bility of nano silver– containing orthodon,c cement. the angle orthodon,st. ding r, qian y, chen m, yi j, zhao z. 2021 feb 11. 19. novel rechargeable nano-caf2 orthodon,c cement with high levels of long-term fluoride release. journal of den,stry. yi j, weir md, melo ma, li t, lynch cd, oates tw, dai q, zhao z, xu hh. 2019 nov 1;90:103214. 20. green synthesis and characteriza,on of ag nanopar,cles from mangifera indica leaves for dental restora,on and an,bacterial applica,ons. progress in biomaterials. sundeep d, kumar tv, rao ps, ravikumar rv, krishna ag. 2017 may;6(1):5766. 21. size and aging effects on an,microbial efficiency of silver nanopar,cles coated on polyamide fabrics ac,vated by atmospheric dbd plasma. acs applied materials & interfaces. zille a, fernandes mm, francesko a, tzanov t, fernandes m, oliveira fr, almeida l, amorim t, carneiro n, esteves mf, souto ap. 2015 jul 1;7(25):13731-44. 22. pear fruit extract-assisted roomtemperature biosynthesis of gold nanoplates. colloids and surfaces b: biointerfaces. ghodake gs, deshpande ng, lee yp, jin e. 2010 feb 1;75(2):5849. 23. green synthesis of silver nanopar,cles toward bio and medical applica,ons: review study. ar,ficial cells, nanomedicine, and biotechnology. mousavi sm, hashemi sa, ghasemi y, atapour a, amani am, savar dashtaki a, babapoor a, arjmand o. 2018 nov 12;46(sup3):s855-72. 24. synthesis of silver nanopar,cles using gum arabic: evalua,on of its inhibitory ac,on on streptococcus mutans causing dental caries and endocardi,s. journal of infec,on and public health. al-ansari mm, aldahmash nd, ranjitsingh aj. 2021 mar 1;14(3):324-30. 25. effec,veness of mentha piperita leaf extracts against oral pathogens: an in vitro study. the journal of contemporary dental prac,ce. raghavan r, devi ms, varghese m, joseph a, madhavan ss, sreedevi pv. 2018 sep 1;19(9):1042-6. 26. biosynthesis of silver nanopar,cles using mint leaf extract (mentha piperita) and their an,bacterial ac,vity. advanced science, engineering and medicine. gabriela ám, gabriela md, luis am, reinaldo pr, michael hm, rodolfo gp, roberto vb. 2017 nov 1;9(11):914-23. 27. mentha piperita as a natural support for silver nanopar,cles: a new an,-candida albicans treatment. colloid and interface science communica,ons. robles-mar|nez m, pa,ñoherrera r, pérez-vázquez fj, montejano-carrizales jm, gonzález jf, pérez e. 2020 mar 1;35:100253. 28. an,bacterial ac,vity and physical proper,es of glassionomer cements containing an,bio,cs. opera,ve den,stry. yesilyurt ce, er k, tasdemir t, buruk k, celik d. 2009 jan;34(1):18-23. 29. addi,on of chlorhexidine gluconate to a glass ionomer cement: a study on mechanical, physical and an,bacterial proper,es. brazilian dental journal. mar, lm, mata md, ferraz-santos b, azevedo er, giro em, zuanon ac. 2014 jan;25:33-7. 30. use of silver nanomaterials for caries preven,on: a concise review. interna,onal journal of nanomedicine. yin ix, zhao is, mei ml, li q, yu oy, chu ch. 2020;15:3181. 31. long-term an,bacterial proper,es and bond strength of experimental nano silvercontaining orthodon,c cements. journal of wuhan university of technology-mater. sci. ed.. li f, li z, liu g, he h. 2013 aug 1;28(4):849-55. 32. the impact of silver nanopar,cles integra,on on biofilm forma,on and mechanical proper,es of glass ionomer cement. journal of esthe,c and restora,ve den,stry. el-wassefy na, mahdy rh, el-kholany nr. 2018 mar;30(2):146-52. in-vitro an+bacterial ac+vity of glass ionomer cements containing silver nanopar+cles synthesized from leaf extract of mentha piperita vol 10 no 1 (2022) doi 10.5195/d3000.2022.267 h#p://den*stry3000.pi#.edu 33. an,bacterial orthodon,c cement to combat biofilm and white spot lesions. american journal of orthodon,cs and dentofacial orthopedics. wang x, wang b, wang y. 2015 dec 1;148(6):974-81. 34. an,bacterial proper,es of nano silver-containing orthodon,c cements in the rat caries disease model. journal of wuhan university of technology-mater. sci. ed.. li f, fang m, peng y, zhang j. 2015 dec;30(6):1291-6. 35. cytotoxic effects of silver diamine fluoride. american journal of den,stry. fancher me, fournier s, townsend j, lallier te. 2019 jun 1;32(3):152-6. 36. negligible par,cle-specific toxicity mechanism of silver nanopar,cles: the role of ag+ ion release in the cytosol. nanomedicine: nanotechnology, biology and medicine. de majeis v, malvindi ma, galeone a, brune� v, de luca e, kote s, kshirsagar p, sabella s, bardi g, pompa pp. 2015 apr 1;11(3):731-9. 37. the stability of silver nanopar,cles in a model of pulmonary surfactant. environmental science & technology. leo bf, chen s, kyo y, herpoldt kl, terrill nj, dunlop ie, mcphail ds, shaffer ms, schwander s, gow a, zhang j. 2013 oct 1;47(19):11232-40. 38. an,microbial ac,vity of glass ionomer cements with chlorhexidine and propolis: an in vitro study. journal of pediatric infec,ous diseases. tuncdemir mt, ugur ar, ozdemir b. 2019 jul;14(04):16870. 39. an,bacterial ac,vity of a glass ionomer containing silver nanopar,cles against streptococcus mutans and streptococcus sanguinis. indian journal of dental research. moshfeghi h, haghgoo r, sadeghi r, niakan m, rezvani mb. 2020 jul 1;31(4):589. 40. inhibi,on of microbial adherence and growth by various glass ionomers in vitro. palenik cj, behnen mj, setcos jc, miller ch. dent mater 1992;8:16-20. 41. growth inhibi,on of glass ionomer cements on mutans streptococci. loyola-rodriguez jp, garcia-godoy f, lindquist r. pediatr dent 1994;16:346-9. 42. enterococcus faecalis hydrolyzes dental resin composites and adhesives. journal of endodon,cs. marashdeh mq, gitalis r, levesque c, finer y. 2018 apr 1;44(4):609-13. 43. an,microbial efficacy of mixtures of nanosilver and zinc oxide eugenol against enterococcus faecalis. the journal of contemporary dental prac,ce. haghgoo r, ahmadvand m, nyakan m, jafari m. 2017 mar 1;18(3):177-81. microsoft word 83 2018.docx   vol  6,  no  1  (2018)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2018.83         http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     comparison  of  root  canal  length  measurement  methods  in  primary   teeth   mine  koruyucu1,  merve  bayram2,  yelda  kasımoğlu1,  figen  seymen3     1research  assistant,  istanbul  university,  faculty  of  den9stry,  department  of  pedodon9cs,  istanbul,  turkey   2assistant  professor,  istanbul  medipol  university,  faculty  of  den;stry,  department  of  pedodon;cs,  istanbul,  turkey   3professor,  istanbul  university,  faculty  of  den.stry,  department  of  pedodon.cs,  istanbul,  turkey     abstract   objec&ves:  the  purpose  of  this  study  was  to  evaluate  the  accuracy  of  conven5onal  radiography,  intraoral  digital  radi-­‐ ovisiography  and  electronic  apex   locator   in  determining  the  working   length  of  root  canals   in  primary  teeth  (in-­‐vivo)   and  to  compare  the  results  with  scanning  electron  microscopy  measurements  (ex-­‐vivo).  materials  and  methods:  this   study  was  conducted  on  50  primary  molar  teeth.  standard  endodon7c  access  cavity  prepara7ons  were  prepared  and   the  actual  length  was  calculated  by  calibrated  inves3gators.  working  lengths  were  determined  by  using  conven5onal   radiography,  rvg  and  apex  locator  in  a  total  of  116  root  canals.  a;er  the  extrac<on  of  the  teeth,  each  canal  length   was  determined  by  using  sem.  data  were  analysed  sta6s6cally  by  using  oneway  anova,  tukey  hds,  student  t  test  and   bonferroni   tests.   results:   the   mean   root   length   measurements   with   conven3onal   radiography   were   significantly   higher  than  apex  locator,  rvg  and  sem.  the  accuracy  of  apex  locator  and  rvg  were  higher  than  conven>onal  radiog-­‐ raphy   in  determining  the  working   length   in  primary  teeth.  conclusions:  the  electronic  apex   locators  provide  an  ac-­‐ ceptable  level  of  accuracy  in  determining  root  canal  length  in  primary  teeth.  clinical  relevance:  the  purpose  of  this   study  was  to  evaluate  the  accuracy  of  conven,onal  radiography,  intraoral  digital  radiovisiography  and  electronic  apex   locator   in  determining  the  working   length  of  root  canals   in  primary  teeth  and,to  compare  the  results  with  scanning   electron  microscopy  measurements.   cita%on:  koruyucu  m,  et  al.  (2018)  comparison   of  root  canal  length  measurement  methods  in   primary  teeth.  den$stry  3000.  1:a001   doi:10.5195/d3000.2018.83   received:  march  2,  2018   accepted:    april  23,  2018   published:    july  13,  2018   copyright:  ©2018  koruyucu  m,  et  al.  this   is   an   open   access   ar!cle   licensed   under   a   crea!ve   commons   a"ribu%on   work   4.0   united   states   license.   email:  mine.yildirim@istanbul.edu.tr         introduction   the  primary  objective  of   pulpectomy  of  primary  teeth  is  to   retain  teeth  with  irreversible  pulp   pathosis  in  a  symptom  free  state   and  maintain  the  integrity  of  the   arch  form  [1,  2].  however,  a  num-­‐ ber  of  reasons,  such  as  the  com-­‐ plex  anatomic  morphology  of  the   root  canal  system  in  primary   teeth,  innate  physiological  root   resorption,  the  close  proximity  of   the  permanent  successor  tooth   and  the  difficulty  of  producing     satisfactory  radiological  images  of   primary  tooth  apices  makes  it  dif-­‐ ficult  to  achieve  proper  treatment   [3].   radiography  is  among  the   most  common  and  widely  used   techniques  for  root  canal  length   determination  [4,  5].  use  of  radi-­‐ ography  to  calculate  root  canal   length  may  not  always  lead  to  ac-­‐ curate  results  especially  in  case  of   physiological  resorption  of  primary   teeth.  instrumentation  and/or   overfilling  becomes  much  more   possible  if  there  is  a  mistake  in   measurement  technique,  thus  the   germ  of  a  permanent  tooth  might   get  damaged  [4,  6,  7].  the  over-­‐ filled  material  can  retain  after   natural  exfoliation  of  the  primary   tooth  [8].  furthermore,  poor  co-­‐ operation  of  children  makes  it  dif-­‐ ficult  to  take  a  radiograph  with   acceptable  diagnostic  value  [4,  5].   proper  detection  of  the   working  length  is  very  important   before  pulpectomy  in  primary   teeth.  due  to  limitations  of  radio-­‐ graphic  interpretation  and  high   possibility  of  over-­‐instrumentation   of  the  unevenly  resorbed  roots   and  subsequent  overfilling,  the   use  of  electronic  apex  locators  is   recommended  regardless  of  the   stage  of  root  resorption  [7].   working  length  determina-­‐ tion  is  an  extremely  relevant  fac-­‐ tor  for  the  success  of  root  canal   treatments.  to  achieve  best  re-­‐ sults,  the  narrowest  part  of  the   root  canal  where  the  width  of   blood  vessels  are  smaller  and  the   possibility  of  healing  is  highest,   namely  apical  constriction  or  api-­‐  comparison  of  root  canal  length  measurement  methods  in  primary  teeth     vol  6,  no  1  (2018)        doi  10.5195/d3000.2018.83    http://dentistry3000.pitt.edu   cal  foramen  are  prepared  [9].  in   theory,  the  canal  terminus  is  re-­‐ garded  as  the  cemento-­‐dentinal   junction,  even  though  it  is  not   possible  to  detect  it  clinically  [10].   therefore,  the  apical  terminus   used  is  minor  foramen  whose  lo-­‐ cation  is  around  0.5–1.0 mm  other   than  the  radiographic  apex  [11-­‐ 13].   generally,  the  convention-­‐ al  radiographic  method  has  been   used  to  determine  the  root  canal   length  [12,  14].  however,  this   method  has  some  disadvantages,   such  as  the  superposition  of  ana-­‐ tomical  structures  and  image  dis-­‐ tortions,  and  the  film  processing   time  [15].  while  producing  imag-­‐ es,  not  a  radiographic  film  but  a   sensor  is  used  in  the  digital  radio-­‐ graphic  method.  this  method  is   more  advantageous  than  the  con-­‐ ventional  one,  as  it  is  faster  in  im-­‐ age  acqusition,  its  amount  of      ra-­‐ diation  is  less  and  it  allows  image   editing,  making  it  easy  to  examine   the  details  [16,  17].   the  apical  foramen  can  be   evaluated  with  an  electronic  apex   locator,  but  only  the  radiographic   apex  can  slightly  be  displayed  by   radiographs  [5,  18].  electronic   apex  locator,  which  helps  to  locate   the  file  position  in  the  canal,  has   been  used  clinically  for  more  than   40 years  [19-­‐21].  several  genera-­‐ tions  of  electronic  apex  loca-­‐ tors  have  evolved  [20].   there  is  no  indication  in  the   literature  whether  there  is   any  difference  in  utilization   the  electronic  apex  locators   in  permanent  or  primary   teeth,  and  as  they  prevent   using  radiation  more  than  needed,   electronic  apex  locators  are  re-­‐ garded  as  safe,  painless,  and  help-­‐ ful.  as  a  result,  its  use  in  primary   teeth  is  recommended  [5].   the  objective  of  this  study   was  to  evaluate  the  accuracy  of   conventional  radiography,  in-­‐ traoral  digital  radiovisiography  and   electronic  apex  locator  in  deter-­‐ mining  the  working  length  of  root   canals  in  primary  teeth  (in-­‐vivo)   and  to  compare  the  results  with   scanning  electron  microscopy   measurements  (ex-­‐vivo).   materials  and  methods   the  study  was  approved  by   the  ethics  committee  of  the  istan-­‐ bul  university,  medical  faculty   (no:2012/1738-­‐1298).  fifty  ex-­‐ tracted  human  primary  molar   teeth  (116  root  canals)  referred   for  extraction  to  istanbul  universi-­‐ ty,  faculty  of  dentistry,  depart-­‐ ment  of  pedodontics  because  it  is   not  possible  to   restore  them   after  caries  are   removed  or   when  there  is  a   root  with  re-­‐ sorption  great-­‐ er  than  two-­‐ thirds  were  se-­‐ lected  follow-­‐ ing  clinical  and   radiographic  examination.  primary   molars  which  had  abnormal  root   morphology  and  evidence  of  frac-­‐ tures  were  excluded  from  the   study.   two  calibrated  pediatric   dentist  (mk,  mb)  carried  out  den-­‐ tal  examinations.  intra-­‐examiner   calibration  was  performed  by  re-­‐ peating  examinations  of  10  teeth   after  an  interval  of  1  week.  kappa   values  for  intra-­‐examiner  con-­‐ sistency  were  95.5%  for  teeth.  the   inclusion  criteria  for  teeth  selec-­‐ tion  were  as  follows:  teeth  have  to   demonstrate  extensive  caries,   show  the  presence  of  two-­‐thirds   of  the  root  length  radiographically,   and  have  adequate  tooth  struc-­‐ ture  to  be  restored.  also  there   should  be  no  radiographic  and   clinical  evidence  of  pulp  pathology   (resorption,  spontaneous  pain,   tenderness  to  percussion  or  palpa-­‐ tion,  swelling,  sinus  tract,  patho-­‐ logic  mobility  etc.).  during  the   treatment,  the  teeth  were  anes-­‐ thetized  and  the  endodontic  ac-­‐ cess  was  performed  using  high   speed  diamond  burs.  the  canals   were  initially  explored  with  #10  k-­‐ files  (mani,  tochigi,  japan).     after  extirpating  the  pulp   with  a  barbed  broach,  the  canals    comparison  of  root  canal  length  measurement  methods  in  primary  teeth     vol  6,  no  1  (2018)        doi  10.5195/d3000.2018.83    http://dentistry3000.pitt.edu   were  irrigated  with  saline  solution   then  dried  with  cotton  pellets   without  any  attempt  to  dry  the   canal.  the  length  of  the  root  ca-­‐ nals  were  measured  with  one  of   the  fitting  k-­‐type  file,  sized  be-­‐ tween  15-­‐40  (mani,  tochigi,  ja-­‐ pan).  after  that,  a  silicone  stop   was  adjusted  to  the  reference,  and   the  distance  between  stop  and  file   tip  was  measured  with  endodontic   ruler  by  magnifying  loupes.  the   reference  point  was  marked  on   the  coronal  portion  of  the  roots   with  a  fine  paint  marker  to  facili-­‐ tate  accurate  reinsertion  of  the   files.    root  canal  length  for  each   tooth  was  measured  with  a  scale   in  milimeters  by  all  methods  (in-­‐ traoral  periapical  radiography,   rvg  and  electronic  apex  locater   before  extraction,  sem  after  ex-­‐ traction)  (picture  1).  the  measur-­‐ ing  precision  was  set  to  0.5  mm   and  the  measurements  were  re-­‐ calculated  three  times  for  each   canal  by  the  same  dentist  and  av-­‐ erage  value  was  recorded.   for  conventional  radiog-­‐ raphy  groups;  intraoral  periapical   radiographs  were  taken  by  x-­‐ray   device  operating  8  ma  and  70  kvp   (kodak,  tokyo,  japan)  using  paral-­‐ leling  technique  the  films  were   then  placed  parallel  to  the  x  ray   tube  (trophy,  tokyo,  japan).   the  apex  locator  was  used   for  the  electronic  measurement   (endo  master,  ems,  switzerland).   the  labial  clip  was  inserted  and  k-­‐ file  attached  to  the  file  holder  was   smoothly  inserted  till  “apex”  ap-­‐ peared  on  the  screen.  then,  the   rubber  stop  was  placed  at  the  cor-­‐ onal  reference,  and  the  measure-­‐ ment  of  root  canal  length  was   done  electronically.    for  rvg   groups,  after  all  intraoral  prepara-­‐ tions  rvg  (kodak,  tokyo,  japan)   were  used  to  assess  the  length  of   the  root  canal.     when  the  eruption  of   permanent  teeth  start,  the  teeth   that  were  mobility  were  extracted.   after  the  teeth  had  been  extract-­‐ ed,  they  were  preserved  in  10%   formaldehyde  solution.  they  were   numbered  and  immersed  in  5.25%   sodium  hypochlorite  solution  for   15  minutes  to  get  rid  of  organic   residues  in  root  surfaces.  then   they  were  scaled  to  remove  any   stain  and  calculus.  in  accordance   with  electron  microscopy  instruc-­‐ tions,  the  snonputter  technique   (polaron  sputter  coater)  for   electron  microscopy  images  was   used  to  coat  the  specimens  with   gold  (150  seconds).  sem  under   x50  magnification  was  used  to   calculate  every  canal  length   (jeol  jsm-­‐5600,  sem).   spss  for  windows,  ver-­‐ sion  10.0  was  run  for  all  statisti-­‐ cal  analyses  (spss,  chicago,   usa).  data  were  analysed  statis-­‐ tically  by  using  oneway  anova,   tukey  hds,  student  t  test  and   bonferroni  tests  post-­‐hoc  analysis.   a  p-­‐value  <0.05  was  considered   statistically  significant.   results   a  statistically  significant   difference  was  found  between  the   mean  root  canal  length  of  the   methods  (p  <0.01)  (table  1).  the   root  canal  measurement  average   of  the  conventional  method  was   found  significantly  higher  than  the   apex  locator  (p:  0.001),  rvg  (p:   0.001)  and  sem  (p:  0.001)  meas-­‐ urements  (p  <0.01).  there  is  no   statistically  significant  difference   between  the  mean  apex  locator,   rvg  and  sem  measurement,  (p>   0.05)  (table  2).   there  is  a  statistically  sig-­‐ nificant  difference  between  apex   locator,  conventional  method,   rvg  and  sem  averages  according   to  age  results  (p<0.001).  the  apex   locator  averages  of  ages  6  and  8   were  found  significantly  higher  in   the  7  years  (p:  0.008)  and  9  years   (p:  0.028)  (p  <0.05,  p  <0.01).  the   conventional  methods  of  9  and  7   years  were  significantly  lower  than    comparison  of  root  canal  length  measurement  methods  in  primary  teeth     vol  6,  no  1  (2018)        doi  10.5195/d3000.2018.83    http://dentistry3000.pitt.edu   5  years  (p:0.008),  6  years  (p:   0.001)  and  8  years  (p:  0.004).  the   conventional  method  avetage  of   the  7  years  was  significantly  lower   than  that  of  6  years  (p:  0.048)   (p<0.05).  rvg  averages  of  6  and  8   years  were  significantly  higher   than  7  years  (p:0.004)  and  9  years   (p:0.002)  (p<0.01).  the  sem  aver-­‐ age  of  the  7  year  was  significantly   lower  than  the  6  year  (p:0.033)   and  the  8  year  (p:0.022)  (table  3).     the  root  canal  measure-­‐ ment  average  of  the  conventional   method  was  significantly  higher   than  the  sem  (p:0.017)  measure-­‐ ments  for  6  years  old  children   (p<0.05).  the  root  canal  meas-­‐ urement  average  of  the  conven-­‐ tional  method  was  found  signifi-­‐ cantly  higher  than  the  apex  locator   (p:0.001),  rvg  (p:  0.011)  and  sem   (p:0.001)  measurements  for  7   years  old  children  (p<0.01).  the   root  canal  measurement  averages   of  rvg  were  significantly  lower   than  the  apex  locator  (p:0.016),   conventional  method  (p:0.016)   and  sem  (p:0.011)  for  9  years  old   children  (p<0.05).  the  root  canal   measurement  average  of  the  con-­‐ ventional  method  was  found  sig-­‐ nificantly  higher  than  the  apex   locator  (p:0.001)  and  rvg   (p:0.032)  for  10  years  old  children   (p  <0.01;  p<0.05).  the  root  canal   measurement  average  of  the  sem   method  was  found  significantly   higher  than  the  apex  locator  (p:   0.002)  (p  <0.01)  for  10  years  old   children  (table  3).   there  is  a  statistically  sig-­‐ nificant  difference  between  the   root  canal  averages  of  the  meth-­‐ ods  according  to  upper  and  lower   jaw  (p<0.01)  (table  5).  the  root   canal  measurement  average  of  the   sem  method  was  found  to  be  sig-­‐ nificantly  lower  than  the  apex   locator  (p:  0.004),  conventional   method  (p:  0.005)  and  rvg  in  the   upper  jaw  (p:  0.036)  (p  <0.05;   p<0.01).  the  root  canal  measure-­‐ ment  average  of  the  conventional   method  was  found  significantly   higher  than  the  apex  locator  (p:   0.001),  rvg  (p:  0.001)  and  sem  (p:   0.001)  measurements  in  the  lower   jaw  (p  <0.01)  (table  6).   discussion   one   of  the  most   significant   steps  to  get   successful   results  in   root  canal   treatment  is   to  determine   the  length  of  root  canal  accurate-­‐ ly.  root  resorption  generally   makes  apex  of  root  canals  in  pri-­‐ mary  teeth  ambigious.     in  determination  of  length   in  a  clinical  setting,  the  only  prac-­‐ tice  that  is  approved  globally,  ac-­‐ cessible  and  trustworthy  is  radiog-­‐ raphy.  however,  repeated  radio-­‐ graphic  exposure  of  pediatric  pa-­‐ tients  before,  during,  and  immedi-­‐ ately  after  the  endodontic  treat-­‐ ment  may  cause  high  concerns.   nevertheless  radiography  can  give   misleading  results  in  resorption   [5].   some  studies  have  report-­‐ ed  that  while  measuring  mean   working  length,  both  digital  radi-­‐ ography  and  apex  locator  demon-­‐ strated  almost  similar  results  with   the  conventional  radiographic   method  in  primary  molars  [5,  22].   likewise,  krishnan  and  sreedharan   study  how  precisely  electronic   apex  locators  and  conventional   radiographic  technique  determine   the  root  canal  length  of  primary   teeth  by  comparing  with  the  ana-­‐ tomic  root  canal  length  of  these   teeth.  the  results  of  this  study   from  conventional  radiography   and  electronic  apex  locator  were   similar;  intra‑class  correlation  co-­‐ efficient  also  showed  that  both    comparison  of  root  canal  length  measurement  methods  in  primary  teeth     vol  6,  no  1  (2018)        doi  10.5195/d3000.2018.83    http://dentistry3000.pitt.edu   the  radiographic  and  electronic   methods  are  reliable  in  determin-­‐ ing  the  root  canal  length  and  there   is  no  need  to  use  them  together   [23].   in  the  study  of  subrama-­‐ niam  et  al,  measured  root  canal   lengths  in  primary  teeth  obtained   from  four  different  techniques  are   compared.  the  conclusion  of  the   study  is  that  reliable  and  precise   root  canal  length  assessment  in   primary  teeth  is  possible  by  the   digital  radiographic  and  apex  loca-­‐ tor  technology.  moreover,  these   methods  increase  both  the  safety   of  endodontic  treatment  and  com-­‐ fort  in  children  [24].   in  one  laboratory  study,   mello-­‐moura  et  al  compared  the   accuracy  of  four  different  methods   in  determining  root  canal  length.   the  study  demonstrated  that   among  all  the  methods  the  elec-­‐ tronic  apex  locators  provided  the   most  accurate  root  canal  length   results,  however  in  case  electronic   resources  are  unavailable  the   combination  of  radiographic  and   tactile  sense  methods  might  be  an   alternative  as  the  results  were  sat-­‐ isfactory  [25].   the  existence  of  root  re-­‐ sorption  is  an  important  charac-­‐ teristic  of  pulpec-­‐ tomy  in  primary   teeth.  it  is  hard   to  radiologically   asses  the  small   areas  of  resorp-­‐ tion  especially  if   the  resorption  is   on  buccal/lingual   aspects  of  the   root.  to  discard  the  disadvantages   of  radiographic  assessment  in  the-­‐ se  cases,  electrical  root  length  de-­‐ termination  may  be  used.     mente  et  al.  evaluated  the   accuracy  of  an  electronic  apex  lo-­‐ cater  device  in  initial  resorption  of   primary  incisors  and  concluded   that  whether  there  was  resorption   in  primary  teeth  did  not  affect   how  accurate  the  electrical  meas-­‐ urement  of  root  canal  length  in-­‐ vitro  [26].  another  study  compar-­‐ ing  two  different  root  canal  meas-­‐ uring  device  in  primary  teeth  with   and  without  resorption  concluded   that  these  devices  did  not  provide   reliable  data.  however,  these  apex   locators  may  be  useful  in  primary   root  canal  therapy  in  case  other   diagnostic  measures  support   them.  additional  in  vivo  assess-­‐ ments  of  them  in  primary  teeth   need  to  be  performed  [27].   apex  locator  was  more   likely  to  miscalculate  root  length   in  primary  molars  with  root  re-­‐ sorption  than  direct  canal  meas-­‐ urement,  yet  root  zx  (morita,   usa)  type  apex  locator  calculated   accurately  in  cases  in  which  root   resorption  was  less  than  one  third   of  root  length  in  primary  molar   teeth  [28].  in  the  study  of  bel-­‐ trame  et  al,  the  root  zx  apex  loca-­‐ tor  was  studied  in  primary  molar   teeth  (with  or  without  root  re-­‐ sorption)  to  analyze  its  in  vivo  and   ex  vivo  accuracy.  as  a  conclusion,   it  was  not  significant  that  whether   there  was  root  resorption  or  not,   as  in  both  cases  the  root  zx  apex   locator  measured  the  root  canal   working  length  ±1  mm  in  primary   molars,  in  vivo  and  ex  vivo  [29].     this  conclusion  backs  up  other  ex   vivo  studies  as  its  results  con-­‐ firmed  that  electronic  apex  loca-­‐ tors  can  be  used  to  measure  canal   length  in  primary  teeth  [25,  50].  in   addition  to  that,  bahrololoomi  et   al.  showed  that  the  accuracy  of   root  zx  electronic  apex  locator   was  high  in  the  primary  anterior   teeth  despite  root  resorption.   therefore,  using  this  device  as  an   adjunct  is  recommended  for  root   canal  length  measurements  in   primary  anterior  teeth  [4].   leonardo  et  al.  evaluated   ex  vivo  accuracy  of  two  different   root  canal  measuring  devices  in   measuring  root  canal  length  of   primary  incisors  and  molar  teeth   that  have  physiological  root  re-­‐ sorption.  they  concluded  that   electronic  apex  locators  are  effec-­‐ tive  and  correct  when  root  canal   length  of  primary  incisor  or  prima-­‐ ry  molar  teeth  (whether  or  not   they  have  root  resorption)  are  de-­‐ termined  because  of  the  results  of   the  electronic  ones  were  almost   perfectly  parallel  to  the  actual  root   canal  length  measurements  [30].    comparison  of  root  canal  length  measurement  methods  in  primary  teeth     vol  6,  no  1  (2018)        doi  10.5195/d3000.2018.83    http://dentistry3000.pitt.edu   in  addition  to  clinical  and   laboratory  studies  in  primary   teeth,  studies  have  been  carried   out  on  permanent  teeth  and  dif-­‐ ferent  results  have  been  obtained.   esmaeili  et  al.  aimed  to  compare   the  accuracy  of  digital  and  conven-­‐ tional  radiographic  techniques  in   the  assessment  of  the  endodontic   working  length  in  premolar  teeth   and  concluded  that  the  accuracy   of  digital  and  conventional  radiog-­‐ raphy  techniques  were  similar  in   determination  of  working  length   [31].  according  to  another  study   findings  showed  that  although   there  was  a  significant  difference   between  experimental  method   and  actual  working  length  but   electronic  apex  locator  showed   the  most  accurate  reading  when   compared  to  actual  working   length  in  premolar  teeth  [32].   orosco  et  al  compared   conventional  and  digital  radio-­‐ graphic  methods  in  measurement   of  the  root  canal  working  length  of   maxillary  incisor  or  canine  teeth.   they  concluded  that  the  root  ca-­‐ nal  working  length  measured  with   conventional  radiographic  method   were  more  accurate  than  the  ones   obtained  with  the  digital  radio-­‐ graphic  method  [15].  diwanji  et  al.   compared  radiographic  method   and  electronic  method  with  apex   locator  in  determining  exact  work-­‐ ing  length  of  young  permanent   root  canal.  then,  the  results  from   these  methods  were  compared  to   the  actual  length  from  histological   sections  under  stereomicroscope.   in  the  light  of  these  comparisons,   apex  locator  was  decided  to  be   more  reliable  and  precise  than  the   digital  radiography  [33].   mittal  et  al.  compared  the   accuracy  of  digital  radiograph  ex-­‐ posed  with  paralleling  technique,   6th  generation  apex  locator,  tac-­‐ tile  method,  paper  point  method,   combined  electronic  and  radio-­‐ graphic  working  length  and  com-­‐ bined  electronic,  tactile  and  paper   point  working  length  method  to   determine  the  working  length  in   permanent  teeth  with  open  apex.   in  the  end  they  found  that  a  com-­‐ bination  of  electronic  tactile  and   paper  point  methods  in  open  apex   increases  the  accuracy  of  working   length  determination  [34].   martins  et  al.  published  a   review  of  compared  the  radio-­‐ graphic  apex  locators  and  radio-­‐ graphs  in  permanent  dentition.   they  reported  that  electronic  apex   locators  were  mostly  more  useful     than  radiography  when  there  is  a   respective  anatomic  reference   point  (for  example,  the  apical  con-­‐ striction  or  apical  foramen)  and   when  radiography  was  only  meth-­‐ od  in  determining  working  length,   most  of  the  times  more  radio-­‐ graphs  were  needed  than  using   electronic  apex  locator.  this  study   shows  that  in  primary  teeth  (with   and  without  initial  root  resorption)   interexaminer  reproducibility  of   electrical  assessment  of  root  canal   length  in  vitro  was  high.  in  com-­‐ parison  with  the  radiographic  re-­‐ sults,  the  accuracy  of  the  working   length  was  higher  [35].   this  study  evaluates  the   accuracy  of  conventional  radiog-­‐ raphy,  intraoral  digital  radiovisiog-­‐ raphy  and  electronic  apex  locator   in  determining  the  working  length   of  root  canals  in  primary  teeth  (in   vivo)  and  to  compare  the  results   with  scanning  electron  microscopy   measurements  (ex  vivo).  the  dif-­‐ ference  of  this  study  from  the   figure  1.  root  canal  length  measurement  for  three  methods.    comparison  of  root  canal  length  measurement  methods  in  primary  teeth     vol  6,  no  1  (2018)        doi  10.5195/d3000.2018.83    http://dentistry3000.pitt.edu   other  studies  is  that  there  is  no   study  which  is  compared  with  sem   in  the  literature.  in  addition,  the   findings  of  comparison  of  methods   according  to  age  and  jaws  were   not  found  in  other  literature.   besides  different  results   are  obtained  according  to  the   methodological  differences  in  the   research  it  can  be  concluded  that   electronic  apex  locators  provide   an  acceptable  level  of  accuracy  in   determining  root  canal  length  in   primary  teeth.  in  this  study  the   accuracy  of  apex  locator  and  rvg   were  higher  than  conventional   radiography  in  determining  the   working  length  in  primary  teeth.     conclusion   the  results  confirm  that   electronic  apex  locaters  can  accu-­‐ rately  determine  the  root  canal   length  in  primary  teeth.   acknowledgements   we  would  like  to  thank  the   dt.  pelin  barlak  for  his  contribu-­‐ tion  to  the  research.   references   1. american  academy  of  pe-­‐ diatric  dentistry  clinical  af-­‐ fairs  committee-­‐-­‐pulp   therapy  subcommit-­‐ tee;  american  academy  of   pediatric  dentistry  council   on  clinical  affairs  (2008-­‐ 2009)  guideline  on  pulp   therapy  for  primary  and   immature  permanent   teeth.  pediatr   dent    30(7):170-­‐4.   2.  rodd  hd,  waterhouse   pj,  fuks  ab,  fayle   sa,  moffat  ma;  british  so-­‐ ciety  of  paediatric  dentis-­‐ try  (2006)  pulp  therapy  for   primary  molars.  int  j  pae-­‐ diatr  dent  16(1):15-­‐23.   3. carotte  p  (2005)  endodon-­‐ tic  treatment  for  children.   british  dental  journal   198(1):9-­‐15.   4. bahrololoomil  z,  soleymani   aa,  modaresi  j,  imanian  m,   lotfian  m  (2015)  accuracy   of  an  electronic  apex  loca-­‐ tor  for  working  length  de-­‐ termination  in  primary  an-­‐ terior  teeth.  jornal  of  den-­‐ tistry,  tehran  university  of   medical  sciences   12(4):243-­‐8.   5. oznurhan  f,  unal  m,   kapdan  a,  ozturk  c,  aksoy   s  (2015)  clinical  evaluation   of  apex  locator  and  radiog-­‐ raphy  in  primary  teeth.  int   j  paediatr  dent  25:199-­‐ 203.   6.  jerrel  rg,  ronk  sl  (1982)   develeopmental  arrest  of   succedaneous  tooth  fol-­‐ lowing  pulpectomy  in  a   primary  tooth.  j  pedod   6(4):337-­‐42.   7. ahmed  hma  (2013)  ana-­‐ tomical  challenges,  elec-­‐ tronic  working  length  de-­‐ termination  and  current   developments  in  root  canal   preparation  of  primary  mo-­‐ lar  teeth.  int  endod  j   46:1011-­‐1022.   8. chawla  hs,  setia  s,  gupta   n,  gauba  k,  goyal  a  (2008)   evaluation  of  a  mixture  of   zinc  oxide,  calcium  hydrox-­‐ ide,  and  sodium  fluoride  as   a  new  root  canal  filling  ma-­‐ terial  for  primary  teeth.  j   indian  soc  pedod  prev   dent  26(2):53-­‐8.   9. ricucci  d,  langeland  k   (1998)  apical  limit  of  root   canal  instrumentation  and   obturation,  part  2.  a  histo-­‐ logical  study.  int  endod  j   31:394–409.   10. burch  jg,  hulen  s  (1972)   the  relationship  of  the  api-­‐ cal  foramen  to  the  anatom-­‐ ic  apex  of  the  tooth  root.   oral  surg  oral  med  oral   pathol  34:262–268.   11. kuttler  y  (1958)  a  precision   and  biologic  root  canal  fill-­‐ ing  technic.  j  am  dent  as-­‐ soc  56:  38–50.   12. katz  a,  tamse  a,  kaufman   ay  (1991)  tooth  length  de-­‐ termination:  a  review.  oral   surg  oral  med  oral  pathol   72:238–242.   13. morfis  a,  sylaras  sn,  geor-­‐ gopoulou  m,  kernani  m,   prountzos  f  (1994)  study   of  the  apices  of  human   permanent  teeth  with  the   use  of  a  scanning  electron   microscope.  oral  surg  oral   med  oral  pathol  77:172– 176.   14. stein  tj,  corcoran  jf  (1992)   radiographic  ‘working   length’  revisited.  oral  surg    comparison  of  root  canal  length  measurement  methods  in  primary  teeth     vol  6,  no  1  (2018)        doi  10.5195/d3000.2018.83    http://dentistry3000.pitt.edu   oral  med  oral  pathol   74:796–800.   15. orosco  fa,  bernardineli  n,   garcia  rb,  bramante  cm,   duarte  mah,  moraes  ig   (2012)  in  vivo  accuracy  of   conventional  and  digital   radiographic  methods  in   confirming  root  canal   working  length  determina-­‐ tion  by  root  zx.  j  appl  oral   sci  20(5):522-­‐525.   16. ellingsen  ma,  hollender   lg,  harrington  gw  (1995)   radiovisiography  versus   conventional  radiography   for  detection  of  small  in-­‐ struments  in  endodontic   length  determination.  ii.  in   vivo  evaluation.  j  endod   21(10):516-­‐20.   17. shearer  ac,  horner  k,  wil-­‐ son  nh   (1990)  radiovisiography   for  imaging  root  canals:  an   in  vitro  comparison  with   conventional  radiog-­‐ raphy.  quintessence  int   21(10):789-­‐94.   18. kobayashi  c.  electronic  ca-­‐ nal  length  measurement   (1995)  oral  surg  oral  med   oral  pathol  oral  radiol  en-­‐ dod  79:226–231.   19. alves  am,  felippe  mc,  fe-­‐ lippe  wt,  rocha  mj  (2005)   ex  vivo  evaluation  of  the   capacity  of  the  tri  auto  zx   to  locate  the  apical  fora-­‐ men  during  root  canal  re-­‐ treatment.  int  endod  j   38:718–724.   20. ebrahim  ak,  wadachgi  r,   suda  h  (2007)  electronic   apex  locators-­‐review.  j   med  dent  sci    54:125–136.   21. goldberg  f,  marroquin  bb,   frajlich  s,  dreyer  c  (2005)   in  vitro  evaluation  of  the   ability  of  three  apex  loca-­‐ tors  to  determine  the   working  length  during  re-­‐ treatment.  j  endod   31:676–678.   22. neena  ie,  ananthraj  a,   praveen  p,  karthik  v,  rani   p  (2011)  comparison  of   digital  radiography  and   apex  locator  with  the  con-­‐ ventional  method  in  root   length  determination  of   primary  teeth.  j  indian  soc   pedod  prev  dent   29(4):300-­‐304.   23. krishnan  is,  sreedharan  s   (2012)  a  comparative  eval-­‐ uation  of  electronic  and   radiographic  determination   of  root  canal  length  in  pri-­‐ mary  teeth:  an  in  vitro   study.  contemporary  clini-­‐ cal  dentistry  3(4):416-­‐420.     24. subramaniam  p,  konde  s,   mandanna  dk  (2005).  an  in   vitro  comparison  of  root   canal  measurement  in  pri-­‐ mary  teeth.  j  indian  soc   pedod  prev  dent   23(3):124-­‐125.   25. mello-­‐moura  acv,  moura-­‐ netto  c,  araki  at,  guedes-­‐   pinto  ac,  mendes  fm   (2010)  ex  vivo  performance   of  five  methods  for  root   canal  length  determination   in  primary  anterior  teeth.   int  endod  j  43:142–147.   26. mente  j,  seidel  j,  buchalla   w,  koch  mj  (2002)  elec-­‐ tronic  determination  of   root  canal  length  in  prima-­‐ ry  teeth  with  and  without   root  resorption.  int  endod  j   35:447–452.   27. bodur  h,  odabaş  m,  tulu-­‐ noglu  o,  tinaz  ac  (2008).   accuracy  of  two  different   apex  locators  in  primary   teeth  with  and  without   root  resorption.  clin  oral   invest  12:137-­‐141.     28. angwaravong  o,  panitvisai   p  (2009)  accuracy  of  an   electronic  apex  locator  in   primary  teeth  with  root  re-­‐ sorption.  int  endod  j   42:115-­‐121.   29. beltrame  apca,  triches  tc,   sartori  n,  bolan  m  (2011)   electronic  determination  of   root  canal  working  length   in  primary  molar  teeth:  an   in  vivo  and  ex  vivo  study.   int  endod  j  44:402–406.   30. leonardo  mr,  silva  lab,   nelson-­‐filho  p,  silva  rab,   raffaini  msgg  (2008)  ex   vivo  evaluation  of  the  ac-­‐ curacy  of  two  electronic   apex  locators  during  root   canal  length  determination   in  primary  teeth.  int  endod   j  41:317–321.   31. esmaeili  f,  akbari  fa,   zarandi  a  (2016)  compari-­‐ son  of  accuracy  of  digital   and  conventional  radiog-­‐  comparison  of  root  canal  length  measurement  methods  in  primary  teeth     vol  6,  no  1  (2018)        doi  10.5195/d3000.2018.83    http://dentistry3000.pitt.edu   raphies  in  determining  en-­‐ dodontic  working  length.   sadj  71(9):  395-­‐397.     32. mittal  r,  singla  mg,  sood   a,  singla  a  (2015)  compar-­‐ ative  evaluation  of  working   length  determination  by   using  conventional  radiog-­‐ raphy,  digital  radiography   and  electronic  apex  loca-­‐ tor.  journal  of  restorative   dentistry  3(3):70-­‐75.   33. diwanji  a,  rathore  as,  aro-­‐ ra  r,  dhar  v,  madhusudan   a,  doshi  j  (2014)  working   length  determination  of   root  canal  of  young  per-­‐ manent  tooth:  an  invitro   study.  annals  of  medical   and  health  sciences  re-­‐ search  4(4):554-­‐558   34. mittal  p,  jadhav  gr,  logani   a  (2016)  accuracy  of  dif-­‐ ferent  methods  to  deter-­‐ mine  working  length  in   teeth  with  open  apex-­‐an  ex   vivo  comparative  study.  j   dent  specialities  4(1):39-­‐ 45.   35. martins  jnr,  marques  m,   mata  a,  carames  j  (2014)   clinical  efficacy  of  electron-­‐ ic  apex  locators:  systematic   review.  j  endod  40(6):759– 77.     microsoft word 75 2017.docx   vol  5,  no  1  (2017)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2017.75           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     familial  inheritance  of  tmd:  a  case  report   maria  genello1      1university  of  pi.sburgh,  school  of  dental  medicine abstract   the  tmj  is  a  joint  that  can  perform  both  hinge  and  sliding  mo'ons  and  whose  proper  func'oning  depends  on  the  ac-­‐ !ons  of  the  joint  and  joint  capsule,  the  muscles  of  mas!ca!on,  and  the  condi!on  and  ac&on  of  the  mandibular  con-­‐ dyle.   if  any  of  these  components  deviate  from  their  normal  structure  or  func0on,  then  tmd  can  result.  there  have   been  many  reported  contribu"ng  factors  to  tmd  development,  many  of  which  include  environmental  influences  such   as  bruxism,  trauma,  and  other  oral  habits.  however,  a  gene7c  component  can  also  play  a  role.  this  paper  reports  a   case  of  tmd  occurring  within  three  consecu3ve  genera3ons  of  a  family  and  explores  the  possibility  of  this  disorder   exhibi!ng  familial  inheritance.  members  of  the  family  affected  all  displayed  manifesta'ons  of  joint  laxity  in  other  are-­‐ as   of   the   body,  migraines,   pain,   and   had   smaller   craniofacial   dimensions  with   a   narrower  mandible.   these   factors   were  found  to  have  a  gene/c  influence  and  these  genes  can  also  be  -ed  to  tmd,  thus  suppor-ng  the  argument  that   the  cases  of  tmd  seen  in  this  family  are  in  fact  due  to  inheritance.  if  tmd  can  be  shown  to  have  a  gene;c  component   and  be   inherited,   then  dental  prac//oners  would  be  able  to   iden/fy  high  risk  pa,ents  and  help  to  modify  environ-­‐ mental  factors  early  on  in  order  to  help  prevent  the  onset  of  tmd  in  those  individuals.   cita%on:  genello,  m.  (2017)  familial  inheritance   of  tmd:  a  case  report.  den$stry  3000.  1:a001   doi:10.5195/d3000.2017.75   received:    may  15,  2017   accepted:    may  26,  2017   published:    june  16,  2017   copyright:   ©2017   genello,   m.   this   is   an   open   access   ar!cle   licensed   under   a   crea!ve   com-­‐ mons   a"ribu!on   work   4.0   united   states   li-­‐ cense.   email:  mkg35@pi).edu   introduction   the  tmj  is  a  unique  bilateral   joint  that  connects  the  mandible  to   the  temporal  bone  [1].  it  performs   both  hinge  and  sliding  motions  which   allows  a  person  to  talk,  chew,  yawn,   and  open  the  mouth.  some  of  the   component  parts  of  the  tmj  include   the  mandibular  condyle,  the  glenoid   fossa  of  the  temporal  bone,  the  artic-­‐ ular  eminence  which  the  condyle   slides  down,  the  articulating  disc  be-­‐ tween  the  condyle  and  temporal   bone,  and  the  joint  capsule.  during   function,  the  condyle  glides  forward   from  the  fossa  and  down  the  articular   eminence.  the  disc  remains  between   the  condyle  and  these  other  elements   in  order  to  act  as  a  shock  absorber   [1].  a  disorder  of  the  tmj,  termed   tmd,  can  affect  one  or  both  of  the   joints.  manifestations  of  tmd  in-­‐ clude,  but  are  not  limited  to,  pain  in   the  face,  limited  movement  and  stiff-­‐ ness  of  the  jaw,  locking  of  the  jaw,   and  popping  or  clicking  of  the  joint   upon  opening  [1].  the  cause  of  tmd   has  been  attributed  to  many  different   factors,  a  large  portion  being  envi-­‐ ronmental.  these  include  trauma,   bruxism,  and  other  oral  habits.  a  ge-­‐ netic  component  is  being  explored,   and  many  different  genes  have  been   proposed  as  possible  candidates.     case  report   three  generations  of  a  family   from  northeastern  pennsylvania  were   analyzed  for  tmd.  the  disorder  ap-­‐ peared  in  each  generation,  and  ap-­‐ peared  to  affect  predominately  the   females  in  the  family.  in  generation   iii,  two  males  appear  to  be  affected.   all  of  those  that  report  having   manifestations  of  tmd  have  the   common  feature  of  clicking  and   popping  upon  jaw  opening.  in   generation  one,  (will  refer  to  as   “female  a”),  the  affected  female  a   reports  having  symptoms  of  pop-­‐ ping  and  clicking,  along  with  mild   hip  and  knee  joint  laxity.  she  also   reports  mild  intermittent  jaw  pain   and  headaches.         in  generation  two,   three  females  (females  b,  c,  and  d   respectively)  are  affected  out  of   four,  and  50%  of  the  offspring  are   affected.  the  affected  females  all   report  jaw  clicking  and  popping   upon  opening,  with  female  b  re-­‐ porting  mild  intermittent  head-­‐ aches  and  occasional,  mild  jaw   pain.  female  c  reports  clicking  and   popping  as  well.  she  suffers  from   migraine  headaches  that  occur   every  few  months.  she  has  laxity   in  her  left  knee  joint,  and  reports   frequent  jaw  pain  that  oftentimes   is  severe.  her  jaw  mobility  fre-­‐ quently  becomes  restricted,  caus-­‐ ing  her  to  have  limited  opening.   female  d  reports  clicking  and   popping,  along  with  mild  jaw  dis-­‐ location  when  she  opens  wider   than  her  normal  functional  range   (for  speaking,  chewing,  etc).  she   familial  inheritance  of  tmd:  a  case  report   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.75    http://dentistry3000.pitt.edu   exhibits  mild  intermittent  jaw   pain.  this  same  female  d  also  re-­‐ ports  laxity  in  her  intervertebral   discs,  and  has  suffered  two   “slipped”  discs.  she  also  reports   more  severe  intermittent  head-­‐ aches.       in  generation  three,   four  members  are  affected,  two  of   which  are  males.  male  a  is  the  off-­‐ spring  of  female  b,  and  presents   with  only  occasional  mild  clicking   and  popping  upon  opening.  male   b  is  the  offspring  of  female  c,  and   reports  only  occasional  clicking   and  popping  that  is  mild.  female   e,  who  is  the  offspring  of  female  c,   presents  with  clicking  and  popping   when  opening  the  jaw,  along  with   severe  left  shoulder  joint  laxity.   she  also  reports  occasional  mod-­‐ erate  headaches  and  mild,  inter-­‐ mittent  jaw  pain.  female  f,  who  is   the  offspring  of  female  d,  reports   clicking  and  popping,  along  with   occasional  migraine  headaches   and  intermittent,  more  moderate   jaw  pain.  when  opening  past  func-­‐ tional  limits,  the  jaw  often  be-­‐ comes  dislocated.  she  also  exhib-­‐ its  laxity  to  her  intervertebral  discs   and  has  suffered  a  herniation,   along  with  laxity  to  her  hip  joint.     of  the  affected  individuals,   all  but  male  b  have  smaller  vertical   and  horizontal  craniofacial  meas-­‐ urements,  with  a  narrow  mandi-­‐ ble.  male  b  exhibits  a  wider,  less   constricted  mandible.       discussion   tmd  proves  to  be  a  disor-­‐ der  of  variable  expression  and  has   many  different  manifestations.   many  cases  of  tmd  have  a  strong   environmental  component,  how-­‐ ever,  genetic  elements  to  the  dis-­‐ ease  are  being  studied  and  sug-­‐ gested.  the  tmd  evident  in  this   family  appears  to  have  a  genetic   influence.    when  you  look  at  fig-­‐ ure  1,  one  sees  how  tmd  appears   to  be  present  in  all  three  genera-­‐ tions.  mainly  females  are  affected,   with  the  ratio  being  6:2  females  to   males.  this  is  expected,  since  it  is   found  that  females,  especially   those  of  child  bearing  age,  are   more  susceptible  to  developing   tmd  and  have  more  severe  symp-­‐ toms  due  to  hormonal  differences   [2].     if  we  were  to  propose  the   most  common  mode  of  inher-­‐ itance  for  tmd,  it  would  most  like-­‐ ly  be  polygenic  inheritance  with   environmental  influences.  since   the  tmj  function  depends  on  a   multitude  of  factors  such  as  masti-­‐ catory  musculature,  articular  disc   formation  and  health,  integrity  of   the  joint  capsule,  shape  and  angle   of  the  condyle,  mandibular  size   and  development,  shape  of  the   glenoid  fossa,  integrity  of  the  car-­‐ tilage,  etc.,  any  variation  in  these   or  other  structures  that  influence   its  function  can  result  in  a  devia-­‐ tion  from  normal  and  lead  to  tmd.   since  these  traits  are  all  controlled   by  many  and  different  genes  work-­‐ ing  together  in  order  to  achieve   proper  development  and  function,   it  would  be  safe  to  say  that  any   tmd  with  a  strong  genetic  com-­‐ ponent  would  be  polygenic,  mean-­‐ ing  there  would  be  variations  in   multiple  genes  that  influence  the   structures  relevant  to  the  tmj.   this  can  also  account  for  the  vari-­‐ able  expression  of  tmd.  since   many  different  genes  can  have   variations  that  lead  to  tmd,  not   just  a  single  gene,  and  different   environmental  factors,  and  not   just  a  single  environmental  factor,   can  aid  in  the  development  of   tmd,  then  the  expression  seen  in   the  population  of  tmd  would  be   variable.     in  this  family’s  case,  there-­‐ fore,  the  inheritance  is  most  likely   polygenic.  those  affected  appear   to  have  a  myriad  of  symptoms   that  deal  with  different  aspects  of   the  tmj,  and  the  expression  of   tmd  within  the  family  isn’t  com-­‐ pletely  uniform,  but  is  rather   somewhat  variable,  leading  us  to   believe  in  the  polygenic  theory.   now,  if  we  are  speaking  in  terms   of  autosomal  dominance  and  re-­‐ cessiveness,  or  x-­‐linked  domi   figure  1.  pedigree  of  the  affected  family.  blue  color  indicates  those  that  have  any  symptoms  pertaining  to   tmd,  whether  they  be  mild,  moderate,  or  severe.  letters  are  used  as  identifiers  when  discussing  the  case.     familial  inheritance  of  tmd:  a  case  report   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.75    http://dentistry3000.pitt.edu   nance  and  recessiveness,  it   is  most  likely  an  autosomal  domi-­‐ nant  inheritance  form.  it  appears   in  every  generation,  ruling  out  a   recessive  inheritance  pattern,  and   it  affects  both  males  and  females,   with  the  females  at  a  higher  in-­‐ stance,  leading  one  to  believe  that   it  is  not  x-­‐linked.       now  that  we  have  estab-­‐ lished  that  it  is  likely  polygenic   with  an  autosomal  dominant  in-­‐ heritance  pattern,  we  must  make   the  case  that  it  is  in  fact  genetic   variations  that  exist  in  this  family   that  are  causing  these  clinical   manifestations  of  tmd,  and  these   variations  are  being  passed  from   one  generation  to  the  next.  the   genetics  behind  pain  perception   and  chronic  pain  have  become  an   area  of  interest  with  regards  to   tmd.  a  few  candidate  genes  have   been  highlighted,  which  includes   the  serotonin  5-­‐ht  transporter   gene  and  the  comt  gene,  whose   activity  is  inversely  correlated  to   pain  sensitivity  and  the  develop-­‐ ment  of  tmd.  it  was  found  that   nearly  a  third  of  new  tmd  cases   reported  could  be  attributed  to   having  a  variation  in  the  comt   gene  [2].  in  our  subjects  here,  we   saw  that  pain  was  a  common  fea-­‐ ture  in  all  three  generations,  rang-­‐ ing  from  mild  to  severe.  it  is  im-­‐ portant  to  note  that  those  with   more  manifestations  of  the  disor-­‐ der,  such  as  female  c  and  female   f,  experienced  more  severe  pain   than  the  others  that  were  affect-­‐ ed,  indicating  a  possible  correla-­‐ tion  between  pain  sensitivity  and   tmd  severity.     another  important  finding   in  this  family  and  that  deals  with   chronic  pain  was  the  presence  of   headaches.  numerous  studies   have  been  done  on  the  association   of  certain  genes  and  headaches.   through  these  studies,  it  was   found  that  some  of  these  genes   not  only  predisposed  a  person  to   headaches  and  migraines,  but  to   tmd  as  well.  comt  was  again  part   of  these  studies.  comt  genetic   variations  were  involved  with  both   migraines  and  tmd.  drd4  was   another  gene  that  is  involved  with   neurotransmission  and  that  was   associated  with  both  the  presence   of  headaches  and  the  manifesta-­‐ tion  of  tmd  [3].  another  gene  of   interest  was  the  esr1  gene,  which   variations  lead  to  the  presence  of   chronic  headaches  and  was  also   associated  with  tmd  manifesta-­‐ tions.  studies  show  that  up  to  50%   of  migraine  and  chronic  headache   etiology  was  due  to  genetic  vari-­‐ ants  [3].  headaches  were  present   in  all  female  cases  in  this  family,   and  ranged  from  mild  to  severe.   the  presence  of  this  shared  trait  in   all  of  the  affected  female  individu-­‐ als  could  be  due  to  genetic  varia-­‐ tion,  specifically  a  gene  that  not   only  deals  with  the  presence  of   headaches,  but  can  also  be  associ-­‐ ated  with  tmd  (like  comt,  drd4,   and  esr1),  which  would  explain   the  concurrent  expression  of  both   tmd  and  chronic  headaches  in  all   of  the  affected  female  individuals.     table  1.  summary  of  clinical  findings.   affected   member   m/f   clicking/popping   headaches   (mild,   mod.,   severe)   systemic  joint   laxity/location   pain   (mild,   mod.,   severe)   movement   restrictions   a   female   yes   yes;  mild   yes;  hip  and   knee   yes;  mild   no   b   female   yes   yes;  mild   no   yes;  mild   no   c   female   yes   yes;  se-­‐ vere   yes;  knee   yes;   severe   yes   d   female   yes   yes;  se-­‐ vere   yes;  iv  discs   yes;  mild   yes   a   male   yes;  mild  and   occasional   no   no   no   no   b   male   yes;  mild  and   occasional   no   no   no   no   e   female   yes   yes;  mod-­‐ erate   yes;  shoulder   yes;  mild   no   f   female   yes   yes;  se-­‐ vere   yes;  hip  and   iv  discs   yes;   moderate   yes   familial  inheritance  of  tmd:  a  case  report   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.75    http://dentistry3000.pitt.edu   a  trait  that  many  of  the  af-­‐ fected  individuals  of  this  family   shared  was  that  of  systemic  joint   laxity.  joints  affected  ranged  from   the  knee  and  hip  joints,  to  the   shoulder  joint  and  intervertebral   discs.  systemic  laxity  of  joints  has   a  genetic  component.  there  are   certain  genes  that  control  the   amount  and  type  of  collagen  pro-­‐ duced  in  the  body.  skin  biopsies  of   patients  with  joint  laxity  found   them  to  have  less  overall  collagen   and  a  higher  ratio  of  type  iii  colla-­‐ gen  to  type  iii+i  [4].  those  with   systemic  joint  laxity  and  hypermo-­‐ bility  had  a  higher  incidence  of   tmd.    this  is  due  to  the  fact  that  if   the  joints  in  the  body  are  more   mobile  and  lax,  then  the  capsule   of  the  tmj  would  also  be  lax,  mak-­‐ ing  the  disc  vulnerable  to  dis-­‐ placement  and  the  condyle  prone   to  hypermobility  [5].  so,  a  genetic   component  can  be  involved  in  the   joint  laxity  and  subsequent  tmd   seen  in  each  generation  of  this   family.   another  important  feature   to  note  in  this  particular  family   was  the  smaller  craniofacial  di-­‐ mensions  and  narrower  mandible.   all  of  the  affected  individuals  but   one  have  a  narrower  and  smaller   face.  a  high  score  on  the   helkimo’s  clinical  dysfunction  in-­‐ dex,  which  is  a  tool  used  to  assess   the  presence  and  severity  of  tmd,   was  found  in  patients  with  smaller   vertical  and  horizontal  facial  di-­‐ mensions  [6].  facial  development   and  size  is  under  strict  genetic   control.  any  variation  in  these  de-­‐ velopmental  genes  can  cause  vari-­‐ ations  in  the  size  and  shape  of  the   craniofacial  bones  and  their  rela-­‐ tionship  to  each  other,  which  can   lead  to  a  tmd.  the  variations  in   the  gene  pool  of  this  family  for  the   size  and  width  of  the  craniofacial   bones  could  be  a  factor  in  their   shared  tmd.   conclusion   the  genetic  component  of   tmd,  while  not  as  extensively  ex-­‐ plored  as  the  environmental  as-­‐ pect,  presents  with  some  compel-­‐ ling  support.  many  candidate   genes  have  been  identified  to  be   associated  with  the  development   of  tmd.  the  family  involved  in  this   case  report  saw  tmd  reported  in   three  consecutive  generations.   there  was  some  variation  in  the   expression  of  the  disorder,  but   most  of  those  affected  had  clicking   and  popping  of  the  joint,  along   with  systemic  joint  laxity,  head-­‐ aches,  and  pain.  smaller  craniofa-­‐ cial  dimensions  were  also  a  com-­‐ mon  feature  seen.  all  of  these  fea-­‐ tures  have  a  genetic  component   that  can  also  be  tied  to  tmd.   therefore,  the  conclusion  is  that   the  tmd  seen  in  three  consecutive   generations  of  this  family  is  the   result  of  genetic  variation  and  is   inherited.  furthermore,  by   demonstrating  a  genetic  basis  for   many  cases  of  tmd,  gene  mapping   can  be  done  in  those  families  or  in   individuals  thought  to  have  a   higher  susceptibility  for  tmd,  and   thus  analyze  the  genes  involved   and  the  risk  of  either  developing  a   tmd  or  the  risk  of  offspring  ac-­‐ quiring  tmd.  if  a  dental  practi-­‐ tioner  is  made  aware  of  a  patient’s   risk  of  tmd,  then  steps  can  be   taken  early  on  to  modify  said  pa-­‐ tient’s  environmental  factors  (such   as  reducing  bruxism,  clenching  of   teeth,  etc.)  in  an  effort  to  try  to   prevent  or  delay  the  onset  of   tmd.     references   1.  tmj  disorders.  national  insti-­‐ tute  of  dental  and  craniofacial  re-­‐ search.  [cited  march  4,  2017]   available  from   https://www.nidcr.nih.gov/oralhe alth/topics/tmj/tmjdisorders.htm# symptoms.     2.  the  many  faces  of  the  genetics   contribution  to  temporomandibu-­‐ lar  joint  disorder.  oakley  m,  vieira   ar.  orthod  craniofac  res.  2008   aug;11(3):125–135.  pmid:   18713149.   3.  genetic  predictors  of  human   chronic  pain  conditions.  zorina-­‐ lichtenwalter  k,  meloto  cb,   khoury  s,  diatchenko  l.  neurosci-­‐ ence.  2016  dec;338:36–62.  pmid:   27143481.   4.  temporomandibular  joint  dys-­‐ function  and  systemic  joint  laxity.   westling  l.  swedish  dental  journal   supplement.  1992;81:1-­‐79.  pmid:   1621231   5.  the  role  of  systemic  hypermo-­‐ bility  and  condylar  hypermobility   in  temporomandibular  joint  dys-­‐ function.  kavuncu,  v.,  sahin,  s.,   kamanli,  a.  et  al.  rheumatol  int.   2006  jan;26(3):  257-­‐60.  pmid:   15988598   6.  temporomandibular  disorders   in  relation  to  craniofacial  dimen-­‐ sions,  head  posture  and  bite  force   in  children  selected  for  orthodon-­‐ tic  treatment.  liselotte  sonnesen,   merete  bakke,  beni  solow.  eur  j   familial  inheritance  of  tmd:  a  case  report   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.75    http://dentistry3000.pitt.edu   orthod.  2001  apr;23(2):  179-­‐192.   pmid:11398555   microsoft word 225 2022.docx effect of aloe vera hydrogel on the scarring and healing of free gingival gra4: randomized controlled trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.225 h#p://den*stry3000.pi#.edu effect of aloe vera hydrogel on the scarring and healing of free gingival graft: randomized controlled trial walaa alawad1, majed othman1, jameela hsaian2, laith alsabek3 1 periodontology department, faculty of dentistry, damascus university, damascus, syria 2 pharmaceutics and pharmaceutical technology department, faculty of pharmacy, damascus university, syria 3 clinical science institute, the national university of ireland – galway, ireland abstract objective: several studies have used aloe vera hydrogel to enhance various ;ssues regenera;on to reduce scar forma;on in wounds. this study aimed to prepare a hydrogel specified for the oral cavity and observe scar forma;on and healing of free gingival gra4 a4er its applica;on compared with the natural healing and scaring of the gra4 without any material. materials and methods: this study is a randomized controlled clinical trial that included 22 pa;ents with a width of acached gingiva less than 2 mm. a4er preparing the aloe vera hydrogel and comple;ng the laboratory stage, the hydrogel was randomly applied for 11 pa;ents a4er the free gingival gra4 procedure while the other 11 pa;ents did not use any material a4er the same surgery. landry’s scale was used to assess the healing index, scarring was evaluated using mucosal scarring index. results: the healing index was higher in the interven;on group with a sta;s;cal difference p<0.05 than the control group a4er three weeks. moreover, scar forma;on in the interven;on group was less in the interven;on group with a significant difference p<0.05 a4er 1,2,3,6 months. conclusion: within the limita;ons of our study, the aloe vera hydrogel can be considered as a good topical treatment a4er oral surgery due to its ability to reduce scar forma;on and speeding up the healing in the receiving area in free gingival gra4 procedures. keywords: free gingival gra4; aloe vera; hydrogel; scarring cita8on: alawad, w et al. (2022) effect of aloe vera hydrogel on the scarring and healing of free gingival graj: randomized controlled trial den8stry 3000. 1:a001 doi:10.5195/d3000.2022.225 received: august 15, 2021 accepted: october 15, 2021 published: may 13, 2022 copyright: ©2022 alawad, w et al. this is an open access ar8cle licensed under a crea8ve commons auribu8on work 4.0 united states license. email: alsabeklaith@gmail.com introduction several procedures were proposed to increase the width of the attached gingiva such as apically repositioned flap and subepithelial connective tissue graft. however, the free gingival graft is the gold standard for obtaining an adequate amount of it around teeth and implants, 1 but free gingival graft is associated with several complications in the receiving area, such as graft shrinkage after several months and lack of color matching between the graft and the surrounded area, where the graft appears as a patch giving a result that is not satisfying for patients. 2 the traditional gingival dressings were initially used as a mechanical barrier only, which helped to prevent external factors from the wound area, but these dressings don’t affect cell activity or have any role in the biological events that occur during wound healing (review,n.d.). furthermore, these dressings do not effect of aloe vera hydrogel on the scarring and healing of free gingival gra4: randomized controlled trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.225 h#p://den*stry3000.pi#.edu reduce pain after surgery or affect the speed of healing, as some studies had shown that healing without applying a dressing was better than applying it.4 therefore, studies have tended to find substances that enhance healing and reduce following complications. in addition, the indiscriminate use of drugs and the development of many antibiotic-resistant strains of germs directed scientists to look for alternative natural treatments and developed the ability to apply them topically.5 the number of orally topically applied drugs is very small, and they have been formulated similarly to those applied in dermatological treatments, without paying attention to the structural, environmental, and functional characteristics of the oral cavity. in addition to the presence of saliva and its role in the instinctive cleaning in the oral cavity, which leads to decreasing the drug dosage which has been applied. therefore, it is necessary to obtain medicinal materials capable of adapting to the specificity of the oral cavity.6 hydrogel is a three-dimensional hydrophilic mesh that absorbs a large amount of water and vital fluids.7 aloe vera has been used for centuries because it has many advantages and medicinal properties, because it is composed of a mixture rich in materials that accelerate healing, such as (vitamins, minerals, enzymes, sugars) but the therapeutic properties of each of its seventy-five components alone have not been determined yet.8 topical application of aloe vera gel stimulates the activity of fibroblasts and collagen proliferation, 9 because they contain glucomannan (a polysaccharide-rich in mannose) and gibberellin (a growth hormone) that interact with the growth factor receptors on the fibroblasts and increase their ability to reproduce.10 hydrogels are commonly used in the biomedical fields and due to their ability to absorb different amounts of water, their properties have been modified in laboratory studies to be compatible with various biological systems.11 the importance of this research comes from the hydrogel was used in it. because it was specially formulated to comply with the oral cavity particularity, and it could stick to the mucosa so the desired effect of aloe vera increased without disturbed the patient or confused the treating doctor while dealing with traditional dressings. this research aimed to study the effectiveness of aloe vera hydrogel on free gingival graft healing and reducing scarring in the receiving area. material and methods the laboratory stage hydrogel components aloe vera, vitamin e, glycerin, water of injection, carpobol 940, hydroxypropyl methylcellulose (hpmc), acacia, sodium benzoate, sorbate potassium, naoh. aloe vera hydrogel preparing two laboratory forms of aloe vera hydrogel were prepared, the excipients made from [glyserin (0.38% ), water of injection(14.3 %), carpobol 940 (0.97%), hydroxypropyl methylcellulose (hpmc) (0.24%), acacia (0.24%), sodium benzoate(0.38%), sorbate potassium(0.2%)], these components were weighed by an electronic scale (precisa company/ uk) according to specific quantities for each one then naoh was added, two equal quantities were prepared and sterilized in autoclave (jsr company, india) in 121c for 15 minutes, then the aloe vera and vitamin e mix were added to the excipients to made 100 ml from hydrogel, vitamin e percentage was (0.52%). the excipients and aloe vera were mixed in a planting tent (class ii, jsr company, india) to maintain the sterility of the material in two ways: the first quantity of excipients was mixed with the filtered aloe vera gel using sterile filters (isolab®) and mix the second amount of excipient with aloe vera gel without filtering. the effect of aloe vera hydrogel on the scarring and healing of free gingival gra4: randomized controlled trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.225 h#p://den*stry3000.pi#.edu hydrogel ph was measured by (ph 211, hanna, italy) and it was 6.35, the viscosity measured by (myr rotary viscometer vr 3000, spain) and it was 3333 centi boaz, the aloe vera percentage in the hydrogel was 82.52%. a sterility test was performed for the resulting forms, and the two forms did not initiate any bacterial growth. the hydrogel was filled with listed syringes and kept in a cool dry place to preserve its properties. the hydrogel stuck to the work area for about half an hour, and the tissues absorbed it during this period. the hydrogel was approved without filtration to facilitate the pre-clinical preparation. the laboratory stage was carried out at the faculty of pharmacy, damascus university. clinical stage the study was registered in isrctn.com (registration number isrctn58370471) and conducted following the helsinki declaration of 1975 and was approved by the internal ethical committee of damascus university no. 3679. written informed consent was obtained from all patients who participated in this project. the sample size was determined based on the null hypothesis, which states that the aloe vera hydrogel has no effect on free gingival graft healing and reducing scar formation in the receiving area. the confidence level was determined by 95%, the desired sample power was 95%, then g power (version 3.1.9) was used, and the required sample size was 22 patients the consort statement has been used, and the consort flow diagram illustrates the research process (figure 1). statistical analysis in this study, the level of significance (p-value) and power of the study was set at 0.05% and 95%, respectively. mann-whitney u test was done to evaluate mucosal scarring index and wound healing index. all statistical analyses were conducted using spss 25.0 for windows (spss inc. chicago, il, usa). 2.3 clinical study design: this study is a randomized clinical trial (rct) which was conducted on patients from the department of periodontics, faculty of dentistry, damascus university, syria. with a total number of 22 patients. the procedure was explained to patients, written and verbal consent was obtained for the surgery and followups. inclusion and exclusion criteria the recruitment process began in (1/11/2020) and follow-ups were done for six months respectively after the placement of test materials. 22 participants were selected from patients attending the periodontal department at the faculty of dentistry, damascus university that matched the following inclusion criteria a) attached gingiva width is less than 2mm b) age is over 18 years old d) good oral hygiene. subjects were excluded if they had a) teeth mobility at graft place b) periodontitis patient c) patients with systemic diseases d) smokers e) pregnancy/lactation. clinical study phases pre-surgical phase all patients received a full intra-oral examination, periodontal tissues were examined, and the following clinical index was recorded: plaque index, gingivitis, bleeding on probing, and attached gingiva width. as all this information was recorded on a special sheet for each patient, which also contains his personal information. scaling and root planning were done for each patient and oral hygiene instructions were given; this session took place three weeks before the surgical procedure. surgical procedure local anesthesia was done using lidocaine 2% containing 1: 80,000 adrenaline. the flap design started with a horizontal incision following effect of aloe vera hydrogel on the scarring and healing of free gingival gra4: randomized controlled trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.225 h#p://den*stry3000.pi#.edu the mucogingival junction (mgj) and measuring approximately 12 mm. mesiodistally, 8 mm apico-coronally. a periosteal bed was prepared. gauze moistened with saline was placed over the recipient bed until graft placement. obtain the graft from the donor site: the epithelial gingival graft is harvested from the palate with dimensions (12 x 8) using a #15 surgical blade from the side of the canine to the first molar at a distance of 3 mm from the free gingival margin. graft placement: the graft was positioned and firmly adapted to the recipient area and stabilized with suspensory periosteal sutures (4-0 nylon monofilament), the sutures figure 1. consort 2010 flow diagram effect of aloe vera hydrogel on the scarring and healing of free gingival gra4: randomized controlled trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.225 h#p://den*stry3000.pi#.edu were made with two sutures on both ends of the graft and an x-shaped suture minimizing any suturingrelated trauma to the graft (figure 2). post-surgical care each patient was given post-surgery instructions orally and written. and to avoid hard food in the first week, cold and hot food and drinks within 3 days, avoid lip, cheek, or lip lifting. each patient was given prescription: 1amoxicillin mg three times daily for five days 2panadol 500 mg three times daily for five days each patient in the test group was given the following instructions for using aloe vera hydrogel: patients were recommended to apply 1/2 ml of hydrogel two times daily for ten days, and to store the syringe in a cool and dark place (figure 3,4). recalls took place after 1, 2, 3, 4 weeks and after 2, 3, 6 months. mucosal scarring index (msi) is based on five parameters: width, height/contour, color, suture marks, and overall appearance. each parameter was assessed with a 0–1–2 score, yielding an msi score ranging from 0 (no scar) to 10 (most extreme scar). 12 the scar was evaluated at all followup periods, and it was mild if the total score was less than 3, moderate if the score was between 3 and 7, and severe if it was more than 7 the wound healing index was evaluated using landry`s et al index 1985.13 results 22 patients participated in this study, randomly divided into two groups using a coin. control group to observe natural healing (11 patients) while in the intervention group aloe vera hydrogel was applied (11 patients). all patients continued all the followups and there was no dropout. the mean of patient's ages was 35 years, while the percentage of males was 27.3% and females were 72.3%. the plaque index was less than 1, attached gingiva width was less than 2 mm at baseline. statistical package for the social sciences (spss 25.0 for windows) was used to analyze data, statistical figure 2. hydrogel syringe with special heads figure 3. free gingival graft surgical procedure a: before the surgery b: periosteal bed c: graft placement figure 4. free gingival graft (fgg) healing and scarring {a, b, c, d, e: test group} a: before (fgg), b: after three weeks, c: after one month, d: after three months, e: after six months {f, g, h, i, j: control group} f: before (fgg), g: after three weeks, h: after one month, i: after three months, j: after six months effect of aloe vera hydrogel on the scarring and healing of free gingival gra4: randomized controlled trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.225 h#p://den*stry3000.pi#.edu difference was considered in value less than 0.05. table 1 contains the results of mucosal scarring index (msi) after 1, 2, 3, 4 weeks and after 2, 3, 6 months. table 1 contains the results of mucosal scarring index (msi) after 1, 2, 3, 4 weeks and after 2, 3, 6 months. a positive significant difference was observed between the two groups after one month (p= 0.008, effect size r= 0.6), after two months (p= 0.001, effect size r= 0.8), and after 3 months (p= 0.001, effect size r= 0.8), and after 6 months (p= 0.001, effect size r= 0.6) where the intervention group surpass the control group with less scar formation (table 1). table 2 contains the results for the healing index after 1, 2, 3, 4 weeks and after 2, 3, 6 months. a positive significant difference was observed after 3 weeks (p= 0.031 effect size r= 0.4), the intervention group surpassed the control group. while no statistical difference was observed between the groups at 1, 2, 4 weeks, and at 2, 3, 6 months. discussion the growing interest in maintaining healthy gingiva is due to its great study groups numbe r ranks average total ranks p value z effect size r scar after one week test 11 11.50 126.50 1.000 0 0 control 11 11.50 126.50 scar after two weeks test 11 10.18 112.00 0.298 -1.04 0.2 control 11 12.82 141.00 scar after 3 weeks test 11 9.95 109.50 0.213 -1.244 0.3 control 11 13.05 143.50 scar after a month test 11 8.23 90.50 0.008 -2.635 0.6 control 11 14.77 162.50 scar after 2 months test 11 6.36 70.00 0.001 -4.056 0.8 control 11 16.64 183.00 scar after 3 months test 11 6.36 70.00 0.001 -4.056 0.8 control 11 16.64 183.00 scar after 6 months test 11 6.36 70.00 0.001 -4.056 0.8 control 11 16.64 183.00 table 1: scarring index in the receiving area effect of aloe vera hydrogel on the scarring and healing of free gingival gra4: randomized controlled trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.225 h#p://den*stry3000.pi#.edu effect on increasing the ability of patients to control dental plaque, which contributes to improving the oral health of individuals, so the best treatment technique must be chosen to achieve the best results and obtain study groups number ranks average total ranks p value z effect size r healing after one week test 11 10.59 116.50 0.328 -0.978 0.2 control 11 12.41 136.50 healing after two weeks test 11 12.36 136.00 0.505 -0.667 0.14 control 11 10.64 117.00 healing after three weeks test 11 13.50 148.50 0.031 -2.160 0.4 control 11 9.50 104.50 healing after a month test 11 12.50 137.50 0.147 -1.449 0.3 control 11 10.50 115.50 healing after two months test 11 11.50 126.50 1.000 0 0 control 11 11.50 126.50 healing after 3 months test 11 11.50 126.50 1.000 0 0 control 11 11.50 126.50 healing after 6 months test 11 11.50 126.50 1.000 0 0 control 11 11.50 126.50 table 2: healing index in the receiving area effect of aloe vera hydrogel on the scarring and healing of free gingival gra4: randomized controlled trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.225 h#p://den*stry3000.pi#.edu the patient’s satisfaction with attention to surgical complications and consider them while making a treatment plan.14 this hydrogel was formulated to avoid the drawbacks of topical application of drugs, especially in the oral cavity, which considers as a part of the mucous adhesive that secures adhesion to the oral mucosa, and leads to enhance the bioavailability of the drug in the treated area15, increases the drug contact with the tissues and reduces the frequency of drug administration and facilitates dose control and drug termination. 16 several recent attempts have been made to investigate the benefits of applying hydrogels containing aloe vera for tissue regeneration. 17 18 and this was the first study that applied hydrogel after free gingival graft to promote healing and reduce scarring in the area. the scar formation was severe after a week (90.9%) in the test group, and it became mild (90.9%) after two months. while it was severe by (90.9%) in the control group by one week, and after 3 months it remained severe by (27.3%), and moderate by (72.7%). the absence of statistically significant differences in the first weeks is due to the irregularity of collagen fibers in the area, where the collagen fibers are at the beginning of the third stage of healing stages and the amount of glucosaminoglycans is large and the fibroblast cells turn into fibrous cells and the small-diameter blood vessels disappear, which forms a scar in the area. during the healing process and after applying aloe vera to the area, the glucosaminoglycan content in the area decreases, the weakly structured collagen type iii is replaced with collagen type i, the fibers regularity increases, the type of cross-linking improves, and the distance between them is reduced, which lead to reducing scarring in the treated area.19 20 aloe vera improved the ulcer’s appearance. dimension and redness reduced and its color was similar to the adjacent mucosa color after healing in the study.21 (ar pradeep et al, 2015) study results showed that the usage of aloe vera hydrogel improved clinical indices of patients with periodontitis in clinical attachment level, as well as decreasing probing depth and bleeding on probing, due to the healing properties of aloe vera.22 it is noticed that the healing was weak in (81.8%) after one week in the test group, and recovery was completed in all samples (100%) after 3 weeks in the test group. whereas healing was weak after a week (81.8%) in the control group, and healing was not completed at 100% after two months of surgery in the control group, which showed the superiority of the test group with statistically significant differences pvalue < 0.005. this is attributed to the mechanism of aloe vera in improving wound healing by maintaining wound hydration, increasing epithelial cell migration, accelerating collagen maturation, enhancing collagen cross-linking, as well as increasing blood flow to the area.23 the precise mechanism of the effect of aloe vera in wound healing is also attributed to the acceleration of the healing stages compared with natural healing, as it speeds up the end of the inflammatory phase by increasing the blood flow by accelerating the formation of new blood vessels in the area and reducing the amount of edema. the number of phagocytes increases in the area, which speeds up the process of phagocytosis and damaged tissues removal and reduces the lifespan of fibrin clot, 24 the polysaccharides present in aloe vera such as acemmane and mannose play a major role in increasing the number of phagocytes and cytokines.25 glucomannan and gebberlin also interact with growth factor receptors on the fibroblasts and stimulate their activity and proliferation, which increases collagen formation.9 our study results were similar to (ghada mansour et al, 2014) study where the use of an aloe vera mucoadhesive gel accelerated the healing of aphthous ulcers and the location of the ulcers after healing was similar to the color of the adjacent mucosa.21 conclusion effect of aloe vera hydrogel on the scarring and healing of free gingival gra4: randomized controlled trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.225 h#p://den*stry3000.pi#.edu in this study limits, the aloe vera hydrogel can be considered as a good topical treatment after oral surgery due to its ability to reduce scar formation and speeding up the healing in the receiving area in free gingival graft procedures. ethics statement/confirmation of patients’ permission ethics permission was taken from damascus university board under number 3679. the patients gave permission for the procedure. references 1. zuhr o, bäumer d, hürzeler m. the addi8on of so= 8ssue replacement gra=s in plas8c periodontal and implant surgery: cri8cal elements in design and execu8on. journal of clinical periodontology. 2014;41 suppl s:s123–s142. doi:10.1111/jcpe.12185 2. bertl k, melchard m, pandis n, müller-kern m, stavropoulos a. so= 8ssue subs8tutes in nonroot coverage procedures : a systema8c review and metaanalysis. clinical oral inves8ga8ons. 2017. doi:10.1007/s00784-016-2044-4 3. review a. of early healing events periodontal repair : a review significance. :158–165. 4. murthykumar k, rajasekar a, kaarthikeyan g. assessment of healing a=er periodontal flap surgery with and without periodontal pack. interna8onal journal of pharmaceu8cal research. 2020;12(august):125– 130. doi:10.31838/ijpr/2020.sp1.020 5. buggapa8 l. herbs in den8stry. 2016;5(6):7–12. 6. paderni c, compilato d, giannola li, campisi g. oral local drug delivery and new perspec8ves in oral drug formula8on. oral surgery, oral medicine, oral pathology and oral radiology. 2012;114(3):e25–e34. doi:10.1016/j.oooo.2012.02.016 7. peppas na, bures p, leobandung w, ichikawa h. hydrogels in pharmaceu8cal formula8ons. 2000;50. 8. mehta i. ’history of aloe vera’(a magical plant). iosr journal of humani8es and social science (iosr-jhss. 2017;22(8):21–24. doi:10.9790/0837-2208162124 9. hamman jh. composi8on and applica8ons of aloe vera leaf gel. molecules. 2008;13(8):1599– 1616. doi:10.3390/molecules13081599 10. eshun k, he q. aloe vera: a valuable ingredient for the food, pharmaceu8cal and cosme8c industries a review. cri8cal reviews in food science and nutri8on. 2004;44(2):91–96. doi:10.1080/1040869049042469 4 11. mateescu a, wang y, dostalek j, jonas u. thin hydrogel films for op8cal biosensor applica8ons. membranes. 2012;2(1):49–69. doi:10.3390/membranes201004 0 12. wessels r, de roose s, de bruyckere t, eghbali a, jacquet w, de rouck t, cosyn j. the mucosal scarring index: reliability of a new composite index for assessing scarring following oral surgery. clinical oral inves8ga8ons. 2019;23(3):1209–1215. doi:10.1007/s00784-018-2535-6 13. landry rg. effec8veness of benzydamine hc1 in the treatment of periodontal postsurgical pa8ents. 1985. 14. professor sa, tanwar post graduate student e, graduate student p, tanwar e, bhat d. post-opera8ve complica8ons a=er periodontal surgery. ~ 152 ~ interna8onal journal of applied dental sciences. 2018;4(4):152–156. 15. mathew ak. oral local drug delivery: an overview. pharmacy and pharmacology research. 2015;3(1):1–6. 16. ludwig a. the use of mucoadhesive polymers in ocular drug delivery. advanced drug delivery reviews. 2005;57(11):1595–1639. doi:10.1016/j.addr.2005.07.005 17. pereira r, mendes a, bártolo p. alginate/aloe vera hydrogel films for biomedical applica8ons. procedia cirp. 2013;5:210–215. doi:10.1016/j.procir.2013.01.04 2 18. pereira r, carvalho a, vaz dc, gil mh, mendes a, bártolo p. development of novel alginate based hydrogel films for wound healing applica8ons. interna8onal journal of biological macromolecules. 2013;52(1):221–230. doi:10.1016/j.ijbiomac.2012.09. 031 19. chithra p, sajithlal gb, chandrakasan g. influence of aloe vera on the glycosaminoglycans in the matrix of healing dermal wounds in rats. journal of ethnopharmacology. 1998;59(3):179–186. doi:10.1016/s03788741(97)00112-8 20. maenthaisong r, chaiyakunapruk n, niruntraporn s, kongkaew c. the efficacy of aloe vera used for burn wound healing: a systema8c review. effect of aloe vera hydrogel on the scarring and healing of free gingival gra4: randomized controlled trial vol 10 no 1 (2022) doi 10.5195/d3000.2022.225 h#p://den*stry3000.pi#.edu burns. 2007;33(6):713–718. doi:10.1016/j.burns.2006.10.384 21. mansour g, ouda s, shaker a, abdallah hm. clinical efficacy of new aloe veraand myrrh-based oral mucoadhesive gels in the management of minor recurrent aphthous stoma88s: a randomized, double-blind, vehicle-controlled study. journal of oral pathology and medicine. 2014;43(6):405–409. doi:10.1111/jop.12130 22. pradeep ar, garg v, raju a, singh p. adjunc8ve local delivery of aloe vera gel in pa8ents with type 2 diabetes and chronic periodon88s: a randomized, controlled clinical trial. journal of periodontology. 2016;87(3):268–274. doi:10.1902/jop.2015.150161 23. gupta v, malhotra s. pharmacological atribute of aloe vera: revalida8on through experimental and clinical studies. ayu (an interna8onal quarterly journal of research in ayurveda). 2012;33(2):193. doi:10.4103/0974-8520.105237 24. vázquez b, avila g, segura d, escalante b. an8inflammatory ac8vity of extracts from aloe vera gel. journal of ethnopharmacology. 1996;55(1):69–75. doi:10.1016/s03788741(96)01476-6 25. kim j, lee i seok, park s, choue r. effects of scutellariae radix and aloe vera gel extracts on immunoglobulin e and cytokine levels in atopic derma88s nc/nga mice. journal of ethnopharmacology. 2010;132(2):529–532. doi:10.1016/j.jep.2010.08.049 706 2024 assessment of iraqi dental practitioners' and undergraduate students' knowledge regarding dentin hypersensitivity management: a cross-sectional study vol 13 no 2 (2024) doi 10.5195/d3000.2024.706 h#p://den*stry3000.pi#.edu assessment of iraqi dental practitioners’ and undergraduate students’ knowledge regarding dentin hypersensitivity management: a cross-sectional study zaidoon hasan mohammed 1, thanaa ghani nema 1, sohaib fadhil mohammed 2, hamsa zaki al-assadi 1, ala mahdi muhamed ali 1, aya alaa hussein 1 college of dentistry, kerbala university, kerbala, iraq 2 college of dentistry, anbar university, anbar, iraq abstract objec&ve: the characteris+c of den+n hypersensi+vity is brief, intense pain that results from exposed den+n in reac+on to a variety of s+muli, including mech anical, thermal, osmo+c, or chemical factors. this study aimed to evaluate the level of knowledge among undergraduate students and prac+cing den+sts in karbala city, iraq, concerning the mechanisms, e+ology, and treatment of den+n hypersensi+vity (dh). methods: 102 surveys were distributed via google forms to general prac++oners and fourthand filhyear dental students at the kerbala college of den+stry in iraq. the data were inpumed into microsol excel and analyzed using spss 22.0 for windows as frequency distribu+on tables and figures. results: despite the differences in comprehension and exper+se between students and den+sts, a compara+ve analysis of their responses to various ques+ons revealed several similari+es. according to the survey, 28.8% of students reported that 10% of pa+ents suffered from dh, while 32% of den+sts reported the same. addi+onally, 72% of den+sts and 71.2% of students stated that dh is a severe problem for pa+ents. both den+sts, 30%, and 26.9% of students, indicated that dh lasted for less than 2 weeks. according to den+sts and students, females are more affected by dh (74%; n=37) and (71%; n=37), respec+vely. conclusion: students and den+sts showed a similar level of knowledge on dh. keywords: den+n sensi+vity; students; diagnosis, surveys and ques+onnaires. cita;on: mohammed zh, et al. (2024) assessment of iraqi dental prac;;oners’ and undergraduate students’ knowledge regarding den;n hypersensi;vity management: a cross-sec;onal study. den;stry 3000. 2:a001 doi:10.5195/d3000.2024.706 received: august 15, 2024 accepted: august 17, 2024 published: september 25, 2024 copyright: ©2024 mohammed zh, et al. this is an open access ar;cle licensed under a crea;ve commons azribu;on work 4.0 united states license. email: zaidoon.h@uokerbala.edu.iq introduction dentin hypersensitivity (dh) is a distinct dental condition characterized by pain induction in response to non-painful stimuli focused on the exposed dentin. in healthy teeth, pain is not a typical consequence of this condition. this disorder, which cannot be linked to any other dental defect or illness, is marked by brief, acute pain that arises when exposed dentin reacts to tactile, thermal, osmotic, evaporative, or chemical stimuli. dentin exposure can occur due to chemical erosion, mechanical abrasion, or loss of cementum due to gingival recession [1-3]. dh is a prevalent condition among patients seeking treatment at dental clinics. it is often confused with other clinical conditions, such as dental caries, microleakage, cracked teeth, or fractured restorations. it is essential to differentiate dh disease from the conditions mentioned above, as they may exhibit similar symptoms at different stages of progression [4,5]. modern lifestyles often involve consuming acidic foods and drinks. this can lead to tooth wear and exposure to dentin, a problem known as dh. dh can significantly affect the quality of life of adults, causing them to avoid certain activities and foods and skip brushing areas that cause pain. even routine dental treatments like scaling and polishing can trigger dh assessment of iraqi dental practitioners' and undergraduate students' knowledge regarding dentin hypersensitivity management: a cross-sectional study vol 13 no 2 (2024) doi 10.5195/d3000.2024.706 h#p://den*stry3000.pi#.edu and make regular dental visits unpleasant and distressing for patients. a hydrodynamic theory can explain dh, suggesting that fluid flow within dentin tubules triggers baroreceptor activation. this activation causes a discharge of neural signals that cause the sensation of pain [6, 7]. females have a slightly higher incidence of the condition than males, with individuals between 20 and 50 years being the most vulnerable [8, 9]. the management of dh involves taking a detailed clinical and dietary history. dh should be distinguished from other dental pain conditions, and its underlying causes should be addressed. before starting treatment for dh, clinicians must exclude several different disorders that may be causing similar symptoms. dental lasers, in-office procedures, gratuitously desensitizing toothpaste and mouthwashes, and home remedies are among the treatment options [5]. before selecting a treatment for dh, the clinician must consider an exclusive differential diagnosis [10]. dh can be caused by various conditions that are different from those that cause other dental issues. chipped enamel, broken restorations, dental cavities, marginal restoration leaks, cracked tooth cusps, and evaporative stimuli are a few of these issues. when a liquid moves over a sensitive area, it can generate mechanical receptors related to sensitivity to fluid pressure. as a result, signals get transmitted into the pulpal nerves, developing a pain reaction [11, 12]. this study aimed to evaluate the level of knowledge among undergraduate students and practicing dentists in karbala city, iraq, concerning the mechanisms, etiology, and treatment of dentin hypersensitivity (dh). methods from november 2022 to february 2023, 102 questionnaires were distributed to 4thand 5th-year dental students and general dentists at karbala college of dentistry in iraq. the questionnaire consisted of 24 predetermined questions and was administered via google forms. microsoft excel was used to gather and arrange the responses, while spss 22.0 for windows was used for analysis. figures and frequency distribution tables were used to present the findings. results of the total participants, 102 participated in the survey, of which 52 were students (51%) and 50 were dentists (49%). in response to question 2 of whether they had examined patients with dh in the previous (2-4) weeks or months, 52.6% (n = 30) of students and 47.4% (n = 27) of dentists responded affirmatively. the following information was obtained in response to question 3, which requested an estimate of the percentage of patients receiving care at the dental hospital who suffered from dh: 10% of patients, according to 28.8% (n = 15) of the students, were thought to have dh, while 15% of patients were believed to have it. furthermore, 10% of patients had dh, as reported by 32% (n = 16) of dentists. in response to question 4 concerning the initiator of the conversation before the clinical examination and dh diagnosis, 52% (n = 26) of the dentists and 59.6% (n = 31) of the students reported that the clinician initiated the conversation. on the other hand, 48% (n = 24) of dentists and 40.4% (n = 21) of students stated that patients initiated the conversation. dentists (82%; n = 41) and students (78.8%; n = 41) reported observing signs of dh in response to question 5. regarding question 6, 72% (n = 36) of dentists considered dh an urgent clinical condition in their patients, while students (71.2%; n = 37) expressed the same opinion. regarding question 7, 30% (n = 15) of dentists and 26.9% (n = 14) of students indicated that the dh lasted for >2 weeks. regarding questions 8 and 9 about the effect of dh on the patient's quality of life, both dentists (72%; n = 36) and students (76.9%; n = 40) reported that it had a significant impact. on the other hand, dentists (82%; n = 41) and students (76.9%; n = 40) indicated that it had mild to moderate effects. assessment of iraqi dental practitioners' and undergraduate students' knowledge regarding dentin hypersensitivity management: a cross-sectional study vol 13 no 2 (2024) doi 10.5195/d3000.2024.706 h#p://den*stry3000.pi#.edu in response to question 10, 68% (n = 34) of dentists and 75% (n = 39) of students indicated that they were asked. some variations between the dentist and student responses were highlighted in question 11 concerning the etiology of dh. according to dentists and students, the top dental issues were as follows: for dentists, 38% (n = 19) reported exposed dentin, 12% (n = 6) reported gingival recession, 10% (n = 5) reported fluid movement, 8% (n = 4) reported loss of enamel, and 6% (n = 3) reported abrasion. for the students, 26.9% (n = 14) reported exposed dentin, 15.3% (n = 8) reported gingival recession, 15.3% (n = 8) reported a loss of enamel, 13.4% (n = 7) reported needing periodontal treatment, and 7.6% (n = 4) reported having caries, as illustrated in table 1. environment, as well as perceived stress and career future (table 1). in response to question 12, when asked about the steps followed to clinically diagnose a patient with dentin hypersensitivity (dh), dentists (38%; n = 19) and students (34.6%; n = 18) mentioned clinical sensitivity to cold as the first diagnostic technique, as shown in table 2. as for question 14 about other dental causes or conditions that should be taken into consideration regarding the diagnosis of dh, they provided the following primary responses: 1) cracked syndrome (20%; n = 10) (19.2%; n = 10); and 2) post-operative sensitivity (18%; n = 9) (19.2%; n = 10). responses to question 15 suggested that dentists were more confident than students in correctly identifying dh compared to other painful dental diseases. out of all the dentists surveyed, 10% (n = 5) were very confident in diagnosing dh, 52% (n = 26) felt confident, and 36% (n = 18) were somewhat confident. on the other hand, only 7% (n = 4) of the students felt very confident in diagnosing dh, whereas 50% (n = 26) were confident, and 25% (n = 13) were somewhat confident. interestingly, a higher percentage of students (17.3%; n = 9) were not very confident compared to dentists (2%; n = 1), as depicted in figure 1. during the survey, dentists and dental students received questions regarding the currently accepted theory of dentinal hypersensitivity. the survey illustrated that 73% (n = 38) of students and 62% (n = 31) of dentists stated they currently subscribed to the hydrodynamic theory. however, some students (26.9%; n = 14) indicated other theories, such as direct innervation theory and odontoblast receptor, compared to dentists who had other answers (38%; n = 19). figure 2 displays the results of a survey conducted among dentists and students about their methods for evaluating patients with dh in a clinical setting. the most used assessment methods, according to the responses of dentists and students, were as follows: 1) a dental exam (54% of dentists and 46% of students) and 2) thermal tests (18% of dentists and 28.8% of students). in question 18, both dentists and students concurred on the following suggestions for patients with dh: 1) use of home desensitizing toothpaste (34%; n = 17) (95.1%; n = 39); and 2) education on proper tooth brushing techniques (34%; n = 17) (87.8%; n = 36). in response to question 19 on recommending at-home materials for dh, dentists and students had varying confidence levels: 10% of the dentists were very confident, 56% were confident, 22% were somewhat confident, and 12% were not. on the other hand, 13.4% of the students were very confident, 25% were confident, 50% were somewhat confident, and 11.5% were not very confident. regarding the question about further details on any non-dental issues associated with dh, dentists and students reported different responses. for dentists, the three most common responses were psychological stress (30%; n = 15), bruxism (22%; n = 11), and clenching (22%; n = 11). on the other hand, students listed bruxism (23%; n = 12), clenching (23%; n = 12), and psychological stress (19.2%; n = 11) as their top three responses. assessment of iraqi dental practitioners' and undergraduate students' knowledge regarding dentin hypersensitivity management: a cross-sectional study vol 13 no 2 (2024) doi 10.5195/d3000.2024.706 h#p://den*stry3000.pi#.edu table 1. selected responses from question 11 regarding an understanding of the etiology of dh. the e%ology of dh (selected variables) staff (n) students (n) exposed den%n 19 14 gingival recession 6 8 wrong (incorrect) brushing 3 0 fluid movement 5 3 alri%on 0 2 abrasion 3 0 enamel fracture 1 0 bleaching techniques 1 2 periodontal disease 2 0 loss of enamel 4 8 caries 2 4 leaking restora%on 1 3 erosion 1 1 periodontal treatment 2 7 table 2. selected responses to question 12 concerning the steps taken to diagnose a patient with dh. figure 1. comparison of confidence levels of dentists and students in diagnosing dh. figure 2. the responses of dentists and students to question 17 regarding the assessment and evaluation of dh in the surgical environment. 0 10 20 30 40 50 60 very confident confident somewhat confident not very confident dentists students 0 10 20 30 40 50 60 med ica l h ist ory den tal exa m probing d epths th erm al tes ts perc ussi on te sts pupal tests den tal ra diogra ph diet a naly sis dentists studens what steps would you take to clinically diagnose a patient with dh (selected variables)? staff (n) students (n) clinical sensitivity to cold 19 18 clinical examination 12 5 dh history 11 13 vitality test 4 3 aggravating factor 0 1 eliminate the cause of dh 0 4 assess recession 0 1 take a radiograph 0 2 apply a bonding agent 1 2 provision of a desensitizing toothpaste 1 0 diet history 1 2 medical history 1 1 assessment of iraqi dental practitioners' and undergraduate students' knowledge regarding dentin hypersensitivity management: a cross-sectional study vol 13 no 2 (2024) doi 10.5195/d3000.2024.706 h#p://den*stry3000.pi#.edu in response to inquiries regarding the adherence of their patients to the professional recommendations for the treatment and control of dh, a significant proportion of dentists and dental students, constituting 64% (n = 32) and 63.4% (n = 33), respectively, indicated that their patients adhered to the advice dispensed. conversely, 36% (n = 18) of dentists and 36.5% (n = 19) of students reported instances where their patients did not adhere to the provided guidance. in response to question 22 regarding the necessity of additional information in the form of a leaflet to prevent further occurrences of dh, 86% of dentists (n = 43) and 86.5% of students (n = 45) responded affirmatively. on the other hand, 14% of dentists (n = 7) and 13.5% of students (n = 7) stated that further information about the prevention of dh was unnecessary. both dentists and students noted that females were more affected by dh (74%; n = 37) and (71%; n = 37), respectively, compared to males (26%; n = 13) and (29%; n = 15), respectively. the teeth that were most affected by dh, according to both dentists and students, were incisors (46%; n = 23) (42%; n = 21), while the least affected were canines (4%; n = 2), (3.8%; n = 2). in response to the last question regarding the potential impact of oral hygiene on dh, 82% of dentists (n = 41) and 80.7% of students (n = 42) agreed that it might have an effect. discussion this study's questionnaire was constructed based on a previous survey conducted in the united kingdom. the initial questionnaire was modified from a survey used in a study by schuurs et al.[13], and this adaptation was confirmed in a study by hatton et al. [14] numerous prior studies and reviews have indicated that dentists may exhibit uncertainty regarding the etiology, diagnosis, and optimal treatment of dh [1417]. insufficient knowledge or understanding of dh often leads to the failure of dentists to screen for dh routinely. consequently, this may affect the dentist's confidence in managing the condition and impact the successful treatment of dh to the patient's satisfaction. moreover, there is an inability to perform a differential diagnosis to discard other clinical conditions with characteristics similar to dh [14]. despite differences in understanding and knowledge, both groups exhibited similar responses. for instance, 10% of patients experienced dh, according to 32% of dentists and 28.8% of students. additionally, both dentists (72%) and students (71.2%) acknowledged the severity of dh for patients. moreover, dh persisted for more than two weeks for 30% of dentists and 26.9% of students. most of both dentists (72%) and students (76.9%) concurred that dh negatively affects people's quality of life (qol). they described it as mild to moderate (82% of dentists and 76.9% of students). the responses regarding dh's causes, diagnosis, and treatment were in line with other published studies [14, 15]. a previous study identified a problem of insufficient understanding of the primary mechanism of dh. the current survey discovered that 73% of students and 62% of dentists identified the hydrodynamic theory as dh's prevailing mechanism of action [16]. recent research has validated that clinicians better understand the mechanisms that trigger dh [15, 17]. the study revealed that dentists had greater confidence in managing dh than students. this suggests dentists are more proficient in dealing with dhrelated issues than students. regarding recommendations for dh and at-home desensitizing products, it was discovered that both dentists and students had the same degree of confidence. however, when asked about the non-dental factors that may impact dh, such as stress, the responses generally agreed. still, specific factors were limited to anxiety, bruxism, and clenching. furthermore, the participants agreed that there was a lack of knowledge about dh and its treatment options, stressing the need for additional information in a leaflet [14]. assessment of iraqi dental practitioners' and undergraduate students' knowledge regarding dentin hypersensitivity management: a cross-sectional study vol 13 no 2 (2024) doi 10.5195/d3000.2024.706 h#p://den*stry3000.pi#.edu according to several studies, dh incidence is significantly higher in females than males. this may be attributed to women's nutritional habits, which render them more vulnerable to tooth sensitivity. in addition, more women tend to seek tooth whitening treatments, which can further aggravate the issue, leading to even more significant tooth sensitivity [2, 18]. it is essential to note that these findings are significant, as they may have implications for dental professionals who treat patients with dh. given the higher incidence of dh in female patients, dental professionals should consider this factor when developing treatment plans and providing recommendations. furthermore, additional research may be necessary to elucidate the underlying factors and develop more effective treatment strategies for patients who experience dh [19]. according to dental professionals and students, the teeth most commonly affected by dh are the incisors and premolars [2, 20], with 46% of dentists and 42% of students reporting this trend. poor oral hygiene is the primary cause of dh, as reported by 82% of dentists and 80.7% of students. this condition can result from incorrect brushing techniques or other detrimental oral hygiene habits, which contribute to gingival recession and the subsequent exposure of teeth to hypersensitivity [21]. understanding the etiology of dh is critical to its effective management and treatment [22]. conclusion students and dengsts showed a similar level of knowledge on dh. conflicts of interest the authors declare no competing interests. references 1. solé-magdalena, a., et al., molecular basis of dental sensigvity: the odontoblasts are mulgsensory cells and express mulgfuncgonal ion channels. 2018. 215: p. 20-29. pmid: 28954208 2. demirci, m., et al., the prevalence, clinical features, and related factors of dentin hypersensitivity in the turkish population. 2022. 26(3): p. 27192732. pmid: 35083586 3. silva, m.s., et al., prevalence and predictive factors of dentin hypersensitivity in brazilian adolescents. 2019. 46(4): p. 448-456. pmid: 30825378 4. torres, a.d.s., et al., effecgveness of cyanoacrylate in the treatment of dengn hypersensigvity: a systemagc review. 2023. 2023(1): p. 1465957. pmid: 37663787 5. liu, x.-x., et al., pathogenesis, diagnosis and management of dengn hypersensigvity: an evidencebased overview for dental pracggoners. 2020. 20: p. 1-10. pmid: 32762733 6. zeola, l.f., p.v. soares, and j.j.j.o.d. cunha-cruz, prevalence of dengn hypersensigvity: systemagc review and metaanalysis. 2019. 81: p. 1-6. pmid: 30639724 7. jalaluddin, m., et al., assessment of the efficacy of different desensitizing agents on dentinal tubules occlusion-an in vitro study. 2022. 14(suppl 1): p. s585-s588. pmid: 36110588 8. agheli, n., et al., dentists’ education, knowledge, and professional behavior concerning the diagnosis and treatment of dentin hypersensitivity: an exploration. 2023. 87(12): p. 17051717. pmid: 37650366 9. das, a., et al., effect of iontophoresis on the effecgveness of nanohydroxyapagte and pro-argin in in-office treatment of dengn hypersensigvity: a split-mouth randomized clinical trial. 2023. 15(12). pmid: 38259360 10. acharya, a.b., et al., a short term comparative evaluation of the efficacy of diode laser with desensitizing toothpastes and mouthwashes in the treatment of assessment of iraqi dental practitioners' and undergraduate students' knowledge regarding dentin hypersensitivity management: a cross-sectional study vol 13 no 2 (2024) doi 10.5195/d3000.2024.706 h#p://den*stry3000.pi#.edu dentinal hypersensitivity. 2022. 14(3): p. e229. pmid: 35317290 11. frank, a.c., et al., comparison of the bleaching efficacy of different agents used for internal bleaching: a systematic review and meta-analysis. 2022. 48(2): p. 171-178. pmid: 34762968 12. goh, v., e.f. corbet, and w.k.j.j.o.c.p. leung, impact of dentine hypersensitivity on oral health-related quality of life in individuals receiving supportive periodontal care. 2016. 43(7): p. 595-602. pmid: 27028655 13. schuurs, a., et al., dentists' views on cervical hypersensitivity and their knowledge of its treatment. 1995. 11(5): p. 240-244. pmid: 8625939 14. hatton, j., et al., knowledge of uk dental undergraduates and dentists in treating dentine hypersensitivity. 2020. pmid: 31537018 15. exarchou, c., et al., a survey of dentists in the management of dentine hypersensitivity: a questionnaire-based study. 2019. 13(03): p. 383-390. pmid: 31537018 16. dionysopoulos, d., o. gerasimidou, and c.j.a.s. beltes, dentin hypersensitivity: etiology, diagnosis and contemporary therapeutic approaches—a review in literature. 2023. 13(21): p. 11632. pmid: 24724135 17. mosquim, v., et al., knowledge and attitudes on preventing and treating dentin hypersensitivity and its predicting factors: a cross-sectional study with brazilian citizens. 2023. 17(03): p. 855-862. pmid: 36513338 18. miglani, s., v. aggarwal, and b.j.j.o.c.d. ahuja, dentin hypersensitivity: recent trends in management. 2010. 13(4): p. 218224. pmid: 21217949 19. rocha, m.o.c., et al., sensitivity and specificity of assessment scales of dentin hypersensitivity–an accuracy study. 2020. 34: p. e043. pmid: 32401933 20. barroso, n.f.f., et al., prevalence of self-reported versus diagnosed dentinal hypersensitivity: a cross-sectional study and roc curve analysis. 2019. 77(3): p. 219-223. pmid: 30646808 21. mascardo, k.c., et al., risk indicators for gingival recession in the esthegc zone: a crosssecgonal clinical, tomographic, and ultrasonographic study. 2024. 95(5): p. 432-443. pmid: 38196327 22. biagi, r., et al., laserassisted treatment of dengnal hypersensigvity: a literature review. 2015. 6(3-4): p. 75. pmid: 26941892 microsoft word 375 2022.docx oral symptoms of pa-ents tested posi-ve for covid-19 with mild severitya cross-sec-onal study vol 10 no 1 (2022) doi 10.5195/d3000.2022.375 h#p://den*stry3000.pi#.edu oral symptoms of patients tested positive for covid-19 with mild severitya cross-sectional study fatemeh tavakoli1, fatemeh lavaee2, maryam tazarvifardshirazi1, shirin fa7ahi3 1graduate student, oral and maxillofacial disease department, school of dentistry, shiraz, university of medical sciences, shiraz, iran 2dmd, associate professor of oral and dental disease research center, oral and maxillofacial disease department, school of dentistry, shiraz university of medical sciences, ghasrdasht street, shiraz, iran 3assistant professor, department of pathology, school of dencstry, tabriz university of medical sciences, tabriz, iran abstract background: the pandemic of coronavirus disease (covid-19) has a major effect on the world. it has several signs and symptoms, and the cause of some oral lesions may be related to this virus. objectives: the aim of the study is to determine the propor-on and evaluate the different oral symptoms and their -me of presenta-on in pa-ents with mild covid-19 infec-on. materials and methods: the present study included 55 par-cipants (40%) females and (60%) males, with an average age of 38.52 ± 10.46 years. results: about 80% (43 pa-ents) had oral symptoms such as xerostomia, recurrent herpes simples, aphthous stoma--s, gingival pain, or other symptoms. conclusion: covid-19 has been shown to have some effects on the mouth because a large propor-on of covid-19 pa-ents had oral symptoms. clinical significance: this research showed a high percentage of covid-19 pa-ents experienced dry mouth, auer that the most likely lesions were herpes; also, other lesions may occur. in addi-on, den-sts should be conscious of oral manifesta-on, par-cularly during the coronavirus pandemic. keywords: covid-19; recurrent herpes; xerostomia cita9on: tavakoli, f et al. (2022) oral symptoms of pa9ents tested posi9ve for covid-19 with mild severity a cross-sec9onal study den9stry 3000. 1:a001 doi:10.5195/d3000.2022.375 received: august 5, 2022 accepted: october 1, 2022 published: october 14, 2022 copyright: ©2022 tavakoli, f et al. this is an open access ar9cle licensed under a crea9ve commons atribu9on work 4.0 united states license. email: ktavakoli766@gmail.com introduction sars-cov-2 was first reported in wuhan, at the end of 2019, it rapidly became a pandemic disease and spread globally. many patients with this virus have not exhibited any serious signs or symptoms [1]. this disease is known as covid-19. the most common clinical symptoms are sore throat, fever, headache, dry throat, dyspnea, cough, abdominal pain, vomiting, and diarrhea [2]. the virus can transmit between humans by respiratory droplets. the median incubation time typically ranges from four to five days, and it may extend to fourteen days [3]. the severity of covid-19 in most people is mild (80%), whilst 20% of infected patients have experienced the severe type of this disease, and 5% of patients may become seriously sick with pneumonia or acute respiratory distress syndrome, which needs ventilation and hospitalization at icu ward [4]. the mouth forms an entry towards the outer environment and has a significant role in the development of sars-cov-2 [5]. angiotensin-converting enzyme 2 (ace 2) receptor is found in the lung, liver, gastrointestinal (gi), kidney, and even the epithelial surfaces of sweat glands and endothelium part of dermal papillary vessels [6,7] is a known receptor for sars-cov-2. up oral symptoms of pa-ents tested posi-ve for covid-19 with mild severitya cross-sec-onal study vol 10 no 1 (2022) doi 10.5195/d3000.2022.375 h#p://den*stry3000.pi#.edu to now, different cutaneous manifestations of covid-19 disease have been described encompassing varicelliform lesions, pseudochilblain, urticaria form, erythema multiforme (em)-like lesions, petechiae and purpura, maculopapular, mottling, and livedo reticularis lesions [8]. although not approved, there may be an association between epithelial expression and localization of the ace2 protein in the oral and nasal mucosa. it has been shown that the ace2 protein in the nasopharynx may be the functional receptor for the sars coronavirus [9]. the virus itself can act as a trigger factor for other virus reactivation thereby leading to oral lesions. over activation of t-cells, shown by enhancing of th17, lymphocytopoenia, and high cytotoxicity of cd8 tcells were seen and reported in covid-19 patients [10]. the available data have not yet introduced an efficient and safe pharmacologic treatment against covid-19 and the potential effects are associated with multiple adverse reactions [11]. remdesivir, protease inhibitors, hydroxychloroquine, and interferon regimen are introduced as potential drugs for treating covid-19 which may affect oral health [12]. the most related oral sign of covid19 is taste disorders, unspecific oral ulcerations are desquamative gingivitis, petechiae, and coinfections such as candidiasis, however, these symptoms have not been confirmed yet. these manifestations might be directional signs and clinical patterns of sars-cov-2 infection, or they are just consequences of a weak immune system and adverse medication effects [13]. the first recognized oral symptom of covid-19 which was seen in 38% of patients with mildmoderate severity was dysgeusia [2]. in this study, we aim to find oral symptoms of covid-19 patients, then what those symptoms are, and which symptoms are more likely to occur among patients. material and methods this cross-sectional study was conducted in the west health center of karaj of iran country from january 2021 to march 2021 in the middle of the omicron pandemic. the study was done according to the ethical principles of helsinki [14]. 55 covid19 positive patients, including 22 females and 33 males who were diagnosed by reverse transcriptionpolymerase chain reaction assay (rtpcr) from oro-nasopharyngeal swab specimens, were enrolled. the patients had mild to moderate symptoms, due to the latest who joint report [15] (mild symptoms consist of every symptom of covid-19 except shortness of breath and difficulty breathing, furthermore moderate symptoms mean blood oxygen levels remain at 94 percent or higher). all the patients were informed and signed the consent form. in this study, children, patients with any systemic diseases or patients using any medication which can cause oral symptoms or lesions, and also pregnant women were excluded from the study. all the enrolled patients had symptoms of coronavirus disease such as fever, cough, and body pain. patient’s demographic details were recorded comprising their age, gender, significant previous medical history, and use of any medications. patient covid-19 positive infection state was also evaluated. they were enquired about the time when they felt sick and their presenting symptoms. further, any significant changes within the oral cavity such as burning sensation, loss of taste sensation, changes in saliva flow, dryness of mouth, any redness, difficulty in brushing, swallowing, and maintaining oral hygiene also formed the part of the evaluation. if any complaints were reported, their first appearance and the duration were also recorded. apart from questioning the patients, oral examination comprising both hard tissue and soft tissue was conducted on all the participants of the study. any changes in the colour, moistness, continuity of the oral mucosa, flow and thickness of saliva, papilla of the tongue were all clinically examined and recorded. if any changes of significance were observed, their location, size, number, colour, texture and relevant information were oral symptoms of pa-ents tested posi-ve for covid-19 with mild severitya cross-sec-onal study vol 10 no 1 (2022) doi 10.5195/d3000.2022.375 h#p://den*stry3000.pi#.edu recorded. the oral findings of significance were brought to patients’ notice to confirm their appearance during the period of covid-19 infection. any oral presentations that were present prior to the infection were excluded. all the patients were followed throughout their treatment period until recovery at regular time intervals. the oral examination was conducted by a fully trained vaccinated dentist, following all the necessary protocolscomplete body protection suit, n95 mask, sterile gloves, and face shield. the oral examination was performed by utilizing sterilized mouth mirrors and probes. results a total number of 55 patients (33 males and 22 females) were enrolled in this cross-sectional study. all the participants had covid-19 symptoms like fever, shivering body ache, and headache. the mean age of the patients was 38.52 ± 10.46 years and the average time that they felt sick and were positive for covid-19 was on day 4.26 of their disease period. the data of patients have shown in table 1. a week after the oral examination that was done by a dentist, patients were followed and asked about how long their oral symptoms lasted. it was claimed by the patients on average, 6.21 days after the examination day their symptoms disappeared. parameters age (mean ± sd) 38.52 ± 10.466 years old gender females 22 (40%) patients males 33 (60%) patients the average of the first day that the examination of patients began 4.29 a high proportion of the patients had oral symptoms and the most common symptoms were xerostomia, recurrent herpes, (rhl), and concentrated salvia. it was observed that the oral manifestations could present alone or in combination with other oral signs and symptoms (table 2). 12 participants did not have any oral symptoms, although 9 participants just had xerostomia. 24 of the participants had xerostomia with other symptoms such as recurrent aphthous stomatitis, bitter taste, burning mouth and throat, and concentrated salvia. in 5 patients, the only symptom was recurrent herpes (figure 1), however, in 8 persons, they had recurrent herpes labialis (rhl) and other oral manifestations which were mentioned before. 7 patients had both rhl and xerostomia, and 3 of them experienced recurrent aphthous stomatitis (figure 2). table 1. the data of patients. figure 1. recurrent herpes in patients with covid-19 positive infection: a) herpes lesion on the labial surface of the lower lip; b) herpes lesions on the labial surface of the lower lip; c) herpes lesion on the labial surface of the upper; d) herpes lesions on the hard palate. figure 2. aphthous stomatitis in patients with covid-19 positive infection: a) aphthous lesion on the labial surface of the lower lip; b) aphthous lesion on the labial surface of the lower lip. oral symptoms of pa-ents tested posi-ve for covid-19 with mild severitya cross-sec-onal study vol 10 no 1 (2022) doi 10.5195/d3000.2022.375 h#p://den*stry3000.pi#.edu the accurate percentage of each item that was observed in the mouth area was mentioned in table 2. all these symptoms are only observed, and nothing was done for their treatment. discussion one of the biggest and world-spread pandemics of this century is covid19. this contiguous infection has threatened all people all around the world [16]. it has demonstrated a wide range of symptoms, from asymptomatic to fatal complications [17], but the most common symptoms are coughs, fever, fatigue, and dyspnea [18,19]. there is no definitive treatment for this infection. the most important management is focused on supportive and palliative care [20]. until now, june 2022, over 547,512,638 cases have been reported worldwide [21]. oral findings frequency percent cumulative percent no change 12 21.8 21.8 recurrent aphthous stomatitis 1 1.8 23.6 bitter taste 1 1.8 25.5 concentrated salvia 1 1.8 27.3 recurrent herpes 5 9.1 36.4 recurrent herpes dry throat 1 1.8 38.2 loss of tasteconcentrated salviarecurrent herpes 1 1.8 40.0 xerostomia 9 16.4 56.4 xerostomia bitter taste 1 1.8 58.2 xerostomiarecurrent aphthous stomatitis 1 1.8 60.0 xerostomiabitter taste 4 7.3 67.3 xerostomiabitter tasteburning throat 1 1.8 69.1 xerostomiabitter taste concentrated salviaburning gingiva 1 1.8 70.9 xerostomiabitter taste recurrent herpes 1 1.8 72.7 xerostomiaconcentrated salvia 6 10.9 83.6 xerostomiaconcentrated salvia bitter taste 1 1.8 85.5 xerostomiadry throat 1 1.8 87.3 xerostomiaburning gingiva recurrent aphthous stomatitis 1 1.8 89.1 xerostomiarecurrent herpes 3 5.5 94.5 xerostomiarecurrent herpes bitter taste 1 1.8 96.4 xerostomiarecurrent herpes concentrated salvia 1 1.8 98.2 xerostomialoss of tasteburning gingivarecurrent herpes 1 1.8 100.0 total 55 100.0 table 2. oral findings in patients tested positive for covid-19. oral symptoms of pa-ents tested posi-ve for covid-19 with mild severitya cross-sec-onal study vol 10 no 1 (2022) doi 10.5195/d3000.2022.375 h#p://den*stry3000.pi#.edu in the present study, 55 patients with covid-19 during their period of the disease have been assessed for their overall and oral symptoms. the results showed that most of the patients had oral symptoms even to a very small extent. for example, 43.2% had xerostomia with other symptoms. patients complained about xerostomia more than other signs. one study showed that sarscov-2 targets ace2-localizing salivary glands, this virus impairs the secretion function of those glands, resulting in low salivary flow and xerostomia [22]. however, the most common symptoms seen in the mouth were recurrent herpes, and recurrent aphthous stomatitis respectively. other manifestations such as burning gingiva, bitterness, or loss of taste were observed too. this could be as related to the impaired immune system [23] and immunologic dysregulation and a high level of pro-inflammatory cytokines [24, 25]. it seems that lymphopenia, hyperinflammatory state, and cytokines all have important roles in the pathology of covid-19 [26]. immune cells like macrophages could be part of the disease progression. macrophages have shown a substantial production of il-6, suggesting they may help with the excessive inflammation in covid19 infection [27]. because of the tcell lymphopenia and their decreased function, immunoglobulins delivered by b cells represent the principal arm of the immune system to battle the virus [28]. nonetheless, the exact pathophysiologic mechanism of covid-19 remains still largely unknown. boosting the immune system to help combat the virus is important [29]. research showed clinical olfactory and gustatory symptoms are prevalent in covid-19 patients and mostly emerged in the first three days [30]. the study of chenghao qiu et al. on covid-19 patients showed that olfactory and/or gustatory dysfunction may address early or even the main symptom of sars-cov2 infection in both grown-ups and children. 19% of patients experienced olfactory or gustatory dysfunction preceding some other covid-19 symptoms, also they considered how long did it take to experience improvement [31]. we measure the time for improvement in our study as well. healing of the symptoms was seen after a while. a ganesan et al. found out covid-19 infection certainly has a significant impact on the oral health of the patients and oral findings are prevalent in the severe form of the disease [32]. in the present study, oral health and manifestations have been assessed in patients with mild to moderate covid-19 symptoms with no hospitalization history. like our study, b iranmanesh et al. showed which manifestations probably will be occurred in the mouth area. they listed aphthous-like lesions, herpetiform/zosteriform lesions, white/red plaques, em-like lesions, necrotizing periodontal disease, and some other lesions in their study. the other subject that they demonstrated was, they saw lack of oral hygiene, stress, immunosuppression, vasculitis, opportunistic infections, and hyperinflammatory response secondary to covid-19 are the most important predisposing factors to develop lesions mouth lesions in covid-19 patients [33]. amorim dos santos et al. reported that taste alterations are the most prevalent oral manifestation in covid-19 patients and with moderate certainty of evidence it is associated with mild/moderate severity and female sex [8]. some of the patients in this study reported bitter taste in their mouths along with their sickness. some research suggested some medications such as corticosteroids and antihistamines for their anti-inflammation effects or antiseptic mouthwash. palliative drugs, antifungal, and antibacterial therapy may also prescribe. however, other studies have declined to consider any medication or treatment for mild or asymptomatic patients because of the nature of self-healing. nsaids may worsen the symptoms of covid-19, but there is no evidence yet [34]. carreras-presas cm et al. suggested to treat dysgeusia or taste disorder, multiple ulcers on the palate and internal lip, desquamative gingivitis, and pain in the tongue by valaciclovir 500 mg every 8 h for 10 days, and oral symptoms of pa-ents tested posi-ve for covid-19 with mild severitya cross-sec-onal study vol 10 no 1 (2022) doi 10.5195/d3000.2022.375 h#p://den*stry3000.pi#.edu topical antiseptics with chlorhexidine and hyaluronic acid; within 7-10 days the symptoms will disappear [35]. another case report of a 56-year-old woman for treating dry mouth suggested using anti-inflammatory and antiasthmatic drugs such as montelukast, naproxen and acetaminophen for a period of 7 days [36]. orcina f et al said using phtalox mouth wash for 12 hours to 4 days could be useful for curing aphthous stomatitis. a few publications indicated that oral lesions are caused by reduced immunity due to viral infection, opportunistic or secondary infection or covid-19 treatment [3539]. many patients with covid-19 infection may show oral symptoms, and it might be the earliest one. so, dentists should be conscious of these symptoms. it is possible that the patients do not have covid-19 symptoms, but they have oral signs and symptoms which are related to the virus [39]. these lesions also may be the secondary response to medications. controlling the infection transmission for community and health care staff and also diagnosis of the asymptomatic covid-19 makes these findings important. a further clinical study with a larger sample size and based on gender and age groups is recommended to confirm the link between oral lesions and the covid19 virus. conclusion xerostomia, recurrent herpes, and recurrent aphthous stomatitis are the three most common symptoms in the evaluated patients of this study. the oral symptoms in patients with covid-19 infection might be the earliest symptom even if the patient is asymptomatic and he is not aware of his sickness. references 1. laboratory diagnosis of emerging human coronavirus infec7ons–the state of the art. emerging microbes & infec7ons. loeffelholz mj, tang y-w. 2020;9(1):747-56. 2. cutaneous manifesta7ons and considera7ons in covid-19 pandemic: a systema7c review. dermatologic therapy. seirafianpour f, sodagar s, pour mohammad a, panahi p, mozafarpoor s, almasi s, et al. 2020;33(6):e13986. 3. management of undifferen7ated cri7cally ill covid-19 pa7ents. cri7cal care of covid-19 in the emergency department: springer; tran qk. 2021. p. 11-7. 4. clinical characteris7cs of coronavirus disease 2019 in china. guan w-j, ni z-y, hu y, liang w-h, ou c-q, he j-x, et al. new england journal of medicine. 2020;382(18):170820. 5. the angiotensin-conver7ng enzyme 2 (ace2) receptor in the preven7on and treatment of covid-19 are dis7nctly different paradigms. mclachlan cs. clinical hypertension. 2020;26(1):1-3. 6. review on the oral manifesta7ons of covid-19 disease and mucormycosis. negi p, kumari p, negi s. journal of advanced medical and dental sciences research. 2021;9(6):647. 7. imaging diagnos7cs and pathology in sars-cov-2-related diseases. scimeca m, urbano n, bonfiglio r, montanaro m, bonanno e, schillaci o, et al. interna7onal journal of molecular sciences. 2020;21(18):6960. 8. oral manifesta7ons in pa7ents with covid-19: a living systema7c review. amorim dos santos j, normando a, carvalho da silva r, acevedo a, de luca canto g, sugaya n, et al. journal of dental research. 2021;100(2):141-54. 9. tissue distribu7on of ace2 protein, the func7onal receptor for sars coronavirus. a first step in understanding sars pathogenesis. hamming i, timens w, bulthuis m, lely a, navis gv, van goor h. the journal of pathology: a journal of the pathological society of great britain and ireland. 2004;203(2):631-7. 10. pathological findings of covid19 associated with acute respiratory distress syndrome. xu z, shi l, wang y, zhang j, huang l, zhang c, et al. the lancet respiratory medicine. 2020;8(4):420-2. 11. extensive hard palate hyperpigmenta7on associated with chloroquine use. godinho gv, paz allm, de araújo gomes epa, garcia cl, volpato ler. bri7sh journal of clinical pharmacology. 2020;86(11):2325-7. 12. a comprehensive review on drug reposi7oning against coronavirus disease 2019 (covid19). rameshrad m, ghafoori m, mohammadpour oral symptoms of pa-ents tested posi-ve for covid-19 with mild severitya cross-sec-onal study vol 10 no 1 (2022) doi 10.5195/d3000.2022.375 h#p://den*stry3000.pi#.edu ah, nayeri mjd, hosseinzadeh h. naunyn-schmiedeberg's archives of pharmacology. 2020;393(7):1137-52. 13. covid-19: symptoms and severity [internet]. 18 april 2022. 14. co-infec7ons: poten7ally lethal and unexplored in covid-19. cox mj, loman n, bogaert d, o'grady j. the lancet microbe. 2020;1(1):e11. 15. 50 years on. la clinica terapeu7ca. petrini c. helsinki 2014;165(4):179-81. 16. a new coronavirus associated with human respiratory disease in china. wu f, zhao s, yu b, chen y-m, wang w, song z-g, et al. nature. 2020;579(7798):2659. 17. epidemiological and clinical characteris7cs of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descrip7ve study. chen n, zhou m, dong x, qu j, gong f, han y, et al. the lancet. 2020;395(10223):507-13. 18. determine the most common clinical symptoms in covid-19 pa7ents: a systema7c review and meta-analysis. alimohamadi y, sepandi m, taghdir m, hosamirudsari h. journal of preven7ve medicine and hygiene. 2020;61(3):e304. 19. clinical course and outcomes of cri7cally ill pa7ents with sarscov-2 pneumonia in wuhan, china: a single-centered, retrospec7ve, observa7onal study. yang x, yu y, xu j, shu h, liu h, wu y, et al. the lancet respiratory medicine. 2020;8(5):475-81. 20. the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak–an update on the status. guo y-r, cao q-d, hong z-s, tan y-y, chen s-d, jin h-j, et al. military medical research. 2020;7(1):1-10. 21. world meters. available from: hpps://wwwworldometersinfo/c oronavirus/#countries. 22. inves7ga7on of covid-19related symptoms based on factor analysis. luo y, wu j, lu j, xu x, long w, yan g, et al. ann palliat med. 2020;9(4):1851-8. 23. the impact of coronavirus infec7ous disease 19 (covid-19) on oral health. dziedzic a, wojtyczka r. oral diseases. 2021;27:703-6. 24. characteris7cs of peripheral lymphocyte subset altera7on in covid-19 pneumonia. wang f, nie j, wang h, zhao q, xiong y, deng l, et al. the journal of infec7ous diseases. 2020;221(11):1762-9. 25. covid-19, immune system response, hyperinflamma7on and repurposing an7rheuma7c drugs. tufan a, güler aa, matucci-cerinic m. turkish journal of medical sciences. 2020;50(si-1):620-32. 26. the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage ac7va7on syndrome-like disease. mcgonagle d, sharif k, o'regan a, bridgewood c. autoimmunity reviews. 2020;19(6):102537. 27. covid-19 and the immune system. paces j, strizova z, daniel s, cerny j. physiological research. 2020;69(3):379. 28. sars-cov-2 seroconversion in humans: a detailed protocol for a serological assay, an7gen produc7on, and test setup. stadlbauer d, amanat f, chromikova v, jiang k, strohmeier s, arunkumar ga, et al. current protocols in microbiology. 2020;57(1):e100. 29. immune response in covid-19: a review. chowdhury ma, hossain n, kashem ma, shahid ma, alam a. journal of infec7on and public health. 2020;13(11):1619-29. 30. covid-19 symptoma7c pa7ents with oral lesions: clinical and histopathological study on 123 cases of the university hospital policlinic of bari with a purpose of a new classifica7on. favia g, tempesta a, barile g, brienza n, capodiferro s, ves7to mc, et al. journal of clinical medicine. 2021;10(4):757. 31. olfactory and gustatory dysfunc7on as an early iden7fier of covid-19 in adults and children: an interna7onal mul7center study. qiu c, cui c, hautefort c, haehner a, zhao j, yao q, et al. otolaryngology– head and neck surgery. 2020;163(4):714-21. 32. oral manifesta7ons of covid-19 infec7on: an analy7cal crosssec7onal study. ganesan a, kumar s, kaur a, chaudhry k, kumar p, dup n, et al. journal of maxillofacial and oral surgery. 2022:1-10. 33. oral manifesta7ons of covid-19 disease: a review ar7cle. iranmanesh b, khalili m, amiri r, zartab h, aflatoonian m. dermatologic therapy. 2021;34(1):e14578. 34. unique skin manifesta7ons of covid-19: is drug erup7on specific to covid-19? sakaida t, tanimoto i, matsubara a, nakamura m, morita a. journal of dermatological science. 2020;99(1):62-4. 35. oral vesiculobullous lesions associated with sars-cov-2 infec7on. carreras-presas cm, sánchez ja, lópez-sánchez af, jané-salas e, pérez mls. oral diseases. 2020. oral symptoms of pa-ents tested posi-ve for covid-19 with mild severitya cross-sec-onal study vol 10 no 1 (2022) doi 10.5195/d3000.2022.375 h#p://den*stry3000.pi#.edu 36. covid-19 from the perspec7ve of den7sts: a case report and brief review of more than 170 cases. eghbali zarch r, hosseinzadeh p. dermatologic therapy. 2021;34(1):e14717. 37. oral complica7ons of icu pa7ents with covid-19: caseseries and review of two hundred ten cases. hocková b, riad a, valky j, šulajová z, stebel a, slávik r, et al. journal of clinical medicine. 2021;10(4):581. 38. the effect of xylitol on dental caries and oral flora. nayak pa, nayak ua, khandelwal v. clinical, cosme7c and inves7ga7onal den7stry. 2014;6:89. 39. oral mucosal lesions in a covid19 pa7ent: new signs or secondary manifesta7ons? dos santos ja, normando agc, da silva rlc, de paula rm, cembranel ac, santos-silva ar, et al. interna7onal journal of infec7ous diseases. 2020;97:326-8. microsoft word vieira editorial 2016.docx   vol  4,  no  1  (2016)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2016.65           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     the  issue  of  peer-­‐review   alexandre  rezende  vieira     editor  in  chief   department  of  oral  biology,  university  of  pi4sburgh  school  of  dental  medicine,  pi%sburgh  pa,  usa                 cita%on:  vieira  ar.  (2016)  the  issue  of  peer  re-­‐ view.  den$stry  3000.  1:a001   doi:10.5195/d3000.2016.65   received:  november  30,  2016   accepted:    december  12,  2016   published:    december  14,  2016   copyright:  ©2016  vieira  ar.  this   is  an  open  ac-­‐ cess  ar!cle   licensed  under  a  crea!ve  commons   a"ribu%on  work  4.0  united  states  license.   email:  arv11@pi(.edu                it  is  widely  accepted  that  peer-­‐ review  is  the  process  that  validates   the  published  scientific  literature.   however,  it  is  known  to  be  incon-­‐ sistent,  biased,  onerous,  slow,  and   expensive.  the  typical  journal,  such   as  dentistry  3000,  follows  these   common  steps:   1. the  editor,  upon  receiving  a   manuscript  submission,  will   decide  if  the  paper  should  be   peer-­‐reviewed.  if  the  decision   is  “no,”  the  editor  will  reject   the  paper.  if  the  decision  is  yes,   the  process  of  inviting  individ-­‐ uals  to  revise  the  manuscript   begins.’   2. the  peer-­‐reviewers  provide  a   general  impression  about  the   paper.  typically,  these  include   suggestions  to  revise  the  paper   and  resubmit.     3. the  editor  receives  from  the   authors  a  revised  version  of   the  manuscript  and  he-­‐she  will   decide  if  the  new  version  is  ac-­‐ ceptable  or  not,  and  often  will   send  it  to  peer-­‐review  again,   trying  to  have  the  same  re-­‐ viewers  look  at  the  revised   manuscript.  this  process  can   have  multiple  iterations  with   the  authors.   this  peer-­‐review  process  gives   credibility  to  publications  and  is  not   only  used  for  defining  which  papers   should  be  published,  but  also  for   making  decisions  on  funding  of  re-­‐ search  projects.    additionally,  jour-­‐ nals  quite  often  suggest  to  authors   their  acceptance  rates,  some  proud   to  reject  60%  or  more  of  the  sub-­‐ missions  without  even  sending  to   peer-­‐review.    journals  define  them-­‐ selves,  not  only  thematically,  but   also  on  the  types  of  papers  they   publish.  many  journals  emphasize   originality  and  positive  results.  it  is   more  difficult  to  publish  the  results   of  a  well-­‐designed  clinical  trial   when  they  are  negative.     therefore,  the  challenge  is  in   how  to  improve  the  peer-­‐review   process.  dentistry  3000  is  trying  to   address  that  by  increasing  con-­‐ sistency  among  reviews  and  by  pub-­‐ lishing  independently  from  the  au-­‐ thors  and  their  affiliations.  dentistry   3000  has  published  close  to  70%  of   its  submissions.  the  main  goal  is  to   disseminate  information,  and  pub-­‐ lish  in  all  areas  and  topics  relevant   to  dental,  oral  and  craniofacial  re-­‐ search.  authors  are  not  charged   publication  fees.    once  there  is   enough  material,  dentistry  3000’s   contents  will  be  listed  in  pubmed.  in   the  future,  scientific  publications   will  receive  bigger  emphasis  on  re-­‐ actions  to  papers  after  publishing,   rather  than  the  delayed  peer-­‐review   process,  which  could  be  stream-­‐ lined  to  be  less  demanding,  since   this  process  will  continue  to  rely  on   trust.  alternatives  such  as  evaluat-­‐ ing  raw  data  are  too  demanding  and   time  consuming  to  be  feasible.   726 2024 an#microbial efficiency of hypochlorous acid incorpora#on and its effect on surface proper#es of irreversible hydrocolloid materials vol 13 no 2 (2024) doi 10.5195/d3000.2024.726 h#p://den*stry3000.pi#.edu antimicrobial efficiency of hypochlorous acid incorporation and its effect on surface properties of irreversible hydrocolloid materials shorouq m. abass, bayan s. khalaf, moamin i. issa, aseel mohammed al-khafaji college of dentistry, university of baghdad/ baghdad, iraq abstract objec&ve: conven&onal approaches for disinfec&on, including spraying and immersion, resulted in only surface disinfec&on of impressions. as a result, self-disinfec&ng impression materials incorporated with an&microbial compounds require more extensive studies. the incorpora&on of a disinfectant into irreversible hydrocolloid impression materials could eliminate the need for the disinfec&on step by conven&onal approaches, including spraying and immersion which only result in surface disinfec&on of impressions. the study was aimed to inves&gate the effect of incorpora&on of hypochlorous acid in irreversible hydrocolloid materials on an&microbial efficiency, detail reproduc&on, and dimensional stability. materials and methods: hypochlorous acid (hocl) was used in two concentra&ons, 100 ppm and 200 ppm, and mixed with alginate powder to compare with the control group (dis&lled water mixed with alginate). candida albicans, staphylococcus aureus, and pseudomonas aeruginosa were chosen for assessing the an&microbial ac&vity with the disk diffusion test. in addi&on, the dimensional stability and reproduc&on of details were tested. results: the results revealed that both hocl concentra&ons of 100 ppm and 200 ppm imposed significant an&microbial ac&vity against all three tested microorganisms. there was no significant difference regarding reproduc&on of details, but the addi&on of the an&microbial had a significant adverse effect on the alginate’s dimensional stability. conclusion: it may be concluded that the incorpora&on of hocl into irreversible hydrocolloid impression material resulted in an impression with an&microbial ac&vity. in addi&on, there was no effect on the impression materials ability to reproduce surface details, but the an&microbial addi&on may affect its dimensional stability. keywords: an$microbial efficiency, hypochlorous acid, incorpora$on, irreversible hydrocolloid, self disinfec$ng cita;on: abass sm, et al. (2024) an;microbial efficiency of hypochlorous acid incorpora;on and its effect on surface proper;es of irreversible hydrocolloid materials. den;stry 3000. 2:a001 doi:10.5195/d3000.2024.726 received: september 2, 2024 accepted: september 14, 2024 published: october 7, 2024 copyright: ©2024 hammed ss, et al. this is an open access ar;cle licensed under a crea;ve commons awribu;on work 4.0 united states license. email: shorouq.m.abass@codental.uobaghdad.edu.iq introduction controlling biologically associated risks in medical services and dental care clinic environments is one of the crucial measures necessary for preventing cross contamination among health workers and patients. the dentist requires an efficient, affordable, readily available, harmless, and practical disinfecting agent for controlling infectious diseases [1,2]. prosthetic appliances and their construction steps are a source of contamination, starting from the impression-making procedure, as they are easily polluted by the saliva and/or blood of the patient. as a result, strict measures must be implemented in dental offices and labs to minimize the risk of cross infection [3,4]. several methods have been used for disinfecting dental impressions. some of which are ultraviolet c (uvc) radiation, gaseous ozone, or using chemical disinfectant like 2% glutaraldehyde, 0.5% sodium hypochlorite, or propolis [5-9]. it is recommended that a hospitalgrade disinfecting agent be used to disinfect dental impressions. there is shared agreement that further research is necessary to an#microbial efficiency of hypochlorous acid incorpora#on and its effect on surface proper#es of irreversible hydrocolloid materials vol 13 no 2 (2024) doi 10.5195/d3000.2024.726 h#p://den*stry3000.pi#.edu achieve the best chemical disinfection composition, concentration, and ideal exposure duration. also, there is always a need to evaluate the interaction between impression materials and disinfecting solutions [10,11]. irreversible hydrocolloid impression materials are associated with high bacterial contamination because of their hydrophilicity [12,13]. extensive studies have been done on various disinfecting agents, techniques, exposure durations, and antimicrobial chemicals. most studies advised the use of sodium hypochlorite or glutaraldehyde as disinfectants, although they had adverse effects on the dimensional stability and detail reproduction [14]. also, it is recommended that sodium hypochlorite at 0.5% or iodophors could be used for irreversible hydrocolloids (alginate) impression disinfection by spraying, as prolonged immersion can lead to damage and deterioration [2]. however, there is still a need for a leading global disinfectant. hypochlorous acid (hocl) is one of the most efficient disinfectants against germs while being completely harmless, nontoxic, and natural [15]. hocl has a wide range of uses, such as in the treatment of wounds, dentistry, and as an antibacterial and virucidal agent [16,17]. hocl was shown to have a wide range of antimicrobial activity for the suppression of several gram positive and gram-negative pathogens. at the same time, it had no detrimental side effects such as mucosal irritation or discoloration of the extrinsic tooth surface and restoration [18,19]. in addition, c. albicans, s. aureus, and p. aeruginosa were effectively reduced in count with hypochlorous acid spray disinfection technique [20]. the effectiveness of disinfection on impression materials is widely overlooked in dental practices. since none of the available impression materials were specifically developed for microbial decontamination, disinfecting solutions may have an undesirable effect on the dimensional stability of impressions and the fabrication of the gypsum model. the disinfection procedure for impression materials was evaluated by various studies to assess the surface characteristics and dimensional stability [21,22]. dental impressions were better disinfected with the immersion then the spray disinfection technique [23]. some limitations of the immersion and spray disinfection procedures have been identified, including dimensional changes and inadequate disinfection, respectively. incorporation of disinfectants into irreversible hydrocolloid impression materials has been developed to address the shortcomings of spray and immersion disinfection procedures [24]. therefore, this study was aimed to evaluate the effect of incorporating hocl at two concentration of 100 and 200 ppm on some properties of irreversible hydrocolloid materials in addition to its antimicrobial efficiency. methods this work was approved by the research ethics committee of the college of dentistry, university of baghdad (ref. number 660, project number 660222 at 13.9.2022). the participants gave a written informed consent for the study. specimens were prepared from an irreversible hydrocolloid impression material (alginate, zhermack, tropicalgin, italy) and according to the requirements for each test. the powder/liquid ratio for mixing the impression material was as recommended by the manufacturer. however, for the test groups, instead of distilled water (dw), hocl was added at an#microbial efficiency of hypochlorous acid incorpora#on and its effect on surface proper#es of irreversible hydrocolloid materials vol 13 no 2 (2024) doi 10.5195/d3000.2024.726 h#p://den*stry3000.pi#.edu concentrations of 100 and 200 ppm. specimens’ grouping the total number of specimens included in this study were 75 specimens. they were subdivided according to the test into 45 specimens for the antimicrobial test and 30 specimens for reproduction of details test and dimensional stability test. the following represent the three main specimen groups for each test: dw (control group): alginate mixed with distilled water (no additive). hocl 100: alginate mixed with 100 ppm of hocl. hocl 200: alginate mixed with 200 ppm of hocl. hypochlorous acid disinfectant solution preparation the hypochlorous acid disinfection solutions were produced on-site with the use of the eco one system (ecoloxtech, west palm beach, fl., usa) by adding one teaspoon of white vinegar (5%) and 2 g of non-iodized salt (kosher salt) to one liter of water. the manufacturer’s instruction were followed for producing the two hocl concentrations of 100 ppm and 200 ppm [4]. specimen preparation for antimicrobial test forty-five alginate specimens with 1 mm thickness and 12 mm in diameter were prepared and distributed evenly for the three groups. these specimens were fabricated from alginate powder mixed with sterile distilled water, 100 ppm of hocl, or 200 ppm of hocl for 45 seconds using hand mixing in a rubber bowl and with a spatula corresponding to the instructions of the manufacturer. the end of a 12 mm internal diameter disposable plastic hypodermic syringe was cut off and filled with the alginate mix. the set material was pushed out of the syringe by 1 mm thickness and sliced at the end of the syringe with a number 11 surgical blade [5]. antimicrobial activity test staphylococcus aureus, pseudomonas aeruginosa, and candida albicans were used in this study for testing the antimicrobial activity test. staphylococcus aureus was used as a grampositive bacteria, pseudomonas aeruginosa was chosen as a gramnegative bacteria, and candida albicans for fungi [25]. isolation of the microorganisms was from patients at the teaching hospital of baghdad college of dentistry in accordance with the ethical approval of the ethical committee and a written informed consent obtained from the patients. the vitek 2 was used for identification of the three microorganisms [2628]. the microorganisms were cultivated and incubated for 24 hours in a mueller-hinton broth. the antimicrobial activity for each group was tested separately with the disk diffusion test against the different microorganisms; candida albicans, staphylococcus aureus, and pseudomonas aeruginosa. inoculum suspensions for each of the above-mentioned microorganisms was prepared with a turbidity to the 0.5 mcfarland standard, which corresponds to approximately 1.5× 108 colony forming unit (cfu)/ml. three specimens were positioned evenly spaced apart in each petri dish. then, inoculum suspension for each microorganism (1.5× 108 cfu/ml) was seeded in 15 ml of mueller-hinton broth and poured over the specimens and according to each group. the petri dishes were kept for 60 minutes at room temperature to allow the diffusion of antimicrobial agent. then, they were incubated aerobically at 37°c for 24 hours. the inhibition zones, after incubation, became evident around the specimens. the antibiotic zone scale was used to an#microbial efficiency of hypochlorous acid incorpora#on and its effect on surface proper#es of irreversible hydrocolloid materials vol 13 no 2 (2024) doi 10.5195/d3000.2024.726 h#p://den*stry3000.pi#.edu measure the inhibition zones for evaluation of the antimicrobial effect [29]. preparation of the specimens for the reproduction of details test and dimensional stability test the surface detail reproduction, and dimensional stability of irreversible hydrocolloid impression materials were evaluated with the use of a ruled test block according to iso specification number 1563:1990 for alginate impression material [30]. the alginate impression material was hand mixed according to the manufacturer’s instructions, applied, and maintained against the ruled test block for a minimum of 3 minutes before removal. this ruled test block had three lines (50±8 µm, 20±4 µm and 75±8 µm wide) inscribed on the superior surface with crosslines x and x’, as seen in figure 1. a ring mold surrounded the test block around the border and acted as a mold for the alginate impression material and was used for preparation of thirty specimens, 10 specimens for each test group. figure 1. diagram showing the dimensions of the ruled test block used for dimensional stability and reproduction of details tests. dimensional stability measurement a represented the measurement of the distance between the crossline x to x’ on the ruled test block while the same measurement on the alginate impression specimens was recorded as measurement b. the measurements were made at the edges of the cross lines [30]. measurements were made for each alginate impression specimen in every group and the following formula was used for calculating the dimensional change [31]: dimensional change%(percentage) = (a − b) /a × 100 reproduction of details the alginate impression specimens were inspected visually with lowangle illumination without magnification immediately after separation from the ring mold and test block. the reproduction of details for the intended line (50±8 µm) reproduced in the alginate impression specimen was considered satisfactory if continuous for the full 25 mm between the crosslines, according to iso specification number 1563:1990 [30]. the data for all tests were statistically analyzed with graphpad prism version 10.2.0 (392) results all the tested groups showed inhibition zones for all tested microorganisms with an increase in the diameter of the zone with the higher concentration of hocl (figure 2). candida albicans was the most sensitive to hocl. in contrast, the control group (dw) did not show any inhibition zone for any of the tested microorganisms. there was a significant difference between the groups for each of the different microorganisms when tested for antimicrobial activity (figures 3-5). further post hoc test with tukey's hsd showed significant differences between the control group and each of the tested groups with exception for staphylococcus aureus microorganism which was not significant (p= 0.1591) between an#microbial efficiency of hypochlorous acid incorpora#on and its effect on surface proper#es of irreversible hydrocolloid materials vol 13 no 2 (2024) doi 10.5195/d3000.2024.726 h#p://den*stry3000.pi#.edu the hocl 100 and hocl 200 test groups. dimensional stability of alginate was affected by the addition of hocl. statistical analysis for the dimensional stability test with one way anova and the post hoc test (tukey's hsd) showed significant differences p˂ 0.0001 between the control group and each of the tested groups (hocl 100 and hocl 200). it was not significant (p=0.6784) between the hocl 100 and hocl 200 test group as shown in figure 6. the results of details reproduction test presented a well-defined, sharp detail and continuous line for the full 25 mm between cross lines for all specimens in control group and tested groups (100 and 200 ppm hocl). the means and standard deviations can be seen in figure 7 for the values of details reproduction for all groups. figure 2. inhibition zone around test group specimens for a) c. albicans, b) s. aureus, c) p. aeruginosa in which dw represent control group, 100 represent hocl 100, and 200 represent hocl 200 test group. an#microbial efficiency of hypochlorous acid incorpora#on and its effect on surface proper#es of irreversible hydrocolloid materials vol 13 no 2 (2024) doi 10.5195/d3000.2024.726 h#p://den*stry3000.pi#.edu figure 3. means and standard deviations of inhibition zones (in mm), one way anova and tukey's hsd statistical analysis of disk diffusion test for candida albicans. (****) represented p<0.0001 and (**) represent p= 0.0032. figure 4. means and standard deviations of inhibition zones (in mm), one way anova and tukey's hsd statistical analysis of disk diffusion test for staphylococcus aureus. (****) represented p<0.0001 and (ns) represent p value= 0.1591. figure 5. means and standard deviations of inhibition zones (in mm), one way anova and tukey's hsd statistical analysis of disk diffusion test for pseudomonas aeruginosa. (****) represented p<0.0001 and (***) represented p= 0.0007. an#microbial efficiency of hypochlorous acid incorpora#on and its effect on surface proper#es of irreversible hydrocolloid materials vol 13 no 2 (2024) doi 10.5195/d3000.2024.726 h#p://den*stry3000.pi#.edu figure 6. means of dimensional stability in (%) and standard deviations, one way anova and tukey's hsd statistical analysis of dimensional stability test. (****) represented p<0.0001 and (ns) represented p= 0.6784. figure 7. means and standard deviations of details reproduction test. discussion disinfection is necessary for the different steps in prosthodontic treatment to prevent cross contamination starting from diagnosis to delivery of the prosthesis. this includes different impression materials, dental casts, occlusal rims, and dentures. they may be disinfected by different methods like microwave irradiation, immersion, or spraying with a disinfectant such as solo and sodium hypochlorite, or incorporating chemicals into gypsum materials like povidone iodine, glutaraldehyde, or sodium hypochlorite. these methods of disinfection and chemicals may have some influence on the properties of the materials being disinfected [4,32-38]. disinfection of irreversible hydrocolloid impression material was generally conducted by spraying or immersing the impression in disinfectant solutions. however, oral microbes are easily integrated into the impression materials during the setting step, so spraying or soaking the impression provides merely surface decontamination. as a result, there is a need for the development of self-disinfecting impression materials [39,40]. an ideal disinfecting agent should be harmless to the surface of the impressions, noncorrosive, efficient in a variety of forms, and reasonably priced [20]. hocl meets all those requirements in addition to that it exhibits broadspectrum antibacterial, antiviral, and antifungal properties [41,42]. in this study hocl was incorporated into irreversible hydrocolloid impression material to evaluate its antimicrobial efficacy and its effect on the impression material’s dimensional stability and reproduction of details. the incorporation of hocl in alginate in two concentrations of 100 ppm and 200 ppm resulted in anti-microbial properties of the alginate material against the different microorganisms used in this study. this could be explained by the microbicidal activity of hocl against multiple grampositive bacteria, gram-negative bacteria, and yeasts [43]. this microbicidal activity resulted from interaction of hocl with different functional protein groups, an#microbial efficiency of hypochlorous acid incorpora#on and its effect on surface proper#es of irreversible hydrocolloid materials vol 13 no 2 (2024) doi 10.5195/d3000.2024.726 h#p://den*stry3000.pi#.edu involving chlorination of amino groups and oxidation of thiol groups of cysteine residues, thioether groups of methionine residues, disulfide groups, tryptophan residues, and heme groups [44-46]. these interactions impair the chain of electron transport and cause a reduction in the amount of adenine nucleotides [44], which are the major contributors in energy storage and transfer. others reported that hocl specifically bonds to the unsaturated lipid layer which subsequently results in disrupting cellular integrity [18, 41]. the antimicrobial efficacy of hocl in the specimens was more effective with the higher concentration. the hocl 200 group was significantly more effective against all tested microorganisms than hocl 100 group and this comes in agreement with chen et al. [47]. in this study it was found that significant dimensional changes resulted from hocl incorporation. this could be attributed to the presence of hocl that may have dissociated in aqueous solution forming hydrogen ion h+ and the hypochlorite group ocl¯. the hypochlorite has high affinity to bond with sodium ions and calcium ions [41], which are present in alginate powder. this resulted in formation of sodium hypochlorite and calcium hypochlorite which may have led to depletion of the water in the mix and affected the powder/water ratio. the water is important for dissociation of calcium sulfate dihydrate reactor present in the alginate powder. the importance of this reactor is to convert the alginate compound from soluble (sodium or potassium alginate) to insoluble (calcium alginate) [48,49]. so, this process may have affected the crosslinking and gelation of alginate, and this may have resulted in permanent deformation of alginate specimens and dimensional changes. the implication of these results is that such dimensional changes in the impressions may lead to formation of dental casts that are dimensionally different from the that of the oral cavity and this may have an unsatisfactory result. however, further investigations need to be done to evaluate the clinical significance of these changes by using different measuring procedures like pouring a simulated clinical cast and measuring the resulted dimensional accuracy [50], or measuring the trueness and precision of the impression using a laser scanner with threedimensional superimposition software [51]. different studies evaluated the effects of disinfectants on the dimensional stability of the alginate impression material with different outcomes. most of them found that disinfection didn’t affect the dimensional accuracy of the alginate. ravishankar et al. [39] reported a non-significant difference in the dimensional stability after incorporation of different concentrations of chlorhexidine, povidone iodine, and benzydamine into alginate. also, a study conducted by benakatti et al. [52] showed no dimensional alterations in alginate after mixing with 0.12% and 0.2% chlorhexidine. other investigations, however, showed dimensional alteration after incorporating disinfecting agents like ismail et al. [48] who found that the addition of different percentages of pvp-iodine powder to alginate impression material resulted in dimensional changes. there was no effect of incorporation of hocl in two concentrations of 100 ppm and 200 ppm on the reproduction of details. there was no loss in the sharpness or continuity of the lines. this could be attributed to the fact that there wasn’t any change observed in the flowability of the unset irreversible hydrocolloid when placed over the an#microbial efficiency of hypochlorous acid incorpora#on and its effect on surface proper#es of irreversible hydrocolloid materials vol 13 no 2 (2024) doi 10.5195/d3000.2024.726 h#p://den*stry3000.pi#.edu mold which was considered to be the most important factor affecting details reproduction [39]. this study has some limitations, one of which was the need for a positive control for evaluating the efficacy of hocl with a well-established disinfectant for the alginate impression material. the microorganisms used were limited to bacteria and fungi and no antiviral activities were tested. the other properties of the alginate material were not evaluated and need to be tested after the incorporation of hocl including the working time, setting time, wettability, change in color, tear strength, and compatibility with the gypsum products. in addition, other antimicrobial tests could be used as the viable count method and evaluating the microbial adhesion and antibiofilm effect after hocl incorporation. also, the antiviral activity of the incorporation method could be evaluated against different orally transmitted viruses as herpes simplex virus 1 (hsv1) and corona virus (sars-cov-2). conclusions the incorporation of 100 ppm and 200 ppm hocl into irreversible hydrocolloid impression material results in an impression material with antimicrobial activity against candida albicans, staphylococcus aureus and pseudomonas aeruginosa. this incorporation adversely affects the dimensional stability of the impression material. therefore, this restricts the use of hocl as a disinfecting agent for alginate impressions until further investigations could evaluate the clinical significance of this effect or overcomes such a drawback. the ability of the irreversible hydrocolloid impression material to reproduce details was not affected by the incorporation of hocl into the impression material. references 1. environmental disinfection of a dental clinic during the covid19 pandemic: a narrative insight. scarano a, inchingolo f, and lorusso f.biomed res int. 2020:8896812. doi: 10.1155/2020/8896812. pmid:33145359. 2. recommendations for infection control in dental laboratories during covid-19 pandemic and normalization period. basmacı f, kilicarslan m, and şenel f.journal of basic and clinical health sciences. 2021 mar;5. doi: 10.30621/jbachs.862910. 3. the effect of water and sodium hypochlorite disinfection on alginate impressions. correiasousa j, tabaio a, silva a, pereira t, sampaio-maia b, and vasconcelos m.revista portuguesa de estomatologia, medicina dentária e cirurgia maxilofacial. 2013 mar;54:8–12. doi: 10.1016/j.rpemd.2012.12.003. 4. the effect of hypochlorous acid disinfectant on the reproduction of details and surface hardness of type iii dental stone. jasim zm and abass sm.cureus. 2022 nov;14(11):e32061. doi: 10.7759/cureus.32061. pmid:36600860. 5. the effect of three different disinfection materials on alginate impression by spray method. badrian h, ghasemi e, khalighinejad n, and hosseini n.isrn dentistry. 2012 jul;2012:695151. doi: 10.5402/2012/695151. 6. disinfection of dental impression materials and its effects on dimensional changes: a literature review. dapello-zevallos gm, san miguel-ramírez knm, febre-cuibin ks, gutiérrez-obando da, and tinedo-lópez pl.revista odontológica mexicana. 2022 ;25(2):154-159. 7. effects of various disinfection methods on the material properties of silicone dental impressions of different types and viscosities. wezgowiec j, paradowska-stolarz a, malysa a, orzeszek s, seweryn p, and wieckiewicz m.international journal of molecular sciences. an#microbial efficiency of hypochlorous acid incorpora#on and its effect on surface proper#es of irreversible hydrocolloid materials vol 13 no 2 (2024) doi 10.5195/d3000.2024.726 h#p://den*stry3000.pi#.edu 2022 ;23(18):10859. doi:10.3390/ijms231810859. 8. evaluating the effect of propolis on surface roughness and wettability properties of the addition silicon impression material. anooz n and abass s.kufa medical journal. 2023 jun;19:8694. doi: 10.36330/kmj.v19i1.12105. 9. effect of disinfection with hypochlorous acid on wettability and surface roughness of polyether impression material. kadhim haa and abass sm.journal of medicinal and chemical sciences. 2023 ;6(5):938-945. doi: 10.26655/jmchemsci.2023.5.1. 10. a comparative study between microwave irradiation and sodium hypochlorite chemical disinfection: a prosthodontic view. goel k, gupta r, solanki j, and nayak m.j clin diagn res. 2014 apr;8(4):zc42-6. doi: 10.7860/jcdr/2014/8578.4274. pmid:24959515. 11. surface roughness of polyvinyl siloxane impression materials following chemical disinfection, autoclave and microwave sterilization. al kheraif aa.j contemp dent pract. 2013 may;14(3):483-7. doi: 10.5005/jpjournals-10024-1349. pmid:24171994. 12. carriage of oral flora on irreversible hydrocolloid and elastomeric impression materials. samaranayake lp, hunjan m, and jennings kj.j prosthet dent. 1991 feb;65(2):244-9. doi: 10.1016/0022-3913(91)90169-w. pmid:2051359. 13. efficacy of different disinfectant systems on alginate and addition silicone impression materials of indian and international origin: a comparative evaluation. samra rk and bhide sv.j indian prosthodont soc. 2010 sep;10(3):182-9. doi: 10.1007/s13191-010-0040-y. pmid:21886411. 14. disinfection efficacy of sodium hypochlorite and glutaraldehyde and their effects on the dimensional stability and surface properties of dental impressions: a systematic review. qiu y, xu j, xu y, shi z, wang y, zhang l, et al.peerj. 2023;11:e14868. doi: 10.7717/peerj.14868. pmid:36846444. 15. electrolyzed water as a novel sanitizer in the food industry: current trends and future perspectives. rahman s, khan i, and oh dh.compr rev food sci food saf. 2016 may;15(3):471490. doi: 10.1111/15414337.12200. pmid:33401818. 16. evaluation of liquidand fog-based application of sterilox hypochlorous acid solution for surface inactivation of human norovirus. park gw, boston dm, kase ja, sampson mn, and sobsey md.appl environ microbiol. 2007 jul;73(14):4463-8. doi: 10.1128/aem.02839-06. pmid:17483283. 17. sarada bv, vijay r, johnson r, rao tn, and padmanabham g, fight against covid-19: arci’s technologies for disinfection. trans indian natl. acad. eng. 2020;5(2):349-54. doi: 10.1007/s41403-020-00153-3. 18. hypochlorous acid as a potential wound care agent: part i. stabilized hypochlorous acid: a component of the inorganic armamentarium of innate immunity. wang l, bassiri m, najafi r, najafi k, yang j, khosrovi b, et al.j burns wounds. 2007 apr ;6:e5. pmid:17492050. 19. taurine chloramine produced from taurine under inflammation provides antiinflammatory and cytoprotective effects. kim c and cha yn.amino acids. 2014 jan;46(1):89-100. doi: 10.1007/s00726-013-1545-6. pmid:23933994. 20. antimicrobial efficiency of hypochlorous acid and its effect on some properties of alginate impression material. khalaf b, abass s, al-khafaji a, and issa m.int j dent. 2023 nov;2023:1-7. doi: 10.1155/2023/8584875. 21. dental impression materials and techniques. punj a, bompolaki d, and garaicoa j.dent clin north am. 2017 oct;61(4):779796. doi: 10.1016/j.cden.2017.06.004. pmid:28886768. 22. effectiveness of disinfectants on antimicrobial and physical properties of dental an#microbial efficiency of hypochlorous acid incorpora#on and its effect on surface proper#es of irreversible hydrocolloid materials vol 13 no 2 (2024) doi 10.5195/d3000.2024.726 h#p://den*stry3000.pi#.edu impression materials. demajo jk, cassar v, farrugia c, millan-sango d, sammut c, valdramidis v, et al.int j prosthodont. 2016 janfeb;29(1):63-7. doi: 10.11607/ijp.4358. pmid:26757331. 23. guidelines for infection control in dental health care settings--2003. kohn wg, harte ja, malvitz dm, collins as, cleveland jl, and eklund kj.j am dent assoc. 2004 jan;135(1):33-47. doi: 10.14219/jada.archive.2004.0019. pmid:14959873. 24. antimicrobial efficacy of irreversible hydrocolloid impression impregnated with silver nanoparticles compared to surface disinfected impressions an in vivo study. rajendran v, suma k, ali sa, karthigeyan r, and kalarani g.j pharm bioallied sci. 2021 jun;13(suppl 1):s532-s536. doi: 10.4103/jpbs.jpbs_565_20. pmid:34447148. 25. the efficacy of three types of disinfectants on the microbial flora from the surface of impression materials used in dentistry—in vitro study. cerghizan d, jánosi km, ciurea cn, popelea o, baloș md, crăciun ae, et al.appl. sci. 2023 ;13(2):1097. doi:10.3390/app13021097. 26. identification and antifungal susceptibility testing of candida species: a comparison of vitek-2 system with conventional and molecular methods. kaur r, dhakad ms, goyal r, haque a, and mukhopadhyay g.j glob infect dis. 2016 oct-dec;8(4):139-146. doi: 10.4103/0974-777x.192969. pmid:27942193. 27. evaluation of the vitek 2 system for identification and antimicrobial susceptibility testing of medically relevant gram-positive cocci. ligozzi m, bernini c, bonora mg, de fatima m, zuliani j, and fontana r.j clin microbiol. 2002 may;40(5):1681-6. doi: 10.1128/jcm.40.5.16811686.2002. pmid:11980942. 28. performance of tdr-300b and vitek®2 for the identification of pseudomonas aeruginosa in comparison with vitek®-ms. moehario lh, tjoa e, putranata h, joon s, edbert d, and robertus t.journal of international medical research.2021 feb;49(2):0300060521989893. doi: 10.1177/0300060521989893. 29. comparison of antimicrobial activities and compressive strength of alginate impression materials following disinfection procedure. alwahab z.j contemp dent pract. 2012 jul;13(4):431-5. doi: 10.5005/jpjournals-10024-1163. pmid:23151687. 30. organization. is, specification for alginate impression material-1563. 1990, geneve: iso. 31. surface detail reproduction and dimensional stability of contemporary irreversible hydrocolloid alternatives after immediate and delayed pouring. kusugal p, chourasiya rs, ruttonji z, astagi p, nayak ak, and patil a.contemp clin dent. 2018 jan-mar;9(1):20-25. doi: 10.4103/ccd.ccd_676_17. pmid:29599578. 32. the effect of disinfectants on the properties of dental gypsum: 1. mechanical properties. abdelaziz km, combe ec, and hodges js.j prosthodont. 2002 sep;11(3):161-7. pmid:12237796. 33. the effect of addition of calcium hypochlorite disinfectant on some physical and mechanical properties of dental stone. abass sm and ibrahim ik.scientific journal published by the college of dentistry–university of baghdad. 2012:36. 34. the effect of solo and sodium hypochlorite disinfectant on some properties of different types of dental stone. al-khafaji am, abass sm, and khalaf bs.j bagh coll dent. 2013 jun;25(2):817. 35. efficiency of different denture disinfection methods. mahmood ma, khalaf bs, and abass sm.global j bio-sci biotech. 2017 ;6:439-4. 36. effect of disinfection with hypochlorous acid on compatibility with impression materials of type iii dental stone. nasser aa and abass sm.(humanities, social and applied sciences) misan journal of academic studies. 2023 ;22(47):339-347. an#microbial efficiency of hypochlorous acid incorpora#on and its effect on surface proper#es of irreversible hydrocolloid materials vol 13 no 2 (2024) doi 10.5195/d3000.2024.726 h#p://den*stry3000.pi#.edu 37. influence of hypochlorous acid on surface roughness and wettability of addition silicon impression material. mohammed mj and abass sm.the egyptian journal of hospital medicine. 2023 ;90(2):3630-3635. 38. effect of microwave disinfection on some properties of gypsum products. abass sm.j bagh coll dent. 2009 ;21(3):47-52. 39. evaluation of efficacy and properties of irreversible hydrocolloid impression material when mixed with various antimicrobial agents. ravishankar y, himashilpa g, srinivas k, sunitha d, modalavalasa h, and kumar ts.international journal of translational medical research and public health. 2019 jul;6:2731. 40. effect of sodium fluoride addition as a disinfectant on some properties of alginate impression material. hussian am and jassim rk.j bagh coll dent. 2015 mar;27(1):70-76. 41. hypochlorous acid: a review. block ms and rowan bg.j oral maxillofac surg. 2020 sep;78(9):1461-1466. doi: 10.1016/j.joms.2020.06.029. pmid:32653307. 42. status report on topical hypochlorous acid: clinical relevance of specific formulations, potential modes of action, and study outcomes. del rosso jq and bhatia n.j clin aesthet dermatol. 2018 nov;11(11):36-39. pmid:30588272. 43. hypochlorous acid: an ideal wound care agent with powerful microbicidal, antibiofilm, and wound healing potency. sakarya s, gunay n, karakulak m, ozturk b, and ertugrul b.wounds. 2014 dec;26(12):342-50. pmid:25785777. 44. redox reactions and microbial killing in the neutrophil phagosome. winterbourn cc and kettle aj.antioxid redox signal. 2013 feb;18(6):642-60.doi: 10.1089/ars.2012.4827. pmid:22881869. 45. hypochlorous acid and taurine-n-monochloramine in periodontal diseases. mainnemare a, mégarbane b, soueidan a, daniel a, and chapple il.j dent res. 2004 nov;83(11):823-31. doi: 10.1177/154405910408301101. pmid:15505230. 46. reaction of hemoglobin with hocl: mechanism of heme destruction and free iron release. maitra d, byun j, andreana pr, abdulhamid i, diamond mp, saed gm, et al.free radic biol med. 2011 jul;51(2):374-86. doi: 10.1016/j.freeradbiomed.2011.04. 011. pmid:21549834. 47. effectiveness of hypochlorous acid to reduce the biofilms on titanium alloy surfaces in vitro. chen cj, chen cc, and ding sj.int j mol sci. 2016 jul;17(7). doi: 10.3390/ijms17071161. pmid:27447617. 48. a self-disinfecting irreversible hydrocolloid impression material mixed with povidone iodine powder. ismail ha, asfour h, and shikho sa.eur j dent. 2016 oct-dec;10(4):507-511. doi: 10.4103/1305-7456.195172. pmid:28042266. 49. restorative dental materials. craig rg and ward m.saunders co. 10th edi. 1997:7783 50. disinfection procedures: their efficacy and effect on dimensional accuracy and surface quality of an irreversible hydrocolloid impression material. rentzia a, coleman dc, o'donnell mj, dowling ah, and o'sullivan m.j dent. 2011 feb;39(2):133-40. doi: 10.1016/j.jdent.2010.11.003. pmid:21093528. 51. a new method to evaluate trueness and precision of digital and conventional impression techniques for complete dental arch. seo k and kim s.appl. sci. 2021 ;11(10):4612. doi:10.3390/app11104612. 52. evaluation of antibacterial effect and dimensional stability of self-disinfecting irreversible hydrocolloid: an in vitro study. benakatti vb, patil ap, sajjanar j, shetye ss, amasi un, and patil r.j contemp dent pract. 2017 oct;18(10):887-892. doi: 10.5005/jp-journals-10024-2144. pmid:28989125. associa'on of dental caries with different abo blood groups vol 12 no 1 (2024) doi 10.5195/d3000.2024.563 h#p://den*stry3000.pi#.edu association of dental caries with different abo blood groups saif saad kamil1, aseel taha khaudhair1, hind thyab hamid1 1college of dentistry, tikrit university, tikrit, iraq abstract background: abo blood type affects general and oral health, as salivary physiochemical features are different among various blood kinds. thus, these may affect the dental caries severity. this work studied the caries experience prevalence in individuals displaying various blood types. this work tested the associa'on between abo blood group and dental caries. also, this work evaluated which abo groups are associated with a higher vulnerability to dental caries. materials and methods: sixty tikrit university students were randomly selected and were put into four groups based on blood type, and dental caries experience was diagnosed and scored based on the index of decay missing filling teeth and surfaces (who, 2013). this index scores caries lesions by severity. results: the blood type o was the most frequent (37%), followed by b, a, and ab types. caries experience (dmfs and dmft) was sta's'cally significantly higher among the b blood group (p<0.05). conclusion: abo blood group was associated with dental caries disease. keywords: dental caries; abo blood group; correla'on; tikrit; oral health; dmfs cita:on: kamil, ss et al. (2024) associa:on of dental caries with different abo blood groups den:stry 3000. 1:a001 doi:10.5195/d3000.2024.563 received: august 12, 2023 accepted: august 25, 2023 published: february 13, 2024 copyright: ©2024 kamil, ss et al. this is an open access ar:cle licensed under a crea:ve commons awribu:on work 4.0 united states license. email: saif1990@tu.edu.iq introduction dental caries (tooth decay) is a common human chronic disease, if not treated, it will destroy the teeth [1]. dental plaque, oral hygiene and diet rich in sugars are significant etiological factors, and other causes as inherited factors, including blood groups, may play a role [2]. abo blood type is the commonest system for clinical decisions, such as blood transfusion and tissue histocompatibility for grafting of tissue and organ transplants. this system includes a, b, ab, and o [3-5]. the o group is known as universal donor, with no antigen, while having a and b antibodies. the ab group does not have antibodies but a and b antigens, so it is known as the universal recipient. the rhesus factor (rh) is usually measured along the abo system. it depends on red blood cell surface proteins [6]. antigens a and b appear on the erythrocyte cell membranes and the antibodies of the plasma [7]. the abo system distribution is complex universally [8]. some differences could appear in various areas in a small country [9]. although the amount of work that was done for several areas of medicine, abo blood types and oral diseases have been less studied [10]. also, the investigation of dental caries' relationship with abo blood group and salivary secretor status has been a lonesome field. yet, some scholars http://dentistry3000.pitt.edu/ associa'on of dental caries with different abo blood groups vol 12 no 1 (2024) doi 10.5195/d3000.2024.563 h#p://den*stry3000.pi#.edu reported low dental caries in secretors’ group than those of nonsecretors [11,12]. chung et.al. in 1965, reported that salivary secretor status and caries score are not related [13]. mazumdar et.al. in 2014 found no significant difference in blood group distributions by dental caries experience [14]. previous studies in iraqi [15,16] showed that the o blood type is the most frequent, followed by b and a, and then the less frequent ab type. blood groups have been employed as genetic markers to investigate the links between certain blood types and certain disorders [17,18]. despite being previously referred to as red cell antigens, these biological processes have nothing to do with red blood cells. it is the antigens on the exterior erythrocyte carbohydrate coating (glycocalyx) that determine whether germs, parasites, and immunological proteins and variation in abo blood types are recognized and attacked [19-23]. different abo blood types appear in different populations, suggesting that some blood types benefit from natural selection in the form of resistance to infectious diseases [24]. aitchison and carmichael were the first to study the distribution of blood groups of randomly selected patients in a dental hospital and people with rampant caries showing that those with o blood suffered more from dental caries and people with the a type were the least affected [25]. in addition, o’rark and lyschon reported a statistical significant difference in the distribution of the blood category in relation to caries experience [26]. in contrast, janghorbani showed that dental caries was less prevalent among 427 soldiers in kerman whose ages were 19 years and had the b blood had the lowest dmf index, whereas soldiers with the ab type had the highest [27]. in addition, the secretors of a had the fewest cavities [28]. yet, barros and witkop studying chileans reported no association between the dmfs scores and abo groups [29]. here, we generated data on the association between dental caries experience and abo blood types in iraqis, which are historically a population less studied. material and methods sixty students in good health from the dentistry college, university of tikrit participated in the study. they had their blood type determined and received an oral exam. we obtained informed consent from the participants before collecting any data. the participant’s age ranged from 18 to 28 years and 22 were males and 38 were females. oral test was conducted in standardized circumstances based on the usual methods of oral health surveys (who, 2013) [30]. oral health examination was performed in an appropriate chair by portable lamps for artificial illumination. sterile disposable mouth mirrors and sterilized dental probe for visual examination of the incisal, occlusal, buccal and lingual caries on the surfaces was used. dmft and dmfs indexes were used to measure caries experience in permanent teeth. determination of blood types was done by agglutination, which is a method that collects distinct particles like red blood cells into masses. determination of blood types was performed at a private laboratory. blood samples were obtained with a punch in the tip of the thumb with lancet needles. three drops of blood were set in a clean glass slide and antiserum a, b, and d were sequentially added to one drop each. antisera were mixed with blood with the tip of toothpicks and let them stand for 2 to 3 minutes noting the result according to the clump formation. results blood group distribution for the sample was 23 participants with type o, 13 with type a, and 12 b and ab each. caries experience (dmfs) mean value per blood group was reported in table 1. individuals type b showed the highest mean dmfs value (14.5; p<0.05). http://dentistry3000.pitt.edu/ associa'on of dental caries with different abo blood groups vol 12 no 1 (2024) doi 10.5195/d3000.2024.563 h#p://den*stry3000.pi#.edu blood group mean dmfs mean dmft group a 7.67 4.74 group b 14.5 6.44 group ab 8.75 5.42 group o 10.1 5.62 p-value 0.04 0.39 discussion the association between blood types and systemic disorders such diabetes mellitus, cancer, genetic disease, skin disease, cardiovascular disease, and dental caries has been the subject of several studies [31-34]. here we found that individuals with abo blood type b had the highest caries experience. evidence suggests that blood groups are critical in the ability or resistance to differentiating the infectious and non-infectious diseases [35,36]. distribution of blood types in the study group with o most prevalent is similar to other geographic areas [37] and contrasted to others as expected [38,39]. individuals with the blood type a had significantly lower dmfs values than those with blood type b and this agrees with a past study that showed dmf index was the lowest in a type and the highest in b [39]. other study, however, found that individuals of blood group b had the lowest numbers of cavities [40]. this discrepancy is perhaps because of differences in sample size and measuring methods of dental caries, aside from geographical, racial, and ethnic differences in blood type distributions. in conclusion, blood type a appears to protect against dental caries. references 1. featherstone jd. dental caries: a dynamic disease process. australian dental journal. 2008 sep;53(3):286-91. 2. al ghamdi as. associagon between abo blood groups and severity of chronic periodonggs. medical science. 2009;16(3). 3. skripal ig. abo system of blood groups in people and their resistance to certain infecgous diseases (prognosis). mikrobiolohichnyi zhurnal (kiev, ukraine: 1993). 1996 mar 1;58(2):102-8. 4. mazumdar p, das uk, goswami s. correlagon between blood group and dental caries in 20-60 years age group: a study. internagonal journal of advanced research. 2014;2(11):413-24. 5. cypriano s, sousa md, wada rs. evaluagon of simplified dmft indices in epidemiological surveys of dental caries. revista de saude publica. 2005;39:28592. 6. skripal ig. abo system of blood groups in people and their resistance to certain infecgous diseases (prognosis). mikrobiolohichnyi zhurnal (kiev, ukraine: 1993). 1996 mar 1;58(2):102-8. 7. kolmakova gn, kononova ll. sudebno-meditsinskaia ekspergza. the prevalence of abo blood groups among persons of nagve nagonality in buryaga. 1999 mar 1;42(2):15-6. 8. vivek s, jain j, simon s, bacur h, supreetha s, haridas r. associagon of abo blood group and rh factor with periodontal disease in a populagon of virajpet, karnataka: a crosssecgonal study. j int oral health. 2013;5(4):30–4. 9. arneberg p, kornstad l, nordbö h, gjermo p. less dental caries among secretor than among non-secretors of blood group substance. european journal of oral sciences. 1976 dec;84(6):362-6. 10. kárpág k, braunitzer g, toldi j, turzó k, virág k, reiche wt, rakonczay z, nagy k. caries and abo secretor status in a hungarian populagon of table 1: caries experience among different blood groups. http://dentistry3000.pitt.edu/ associa'on of dental caries with different abo blood groups vol 12 no 1 (2024) doi 10.5195/d3000.2024.563 h#p://den*stry3000.pi#.edu children and adolescents: an exploratory study. caries research. 2014 apr 1;48(3):17985. 11. chung cs, witkop jr cj, wolf ro, brown ks. dental caries in relagon to ptc taste sensigvity, secretor status, and salivary thiocyanate level. archives of oral biology. 1965 jul 1;10(4):645-53. 12. mazumdar p, das uk, goswami s. correlagon between blood group and dental caries in 20-60 years age group: a study. internagonal journal of advanced research. 2014;2(11):413-24. 13. salih ha. frequency distribugon of abo blood groups and rh phenotypes of blood donors in babylon governorate-iraq. medical journal of babylon. 2009;6(2):268-75. 14. taha jy. frequency of various rh anggens in basrah province, iraq. the medical j basrah university. 2005; 23(1): 53-5. 15. kaya h, gündoğdu m, akarsu e, kiki i, tekin b. the distribugon of blood groups in erzurum. medical j atatürk university 1999; 31: 20–2. 16. turkish red crescent society. www.kizilay.org.tr. 2006. 17. lowe jb. carbohydrateassociated blood group anggens: the abo, h/se and lewis loci. immunobiology of transfusion medicine. 1994:3-6. 18. campi c, escovich l, valdés v, garcía borrás s, racca l, racca a, cotorruelo c, biondi c. secretor status and abh anggens expression in pagents with oral lesions. medicina oral, patología oral y cirugía bucal (internet). 2007 oct;12(6):431-4. 19. mahajan nm, kohad rm. associagon between abo blood groups and periodontal diseases. jisp. 1998;1(2):62-3. 20. oriol ra, le pendu ja, sparkes rs, sparkes mc, crist mi, gale rp, terasaki pi, bernoco m. insights into the expression of abh and lewis anggens through human bone marrow transplantagon. american journal of human genegcs. 1981 jul;33(4):551. 21. oriol r, le pendu j, mollicone r. genegcs of abo, h, lewis, x and related anggens. vox sanguinis. 1986 nov;51(3):161-71. 22. trickec lp, evans pr, maciver ag, smith jl, slapak m. variable localizagon of blood group anggen in group a kidneys. brigsh journal of experimental pathology. 1983 apr;64(2):137. 23. dean l. the mns blood group. in blood groups and red cell anggens . 2005. nagonal center for biotechnology informagon (us). 24. ishikawa i. host responses in periodontal diseases: a preview. periodontol 2007; 43: 9–13. 25. aitchison j. the relagonship between the abo blood mutagons and dental caries. dent prac. 1962;13:93-5. 26. o'roark wl, leyschon c. dentalcaries prevalence as related to blood groups. journal of dental research. 1963 nov;42(6):1530-. 27. janghorbani m, akhavanmahdavi s, masoudi hr. the relagonship between abo blood groups and rh factor with dental caries in soldiers of a military base in kerman. journal of kerman university of medical sciences. 1996 mar 1;3(2):87-91. 28. cypriano s, sousa md, wada rs. avaliação de índices cpod simplificados em levantamentos epidemiológicos de cárie dentária. revista de saúde pública. 2005 apr;39(2):285-92. 29. barros l, witkop jr cj. oral and genegc study of chileans 1960— iii: periodontal disease and nutrigonal factors. archives of oral biology. 1963 mar 1;8(2):195-in27. 30. oral h. survey, basic methods. world health organizagon. 1997. 31. rai p, acharya s, hallikeri k. assessment of abo blood grouping and secretor status in the saliva of the pagents with oral potengally malignant disorders. journal of oral and maxillofacial pathology: jomfp. 2015 may;19(2):164. 32. pai gp, dayakar mm, shaila m, dayakar a. correlagon between “abo” blood group phenotypes and periodontal disease: prevalence in south kanara district, karnataka state, india. journal of indian society of periodontology. 2012 oct;16(4):519. 33. salih k, jouda j, al-jaff s, elhaboby b, shaker e. frequency of abo blood groups and secretor status and their relagon with dental decay in a secgon of students and employees of almustansiriyiah university in iraq. world j exp biosci. 2015;3(2):108-2. 34. moon t. an examinagon of the relagonship of abo blood group and lifespan in a hospitalized populagon in the southeastern united states. 35. mondal b, maig s, biswas bk, ghosh d, paul s. prevalence of hemoglobinopathy, abo and rhesus blood groups in rural areas of west bengal, india. journal of research in medical sciences: the official journal of isfahan university of medical sciences. 2012 aug;17(8):772. 36. kadhum rf, diab bs. abo blood type in relagon in relagon to http://dentistry3000.pitt.edu/ associa'on of dental caries with different abo blood groups vol 12 no 1 (2024) doi 10.5195/d3000.2024.563 h#p://den*stry3000.pi#.edu caries experience and salivary physicochemical characterisgc among college students at aldiwania governorate in iraq. journal of baghdad college of dengstry. 2015;27(4):125-31. 37. dawes c. salivary flow pacerns and the health of hard and so| oral gssues. the journal of the american dental associagon. 2008 may 1;139:18s-24s. 38. kaur s, maykanathan d, lyn nk. factors associated with dental caries among selected urban school children in kuala lumpur, malaysia. archives of orofacial science. 2015 jun 1;10(1). 39. mangal skc, preetha s. relagonships between blood groups and dental carries. int j cur adv res 2017; 6:3082-3083. 40. govindaraju l, jeevanandan g, subramanian em. abo blood grouping: a potengal risk factor for early childhood caries-a cross-secgonal study. indian journal of dental research. 2018 may 1;29(3):313. http://dentistry3000.pitt.edu/ effects of a novel dye containing toothpaste versus regular fluoridated toothpaste for supragingival biofilm reduction in patients with chronic gingivitis: a randomized controlled trial vol 9 no 1 (2021) doi 10.5195/d3000.2021.211 http://dentistry3000.pitt.edu erratum to: effects of a novel dye containing toothpaste versus regular fluoridated toothpaste for supragingival biofilm reduction in patients with chronic gingivitis: a randomized controlled trial. rath, a., chong, j.n., hr, p., teo, b., zhen, n., & yin, o. (2021). dentistry 3000 2021; 9(1). doi: https://doi.org/10.5195/d3000.2021.211 abstract the name of one of the authors was misspelled by the author during the submission process as well as a name of a collaborator in the acknowledgement section. the author’s name chong junning was corrected to chong jun ning. in the acknowledgment section mr. che vern was corrected to mr. yeoh chin vern. the original manuscript has been updated to reflect the aforementioned changes. http://dentistry3000.pitt.edu/ prevalence of dehiscence and fenestration in iraqi population: cbct-based analysis vol 12 no 1 (2024) doi 10.5195/d3000.2024.620 http://dentistry3000.pitt.edu prevalence of dehiscence and fenestration in iraqi population: cbct-based analysis hayder sadiq baker1, hamsa jamal mahdi1, ahmed saad ajeel1 1 al-bayan university , college of dentistry abstract objective: this study utilized the cone-beam computed tomography (cbct) to investigate the prevalence of lower and upper jaw fenestration and dehiscence in individuals with normal patterns, with a particular focus on central incisors to first molars on the two sides. materials and methods: an analysis was conducted on a cohort of 415 patients treated from january 2022 to december 2022 in the periodontal departments of baghdad, sulemania, basra, and najaf. there was a total of 174 males and 241 females that had class i characteristics with normal anterior region patterns. the study focused on the occurrence of fenestration and dehiscence in the dentated area of both the right and left sides. these occurrences were subjected to statistical analysis. results: dehiscence has been seen in 4.89% of the assessed mandibular teeth, whereas fenestration was observed in 0.73% of the same teeth. dehiscences were seen in 9.78% of the examined cases in the upper jaw, while fenestrations were discovered in 5.13% of the cases. these findings indicate a significant difference in the frequency of these conditions between the upper and lower jaws. conclusion: the occurrence of dehiscence in both jaws was found to be greater than the occurrence of fenestration. the observed patterns suggested that these findings are more possibly to be attributed to physiological instead of pathological factors. keywords: periodontal disease, dehiscence, fenestration, cbct, upper jaw, lower jaw. citation: baker, hs et al. (2024) prevalence of dehiscence and fenestration in iraqi population: cbct-based analysis. dentistry 3000. 1:a001 doi: 10.5195/d3000.2024.620 received: november 3, 2023 accepted: january 13, 2024 published: february 13, 2024 copyright: ©2024 baker, hs et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: hayder.sadiq@albayan.edu.iq introduction the health of alveolar bone, which acts as a foundation for the teeth, is commonly overlooked both prior to and following orthodontic treatment. patients with highangle malocclusion have the least amount of alveolar bone thickness [1-4]. misapplied mechanotherapy may lead to alveolar bone thinning, an elongation of the gap between cementoenamel junction (cej) and the alveolar bone (dehiscence), and root penetration into the alveolar bone (fenestration) [5]. the presence of alveolar bone fenestration and dehiscence may result in a reduction in the overall strength of the bone support around teeth. gingival inflammation induced by plaque may have negative implications for both dental and periodontal health when orthodontic movement is performed without adequate bone support [2]. the presence of an undiagnosed buccal alveolar bone deficit may lead to an increased likelihood of treatment relapse [3] and the development of gingival recession, resulting in an aesthetically unfavorable outcome of orthodontic treatment and sensitivity of teeth [4]. bony fenestrations and dehiscences in the alveolar http://dentistry3000.pitt.edu/ mailto:hayder.sadiq@albayan.edu.iq prevalence of dehiscence and fenestration in iraqi population: cbct-based analysis vol 12 no 1 (2024) doi 10.5195/d3000.2024.620 http://dentistry3000.pitt.edu ridge cannot be seen with conventional 2d imaging, but can be assessed by cbct. cbct was utilized for research on alveolar bone abnormalities in kids with cleft lip and palate [5], adults with varying vertical skeletal patterns [6,7], fast maxillary growth in teenagers [8,9], and a range of skeletal malocclusions [10]. although posterior crossbite in adults is common, no research has investigated alveolar bony abnormalities. the comprehensive evaluation of preexisting alveolar bony deficiencies is a crucial diagnostic measure in orthodontic therapy for posterior crossbite, since there is a risk of tooth movement through a thin osseous plate [11, 12] in these patients. to avoid aggravating preexisting periodontal issues, orthodontists should be familiar with the anatomical constraints on tooth mobility [12]. many distinct kinds of fenestration and dehiscence of alveolar bone have been seen in dry skull investigations of people of diverse racial and cultural backgrounds [13-22]. bone dehiscence is seen in 0.99–13.4% of cases, whereas fenestration occurs in 0.23– 16.9% of cases [23]. alveolar bone dehiscence was linked to gingival recession [24], and there is evidence to suggest that orthodontic therapy may exacerbate periodontal disease in patients with alveolar bone shortage [24-27]. in order to mitigate the potential for post-treatment complications, one must critically assess the interaction between roots and bones prior; particularly identifying any abnormalities in alveolar bone. conventional imaging techniques, however--despite their competence in numerous areas--fall short when it comes to reliably detecting alveolar bone dehiscence and fenestration (28-30). contrastingly: cbct emerges as an exceptional tool with a proven track record of accurate identification. both in vivo and in vitro studies have examined cbct's viability as a diagnostic tool for alveolar bone abnormalities; these investigations aim to ascertain its strengths and weaknesses. in cases of both dehiscence and fenestration, cbct robustly demonstrates a negative predictive value; however, its positive predictive value is limited [1,31]. as a result, cbct has the potential to exaggerate bone abnormalities, particularly fenestration. when it comes to orthodontists utilizing bone defect data as a precaution before treatment, however, overestimation might be seen as adding to the side of caution rather than supplying false information. if doctors are aware of the overestimation constraint, they may still utilize the bone defect information obtained by cbct. it is also unclear if such flaws are uncommon or prevalent, or whether they are causally linked to the development of malocclusion. therefore, we set out to use cbct to look at the frequency of bilateral anterior alveolar bone dehiscence as well as fenestration in people with normalcy patterns. materials and methods a total of 415 cases were screened in the periodontal departments of baghdad, sulemania, basra, and najaf from january 2022 to december 2022, and class i persons with normal patterns in anterior area have been chosen. the individuals http://dentistry3000.pitt.edu/ prevalence of dehiscence and fenestration in iraqi population: cbct-based analysis vol 12 no 1 (2024) doi 10.5195/d3000.2024.620 http://dentistry3000.pitt.edu included in the study were selected based on the condition of their upper and lower front teeth. the criteria for evaluating dental occlusion include the following: (1) the presence of bilateral class i molar and canine relationships; (2) an anb angle within the range of 0° to 3°; (3) the absence of abnormal anterior and posterior teeth overjet and overbite; (4) the presence of properly aligned anterior teeth with minimal crowding (<2mm) and negligible spacing between teeth (<0.5mm); (5) a spee curve depth measuring (<2mm); and (6) the absence of significant rotation (<5°) in anterior region. individuals with evident wear were excluded. also, those with defective dentition or supernumerary teeth were not included. patients with periodontal disease or attachment loss exceeding 30% were also excluded from the study. furthermore, people who had previously had restorations, orthodontic treatment, or maxillofacial surgery were excluded from the study. in addition, participants presenting with craniofacial syndromes or evident diseases were also removed from the research. in accordance with the aforementioned criteria, the present study had a sample size of 415 individuals, including 174 males and 241 females. the participants' ages ranged between 17 and 35 years, with a mean age of 26 years. before this study began, all participants received thorough information regarding the utilization of their cbct data. figure 1 illustrates how they offered informed consent by signing the required forms. figure 1. for this research, we utilized cbct data that participants willingly provided their informed consent for by signing pertinent papers. acquisition of cbct data as part of the regular checkup, cbct pictures (kavo, germany) were taken before the dental evaluation and treatment plan were made. ensuring the correct calibration of the ct device, we positioned the patient's head to align with both a parallel frankfort plane and horizontal plane in central occlusion. we utilized version 2.2.1-2019 of our house's 3d planner to examine scan settings; subsequently, we reconstructed data from cbct scans for all 415 cases— generating insightful images in this process. specific reference points namely, the http://dentistry3000.pitt.edu/ prevalence of dehiscence and fenestration in iraqi population: cbct-based analysis vol 12 no 1 (2024) doi 10.5195/d3000.2024.620 http://dentistry3000.pitt.edu cementoenamel junction and most coronal point of alveolar bone on buccal and labial aspects for upper and lower teeth provided views to these images. note the omission of the third molars in our analysis: we based our selection for evaluating alveolar bone dehiscence and fenestration on sun et al. 's methods [18,20]. they chose as did we the largest labiolingual section as a measurement plane; this was applied to both incisors and canines within our sample. we replicated the same measurement variables and reference points from a prior study [32]. after computing and documenting data for all 415 anterior upper and lower teeth, we analyzed the results of both initial measurements in accordance with their respective mean values. statistical analysis: chi-square tests performed within 2018 version of sas have been used for the purpose of determining the statistical significant differences (0.05 and 0.01 probability). results in the lower jaw, we observed a higher prevalence of alveolar bone dehiscence (4.89%) compared to that in the upper jaw (9.78%). likewise, fenestration presented itself more frequently in our findings from the lower jaw (0.73%) than it did within those obtained for the upper jaw (5.13%). we based these conclusions on an analysis involving 415 anterior teeth distributed across both maxilla and mandible regions at our disposal; this provided us with comprehensive insight into this dental phenomenon. the upper jaw exhibited the highest rates of dehiscence and fenestration, while conversely, the lower jaw manifested significantly lower rates. tables 1, 2 and 3 describe the frequency of fenestration and dehiscence in the upper and lower jaws. table 1. comparison between prevalence of lower dehiscence and fenestration in the lower jaw. total sample 409 (%) p-value lower dehiscence 20 (4.89%) 0.0001 lower fenestration 3 (0.73%) -- table 2. comparison between prevalence of upper dehiscence and fenestration in the upper jaw. total sample 409 (%) p-value upper dehiscence 40 (9.78%) 0.0013 upper fenestration 21 (5.1350 -- http://dentistry3000.pitt.edu/ prevalence of dehiscence and fenestration in iraqi population: cbct-based analysis vol 12 no 1 (2024) doi 10.5195/d3000.2024.620 http://dentistry3000.pitt.edu table 3. comparison between upper and lower jaws dehiscence and fenestration. lower upper p-value total sample 409 409 -- dehiscence 20 40 0.0004 fenestration 3 21 0.0001 discussion cbct benefits must be greater than any possible risks related to the exposure to radiations. based on as low as reasonably achievable (alara) principle, each case must have a certain field of view (fov) size that is carefully calculated [33]. although traditional radiographs, like the lateral cephalogram and panoramic xray, could offer crucial diagnostic data, there are situations in which cbct may be recommended when it comes to impacted canines, multiple teeth, unerupted teeth, severe root resorption, and noticeable skeletal abnormalities [28–30]. concerning the selection of participants, individuals who were under the age of 18, often referred to as growing patients, were excluded from the study. this decision was based on prior research findings which indicated that hormonal and functional changes connected with age had an impact on cortical bone thickness [32, 36]. individuals over the age of 35 were deliberately excluded from the research due to the heightened susceptibility to periodontitis within this demographic [37]. this decision was made to mitigate the potential influence of periodontitis-induced bone loss, which might have introduced confounding variables into the study. the study findings indicate that there have been statistically significant differences in the occurrence of total bony defects and dehiscence between individuals with posterior crossbite and those without. moreover, these differences were also observed among the three subgroups. however, it is worth noting that the disparities in prevalence were approximately 10%, which may not have significant clinical implications. statistical differences were not seen in all mean defect size assessments across the three categories; nonetheless, a consistent pattern was observed in all measurements. it is possible to explain the observed phenomena by thinking of teeth's buccolingual inclination as a compensatory mechanism. people who have a posterior crossbite have been shown to have dental compensation for the teeth that are not impacted by the crossbite, whereas the teeth that are affected by the crossbite retain their original position within the bone structure [6, 7]. it is clear from this that the occurrence of bone anomalies in non-crossed teeth was higher in those with crossbite. the reason behind this phenomenon could be that these teeth have been http://dentistry3000.pitt.edu/ prevalence of dehiscence and fenestration in iraqi population: cbct-based analysis vol 12 no 1 (2024) doi 10.5195/d3000.2024.620 http://dentistry3000.pitt.edu positioned outside of bony housing in order to correct occlusal connections. our investigation revealed a noteworthy observation: the frequency of dehiscence and fenestration is higher on the buccal side, in comparison with the lingual side. this significant finding aligns precisely; indeed, it mirrors identical results outlined in an earlier study [12]. the buccal side often exhibits lesser density than its counterparts (the lingual or palatal surfaces). however, potential oversight of a thin layer of bone on buccal side might have led to an exaggeration in the reported prevalence. this mistaken classification as defects could indeed reduce accuracy [1]. the first premolars demonstrated a higher dehiscence frequency compared to other posterior teeth across all categories. this phenomenon may originate from the comparatively limited width of the alveolar bone in this specific location, as opposed to broader dimensions found in other posterior areas. the narrowing pattern of alveolar bone width occurs during progression from posterior towards anterior regions within both maxilla and mandible. two distinct root morphologies, diverging from each other, may contribute to the increased susceptibility of the maxillary first premolar to bone abnormalities. the maxilla had a greater incidence of fenestration compared to the mandible. similar findings have been reported in previous research (8–10,12), which indicate that the maxillary bone has a narrowing pattern from cervical to the apical level of teeth, whereas the mandible demonstrates a thicker cortical bone. a reduced incidence of fenestration inside the maxilla was noted, specifically regarding the second premolars, when put to comparison with other posterior teeth in the maxillary area. the rationale for this is due to the fact that maxillary second premolars exhibit a unique dental trait, since they are the only maxillary posterior teeth that possess a solitary root, devoid of the diverging root prominence often seen in teeth with multiple roots. previous research has shown that cbct tends to exaggerate bone abnormalities due to its poor positive predictive value, particularly in cases involving fenestration [1,38]. the prevalence of bone deformities as reported in this research is likely to be overstated and may include some false-positive numbers. therefore, it is important to reevaluate the proportion of bone abnormalities while considering the potential occurrence of false-positive interpretations. alveolar bone thickness and periodontal biotype are significant confounding variables that need to be considered when detecting bony defects, both within individual patients and across different subjects. the study did not include measures of alveolar bone thickness or clinical evaluation of periodontal bio-type. instead, face data of a vertical nature was used. gracco et al. [39] have shown a statistically significant correlation between vertical face type and alveolar bone thickness. the researchers observed that hypo-divergent individuals had a higher degree of alveolar bone thickness compared to hyper divergent patients. yet, it is important to note that this study just http://dentistry3000.pitt.edu/ prevalence of dehiscence and fenestration in iraqi population: cbct-based analysis vol 12 no 1 (2024) doi 10.5195/d3000.2024.620 http://dentistry3000.pitt.edu concentrated on the assessment of upper incisors. subsequent research has also shown that individuals with hyper divergent and neurodivergent face types have a greater incidence of dehiscence compared to those with a hypo-divergent facial type [9]. limitation as described in the techniques section, we conducted the acquisition of cbct images for this experiment using two unique settings. the existing data suffers from an inherent drawback: a lack of control over past parameters used to capture cbct images by our investigator. however, research suggests that altering cbct settings may not significantly affect alveolar bone measure quantification [31]. the sample selection process encountered a restriction: an exact match in the anterior-posterior skeletal pattern (anb value) between research group and comparison group was not found. nonetheless, it is crucial to emphasize that experts deemed the observed discrepancy of 1.56° as clinically acceptable; this underscores precision within their evaluation. the asymmetrical geometric properties of dehiscence and fenestration potentially lead to varied measurements in the vertical dimension, depending on picture orientation. in an effort to reduce error, we optimally aligned our research's vertical distance measurement with the long axis of a tooth while positioning it perpendicular to its occlusal plane. however, the present study faced significant constraints due to its limited sample size; determining prevalence was our primary aim despite these limitations. a higher sample size would have indeed enhanced the findings' robustness; specifically--the inclusion of confounding variables like occlusal interference and occlusal wear: these additions could have fortified the research. disclosure of interest the authors declare that they have no competing interests. references 1. accuracy and reliability of conebeam computed tomography for measuring alveolar bone height and detecting bony dehiscences and fenestrations. leung cc, palomo l, griffith r, hans mg. am j orthod dentofac orthop. 2010;137(4 suppl):s109–19. 2. periodontal condition in orthodontically treated and untreated individuals ii. alveolar bone losss: radiographic findings. zachrisson bu, alnaes l. angle orthod. 1974;44(1):48–55. 3. trabecular and cortical bone as risk factors for orthodontic relapse. rothe le, bollen a-m, little rm, herring sw, chaison jb, chen csk, et al. am j orthod dentofac orthop. 2006;130(4):476–84. 4. mucogingival considerations in orthodontic treatment. wennström jl. semin orthod. 1996;2(1):46–54. 5. evaluation of dehiscence and fenestration in adolescent patients affected by unilateral cleft lip and palate: a retrospective cone beam computed tomography study. buyuk sk, ercan e, celikoglu m, sekerci ae, hatipoglu m. angle orthod. 2016;86(3):431–6. 6. evaluation of alveolar bone loss following rapid maxillary expansion using cone-beam computed tomography. baysal a, uysal t, veli i, ozer t, karadede i, hekimoglu s. http://dentistry3000.pitt.edu/ prevalence of dehiscence and fenestration in iraqi population: cbct-based analysis vol 12 no 1 (2024) doi 10.5195/d3000.2024.620 http://dentistry3000.pitt.edu korean j orthod. 2013;43(2):83–95. 7. alveolar bone changes after asymmetric rapid maxillary expansion. akin m, baka zm, ileri z, basciftci fa. angle orthod. 2015;85(5):799–805. 8. dehiscence and fenestration in patients with class i and class ii division 1 malocclusion assessed with cone-beam computed tomography. evangelista k, vasconcelos kde f, bumann a, hirsch e, nitka m, silva ma. am j orthod dentofac orthop. 2010;138(2):133.e1–7. 9. dehiscence and fenestration in patients with different vertical growth patterns assessed with cone-beam computed tomography. enhos s, uysal t, yagci a, veli i, ucar fi, ozer t. angle orthod. 2012;82(5):868–74. 10. dehiscence and fenestration in skeletal class i, ii, and iii malocclusions assessed with cone-beam computed tomography. yagci a, veli i, uysal t, ucar fi, ozer t, enhos s. angle orthod. 2012;82(1):67–74. 11. carranza fa. carranza’s clinical periodontology. newman mg, takei hh, klokkevold pr, 12th ed. st. louis, mo: elsevier; 2015. 12. appraisal of the relationship between tooth inclination, dehiscence, fenestration, and sagittal skeletal pattern with cone beam computed tomography. coşkun i̇, kaya b. the angle orthodontist. 2019;89(4):544–51. 13. alveolar defects in human skulls. davies rm, downer mc, hull ps, lennon ma j clin periodontol. 1974; 1:107–111 14. alveolar bone dehiscences and fenestrations: an anatomical study and review. nimigean vr, nimigean v, bencze ma, dimcevici-poesina n, cergan r, moraru s. rom j morphol embryol. 2009; 50(3):391–397 15. alveolar plate fenestrations and dehiscences of the human skull. larato dc (1970) oral surg oral med oral pathol. 1970; 29:816–819 16. alveolar plate defects in children’s skulls. larato dc. j periodontol, 1972 43:502. 17. prevalence of dehiscences and fenestrations in modern american skulls. rupprecht rd, horning gm, nicoll bk, cohen me. j periodontol. 2001; 72:722–729. 18. incidence and distribution of alveolar bony dehiscence and fenestration in dry human egyptian jaws. abdelmalek rg, bissada nf. j periodontol. 1973; 44:586–588. 19. bony defects in dried bantu mandibles. volchansky a, cleaton-jones p. oral surg oral med oral pathol. 1978; 45:647– 653. 20. alveolar bone fenestrations and dehiscences in dry bedouin jaws. edel a. j clin periodontol. 1981; 8:491–499. 21. the correlation between the presence of dehiscence or fenestration and the severity of tooth attrition in contemporary dry japanese adult skulls–part i. ezawa t, sano h, kaneko k, hiruma s, fujikawa k, murai s. j nihon univ sch dent. 1987; 29:27–34 22. dehiscence and fenestration: study of distribution and incidence in a homogeneous population model. urbani g, lombardo g, filippini p, nocini fp. stomatol mediterr, 1991; 11:113–118 23. dehiscence and fenestration in patients with class i and class ii division 1 malocclusion assessed with conebeam computed http://dentistry3000.pitt.edu/ prevalence of dehiscence and fenestration in iraqi population: cbct-based analysis vol 12 no 1 (2024) doi 10.5195/d3000.2024.620 http://dentistry3000.pitt.edu tomography. evangelista k, vasconcelos kdf, bumann a, hirsch e, nitka m, silva mag. am j orthod dentofacial orthop. 2010; 138:131–133 24. depth of alveolar bone dehiscences in relation to gingival recessions. lost c. j clin periodontol. 1984; 11:583–589. 25. evaluation of dehiscence and fenestration in adolescent patients affected by unilateral cleft lip and palate: a retrospective cone beam computed tomography study. buyuk sk, ercan e, celikoglu m, sekerci ae, hatipoglu m. angle orthod. 2016; 86:431–436. 26. the effect of orthodontic treatment on periodontal bone support in patients with advanced loss of marginal periodontium. artun j, urbye ks. am j orthod dentofacial orthop. 1988; 93:143–148. 27. periodontal tissue response to orthodontic movement of teeth with infrabony pockets. wennstrom jl, stokland bl, nyman s, thilander b. am j orthod dentofacial orthop. 1993; 103:313–319. 28. accuracy and reliability of buccal bone height and thickness measurements from cone-beam computed tomography imaging. timock am, cook v, mcdonald t, leo mc, crowe j, benninger bl et al. am j orthod dentofacial orthop. 2011; 140:734–744. 29. accuracy and reliability of cone-beam computed tomography for measuring alveolar bone height and detecting bony dehiscences and fenestrations. leung cc, palomo l, griffith r, hans mg. am j orthod dentofacial orthop. 2010; 137(4 suppl):109–119 30. accuracy of cone-beam computed tomography in detecting alveolar bone dehiscences and fenestrations. sun l, zhang l, shen g, wang b, fang b. am j orthod dentofacial orthop. 2015; 147:313–323 31. accuracy of alveolar bone measurements from cone beam computed tomography acquired using varying settings. cook vc, timock am, crowe jj, wang m, covell da jr. orthod craniofacial res. 2015;18(s1):127–36. 32. changes of alveolar bone dehiscence and fenestration after augmented corticotomyassisted orthodontic treatment: a cbct evaluation. sun l, yuan l, wang b, zhang l, shen g, fang b. prog orthod. 2019; 20:7. 33. clinical recommendations regarding use of cone beam computed tomography in orthodontics.[corrected]. position statement by the american academy of oral and maxillofacial radiology. american academy of o, maxillofacial r. oral surg oral med oral pathol oral radiol. 2013; 116(2):238–57. 34. the prevalence of dehiscence and fenestration on anterior region of skeletal class iii malocclusions: a cone-beam ct study. sun ly, wang b, fang b. shanghai kou qiang yi xue. 2013; 22:418–422 35. evaluation of alveolar bone defects on anterior region in patients with bimaxillary protrusion by using cone-beam ct. zhou l, li wr. beijing da xue xue bao yi xue ban. 2015; 47:514–520 36. prevalence of posterior alveolar bony dehiscence and fenestration in adults with posterior crossbite: a cbct study. choi jy, chaudhry k, parks e, ahn jh. prog orthod. 2020; 21:8 37. incidence and distribution of dehiscences and http://dentistry3000.pitt.edu/ prevalence of dehiscence and fenestration in iraqi population: cbct-based analysis vol 12 no 1 (2024) doi 10.5195/d3000.2024.620 http://dentistry3000.pitt.edu fenestrations on human skulls. jorgic-srdjak k, plancak d, bosnjak a, azinovic z. coll antropol. 1998; 22(suppl):111– 116. 38. accuracy of cone-beam computed tomography in detecting alveolar bone dehiscences and fenestrations. sun l, zhang l, shen g, wang b, fang b. am j orthod dentofac orthop. 2015;147(3):313–23. 39. upper incisor position and bony support in untreated patietns as seen on cbct. gracco a, lombardo l, mancuso g, gravina v, siciliani g. angle orthodontist. 2009;79(4):692–702. http://dentistry3000.pitt.edu/ microsoft word 66 2017.docx   vol  5,  no  1  (2017)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2017.66           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.     could  recurrent  aphthous  stomatitis  be  linked  to  cancer     development?   emily  norton1      1university  of  pi.sburgh,  school  of  dental  medicine abstract   background:  recurrent  aphthous  stoma//s  is  a  common  mul/factorial  oral  mucosal  disorder.  gene$c  and  local  fac-­‐ tors  causing  ras  may  contribute  to  the  development  of  cancer.  this  study  aims  to  explore  this  connec<on  by  examin-­‐ ing  a  family  with  a  history  of  ras  and  ovarian  cancer.    case  descrip+on:  the  family  described  in  the  case  exhibits  a   history  of  minor   recurrent   aphthous   stoma11s.   the   severity   of   this   condi1on   increases  with   each   genera1on.  one   member  of  the  family  has  no  history  of  ras  but  was  diagnosed  with  ovarian  cancer.  gene;c  tes;ng  for  brca  muta-­‐ !ons  was  nega!ve  indica!ng  a  different  gene)c  cause  of  the  cancer.  all  affected  members  of  the  family  indicate  high   levels  of   stress  or  difficulty   responding   to  stressful   situa4ons.  no   treatment   for  ras  was  performed  due   to   its   self-­‐ limi$ng  nature.    prac%cal  implica%ons:  further  research  is  needed  before  den.sts  begin  to  tell  their  pa.ents  with  ras   they  may  be  at  higher  risk  of  developing  cancer.  diligent  oral  cancer  screening  and  stress  management  counseling  can   decrease  the  risk  to  the  pa.ent.     cita%on:  norton,  e.  (2017)  could  recurrent  aph-­‐ thous  stoma))s  be  linked  to  cancer  develop-­‐ ment?  den$stry  3000.  1:a001   doi:10.5195/d3000.2017.66   received:    may  1,  2017   accepted:    may  2,  2017   published:    june  8,  2017   copyright:  ©2017  norton.  this  is  an  open  access   ar!cle   licensed   under   a   crea!ve   commons   a!ribu%on  work  4.0  united  states  license.   email:  emn27@pi).edu   introduction   recurrent  aphthous  stoma-­‐ titis  (ras)  is  the  most  common   oral  mucosal  lesion,  affecting  as   much  as  25%  of  the  world  popula-­‐ tion  [1-­‐3].  this  condition  is  charac-­‐ terized  by  painful  erosions  or  ul-­‐ cers  surrounded  by  an  erythema-­‐ tous  halo  on  the  non-­‐keratinized   oral  mucosa  [2,  4].    the  lesions   may  affect  the  day  to  day  activities   of  the  patients,  especially  eating,   drinking  and  speaking  [3].  aphthae   are  identified  as  one  of  three   types.  the  three  types  differ  in   size  of  ulcers,  healing  time  and   scarring  upon  healing.  the  first   and  most  common  type,  which   comprises  80-­‐90%  of  all  cases,  is   minor  recurrent  aphthous  stoma-­‐ titis  [3].  in  these  cases,  the  lesions   present  are  less  than  1  cm  in  di-­‐ ameter,  heal  within  seven  to  four-­‐ teen  days  and  do  not  scar  [3].  the   second  type  is  known  as  major  re-­‐ current  aphthous  stomatitis  and  is   characterized  by  lesion  greater   than  1  cm  in  diameter  that  heal   within  20-­‐30  days  with  scarring   [3].  the  final  type  is  known  as  her-­‐ petiform  ulcers,  which  presents   are  multiple,  clustered  lesions  1-­‐3   mm  in  diameter  [3].  these  lesions   may  coalesce  to  form  larger  le-­‐ sions  and  usually  heal  within  fif-­‐ teen  days  [3].     despite  being  an  incredibly   common  condition,  the  etiology  of   recurrent  aphthous  stomatitis  re-­‐ mains  unclear.  however,  there  is  a   general  consensus  that  ras  is  a   multifactorial  condition,  with  sev-­‐ eral  factors  implicated  in  its  occur-­‐ rence  [1,  3,  5].  many  common  lo-­‐ cal  factors  are  thought  to  be  in-­‐ volved  such  as  viral  and  bacterial   infections,  allergic  agents  (such  as   food  hypersensitivity),  nutritional   deficiencies  and  stress  and  anxiety   [3-­‐5].  in  addition,  there  are  also   genetic  factors  that  can  predis-­‐ pose  an  individual  to  ras.  certain   hla  alleles  have  been  found  to  be   associated  with  patients  with  ras   as  well  as  specific  racial  and  ethnic   segregations  [2,  5].  ras  is  associ-­‐ ated  with  certain  genetic  syn-­‐ dromes  such  as  behcet’s  syn-­‐ drome  [5].  ras  is  also  known  to   accumulate  in  families,  giving  fur-­‐ ther  strength  to  the  theory  of  a   genetic  cause  [5].  early  studies   proposed  an  autosomal  recessive   or  multigene  mode  of  inheritance   with  environmental  influences  [4].     this  paper  aims  to  explore   the  possible  connection  between   ras  and  cancer  development  by   examining  a  family  with  a  history    could  recurrent  aphthous  stoma11s  be  linked  to  cancer  development?   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.66    http://dentistry3000.pitt.edu   of  ras  as  well  as  cancer.  the  prac-­‐ tical  implications  of  these  findings   in  the  dental  clinic  and  possible   ethical  considerations  will  also  be   discussed.     case  presentation     the  family  studied  exhibits   three  generations  of  reported  re-­‐ current  aphthous  stomatitis  with   varying  degrees  of  severity.  one  of   the  family  members  in  the  third   generation  is  not  affected  by  ras,   but  she  was  diagnosed  with  ovari-­‐ an  cancer.  figure  1  is  the  family’s   pedigree,  showing  which  individu-­‐ als  have  been  affected  by  ras.   three  consecutive  generations   have  been  affected,  as  seen  in  the   pedigree.  the  family  members  in   the  fourth  generation  have  not  yet   shown  as  signs  of  ras,  so  their   status  is  currently  unknown.  each   member’s  presentation  of  ras  and   account  of  other  risk  factors  will   be  discussed  individually.   family  member  i:2   the  initial  presentation  of   ras  is  seen  in  the  first  generation,   in  patient  i:2.  the  family  account   of  her  condition  is  limited  at  this   time.  her  son,  family  member  ii:1,   states  he  remembers  his  mother   getting  “canker  sores  a  few  times”   when  he  was  growing  up.  he  could   not  remember  any  information   about  the  size  of  the  sores  or  the   frequency  of  the  episodes.  no   other  signs  that  would  indicate  a   systemic  disease,  such  as  behcet’s,   were  reported.  family  member   ii:1  indicated  that  his  mother  was   not  under  a  high  level  of  stress  but   did  seem  to  be  easily  upset  in   some  situations.   family  member  ii:1   family  member  ii:1  indi-­‐ cated  that  he  shared  the  same   proclivity  as  his  mother  for  “can-­‐ ker  sores”.  he  stated  that  he   would  get  “attacks”  about  three  or   four  times  a  year,  lasting  for  about   a  week  at  a  time.  he  said  that  the   lesions  were  “mostly  small”  and   based  on  this  information  and   known  prevalence  of  the  different   types  of  aphthae,  it  is  safe  to  as-­‐ sume  he  was  afflicted  with  minor   ras.  again,  no  indications  of  sys-­‐ temic  disease  were  noted.  when   asked  about  his  stress  level,  he   indicated  that  his  job  was  moder-­‐ ately  stressful.  he  also  mentioned   that  a  few  of  his  “attacks”  coincid-­‐ ed  with  times  were  his  job  stress   was  slightly  higher.     family  member  iii:2   family  member  iii:1  is  the   third  member  of  this  family  to  be   affected  by  ras.  she  stated  she   would  have  “frequent  outbreaks   of  canker  sores  four  to  five  times  a   year”.  she  remembered  her  father   being  affected  by  canker  sores  “of   similar  size  and  duration”  as  those   that  she  experienced.  this  indi-­‐ cates  she  also  suffered  from  the   minor  type  of  ras.  she  also  men-­‐ tioned  that  the  “sores”  affected   her  ability  to  eat,  drink  and  carry   on  with  some  day  to  day  activities.   still  no  indications  of  systemic  dis-­‐ ease  were  noted  in  this  family   member.  regarding  daily  stress,   she  stated  that  she  did  not  have  a   stressful  job,  but  admitted  to  “not   always  handling  stressful  situa-­‐ tions  well”.  she  could  not  remem-­‐ ber  specifically  if  her  outbreaks   coincided  with  times  of  stress.   family  member  iii:3   family  member  iii:3  stated   that  she  “has  never  had  an  out-­‐ break  of  canker  sores”  like  her  sis-­‐ ter  or  father.  however,  she  was   diagnosed  with  stage  i  ovarian   cancer  within  the  past  two  years.   she  was  initially  treated  with  six   rounds  of  chemotherapy  with  min-­‐ imal  complications.  during  treat-­‐ ment,  she  was  also  tested  for  mu-­‐ tations  of  the  brca  genes,  none  of   which  were  found.  within  the  last   year,  a  ca125  test  revealed  ele-­‐ vated  values  and  the  family  was   concerned  about  a  possible  recur-­‐ rence,  although  all  scans  were   clear.  subsequent  testing  revealed   no  change  in  the  values  and  the   initial  elevation  was  attributed  to   the  presence  of  multiple  hernias   at  the  initial  incision  site,  rather   than  recurrence.  however,  during   the  time  between  tests,  the  pa-­‐ tient  became  so  stressed  that  she   was  prescribed  xanax  to  help  her   relax.  based  on  this  and  observa-­‐ tions  from  other  family  members,   it  has  been  concluded  that  family   member  iii:3  should  be  character-­‐ ized  as  a  high  stress  individual.     discussion     the  goal  of  this  study  is  to   examine  the  possible  relationship   between  recurrent  aphthous  sto-­‐ matitis  and  cancer.  in  the  family   described,  the  link  between  ras   and  ovarian  cancer  specifically  will   be  discussed.  the  discussion  will   focus  on  the  possible  genetic  fac-­‐ tors  making  members  of  this  fami-­‐ ly  susceptible  to  these  diseases  as   well  as  the  local  factor  of  stress.   also,  the  ethical  concerns  and   possible  implications  of  a  familial    could  recurrent  aphthous  stoma11s  be  linked  to  cancer  development?   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.66    http://dentistry3000.pitt.edu   presentation  such  as  the  one  de-­‐ scribe  in  a  dental  practice  will  also   be  discussed.   inheritance  pattern   examining  figure  1,  it  is   clear  that  this  family  has  three   consecutive  generations  affected   by  ras  and  a  fourth  generation   that  could  show  signs  of  ras  but   have  not  to  date.  also  both  male   and  female  family  members  have   been  affected.  using  this  infor-­‐ mation  alone,  it  may  be  postulat-­‐ ed  that  ras  is  being  inherited  in   an  autosomal  dominant  fashion.   this  would  be  in  direct  contradic-­‐ tion  to  the  initial  proposal  of  auto-­‐ somal  recessive  inheritance  in   1965  [4].  however,  this  family  is   relatively  small,  making  the  sam-­‐ ple  size  too  small  to  reliably  de-­‐ termine  mode  of  inheritance  from   the  pedigree  alone.  also  multiple   members  of  the  family  are  known   to  have  high  stress  levels  or  diffi-­‐ culty  properly  dealing  with  stress-­‐ ful  situations.  this  information   supports  the  widely  accepted  the-­‐ ory  of  a  multifactorial  inheritance   pattern.     genetic  factors   aphthous  ulcers  arise  due   to  an  abnormal  immune  interac-­‐ tion  on  the  oral  mucosal  cells,  cat-­‐ egorizing  it  as  an  autoinflammato-­‐ ry  disease  [5].  it  is  thought  that   disruption  in  both  the  humoral   and  cellular  immune  systems  con-­‐ tribute  to  the  occurrence  of  ras   [4,  5].  specifically,  im-­‐ paired  activation  of  proin-­‐ flammatory  components   such  as  tumor  necrosis   factors  and  interleukin-­‐1   (il-­‐1)  have  been  implicat-­‐ ed  [4,  5].  the  il-­‐1  family   are  primary  activators  of   the  expression  of  endothe-­‐ lial  cell  adhesion  mole-­‐ cules  which  facilitates  the   migration  of  leukocytes   into  tissues  [4].  a  member   of  the  il-­‐1  family,  il-­‐1β,  is   primarily  released  by   monocytes  and  macro-­‐ phages,  as  well  as  some   nonimmune  cells,  such  as   fibroblasts  and  endothelial  cells  at   sites  of  injury  to  stimulate  in-­‐ creased  migration  of  leukocytes   [6].  the  increased  migration  of   leukocytes  caused  by  increased   release  of  il-­‐1β  could  account  for   the  damage  to  mucosal  cells  that   results  in  ras  [4].  gene  polymor-­‐ phisms  associated  with  il-­‐1β  have   been  thought  to  correlate  to  a   higher  incidence  of  ras,  however   studies  of  these  polymorphisms   have  produced  conflicting  results   [4].  because  research  into  specific   il-­‐1  mutations  has  produced  in-­‐ conclusive  results  it  is  difficult  to   say  if  a  mutation  in  il-­‐1β  could  be   responsible  for  the  presentation  of   ras  seen  in  this  family.  however,   it  has  been  previously  concluded   that  multiple  factors  are  involved   in  the  presentation  of  ras  so  a   mutation  of  il-­‐1β  could  be  con-­‐ tributing  to  the  development  of   aphthous  ulcers  in  this  family.  ge-­‐ netic  testing  of  this  family  may  be   helpful  in  determining  if  il-­‐1β  is  a   major  component  of  their  ras  and   could  also  reveal  possible  genes  to   focus  on  for  future  research.  given   the  history  of  cancer  within  this   family  it  seems  likely  that  tumor   necrosis  factor-­‐α  could  have  a   large  influence  on  the  disease   presentation  seen.   tumor  necrosis  factor-­‐α   (tnf-­‐α)  is  another  inflammatory   cytokine  involved  with  ras.  tnf-­‐α   has  been  implicated  as  a  major   contributor  to  the  development  of   ras.  it  is  a  major  proinflammatory   cytokine  involved  with  the  chemo-­‐ taxis  of  immune  components,  such   as  neutrophils  and  major  histo-­‐ compatibility  complexes  (mhc)   [5].    tnf-­‐α  is  involved  in  cell  sig-­‐ naling  pathways,  one  of  which  is   involved  in  apoptosis  of  cells  [6].   increased  production  of  tnf-­‐α   could  explain  the  destruction  of   mucosal  cells  in  ras  through  acti-­‐ vation  of  the  apoptotic  pathway.  it   is  also  effective  in  the  t  cell-­‐ mediated  immune  response,   which  is  responsible  for  a  part  of   the  immune-­‐mediated  pathogene-­‐ sis  of  ras  [5].  it  has  been  found   that  the  number  of  cd4+  t  cells  is   decreased  and  the  number  of   cd8+  t  cells  is  increased  in  pa-­‐ figure  1.  family  pedigree.  individuals  with  ras  are  identified   by  the  shaded  figures.  member  iii:3  presented  with  ovarian   cancer,  marked  oc.  ras  presentation  in  members  iv:1  and   iv:2  has  not  yet  been  diagnosed,  indicated  by  the  “?”.    could  recurrent  aphthous  stoma11s  be  linked  to  cancer  development?   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.66    http://dentistry3000.pitt.edu   tients  with  ras  [5].  tnf-­‐α  has   many  beneficial  functions,  in  addi-­‐ tion  to  its  role  in  the  immune  re-­‐ sponse,  including  maintenance  of   homeostasis  by  regulating  the   body’s  circadian  rhythm  and  pro-­‐ moting  the  remodeling  of  injured   tissue  [7].  however,  prolonged   existence  of  tnf-­‐α  in  the  body  can   produce  negative  effects.  after   remaining  in  the  body  for  a  long   time,  tnf-­‐α  loses  its  anti-­‐tumor   activity,  which  could  result  in  the   growth  of  various  tumors   throughout  the  body  [7].  devel-­‐ opment  of  ras  indicates  an  in-­‐ crease  in  tnf-­‐α  activity,  which   could  lead  to  a  decrease  in  the  an-­‐ ti-­‐tumor  function  of  the  cytokine.   this  would  prevent  tnf-­‐α  from   destroying  the  developing  cancer   cells,  allowing  a  tumor  to  form.  as   tests  for  other  genetic  mutations   associated  with  ovarian  cancer   were  negative,  it  is  possible  that   increased  tnf-­‐α  activity  could  be   a  contributing  factor  to  the  devel-­‐ opment  of  cancer  in  family  mem-­‐ ber  iii:3.   local  factors:  stress   psychological  stress  has   been  implicated  as  a  factor  induc-­‐ ing  ras.  all  members  of  the  family   studied  reported  higher  than   normal  levels  of  stress  or  de-­‐ creased  ability  to  handle  stress.  it   has  been  found  that  patients  with   ras  had  higher  overall  stress  lev-­‐ els  than  patients  without  ras  [8].   also  many  patients  with  ras  indi-­‐ cate  that  their  outbreaks  correlate   to  high  stress  times  in  their  life  [8].   as  previously  described,  the  path-­‐ ogenesis  of  ras  involves  increased   immune  activity  leading  to  de-­‐ struction  of  the  oral  mucosa  [4,  5,   8].  stress  contributes  to  this  activi-­‐ ty  by  increasing  the  number  of   leukocytes  at  sites  of  inflammation   [8].    in  this  family  only  one  mem-­‐ ber,  ii:1,  could  recall  a  correlation   between  stress  levels  and  appear-­‐ ance  of  aphthae.  this  discrepancy   from  the  literature  is  probably  due   to  the  inaccuracy  of  the  patient’s   memory,  rather  than  a  lack  of  as-­‐ sociation  between  stress  and  ras.   also,  stress  is  a  complex  factor   which  affects  each  person  differ-­‐ ently  [8].  this  could  account  for   the  different  levels  of  severity  de-­‐ scribed  by  family  members  ii:1   and  iii:2.     psychological  stress  has   been  found  to  have  an  effect  on   cancer  development  as  well.  it  has   been  shown  the  increased  stress   levels  could  affect  cellular  pro-­‐ cesses,  such  as  those  involved  in   dna  repair  [9].  this  could  lead  to   oncogenic  mutations.  it  has  also   been  found  that  stress  may  in-­‐ crease  the  rate  of  tumor  growth   [9].  family  member  iii:3  and  her   family  indicated  that  she  may  not   handle  stressful  situations  well.   the  situation  mentioned  in  this   study  (increased  ca125  levels  for  a   recovering  cancer  patient)  would   be  an  extremely  stressful  time  for   anyone  and  may  not  be  the  best   indicator  of  this  patient’s  response   to  stress.  however,  the  family  ob-­‐ served  other  situations  in  which   the  patient’s  response  to  stress   was  extreme,  showing  a  pattern   for  this  patient.     practical  implications  and  ethical  con-­‐ cerns   due  to  the  common  nature   of  ras,  it  is  highly  likely  that  a   dentist  will  see  and  diagnose  many   patients  with  this  condition   throughout  their  career.  the  theo-­‐ ry  posed  in  this  study  raises  the   question:  what  should  a  dentist   do  considering  their  patient  with   ras  may  be  at  higher  risk  of  can-­‐ cer?  another  ethical  question  may   be  whether  to  tell  the  patient   about  this  possible  increased  risk.   at  this  point,  there  is  not  enough   research  to  support  this  theory  to   warrant  alarming  a  patient  with   simple  ras.  however,  the  dentist   may  keep  this  theory  in  mind   when  examining  a  patient  with  a   history  of  ras.  it  may  be  prudent   for  the  dentist  to  screen  these  pa-­‐ tients  for  oral  cancer  more  fre-­‐ quently  in  an  attempt  to  discover   any  cancerous  changes  early.  the   dentist  may  also  consider  counsel-­‐ ing  their  ras  patients  regarding   their  stress  level.  this  would  both   reduce  their  risk  of  ras  outbreaks   and  possibly  the  development  of   cancer.  regarding  the  family  stud-­‐ ied,  the  children  in  the  fourth  gen-­‐ eration  have  not  yet  shown  any   signs  of  ras.  however,  given  the   family  history,  it  would  be  logical   to  monitor  them  closely  for  any   signs  of  ras  or  even  cancer   growth.   conclusion     this  study  has  examined   the  possible  connection  between   recurrent  aphthous  stomatitis  and   the  development  of  cancer  by   looking  at  a  family  with  a  history   of  both  conditions.  both  genetic   and  local  factors  contribute  to  the   development  of  ras  as  well  as    could  recurrent  aphthous  stoma11s  be  linked  to  cancer  development?   vol  5,  no  1  (2017)        doi  10.5195/d3000.2017.66    http://dentistry3000.pitt.edu   cancer.  in  this  family,  it  was  sug-­‐ gested  that  increased  tnf-­‐α  activ-­‐ ity  combined  with  high  psycholog-­‐ ical  stress  contributed  to  the  de-­‐ velopment  of  ras  in  family  mem-­‐ bers  i:2,  ii:1  and  iii:2.  these  same   factors,  coupled  with  the  lack  of   other  genetic  mutations,  could   have  contributed  to  the  develop-­‐ ment  of  ovarian  cancer  in  family   member  iii:3.  as  far  as  the  practic-­‐ ing  dentist  is  concerned,  patients   with  ras  should  be  closely  moni-­‐ tored,  but  at  this  point  more  harm   than  good  would  be  done  by  tell-­‐ ing  these  patients  they  could  pos-­‐ sibly  develop  cancer.  more  re-­‐ search  needs  to  be  done  to  solidify   a  connection  between  ras  and   the  development  of  cancer.  this   research  could  also  elucidate  new   genes  to  study  regarding  the   pathogenesis  of  ras.   references   1. nasry  sa,  el  shenawy  hm,   mostafa  d,  ammar  nm.   different  modalities  for   treatment  of  recurrent   aphthous  stomatitis.  a   randomized  clinical  trial.  j   clin  exp  dent.  2016  dec   1;8(5):e517-­‐e522.  ecollec-­‐ tion  2016  dec.  pubmed   pmid:  27957263;  pmcid:   pmc5149084.   2. najafi  s,  mohammadzadeh   m,  zare  bidoki  a,  meighani   g,  aslani  s,  mahmoudi  m,   rezaei  n.  hla-­‐drb  and   hla-­‐dqb  allele  and  haplo-­‐ type  frequencies  in  iranian   patients  with  recurrent   aphthous  stomatitis.  iran  j   allergy  asthma  immunol.   2016  aug;15(4):289-­‐295.   pmid:  27921409.   3. han  m,  fang  h,  li  ql,  cao   y,  xia  r,  zhang  zh.  effec-­‐ tiveness  of  laser  therapy   in  the  management  of  re-­‐ current  aphthous  stomati-­‐ tis:  a  systematic  review.   scientifica  (cairo).   2016;2016:9062430.  doi:   10.1155/2016/9062430.   epub  2016  dec  18.  review.   pubmed  pmid:  28078164;   pmcid:  pmc5203897.   4. ślebioda  z,  krawiecka  e,   rozmiarek  m,  szponar  e,   kowalska  a,  dorocka-­‐ bobkowska  b.  clinical  phe-­‐ notype  of  recurrent  aph-­‐ thous  stomatitis  and  inter-­‐ leukin-­‐1β  genotype  in  a   polish  cohort  of  patients.  j   oral  pathol  med.  2016  dec   9.  doi:  10.1111/jop.12533.   [epub  ahead  of  print]   pmid:  27935125.   5. yigit  s,  tekcan  a,  rustemo-­‐ glu  a,  tumer  mk,  kalkan  g,   yerliyurt  k.  investigation  of   cd40  gene  rs4810485  and   rs1883832  mutations  in   patients  with  recurrent   aphthous  stomatitis.  arch   oral  biol.  2017  feb;74:51-­‐ 54.  doi:   10.1016/j.archoralbio.2016 .11.003.  epub  2016  nov  5.   pmid:  27875792.   6. zhang  jm,  an  j.  cytokines,   inflammation,  and  pain.  int   anesthesiol  clin.  2007   spring;45(2):27-­‐37.  re-­‐ view.  pubmed  pmid:   17426506;  pmcid:   pmc2785020.   7. esposito  e,  cuzzocrea  s.   tnf-­‐alpha  as  a  therapeutic   target  in  inflammatory  dis-­‐ eases,  ischemia-­‐ reperfusion  injury  and   trauma.  curr  med  chem.   2009;16(24):3152-­‐67.   pmid:  19689289.   8. gallo  cde  b,  mimura  ma,   sugaya  nn.  psychological   stress  and  recurrent  aph-­‐ thous  stomatitis.  clinics   (sao  paulo).   2009;64(7):645-­‐8.  doi:   10.1590/s1807-­‐ 59322009000700007.   pubmed  pmid:  19606240;   pmcid:  pmc2710437.   9. chida  y,  hamer  m,  wardle   j,  steptoe  a.  do  stress-­‐ related  psychosocial  fac-­‐ tors  contribute  to  cancer   incidence  and  survival?  nat   clin  pract  oncol.  2008   aug;5(8):466-­‐75.  doi:   10.1038/ncponc1134.  epub   2008  may  20.  review.   pmid:  18493231.           assessment of the alignment accelera1on of the lower incisors using a single aligner appliance modified with nickel-titanium coils: a randomized controlled clinical trial in adults. vol 12 no 1 (2024) doi 10.5195/d3000.2024.532 h#p://den*stry3000.pi#.edu assessment of the alignment acceleration of the lower incisors using a single aligner appliance modified with nickel-titanium coils: a randomized controlled clinical trial in adults. ziad mohamad alhafi1, nada rajeh1, zaher jumaa2, zuhir alasfar3 1department of orthodontics, faculty of dentistry, damascus university, damascus, syria 2department of oral and maxillofacial surgery, faculty of dentistry, damascus university, damascus, syria 3department of periodontology, faculty of dentistry, damascus university, damascus, syria abstract objective: the aim of this study was to inves1gate the effects of a single aligner appliance modified with nickel-1tanium coils in accelera1ng the alignment of crowded lower incisors. materials and methods: 36 pa1ents with mild crowding of their lower incisors were randomly divided into two groups: one group received a modified aligner appliance with nickel-1tanium coils (experimental group), while the other group received tradi1onal orthodon1c brackets (control group). the degree of irregularity in the alignment of the incisors was measured every 2 weeks using liqle's irregularity index, and this was done at different 1me points throughout the study: before treatment (t0), ater 2 weeks (t1), ater 4 weeks (t2), ater 6 weeks (t3), ater 8 weeks (t4). results: a total of 87 pa1ents with anterior mild crowding in the lower dental arch were ini1ally assessed for eligibility. out of these, 64 pa1ents met the inclusion criteria. eventually, 36 par1cipants were assigned to receive the treatment. accordingly, the data from 36 pa1ents were analyzed sta1s1cally. there was no significant difference when comparing the two groups at t0, t1, t3, and t4. however, the difference was significant at t2 (p = 0.037). the differences were sta1s1cally significant at all 1me periods within each group (p < 0.001). conclusion: the use of a modified aligner appliance with nickel-1tanium coils effec1vely treats lower incisors crowding, but it does not accelerate the alignment process compared to the tradi1onal fixed orthodon1c appliance. keywords: aligners; teeth alignment; dental crowding; accelera1on cita9on: alhafi, zm et al. (2024) assessment of the alignment accelera9on of the lower incisors using a single aligner appliance modified with nickel-titanium coils: a randomized controlled clinical trial in adults. den9stry 3000. 1:a001 doi:10.5195/d3000.2024.532 received: july 13, 2023 accepted: july 14, 2023 published: may 9, 2024 copyright: ©2024 alhafi, zm et al. this is an open access ar9cle licensed under a crea9ve commons azribu9on work 4.0 united states license. email: ziad.alhafy@gmail.com introduction the rising focus on appearance in society has resulted in a greater desire among adults to resolve minor dental problems like mild crowding [1-3]. however, instead of choosing conventional braces orthodontic appliances due to the non-aesthetic metallic appearance of wires and brackets, they are increasingly inclined towards reconstructive procedures that may have the potential to harm dental tissues [1, 46]. as a result, there is a growing preference for invisible orthodontic methods, particularly among adults [7]. consequently, numerous http://dentistry3000.pitt.edu/ assessment of the alignment accelera1on of the lower incisors using a single aligner appliance modified with nickel-titanium coils: a randomized controlled clinical trial in adults. vol 12 no 1 (2024) doi 10.5195/d3000.2024.532 h#p://den*stry3000.pi#.edu methods and substances have been created in the field of clinical practice to alleviate these constraints, such as porcelain brackets, orthodontic techniques that are placed on the inner surface of the teeth, and clear aligners [8], but these options tend to be quite costly [9]. the spring aligner appliance, also known as the spring retainer, was introduced by barrer in 1974 as a removable device for aligning the incisors. it has been used for more than 25 years [10]. however, its activation was limited and it was only used to correct simple issues after orthodontic treatment and for retention [11]. in 2001, don inman modified the traditional spring retainer and created the inman aligner, a removable appliance that served multiple orthodontic purposes [4]. this new appliance relied on superelastic open coil springs to apply light and constant forces on both the front and back surfaces of the anterior teeth [11]. after reviewing the medical literature, we found that no clinical studies had been conducted that evaluated the acceleration of mandibular incisor alignment using any type of aligner appliance in the treatment of mild crowding in adult patients. based on this reasoning, the aim of this research was to assess the efficiency of a single aligner appliance modified with nickel-titanium springs, by comparing it with a traditional fixed orthodontic appliance in treating mild crowding of the lower incisors in adults. material and methods study design the study design was a randomized, single-blinded clinical trial with two groups and an equal allocation ratio. the study received approval from the university institutional review board and the ethical review committee of damascus university (approval number: 3166). the trial was also registered in the clinicaltrials.gov database with the identifier nct04988373. sample size estimation minitab® program (version 20.1; minitab, llc, state college, pa) was used to determine the sample size for the study. they considered the average movement of teeth per day with traditional fixed orthodontic appliances and aimed for 80% power at a significance level of 5%. based on previous data, it was determined that a minimum of 17 patients was needed in each group (figure 1). to account for potential dropouts, the number of patients was increased to 18 per group. patient selection, recruitment the study included a total of 36 patients, consisting of 29 females and 7 males. these patients were selected from individuals who sought treatment at the department of orthodontics, faculty of dentistry, damascus university in syria between january 2021 and april 2021. inclusion criteria • adults’ padents between the ages of 16 28 years. • mild crowding (1-4 mm) in the frontal area of their lower dental arch according to liele’s irregularity index (lii). • class i malocclusion. • should be no extracted or congenitally missing teeth, except for third molars. exclusion criteria • previous orthodondc treatment. • severe skeletal discrepancy. • severe protrusion of both upper and lower teeth. • any systemic diseases that could affect tooth movement. • poor oral hygiene. randomization and blinding the participants were divided into both groups using a 1:1 allocation ratio, utilizing the simple computer random method through the minitab® program (version 20.1; minitab, llc, state college, pa). blinding of the examiner and the patient was not possible, but blinding could be implemented during the analysis of the findings to prevent bias in detection. interventions the patients underwent a clinical examination, and impressions were http://dentistry3000.pitt.edu/ assessment of the alignment accelera1on of the lower incisors using a single aligner appliance modified with nickel-titanium coils: a randomized controlled clinical trial in adults. vol 12 no 1 (2024) doi 10.5195/d3000.2024.532 h#p://den*stry3000.pi#.edu made to create study models. additionally, digital lateral cephalometric and panoramic x-rays were requested. in the single aligner appliance modified with nickel-titanium coils group (experimental group), 18 patients were treated a setup was created using the orthodontic panel of blueskyplan® software to align the anterior teeth. the setup was then used to create a modified digital model, which was printed using a resin 3d printer. the bands were placed and the single aligner appliance was fabricated on this model. before placing the aligner appliance, interproximal reduction (ipr) was performed manually using an abrasive strip, which varied depending on the case planning. the amount of ipr was determined using an ipr gauge (ortho technology inc, west columbia, usa). the molars were then fitted with bands and cemented using gic (ivoclar vivadent, enderby, uk). the aligner appliance was subsequently placed. the force applied during the procedure was measured using an intraoral orthodontic force gauge (dtc medical apparatus co, hangzhou, china) and adjusted to a maximum of 80 grams per side, based on the specific needs of each case (figure 2). in the conventional orthodontic appliance (control group), 18 patients were treated using a traditional edgewise with the mbt prescription, 0.022-inch slot (master series, american orthodontics, sheboygan, usa). the archwire sequence used in this group was as follows: 0.012-inch nickel-titanium (niti), 0.014-inch niti, 0.016-inch niti, 0.016 × 0.022-inch niti, 0.016 × 0.022-inch stainless steel (ss), and 0.017 × 0.025-inch ss. interproximal reduction (ipr) was performed in the same manner as described in the experimental group. the visual confirmation of the completion of alignment for both groups was done using little's irregularity index (lii) [12], which figure 1. sample size estimation. figure 2. the applied appliance in the experimental group. http://dentistry3000.pitt.edu/ assessment of the alignment accelera1on of the lower incisors using a single aligner appliance modified with nickel-titanium coils: a randomized controlled clinical trial in adults. vol 12 no 1 (2024) doi 10.5195/d3000.2024.532 h#p://den*stry3000.pi#.edu indicated an equality of zero (figure 3). outcome measures little's irregularity index (lii) was the measure used to assess the outcome in this study. the lii was measured on study models at multiple time points: before treatment (t0), after 2 weeks of treatment (t1), after 4 weeks (t2), after 6 weeks (t3), and after 8 weeks (t4). the measurements were conducted using a digital caliper (insize, insize co, china) with a precision of 0.1 mm. the methodology for measuring lii followed little's guidelines ]12[ . statistical analysis the statistical analysis of the search results was conducted using spss software (version 26.0, ibm corp, armonk, ny). the shapirowilk test was used to assess the normality of the data distribution, and it indicated that the distribution was normal. therefore, parametric tests were employed. the independent t-test was utilized to identify significant differences in the mean values of the little irregularity index between the two groups at each time point. additionally, the paired t-test was used to detect significant differences within each group. the statistical significance level was set at 0.05 with a 95% confidence interval. results consort guideline shows the flowchart of the patients throughout the trial (figure 4). a total of 36 patients (7 males, 29 females) were included in the study and divided into two groups of 18 participants. there were no dropouts or withdrawals during any stage of the trial. figure 3. end of the lower incisor alignment stage. http://dentistry3000.pitt.edu/ assessment of the alignment accelera1on of the lower incisors using a single aligner appliance modified with nickel-titanium coils: a randomized controlled clinical trial in adults. vol 12 no 1 (2024) doi 10.5195/d3000.2024.532 h#p://den*stry3000.pi#.edu the enrollment of participants in this study included a total of 36 patients, as shown in table 1 which presents the basic characteristics of the sample. table 2 provides the descriptive statistics for the variables that were evaluated in the study. the findings regarding changes in lii at different time points are presented in table 3. there were no significant differences between the two groups at t0, t1, t3, and t4 (p = 0.994, p=0.095, p=0.060, p=0.191, respectively). however, a significant difference was found at t2 (p=0.037). table 4 shows that there are statistically significant differences in the mean values of lii between the five studied time periods within each group (p<0.001). figure 4. consort flow diagram http://dentistry3000.pitt.edu/ assessment of the alignment accelera1on of the lower incisors using a single aligner appliance modified with nickel-titanium coils: a randomized controlled clinical trial in adults. vol 12 no 1 (2024) doi 10.5195/d3000.2024.532 h#p://den*stry3000.pi#.edu variable group mean sd min max q1 median q3 liit0 experimental 3.10 0.68 2 4 2.32 3.38 3.58 control 3.09 0.68 2 4 2.5 3.08 3.77 liit1 experimental 1.88 0.56 1 2.70 1.27 2 2.24 control 2.23 0.66 1.23 3.33 1.65 2.15 2.78 liit2 experimental 1.01 0.51 0.22 1.90 0.58 1.06 1.41 control 1.40 0.54 0.67 1.70 0.91 1.30 1.91 liit3 experimental 0.37 0.33 0 1 0 0.35 0.68 control 0.63 0.46 0 1.45 0.19 0.60 1 liit4 experimental 0.10 0.17 0 0.56 0 0 0.21 control 0.18 0.19 0 0.57 0 0.19 0.36 group mean age ±sd min. age max. age p value* gender n (%) p value† experimental 22.56 ± 3.50 18 28 0.130 male 3 (16.7) 0.674 female 15 (83.3) control 20.89 ± 2.90 17 28 male 4 (22.2) female 14 (77. 8) total 21.72 ± 3.28 17 28 male 7 (19.4) female 29 (80.6) table 1: basic sample characteristics table 2: descriptive statistics of little’s irregularity index values (mm) in the two groups. *: employing independent t-test, min: minimum, max: maximum, †: employing chi-square test liit0: little’s irregularity index before treatment; liit1: after 2 weeks of treatment; liit2: after 4 weeks; liit3: after 6 weeks; liit4: after 8 weeks; sd: standard deviation; min: minimum; max: maximum, q1: first quartile; q3: third quartile. http://dentistry3000.pitt.edu/ assessment of the alignment accelera1on of the lower incisors using a single aligner appliance modified with nickel-titanium coils: a randomized controlled clinical trial in adults. vol 12 no 1 (2024) doi 10.5195/d3000.2024.532 h#p://den*stry3000.pi#.edu little's irregularity index group mean (sd) mean difference 95% ci of the difference p-value lower bound upper bound before treatment experimental 3.10 (0.68) 0.001 -0.46 0.46 0.994 control 3.09 (0.68) at 2 weeks experimental 1.88 (0.56) -0.35 -0.77 0.06 0.095 control 2.23 (0.66) at 4 weeks experimental 1.01 (0.51) -0.38 -0.74 -0.02 0.037* control 1.40 (0.54) at 6 weeks experimental 0.37 (0.33) -0.26 -0.53 0.01 0.060 control 0.63 (0.46) at 8 weeks experimental 0.10 (0.17) -0.08 -0.20 0.04 0.191 control 0.18 (0.19) discussion as far as we know, this research is the initial randomized controlled trial to assess the acceleration impact of using a modified single aligner appliance with nickel-titanium springs to align mildly crowded lower incisors. this study is a well-controlled clinical trial with a parallel-group design, and a simple computerized randomization method was used to allocate participants to the two groups to avoid bias in patient selection. it was not possible to blind the researcher and patients during the evaluation period due to the researcher's involvement in the application and periodic monitoring of the orthodontic appliances. however, the researcher was blinded during the plaster cast study phase and measurements were performed by another academic physician who had no connection to the research in group time period mean difference t p-value experimental liit0liit1 1.21 25.51 <0.001** liit1liit2 0.86 17.99 <0.001** liit2liit3 0.64 11.37 <0.001** liit3liit4 0.27 5.54 <0.001** liit0liit4 2.99 21.60 <0.001** control liit0liit1 0.86 24.61 <0.001** liit1liit2 0.83 21.84 <0.001** liit2liit3 0.76 24.04 <0.001** liit3liit4 0.45 6.84 <0.001** liit0liit4 2.91 23.25 <0.001** table 3: the results of the observed lii changes between two groups (in mm)†. †: independent t-test, *: significant at p<0.05; sd: standard deviation. table 4: the results of the observed lii changes within each group †. †: paired t-test, *: significant at p<0.05,**: significant at p<0.001; liit0: little’s irregularity index before treatment; liit1: after 2 weeks of treatment; liit2: after 4 weeks; liit3: after 6 weeks; liit4: after 8 weeks; sd: standard deviation. http://dentistry3000.pitt.edu/ assessment of the alignment accelera1on of the lower incisors using a single aligner appliance modified with nickel-titanium coils: a randomized controlled clinical trial in adults. vol 12 no 1 (2024) doi 10.5195/d3000.2024.532 h#p://den*stry3000.pi#.edu order to avoid bias in the investigation. in order to prevent the oversight of important changes, the assessment was performed every two weeks throughout the duration of the treatment to accurately monitor progress. to avoid discrepancies in metabolic rate across various age groups, participants between the ages of 16 and 28 were chosen for the study. the experimental group had an average age of 22.56 ±3.50 years, while the control group had an average age of 20.89 ±2.90 years [13]. the majority of participants in this study were women, aligning with previous studies that women are more likely to seek orthodontic treatment as adults [14, 15]. digital dental scanning was relied upon to obtain a digital model as it is an accurate and proven effective method [16, 17]. additionally, the device was manufactured based on a 3d printed resin model, which studies have shown to provide clinically acceptable results [17, 18]. the niti springs were activated using a force of 80 g on both sides because each tooth requires a force between 35 and 60 g to create a tipping motion [19]. specifically, a force of 40 g was applied to each incisor, resulting in a total force of 160 g (80 g on each side). in order to ensure accurate results, a fixed type of appliance was utilized in this research, which eliminates the reliance on patient compliance. it has been established in previous studies that achieving optimal treatment outcomes with removable appliances is contingent upon patient compliance [20, 21]. the researchers used lii as a method to measure the progress in alignment because it is a reliable and widely accepted way to accurately measure the difference in length of the front arch. this method has been proven to be consistent and trustworthy in previous studies [22]. the study results showed that the average values of little's index were lower in the experimental treatment group compared to the control group. this can be explained by the difference in the application of force between the two appliances, as the point of force application in the traditional fixed orthodontic appliance is towards the buccal side from the center of resistance [23], while it is towards the lingual side in the application of the aligner appliance. this means that it is closer to the center of resistance of the anterior teeth, which may affect the speed of alignment of the lower incisors as in the lingual orthodontic technique [24]. however, this slight difference is not clinically or statistically significant at all evaluation times except at t2 (p=0.037). this can also be attributed to the magnitude of force applied at the beginning of treatment, as the forces applied in the aligner appliance are calibrated and consistent, while these forces were random and unmeasurable in the control group [25]. therefore, the forces in the aligner appliance gradually dissipate as the treatment progresses and crowding is resolved, which explains the absence of differences in subsequent stages. no study has evaluated the alignment of anterior teeth using any type of spring appliance, and the difference in this current study from other studies limits the possibility of comparison with them [11]. the findings showed a significant difference in the value of the lii between the five time periods studied within both groups (p<0.001). this means that the value of this index decreased significantly as the lower anterior teeth were aligned in both groups. this difference was not due to any problem in the progress of treatment in any of the studied time periods. the application of the single aligner appliance modified with nickeltitanium coils within the lower anterior dental arch was the main limitation of the current study. conclusion the clinical use of a modified aligner appliance with nickel-titanium coils effectively resolves lower incisors crowding, but it does not accelerate the alignment process compared to the conventional fixed orthodontic appliance. references http://dentistry3000.pitt.edu/ assessment of the alignment accelera1on of the lower incisors using a single aligner appliance modified with nickel-titanium coils: a randomized controlled clinical trial in adults. vol 12 no 1 (2024) doi 10.5195/d3000.2024.532 h#p://den*stry3000.pi#.edu 1. the impact of labial fixed appliance orthodontic treatment on patient expectation, experience, and satisfaction: an overview of systematic reviews. yassir ya, mcintyre gt, bearn dr. eur j orthod. 2020 jun; 42(3): 223-230. pmid: 31147683. 2. prevalence of malocclusion, its association with occlusal interferences and temporomandibular disorders among the saudi subpopulation. haralur sb, addas mk, othman hi, shah fk, elmalki ai, al-qahtani ma. oral health dent manag. 2014 jun; 13(2): 164-169. pmid: 24984617. 3. oral health-related quality of life of adolescents after orthodontic treatment. a systematic review. ferrando-magraner e, garcíasanz v, bellot-arcís c, montielcompany jm, almerich-silla jm, paredes-gallardo v. j clin exp dent. 2019 feb; 11(2): e194e202. pmid: 30805125. 4. the inman aligner for anterior tooth alignment. qureshi a. dent update. 2008 oct; 35(8): 569-571, 574-576. pmid: 19055094. 5. assessment of perceived orthodontic appliance attractiveness. ziuchkovski jp, fields hw, johnston wm, lindsey dt. am j orthod dentofacial orthop. 2008 apr; 133(4 suppl): s68-78. pmid: 18407023. 6. relationship between patient discomfort, appliance acceptance and compliance in orthodontic therapy. doll gm, zentner a, klages u, sergl hg. j orofac orthop. 2000 nov; 61(6): 398-413. pmid: 11126015. 7. discomfort associated with invisalign and traditional brackets: a randomized, prospective trial. white dw, julien kc, jacob h, campbell pm, buschang ph. angle orthod. 2017 nov; 87(6): 801-808. pmid: 28753032. 8. the efficacy of orthodontic treatments for anterior crowding with invisalign compared with fixed appliances using the peer assessment rating index. lanteri v, farronato g, lanteri c, caravita r, cossellu g. quintessence int. 2018 jun; 49(7): 581-587. pmid: 29881831. 9. [possibilities and limitations of treatment with clear aligners. an orientation]. doomen ra, aydin b, kuitert r. ned tijdschr tandheelkd. 2018 oct; 125(10): 533-540. pmid: 30317374. 10. protecting the integrity of mandibular incisor position through keystoning procedure and spring retainer appliance. barrer hg. j clin orthod. 1975 aug; 9(8): 486-494. pmid: 1072074. 11. evaluation of the effectiveness of modified aligner appliance with nickel-titanium springs in treatment of mild crowding of lower incisors: a randomized controlled clinical trial. alhafi zm, rajeh n. j world fed orthod. 2022 aug; 11(4): 107113. pmid: 35624004. 12. the irregularity index: a quantitative score of mandibular anterior alignment. little rm. am j orthod. 1975 nov; 68(5): 554-563. pmid: 1059332. 13. factors related to the rate of orthodontically induced tooth movement. dudic a, giannopoulou c, kiliaridis s. am j orthod dentofacial orthop. 2013 may; 143(5): 616-621. pmid: 23631963. 14. adult orthodontics--a review. nattrass c, sandy jr. br j orthod. 1995 nov; 22(4): 331337. pmid: 8580099. 15. a randomized clinical trial comparing mandibular incisor proclination produced by fixed labial appliances and clear aligners. hennessy j, garvey t, al-awadhi ea. angle orthod. 2016 sep; 86(5): 706-712. pmid: 27571371. 16. agreement of treatment recommendations based on digital vs plaster dental models. ko hc, liu w, hou d, torkan s, spiekerman c, huang gj. am j orthod dentofacial orthop. 2019 jan; 155(1): 135-142. pmid: 30591157. 17. accuracy of digital dental models and three-dimensional printed dental models in linear measurements and bolton analysis. suryajaya w, purbiati m, ismah n. f1000res. 2021 sep; 10(2): 180. pmid: 33968366. 18. evaluation of the fused deposition modeling and the digital light processing techniques in terms of dimensional accuracy of printing dental models used for the fabrication of clear aligners. jaber st, hajeer my, khattab tz, mahaini l. clin exp dent res. 2021 aug; 7(4): 591-600. pmid: 33258297. 19. a comparative study of forces in labial and lingual orthodontics using finite element method. mascarenhas r, chatra l, shenoy s, husain a, mathew j, dasnadi s. j indian orthod soc. 2015 mar; 49(1): 15-18. pmid: 158628. http://dentistry3000.pitt.edu/ assessment of the alignment accelera1on of the lower incisors using a single aligner appliance modified with nickel-titanium coils: a randomized controlled clinical trial in adults. vol 12 no 1 (2024) doi 10.5195/d3000.2024.532 h#p://den*stry3000.pi#.edu 20. a comparison between indirect and objective wear-time assessment of removable orthodontic appliances. schott tc, meyer-gutknecht h, mayer n, weber j, weimer k. eur j orthod. 2017 apr; 39(2): 170175. pmid: 27030283. 21. compliance with removable orthodontic appliances and adjuncts: a systematic review and meta-analysis. al-moghrabi d, salazar fc, pandis n, fleming ps. am j orthod dentofacial orthop. 2017 jul; 152(1): 17-32. pmid: 28651764. 22. estimating arch length discrepancy through little's irregularity index for epidemiological use. bernabé e, flores-mir c. eur j orthod. 2006 jun; 28(3): 269-273. pmid: 16648212. 23. on tooth movement. isaacson rj, lindauer sj, davidovitch m. angle orthod. 1993 dec; 63(4): 305-309. pmid: 8297056. 24. concepts on control of the anterior teeth using the lingual appliance. romano r. semin orthod. 2006 sep; 12(3): 178185. pmid: 101053. 25. measuring system for in vivo recording of force systems in orthodontic treatment-concept and analysis of accuracy. friedrich d, rosarius n, rau g, diedrich p. j biomech. 1999 jan; 32(1): 81-85. pmid: 10050954. http://dentistry3000.pitt.edu/ microsoft word vieira editorial 2015.docx   vol  3,  no  1  (2015)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2015.39           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.   rescuing  case  reports   alexandre  rezende  vieira     editor  in  chief   department  of  oral  biology,  university  of  pi4sburgh  school  of  dental  medicine,  pi%sburgh  pa,  usa                 cita%on:  vieira  ar.  (2015)  rescuing  case  re-­‐ ports.  den!stry  3000.  1:a001   doi:10.5195/d3000.2015.39   received:  november  25,  2015   accepted:    november  26,  2015   published:    december  18,  2015   copyright:  ©2015  vieira  ar.  this   is  an  open  ac-­‐ cess  ar!cle   licensed  under  a  crea-ve  commons   a!ribu!on  work  4.0  united  states  license.   email:  arv11@pi$.edu     this  issue  of  dentistry  3000   features  a  series  of  case  reports  that   have  particular  emphasis  on  condi-­‐ tions  that  appear  to  be  aggregating   in  families.  the  decision  to  empha-­‐ size  case  reports  aims  to  address   several  areas  of  concern.     one  of  the  consequences  of   the  shift  towards  evidence-­‐based   practice  is  the  assumption  that  case   reports  are  very  weak  evidence  in   the  hierarchy  of  research  evidence,   since  they  bring  information  that   has  not  been  rigorously  tested.   conversely,  this  shift  has  created  a   high  value  for  randomized  clinical   trials,  for  that  very  reason:  the  im-­‐ plementation  of  testing  by  rigorous   scientific  means.  it  is  interesting  to   say  the  least  to  realize  that  increas-­‐ ingly,  randomized  clinical  trials   have  become  divorced  from  normal   patient  populations  by  their  insist-­‐ ence  on  inclusion  of  patients  de-­‐ fined  by  narrow  criteria  and  ab-­‐ sence  of  comorbidities.  it  is  also  in-­‐ teresting  to  realize  that  the  original   definition  of  evidence-­‐based  prac-­‐ tice  that  included  clinician  experi-­‐ ence  and  characteristics  of  individ-­‐ ual  patients  as  well  as  sound  re-­‐ search  evidence  in  the  decision-­‐ making  process  has  been  hijacked   [1]  to  the  point  that  to  be  consid-­‐ ered  valid  evidence,  the  evidence   needs  to  be  derived  from  research,   eliminating  the  need  to  consider   both  the  clinician  experience  or  the   patient  characteristics.     this  scenario  has  steadily   declined  opportunities  for  scholars   to  publish  case  reports.  it  was   through  short  case  reports  that  for   the  first  time,  trisomy  of  chromo-­‐ some  21  was  linked  to  down  syn-­‐ drome  [2],  or  that  down  syndrome   would  associate  with  leukemia  [3].   almost  simultaneous  reports  in   germany  and  australia  led  to  tha-­‐ lidomide  to  be  identified  as  the   cause  of  phocomelia  in  newborns   [5].  or  a  1981  report  of  five  cases   that  for  the  first  time  described  a   syndrome  that  was  soon  to  be   known  as  aids.  the  report  conclud-­‐ ed:  "the  above  observations  sug-­‐ gest  the  possibility  of  a  cellular-­‐ immune  dysfunction  related  to  a   common  exposure  that  predisposes   individuals  to  opportunistic  infec-­‐ tions."  the  bottom  line  is  that  case   reports  can  provide  insight  to  im-­‐ portant  problems  that  deserve  fur-­‐ ther  study.     finally,  writing  case  reports   is  a  good  exercise  for  young  profes-­‐ sionals  in  training  and  can  serve  as   an  important  tool  to  teach  a  number   of  skills  from  appreciation  and  the   ability  to  critically  evaluate  infor-­‐ mation  to  generate  hypothesis  driv-­‐ en  questions  that  deserve  further   exploration.  this  issue  brings  a   number  of  case  reports  authored  by   young  professionals  and  emphasize   the  genetic  etiology  of  oral,  dental,   and  craniofacial  conditions.   rescuing  case  reports   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.39    http://dentistry3000.pitt.edu   2     dentistry  3000  will  contin-­‐ ue  to  publish  original  case  reports   and  support  the  scholarly  develop-­‐ ment  of  dental  professionals  both   young  and  experienced.   references   1.  evidence-­‐based  case  reports.  bol-­‐ ton  j.  j  can  chiropr  assoc  v.58(1);   2014  mar  pmc3924510   2.  [chromosomic  diagnosis  of  mongo-­‐ lism].  lejeune  j,  turpin  r,  gautier  m.   arch  fr  pediatr.  1959;16:962-­‐3.   french.  pmid:14415503   3.  inheritance  of  an  abnormal  chro-­‐ mosome  in  down's  syndrome  (mon-­‐ golism)  with  leukemia.  german  jl,   demayo  ap,  bearn  ag.  am  j  hum   genet.  1962  mar;14(1):31-­‐43.   pmid:17948467   4.   recognition  of  thalidomide   defects.  smithells  rw,  newman  cg.  j   med  genet.  1992  oct;29(10):716-­‐23.   pmid:1433232   5.  pneumocystis  pneumonia-­‐-­‐los  an-­‐ geles.  centers  for  disease  control   (cdc).  mmwr  morb  mortal  wkly   rep.  1981  jun  5;30(21):250-­‐2.   pmid:6265753     747 2024 five techniques of osseodensifica3on in one case vol 13 no 2 (2024) doi 10.5195/d3000.2024.747 h#p://den*stry3000.pi#.edu five techniques of osseodensification in one case abdulkarim sheikh ibrahim1, mohamad hassan jaafo2, anas abdo3 faculty of dental medicine, damascus university abstract ini$al stability of implants is an important factor for the osseointegra$on of dental implants. ini$al stability of implants can be lost when the bone is insufficient in cases of immediate implanta$on or when the bone density is insufficient, making it difficult to ensure good ini$al stability. many techniques have been used to increase bone density. this study tested the use of five methods to increase bone density in one pa$ent. this study concluded that the five methods used were effec$ve in densifying the bone and achieving clinically acceptable primary stability in low-density bone. keywords: ini$al stability; primary inser$on torque; implant stability quo$ent; densah burs; magne$c mallet. cita$on: ibrahim as, et al. (2024) five techniques of osseodensifica$on in one case. den$stry 3000. 1:a001 doi:10.5195/d3000.2024.747 received: september 29, 2024 accepted: october 1, 2024 published: october 7, 2024 copyright: ©2024. this is an open access ar$cle licensed under a crea$ve commons a[ribu$on work 4.0 united states license. email: abdulkarim.sh.ibrahim@gmail.com introduction primary implant stability is a critical factor for achieving osseointegration of the dental implants. primary implant stability is critical in immediate loading protocols. implant micromovement greater than 50100 μm has been reported to increase peri-implant bone resorption or implant failure [1]. in an in vivo study, a statistically significant association between peri-implant bone density, primary insertion torque, and micromovement was reported. a significant increase in insertion torque and a concomitant decrease in micromovement were observed with increasing bone density values [2]. a systematic review reported no significant difference in maxillary bone resorption and failure rate between implants inserted with high or low insertion torque values [3]. it was also noted the ability of densifying drills to increase the bone volume and bone-to-implant contact (bic) ratio in dental implants inserted in low-density bone compared to conventional densification, which may help promote osseointegration [4]. unlike conventional densification, in densah densification, the bone is not drilled but compressed and the autologous bone is implanted during cutting, thus preserving the vital bone tissue [5]. a novel method for preparing the dental implant site by densifying the recipient's bone is the magnetic mallet. dynamic magnetic technology exploits the physical principles of electromagnetism to apply controlled forces to the body while reducing the time of action. control and stability of applied forces make procedures safe for patients and surgeons [6]. the magnetic mallet consists of a handpiece activated by a power control device, delivering forces according to the timing of application, where different inputs can be attached to the handpiece, which drives a wave on its tip according to the surgical procedure [7]. the present study describes the use of five methods for densifying five techniques of osseodensifica3on in one case vol 13 no 2 (2024) doi 10.5195/d3000.2024.747 h#p://den*stry3000.pi#.edu low-density bone when implanting nine maxillary implants in one individual to test the hypothesis that there are no differences in healing among different techniques. subject and results a 43-year-old female with no systemic diseases and a nonsmoker with complete edentulous upper jaw, except for teeth 11, 13, 21, and 22 that were nonrestorable root remnants, looked for care in our clinics. she underwent a radiological examination using both panoramic imaging and cone beam computed tomography (cbct) to plan for dental implants. after studying the radiograph, it was found that the bone three dimensions were suitable for dental implant placement. however, the bone density around the premolars and maxillary molars was low (class iv according to lekholm and zarb classification), with an average of 250 hounsfield units. local anesthesia was performed with infiltration using 4% articaine with 1:80,000 adrenaline, and extraction of the root remnants was performed (figure 1). after that, a gingival flap was raised (figure 2) over the entire upper jaw, where it was decided that nine dental implants were going to be placed. the maxillary first and second molar implants on the right side were initially prepared using densah densification burs (jksurgical, pakistan). the sites were initially prepared using a 2 mm diameter pilot drill, and were gradually increased using densah burs (figure 3), until they reached a diameter of 3.3 mm. two implants of 4 mm diameter and 8 mm in lenght (anyone, megagen, korea) were placed in the first and second molar sites, and the primary insertion torque was measured using a megagen anyone ratchet. the implant stability quotient (isq) was measured using a mega isq (megagen, korea) device. then the maxillary first and second molar implants on the left side were prepared using a magnetic mallet (meta ergonomica srl; turbigo mi, italy) (figure 4), where they were initially prepared using a 2 mm diameter pilot drill, then gradually increased using heads until they reached a diameter of 3 mm. two implants of 4 mm diameter and 8 mm in length (anyone, megagen, korea) were placed in the first and second molar sites, and the primary insertion torque was measured using a megagen anyone ratchet. the implant stability quotient (isq) was measured using a mega isq (megagen, korea) device. the first premolar implant sites (between the first premolar site and the second premolar site) were prepared on both sides using osteotomes (friadent, usa) (figure 5), after expansion to 2 mm was done using a driving drill. then, the tampers were graduated to a diameter of 3.8 mm. implants with a diameter of 4 mm and a length of 8 mm (anyone, megagen, korea) were placed into the premolar sites. the primary insertion torque was measured using a megagen anyone ratchet, and the implant stability quotient of the dental implants (isq) was measured using a mega isq (megagen, korea) device. then, the right and left canine implant sites were prepared using the bonex expanders (megagen, korea), where they were initially prepared using a 2 mm diameter pilot drill, then mechanical expanders were used to reach a diameter of 3.3 mm. two implants with a diameter of 4 mm and 8 mm in length (anyone, megagen, korea) for the left canine site and 10 mm in length (anyone, megagen, korea) fort the right canine site were placed. the primary insertion torque was measured using a megagen five techniques of osseodensifica3on in one case vol 13 no 2 (2024) doi 10.5195/d3000.2024.747 h#p://den*stry3000.pi#.edu anyone ratchet, and the implant stability quotient of the dental implants isq was measured using a mega isq (megagen, korea) device. eventually, the implant site was prepared in the right incisor site using the undersized preparation technique, and preparation was carried out using a pilot drill with a diameter of 2 mm, reaching 10 mm, then moving to the second drill with a diameter of 2.5 mm, and then to the final drill of 2.8 mm in diameter. an implant with a diameter of 4 mm and 8 mm in length (anyone, megagen, korea) was placed in the right incisor site. the primary insertion torque was measured using a megagen anyone ratchet, and the implant stability quotient of the dental implants isq was measured using a mega isq (megagen, korea) device. the gingival flap was then sutured with continuous and interrupted sutures using 5/0 nylon sutures (figure 6). the patient was given postoperative instructions. the sutures were removed two weeks after surgery and the patient did not suffer from any complications during the healing period up to four months, where the gingival healing abutment was applied (figure 7). interrupted sutures were performed using 5/0 nylon sutures. the sutures were removed after two weeks, and an impression was taken using open transfer (figure 8). a pfm restoration was performed (figure 9). the patient was followed up for 18 months and a panoramic x-ray was performed (figure 10). implant details can be seen in table 1. figure1. the upper jaw after extractions. figure 2. the gingival flap elevation. figure 3. densah burs, bonex expanders, osteotomes, and the anyone implant kit. figure 4. the magnetic mallet handpiece and head. figure 5. densification using osteotome. figure 6. suture after implantation. five techniques of osseodensifica3on in one case vol 13 no 2 (2024) doi 10.5195/d3000.2024.747 h#p://den*stry3000.pi#.edu figure 7. the tissue appearance after healing abutment application. figure 8. the open tray impression. figure 9. the final pfm prosthetic. figure 10. panoramic x-ray after 18 months of implantation. discussion the superiority of the magnetic mallet in primary insertion torque can be explained by the minimal bone removal with the pilot study drill compared to other methods, where the required preparation before starting the densification was just 1.2 mm in size. densah burs were the second most conservative, and this is also due to the lateral densification that these burs perform at the preparation site, as both their designs and their counterclockwise rotations ensure good bone densification. these two techniques outperformed the bone compactors, and this may be attributed to the anatomical compatibility between these two methods and the shape of the implant, while compactors are generally cylindrical or slightly graduated, which means a wider diameter for preparation in the apical region and less apical stability. the superiority of the preparation technique using bone densification burs in isq over other methods can be explained by its special bone densification mechanism, thanks to the unique design of the drill shape and the counterclockwise rotation, which helps in densifying the bone apically and laterally, and enhances the local density, increasing the contact surface area between the implant and the surrounding bone, thus positively improving the stability of the implant. the good isq values in the magnetic hammer preparations and osteodensification burs can also be explained by the lateral and apical bone density achieved by the tools for preparing the implant site using the magnetic hammer device and the minimal bone removal, thus increasing the contact surface area between the implant and the bone. the good implant stability values obtained with the bone expander preparation can be explained by the minimal bone removal in the implant site and the lateral bone density it performs. the undersized preparation technique achieved acceptable isq values for the implants and this is attributed to the significant difference between the diameter of the last drill used to prepare the five techniques of osseodensifica3on in one case vol 13 no 2 (2024) doi 10.5195/d3000.2024.747 h#p://den*stry3000.pi#.edu site and the diameter of the implant and the effect of it on improving the local bone density and increasing the contact surface area between the implant and the surrounding bone. previous work [8] looked at traditional preparation and densah densification burs in increasing implant stability at implant sites prepared in the bone of pigs after slaughter and concluded that densification using densah burs led to a greater insertion torque than the traditional method and to a greater isq than the traditional preparation. densah densification burs with expanders in the upper premolar region, measured immediately after implant placement and after two months, six months, and eight months, showed that the implant stability score immediately after implant placement was statistically significantly greater in favor of the expanders [9]. magnetic mallet was also previously shown to outperform the traditional preparation in achieving greater implant stability [10]. conclusion within the limitations of this clinical case, we conclude that the five methods used were effective in densifying the bone and achieving clinically acceptable primary stability in low-density bone, with both densah burs and magnetic mallet outperforming the other methods. references 1. mello-machado rc, sartoretto sc, granjeiro jm, calasans-maia jda, de uzeda mjpg, mourão cfdab, et al. osseodensification enables bone healing chambers with improved low-density bone site primary stability: an in vivo study. scientific reports. 2021;11(1):15436. 2. trisi p, berardini m, falco a, vulpiani mp. effect of implant thread geometry on secondary stability, bone density, and boneto-implant contact: a biomechanical and histological analysis. implant dentistry. 2015;24(4):384-91. 3. berardini m, trisi p, sinjari b, rutjes aw, caputi s. the effects of high insertion torque versus low insertion torque on marginal bone resorption and implant failure rates: a systematic review with meta-analyses. implant dentistry. 2016;25(4):532-40. 4. lahens b, neiva r, tovar n, alifarag am, jimbo r, bonfante ea, et al. biomechanical and histologic basis of osseodensification drilling for endosteal implant placement in low density bone. an experimental study in sheep. journal of the mechanical behavior of biomedical materials. 2016;63:56-65. 5. meyer e, huwais s. osseodensification is a novel implant preparation technique that increases implant primary stability by compaction and autografting bone. san francisco, ca: american academy of periodontology. 2014. 6. gopi a. magnetic mallet for indirect sinus lift and simultaneous implant placement: a retrospective study. journal of dentistry defense section. 2023;17(2):66-70. 7. oddi c, collina g, ceresoli l, ferri s, grassi a, nagni m. use of the magnet mallet in a case of extraction and post-extraction implant site preparation, in combination with regenerative therapy on hypertensive patient: a case report and literature review. annali di stomatologia. 2023;14(1):29-34. 8. cáceres f, troncoso c, silva r, pinto n. effects of osseodensification protocol on insertion, removal torques, and resonance frequency analysis of biohorizons® conical implants. an ex vivo study. journal of oral biology and craniofacial research. 2020;10(4):625-8. 9. mahmoud riad ah, fahmy a, el khourazaty n. evaluation of implant’s primary stability using densah bur versus expander in five techniques of osseodensifica3on in one case vol 13 no 2 (2024) doi 10.5195/d3000.2024.747 h#p://den*stry3000.pi#.edu patients with missing maxillary premolar (a randomized clinical trial). egyptian dental journal. 2021;67(2):1487-95. 10. sanad a, elsheikh sa, el halawani gn. evaluation of the effect of magnetic mallet versus conventional drilling implant placement protcol on periimplant osseointegration in anterior maxillary region. alexandria dental journal. 2024. table 1. length, diameter, primary insertion torque, and implant stability quotient of the placed implants. implant site implant length (mm) implant diameter (mm) primary insertion torque (n/cm2) implant stability quotient 17 8 4 41 81 16 8 4 39 82 14 8 4 33 75 13 10 4 37 68 11 8 4 32 71 23 8 4 35 67 24 8 4 34 77 26 8 4 40 79 27 8 4 38 80 vol 2, no 1 (2014) issn 2167-8677 (online) doi 10.5195/d3000.2014.26 http://dentistry3000.pitt.edu new electronic apex locator romiapex a-15 presented accuracy for working length determination in permanent teeth etevaldo matos maia filho1, cláudia de castro rizzi2, daniela silva barroso de oliveira3, paulo nelson filho4, raquel assed bezerra da silva4, léa assed bezerra da silva4 1school of dentistry of ceuma university, são luís, maranhão, brazil. 2phd student, department of pediatric clinics, school of dentistry of ribeirão preto, university of são paulo, ribeirão preto, são paulo, brazil; school of dentistry of ceuma university, são luís, maranhão, brazil. 3phd student, department of pediatric clinics, school of dentistry of ribeirão preto, university of são paulo, ribeirão preto, são paulo, brazil; school of dentistry, federal university of alfenas, alfenas, minas gerais, brazil. 4department of pediatric dentistry, school of dentistry of ribeirão preto, university of são paulo, ribeirão preto, são paulo, brazil. abstract purpose: the present study aims to evaluate, ex vivo, the accuracy of electronic apex locators root zx ii and romiapex-15, for working length (wl) determination in permanent teeth. materials and methods: fourteen single-rooted teeth (incisors and canines), with their apices fully formed were used. the dental crowns were removed. the anatomic length of the tooth (real measurement) was visually determined through the insertion of a size 10 k-file until the tip of the instrument could be observed in the apical foramen under a microscope (20x). teeth were fixed in a model of resin and adapted into alginate soaked with saline solution, which was used as an electrical conductor. using a k-file, root canals were measured electronically using both devices. the results obtained for each apex locator were compared to the real measurements. the accuracy between the devices was statistically analyzed using the bland-altman graph, intraclass correlation coefficient (icc), and student’s t-test. results: the mean difference between the measurements using the root zx ii was 0.277mm greater than the real measurement, while the measurements from the romiapex-15 were 0.308mm higher on average. the comparison between root zx ii and romiapex-15 had no significant difference (p= 0.868). conclusion: it was concluded that root zx ii and romiapex-15 had similar accuracy. romiapex-15 could be an option for wl determination in permanent teeth. citation: maia filho em, rizzi cc, oliveira dsb, nelson-filho p, silva rab, silva lab . (2014) new electronic apex locator romiapex a-15 presented accuracy for working length determination in permanent teeth. dentistry 3000. 1:a001 doi:10.5195/d3000.2014.26 received: september 22, 2014 accepted: november 1, 2014 published: december 18, 2014 copyright: ©2014 filho et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: raquel@forp.usp.br introduction working length (wl) determination is a crucial step in endodontic treatment. however, the ideal wl for root canal treatment has been an issue of discussion and controversy. sjogren et al., (1990) [1] performed an epidemiological study and observed that the endodontic treatment had a better prognosis when the apical limit obturation was 2.0mm from the radiographic apex. these results were better explained by ricucci and langeland (1998) [2], who performed a microscopic study and found that the best condition of repair was when the instrumentation and root canal filling remained close to the apical constriction. thus, preferably, the instrumentation and the obturation should stay in the narrow portion of the root canal, i.e. in apical constriction, because it causes less damage and thus provides better conditions for repair [2]. the most popular method for the establishment of the wl is the radiographic method, which is based on the premise that http://dentistry3000.pitt.edu/ new electronic apex locator romiapex a-15 presented accuracy for working length determination in permanent teeth vol 2, no 1 (2014) doi 10.5195/d3000.2014.26 http://dentistry3000.pitt.edu 2 the apical constriction is located between 0.5 to 1.0mm of the radiographic apex in both permanent and primary teeth [3,4]. determination using the radiographic method has several limitations, such as: distortion, shortening and lengthening of the image, variability of interpretation, and it can only represent dimensional structures in a two dimension image. in this way, the radiographic estimation may lead to over or under root canal instrumentation and/or obturation. currently, the method of wl determination considered to be more accurate is electronic, which uses electronic apex locators (eal) [5]. these devices are based on measurements of impedance ratio and are able to determine the distance between the highest foramen and the lowest foramen based on the difference in the file tip and the fluid in the channel at different frequencies through the electrical resistance of the dentin [6]. several models of eal use the principle of impedance to determine apical constriction and published papers have showed their effectiveness in working length determination [7-13]. recently, electronic equipment has been popularized and the device root zx ii (j. morita, tokyo, japan) has been proven to be a reference for new equipment testing due to its good results [14-17]. however, new devices have been introduced to the market and it is mandatory to assess their accuracy to determine wl. thus, considering that there are few studies evaluating the romiapex a-15 [18-20], this ex vivo study aimed to evaluate the accuracy of romiapex15 in the determination of the wl of permanent teeth in comparison to root zx ii – a standard method used in literature. materials and methods this study was approved by the research ethics committee of the university center of maranha o, brazil (protocol number 00519/11). fourteen extracted human teeth, both upper and lower, with a single root (incisors and canines) and with apices fully formed, were selected. the teeth were stored in 2.5% sodium hypochlorite solution for 6 hours and then stored in sterile saline solution. the crowns of the teeth were sectioned horizontally at the cementum-enamel junction in order to simplify access to the root canal and to obtain a reliable occlusal reference point. the canals were irrigated with saline solution and patency was checked with k-file size 10. if remnants of pulp were presented, they were removed. the real measurements of the root canal length were performed using a k-file with a silicone stopper passively introduced into the root canal until the tip was visible in the apical foramen. at this time, the silicone stopper was adjusted in occlusal reference and the file was removed from the root canal. the distance from the tip of the instrument to the stopper was recorded using an electronic caliper with an approximation of 0.01mm (mitutoyo digimatic, kyoto, japan). the measurements, in millimeters, were made by a calibrated examiner (kappa = 0.9) and were recorded in specific charts. for wl determination with the electronic devices, the teeth were fixed in a model of resin and soaked up to 2mm below the cementum-enamel junction in alginate molding (jeltrate ii dentply, petropolis, brazil). the alginate was prepared according to the manufacturer's recommendations. root canals were filled with 1.0% sodium hypochlorite solution (biodynamics, sa o paulo, brazil). cotton pellets were used eliminate excess saline from the pulp chamber. the root canal of all teeth was measured using the electronic apex locators romiapex a-15 (romidan ltd, kiryat ono, israel) and root zx ii (j. morita usa, irvine, ca -usa). initially, a file was gently inserted into the root canal until the appliance emitted a continuous audible tone and the word "apex" flashed on the root zx ii display and “0.0” on the display of the romiapex a-15. then, the file was retracted until the display of both pieces of equipment indicated the position stabilized at 0.5. at this time, the silicone stopper was adjusted in occlusal reference, the file removed from the root canal, and the distance from the tip of the instrument to the stopper was recorded using a digital caliper. all of the measurements were carried out by a single calibrated examiner and the results (in millimeters) obtained from the two pieces of equipment were tabulated. the results were statistically analyzed using the bland-altman graph and the intraclass correlation coefficient (icc) in order to test the null hypothesis that there is no correlation between the measurements and to compare the accuracy between the two figure 1. graphical representation of the difference in values obtained using the electronic apex locator root zx ii and the real measurements. fififd figure 2. graphical representation of the difference in values obtained using the electronic apex locator romiapex a-15 and the real measurements. http://dentistry3000.pitt.edu/ new electronic apex locator romiapex a-15 presented accuracy for working length determination in permanent teeth vol 2, no 1 (2014) doi 10.5195/d3000.2014.26 http://dentistry3000.pitt.edu 3 devices and the real measurements. to assess whether there was a difference between both apex locators, student's t-test was used. all analyses were carried out using the statistical program spss version 19.00 (spss inc., chicago, il, usa) with a significance level of 5%. results table 1 shows the differences between both eal and the real measurement, including the intraclass correlation coefficient (icc) and p values. it was observed that the mean of the differences between the measurements using the root zx ii was 0.277mm greater than the real measurements, while the romiapex-15 measurements were 0.308mm higher on average. figures 1 and 2 are a graphical representation of the differences between the measurement of linear distances obtained from both devices and real measurements. the vertical line represents the mean of the differences. the closer the vertical line is to zero, the greater the accuracy of the devices. the mean of the differences between the measurements obtained with the root zx ii and the romiapex a-15 was 0.030. the student's t-test showed that there was no statistically significant difference between the measurements performed with root zx ii and the romiapex a-15 (p= 0.868). discussion the evaluation of different endodontic equipment is important to guide clinicians in their dental practice. thus, in this study we evaluated two electronic apex locators ex vivo in comparison to real measurements of root canals. in order to maintain the experimental conditions with the smallest number of possible variables, total removal of the dental crown was performed to design a more reliable reference [21]. canals were irrigated with 1.0% sodium hypochlorite and excess in the pulp chamber was removed by aspiration, while keeping the root canals wet, in order to use the devices according the manufacturers’ instructions [22]. also, according to the literature, alginate was used for electrical conduction in ex vivo studies with eal [23]. in this study, the alginate was also used for maintenance of teeth and allowed electrical conduction in the ex vivo evaluation. several studies assessed the accuracy of the eal root zx ii to determine the wl in deciduous and permanent teeth [1417; 24-26], and, due to the excellent results found, this device has been used as parameter for comparison with other equipment. although other previous studies attempted to evaluate romiapex a-15 [18-20], our study adds important information to endodontic literature confirming the accuracy of both tested apex locators. it is recommended to use the technique of apical setback to establish the wl using eal. this consist of the introduction of the file to the foramen (which appears on the devices’ screen as “0.0” or “apex”), and then performing the setback until the apical constriction is reached. this technique allows the user to identify the first contrition toward the apex-crown. according ricucci (1998) [27], this constriction is located 0.5 to 1.0mm coronal to the foramen and is considered an ideal spot for instrumentation and obturation of the root canals. the average difference between the measurements obtained with the romiapex a-15 and the real measurements (0.308mm) was greater than the mean difference obtained with the root zx ii and the real measurements (0.277mm). the variation of ± 0.5mm in wl is acceptable to avoid over-instrumentation [28]. therefore, we can conclude that both devices were within the recommended standards. romiapex a-15 presented a lower dispersion (standard deviation = 0.391mm) compared to the root zx ii (standard deviation = 0.472). in addition, the error ranging was 0.542 for the romiapex a-15, while it was 0.654 for the root zx ii. this means that the measurements obtained with the romiapex a-15 showed a lower oscillation pattern. measurements obtained with the romiapex a-15 were closer to the vertical line than the measurements from the root zx ii. the smallest variation of measurements obtained with romiapex a-15 represents the high correlation with the real measurement showed by the interclass test. however, when the difference in measurements obtained with both devices was assessed, no statistical difference was found. it is important to emphasize that the difference found in this study is not clinically significant for endodontic treatment, which corroborates with vasconcelos et al. (2013) [28]. conclusion although ex vivo studies have some limitations, it is possible to conclude that the electronic apex locator romiapex-15 presented accuracy in the determination of the wl in single-rooted permanent teeth and had results similar to the root zx ii. references 1. factors affecting the long-term results of endodontic treatment. sjogren u, hagglund b, sundqvist g, wing k. j endod. 1990 oct; 16: 498-504. pmid:20844204 2. apical limit of root canal instrumentation and obturation, part 2. a histological study. ricucci d, langeland k. int endod j. 1998 nov; 31: 394-409. pmid:15551607 3. study of the apices of human permanent teeth with the use of a scanning electron microscope. morfis a, sylaras sn, georgopoulou m, kernani m, prountzos f.oral surg oral med oral pathol. 1994 feb; 77: 172-6. pmid:8139836 4. electronic apex locator: a useful tool for root canal treatment in the primary dentition. katz a, mass e, kaufman ay. asdc j dent child. 1996 nov-dec; 63: 414-7. pmid:9017174 5. the fundamental operating principles of electronic root canal length measurement devices. nekoofar mh, ghandi mm, hayes sj, dummer pm. int endod j. 2006 aug; 39: 595-609. pmid:16872454 6. new principle and method for measuring the root canal length. ushiyama j. j endod. 1983 mar; 9(3): 97-104. pmid: 6590776 7. ex vivo evaluation of the ability of four different electronic apex locators to determine the working length in teeth with various foramen diameters. ebrahim ak, wadachi r, suda h. aust dent j. 2006 sep; 51:258-62. pmid:17037894 diferences min. max. average difference standard deviation range error icc p root zx ii real -0.51 1.03 0.277 0.472 ±0.654 0.981 <0.001 romiapex a-15 -real -0.21 1.39 0.308 0.391 ±0.542 0.990 <0.001 table 1. data obtained after the comparison between the differences in measurement of electronic apex locators root zx and romiapex a-15 and real measurements. http://dentistry3000.pitt.edu/ new electronic apex locator romiapex a-15 presented accuracy for working length determination in permanent teeth vol 2, no 1 (2014) doi 10.5195/d3000.2014.26 http://dentistry3000.pitt.edu 4 8. evaluation of precision of length determination with 3 electronic apex locators: root zx, elements diagnostic unit and apex locator, and romiapex d-30. bernardes ra, duarte ma, vasconcelos bc, moraes ig, bernardineli n, garcia rb, baldi jv, victorino fr, bramante cm. oral surg oral med oral pathol oral radiol endod. 2007 oct; 104:e91-4. pmid:17703968 9. electronic working length determination in primary teeth by propex and digital signal processing. nelson-filho p, lucisano mp, leonardo mr, da silva ra, da silva la. aust endod j. 2010 dec; 36:105-8. pmid:21091842 10. accuracy of the ipex multi-frequency electronic apex locator in primary molars: an ex vivo study. nelson-filho p, romualdo pc, bonifa cio kc, leonardo mr, silva ra, silva la. int endod j. 2011 apr;44:303-6. pmid:21166827 11. comparison of accuracy of two electronic apex locators in the presence of various irrigants: an in vitro study. mull jp, manjunath v, manjunath m. j conserv dent. 2012 apr;15:178-82. pmid:22557820 12. an in vivo evaluation of different methods of working length determination. mandlik j, shah n, pawar k, gupta p, singh s, shaik sa. j contemp dent pract. 2013 jul;14:644-8. pmid:24309342 13. efficiency of 2 electronic apex locators on working length determination: a clinical study. koçak s, koçak mm, sag lam bc. j conserv dent. 2013 may;16:229-32. pmid:23833456 14. comparative evaluation of accuracy of two electronic apex locators in the presence of various irrigants: an in vitro study. jain s, kapur r. contemp clin dent. 2012 sep; 3(suppl 2): s140-5. pmid:23230349 15. comparison of working length determination with radiographs and four electronic apex locators. vieyra jp, acosta j. int endod j. 2011 jun; 44(6): 510-8. pmid:21306402 16. clinical reproducibility of three electronic apex locators. miletic v, beljicivanovic k, ivanovic v. int endod j. 2011 aug; 44(8): 769-76. pmid:21599708 17. evaluation of the joypex 5 and root zx ii: an in vivo and ex vivo study. soares rm, silva ej, herrera dr, krebs rl, coutinho-filho ts. int endod j. 2013 oct;46:904-9. pmid:23480176 18. clinical reproducibility of three electronic apex locators. miletic v, beljicivanovic k, ivanovic v. int endod j. 2011 aug; 44(8): 769-76. pmid:21599708 19. accuracy of five electronic foramen locators with different operating systems: an ex vivo study. vasconcelos bc, bueno mm, luna-cruz sm, duarte ma, fernandes ca. j appl oral sci. 2013 mar-apr; 21(2): 132-7. pmid:23739852 20. ex vivo accuracy of root zxii, root zx mini and romiapex a-15 apex locators in extracted vital pulp teeth. silva tm, alves fr. j contemp dent pract. 2014 may; 15(3): 312-4. pmid:25307812 21. accuracy of the justy ii apex locator in determining working length in simulated horizontal and vertical fractures. azabal m, garcia-otero d, de la macorra jc. int endod j. 2004 mar; 37: 174-7. pmid:15009406 22. the ability of two apex locators to locate the apical foramen: an in vitro study. d'assuncao fl, de albuquerque ds, de queiroz ferreira lc. j endod. 2006 jun; 32: 560-22. pmid:16728251 23. influence of embedding media on the assessment of electronic apex locators. baldi jv, victorino fr, bernardes ra, de moraes ig, bramante cm, garcia rb, et al. j endod. 2007 apr; 33: 476-9. pmid:17368343 24. in vitro evaluation of the accuracy of three electronic apex locators. lucenamartin c, robles-gijon v, ferrer-luque cm, de mondelo jm. j endod. 2004 apr; 30: 231-3. pmid:15085053 25. in vitro evaluation of the ability of three apex locators to determine the working length during retreatment. goldberg f, marroquin bb, frajlich s, dreyer c. j endod. 2005 sep; 31: 676-8. pmid:16123705 26. ex vivo accuracy of three electronic apex locators: root zx, elements diagnostic unit and apex locator and propex. plotino g, grande nm, brigante l, lesti b, somma f. int endod j. 2006 may; 39: 408-14. pmid:16640641 27. apical limit of root canal instrumentation and obturation, part 1. literature review. ricucci d. int endod j. 1998 nov; 31: 384-93. pmid:15551606 28. accuracy of five electronic foramen locators with different operating systems: an ex vivo study. de vasconcelos bc, bueno mde m, luna-cruz sm, duarte ma, fernandes ca. j appl oral sci. 2013 mar-apr;21:132-7. pmid:23739852 http://dentistry3000.pitt.edu/ microsoft word black proof 3-23.docx   vol  3,  no  1  (2015)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2015.31           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.   familial  aggregation  of  phenotypic  expression  of  premature  hair  hy-­‐ popigmentation  in  the  craniofacial  region   corey  l.  black1     1  university  of  pittsburgh  school  of  dental  medicine,  pittsburgh  pa,  usa     abstract   there  are  many  pa+ents  who  experience  premature  hypopigmenta+on  of  hair,  but  do  not   understand  the  underlying  causes  and  poten2al  dangers  associated  with  them.  the  causes   range  from  gene*c  predisposi*on  to  environmental  influences  such  as  tobacco  use.  prema-­‐ ture  hypopigmenta/on  of  the  hair  sha2  can  also  be  associated  with  many  syndromes;  some   which  cause  dental  anomalies.  today,  treatment  op6ons  are  limited  for  pa6ents,  although   various  studies  are  being  done  on  mice  to  target  the  underlying  mechanism  of  ac4on.  un-­‐ derstanding  the  differences  between  all  of  the  possible  causes  of  this  par5cular  phenotype   can   help   clinicians   be,er   iden/fy   the   symptoms,   educate   pa/ents,   and   possibly   modify   treatment  to  suit  the  needs  of  each  pa1ent  on  an  individual  basis.       cita%on:  black,  c.  (2015)  familial  aggrega*on  of   phenotypic   expression   of   premature   hair   hy-­‐ popigmenta!on   in   the   craniofacial   region.  den-­‐ !stry  3000.  1:a001  doi:10.5195/d3000.2015.31   received:  march  18,  2015   accepted:  march  ,  2015   published:  april  7  ,  2015   copyright:  ©2015  black.   this   is   an  open  access   ar!cle   licensed   under   a   crea!ve   commons   a!ribu%on  work  4.0  united  states  license.   email:  clb196@pi*.edu     introduction       determination  of  the  color   of  hair  is  a  complicated  process.   there  are  genes  that  dictate  color,   texture,  distribution,  and  number  of   stem  cells  [1].  gray  hair,  also  known   as  canities,  is  a  phenotypic  manifes-­‐ tation  of  a  process  in  while  melano-­‐ cytes  within  hair  follicles  stop  pro-­‐ ducing  melanin.  for  unknown  rea-­‐ sons,  this  process  occurs  with  most   individuals  as  the  aging  process   proceeds.  however,  with  a  fair   number  of  individuals,  the  process   of  pigmentation  of  the  hair  shaft   begins  much  sooner  than  the  aver-­‐ age  individual.    hair  is  said  to  gray   prematurely  when  it  occurs  before   the  age  of  20  in  whites,  25  in  asians   and  30  in  africans  [1].  there  have   been  many  proposed  causes  of  gray   hair  including  stress,  cigarette   smoking,  nutritional  deficiencies,   genetics,  and  a  variety  of  syn-­‐ dromes.  it  is  important  for  a  clini-­‐ cian  to  understand  the  many  possi-­‐ bilities  of  this  phenotype  in  order  to   be  able  to  identify  patients  who  may   display  premature  canities.      case  report           an  18-­‐year-­‐old  african   american  male,  individual  ii.2  (fig-­‐ ure  1)  began  to  experience  canities   prior  to  college  admission  in  july  of   2007.  the  patient  noticed  two  gray   hairs  in  the  center  of  his  scalp.  be-­‐ lieving  that  they  may  have  been   caused  by  stress,  he  left  them  alone   and  started  to  use  stress  reduction   techniques  such  as  exercise  and   constant  breaks  when  studying.  he   also  started  to  take  a  multi-­‐vitamin   to  ensure  that  the  presentation  of   the  gray  hairs  was  not  due  to  a  b-­‐12   or  iron  deficiency.  however,  as  the   years  progressed,  the  number  of   gray  hairs  continued  to  increase  at  a   rate  of  approximately  10-­‐15  hairs   per  year.  at  the  age  of  22,  the  phe-­‐ notypic  expression  of  the  gray  hairs   extended  to  his  facial  hair,  including   a  few  hairs  along  the  jaw  line  and  a   significant  amount  extending  across   the  anterior  region  of  the  neck.   within  a  span  of  8  years,  the  num-­‐ ber  of  hypopigmented  hairs  in-­‐ creased  from  2  to  approximately  80   (figure  2).      familial  aggrega*on  of  phenotypic  expression  of  premature  hair  hypopigmenta/on  in  the  craniofacial  region   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.31    http://dentistry3000.pitt.edu   2     the  patient’s  medical  histo-­‐ ry  was  negative  for  any  major  sys-­‐ temic  disease.  there  were  no  medi-­‐ cations,  other  than  antibiotics  and   multivitamins.  he  annually  visits  a   primary  care  physician  and  had  no   major  hospitalizations.  the  patient   has  a  family  history  of  cancer,  hy-­‐ pertension,  and  diabetes  on  the  ma-­‐ ternal  side.  there  is  a  history  of   heart  and  liver  disease  on  the  pa-­‐ ternal  side.       when  examining  the  family   history  of  the  patient,  it  appears   that  the  male  progeny  within  the   immediate  generation  are  affected   by  hypopigmentation  of  the  hair   follicles  of  the  craniofacial  area.  af-­‐ ter  consulting  with  his  father,  it  was   revealed  that  the  father  of  the  pa-­‐ tient  was  affected  by  the  same  phe-­‐ notype  of  premature  hypopigmen-­‐ tation  at  the  age  of  20.  there  was  a   similar  manifestation  of  the  initial   hypopigmentation  appearing  in  the   scalp  and  subsequent  hypopigmen-­‐ tation  in  the  anterior  region  of  the   neck.       the  individual  ii.1,  a  direct   male  descendant  of  i.1,  also  experi-­‐ enced  a  similar  phonotypical  mani-­‐ festation.  however,  the  hypopig-­‐ mentation  appeared  only  in  the  an-­‐ terior  neck  region  and  began  at  the   age  of  25  (figure  2).  this  may  be   explained  by  variable  expressivity   when  compared  to  the  early  presen-­‐ tation  of  this  phenotype  at  age  18  in   individual  ii.2.  individual  ii.1  also   experienced  male  pattern  baldness   at  the  age  of  22.  therefore,  it  is  un-­‐ clear  whether  the  follicles  on  the   scalp  were  affected  by  hypopigmen-­‐ tation.  individual  i.1  also  has  anoth-­‐ er  son  from  a  previous  marriage   who  is  also  affected  by  hypopig-­‐ mentation.  it  is  unsure  at  what  age   the  phenotype  was  expressed;  how-­‐ ever  it  was  before  the  age  of  30,   which  is  considered  premature.     the  patient’s  sister,  individual  ii.3   does  not  express  any  symptoms  of   hypopigmentation  (figure  1).       discussion     today,  the  exact  mechanism   of  canities  is  unknown.  however,   when  considering  the  underlying   cause  of  premature  hypopigmenta-­‐ tion,  one  must  consider  multiple   mechanisms,  encompassing  envi-­‐ ronmental,  systemic,  and  genetic   factors.  also,  the  expressivity  of  the   phenotype  varies  for  each  individu-­‐ al.  in  this  case,  there  seems  to  be  a   strong  genetic  link,  originating  from   the  paternal  side  of  the  family.     from  a  genetic  standpoint,   premature  graying  of  hair  occurs   more  commonly  without  any  under-­‐ lying  pathology  but  it  is  thought  to   be  inherited  in  autosomal  dominant   pattern  [2].  it  appears  that  the  gen-­‐ otype  is  one  that  includes  multiple   genes  and  peptides  that  affect  the   enzymes  responsible  for  melanin   production.       the  activity  of  the  tyrosi-­‐ nase  enzyme  is  thought  to  be  crucial   in  this  process.  one  study  observed   the  melanin  in  albino-­‐mouse  hair   follicles.  in  this  study,  a  point  muta-­‐ tion  in  the  tyrosinase  gene  was  cor-­‐ rected  in  some  of  the  murine  folli-­‐ cles  with  rna-­‐dna  chimeric  oligo-­‐ figure  1.  a  pedigree  shows  that  the  males   in  generations   ii  and   i  are  affected  by   premature  hypopigmentation.  individual  ii.4  is  also  affected,  regardless  of  the  fact   that  his  maternal  genome  differs  from  the  other  progeny  in  the  second  generation.   the  individual  of  the  case  report,  ii.2  is  demarcated  with  a  “star”.     figure  2.  the  photo  on  the  left  is  individual  ii.1  shown  in  the  pedigree.  the  pho-­‐ to  on  the  right  is  individual  ii.2.          familial  aggrega*on  of  phenotypic  expression  of  premature  hair  hypopigmenta/on  in  the  craniofacial  region   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.31    http://dentistry3000.pitt.edu   3   nucleotides.  a  recombinant  retrovi-­‐ rus  (plmelsn)  from  the  locus  of   streptomyces  antibioticus  genome   was  used  to  induce  melanin  produc-­‐ tion  in  vitro  [3].  these  results  indi-­‐ cate  that  the  tyrosinase  inactivity   may  be  a  possible  link  to  hair  hy-­‐ popigmentation  and  some  factors   may  cause  the  premature  inactiva-­‐ tion  of  these  enzymes.       because  the  hair  cycle  and   growth  process  are  complicated,  it   makes  it  difficult  to  study  specific   aspects  of  the  cycle  –  especially   melanin  production.  however,  oth-­‐ er  genes  are  thought  to  be  involved   in  this  very  complex  process.  it  is   suspected  that  mice  deficient  in  bcl-­‐ 2  and  bcl-­‐x  genes  are  normal  at   birth  but  develop  gray  hair  after  a   few  weeks  [2].  also,  a  germ  line  mu-­‐ tation  in  syntaxin  17  gene  in  horses   causes  premature  gray  hair  in  the   homozygous  genotype  [4].  based  on   the  patient’s  medical  and  family  his-­‐ tory,  there  seems  to  be  a  strong  ge-­‐ netic  link  to  the  cause  of  this  partic-­‐ ular  phenotype.       there  are  many  diseases,   deficiencies,  and  syndromes  that   have  been  linked  to  premature  hy-­‐ popigmentation  of  hair.  some  of   these  include  vitiligo,  werner’s   syndrome,  thyroid  disease,  vitamin   b12,  iron,  and  calcium  deficiencies.   one  syndrome,  known  as  book’s   syndrome,  contains  hypodontia  and   premature  graying  as  phenotypic   traits  [2].  clinicians  should  be   aware  of  this  syndrome  and  its  pos-­‐ sible  physical  manifestations.  the   patient  has  no  known  syndromes  or   deficiencies.  therefore,  it  is  unlikely   that  this  is  the  cause  of  the  pheno-­‐ type.     the  environment  can  also   play  a  crucial  role  in  early  hy-­‐ popigmentation  of  hair.  studies   suggest  that  there  is  a  significant   association  between  tobacco  use   and  aging  on  graying  of  hair  [5].   another  study  suggests  that  prema-­‐ ture  graying  of  the  hair  is  associated   with  premature  cardiovascular  dis-­‐ ease.  it  should  probably  be  regard-­‐ ed  as  a  coronary  risk  factor  and   used  to  identify  patients  at  in-­‐ creased  risk  [5].  these  environmen-­‐ tal  influences  should  be  explained   to  patients  who  may  not  contain   such  relevant  knowledge.  since  car-­‐ diovascular  disease  is  present  in  the   family  medical  history,  it  should  be   explained  to  the  patient  that  there   may  be  a  possible  link  between   premature  canities  and  heart  dis-­‐ ease.  hence,  future  testing  should   be  done.     conclusion     clinicians  must  be  able  to  recognize   premature  hypopigmentation  of   hair  and  understand  the  many   causes  of  it.  the  ultimate  goal  is  to   know  that  the  hypopigmentation   phenotype  can  be  explained  by  a   number  of  factors.  in  order  to  un-­‐ derstand  the  exact  cause,  clinicians   must  ask  the  appropriate  questions,   comprehending  that  the  physical   appearance  of  a  few  premature  gray   hairs  can  provide  information  about   unknown  underlying  factors.    once   the  real  cause  is  known,  treatment   may  be  modified  to  ensure  that  the   patient  receives  the  best  quality  of   care  when  under  the  care  of  that   particular  clinician.       references     1.  epidemiological  and  investigative   study  of  premature  graying  of  hair   in  higher  secondary  and  pre-­‐ university  school  children.  bhat   rm,  sharma  r,  pinto  ac,  dandekeri   s,  martis  j.  int  j  trichology.  2013   jan;5(1):17-­‐21.  doi:  10.4103/0974-­‐ 7753.114706.  pmid:23960391       2.  tobin,  d.  &  trueb,  r.  (2010).  nat-­‐ ural  hair  graying.  in  aging  hair  (pp.   208-­‐210).  new  york,  new  york:   springer  science  &  business  media.     3.  the  hair  follicle  and  its  stem  cells   as  drug  delivery  targets.  hoffman   rm.  expert  opin  drug  deliv.  2006   may;3(3):437-­‐43.  review.   pmid:16640502     4.  polymorphisms  in  the  syntaxin   17  gene  are  not  associated  with   human  cutaneous  malignant  mela-­‐ noma.  zhao  zz,  duffy  dl,  thomas   sa,  martin  ng,  hayward  nk,  mont-­‐ gomery  gw.  melanoma  res.  2009   apr;19(2):80-­‐6.  doi:   10.1097/cmr.0b013e328322fc45.   pmid:19209086     5.  premature  hair  graying:  a  proba-­‐ ble  coronary  risk  factor.  gould  l,   reddy  cv,  oh  kc,  kim  sg,  becker  w.   angiology.  1978  nov;29(11):800-­‐3.   pmid:727561         microsoft word 237 2022.docx hypertrichosis associated with gene2c condi2ons with head and neck altera2ons vol 10 no 1 (2022) doi 10.5195/d3000.2022.237 h#p://den*stry3000.pi#.edu hypertrichosis associated with genetic conditions with head and neck alterations mirelle estéfane de oliveira caixeta1, caroline rodrigues dias1, rafael mar9ns afonso pereira1, thiago de amorim carvalho1, ivania aparecida pimenta santos silva1, rodrigo soares de andrade1 1 centro universitário de patos de minas-unipam abstract introduction: hypertrichosis is a rare disorder in which there is an exaggerated growth of body hair in places that are not necessarily androgen-dependent, a par2cular feature of hirsu2sm. its e2ology is s2ll ques2onable, with an increased incidence when associated with syndromes. this gene2c associa2on leads to numerous significant systemic and craniofacial changes, which should be recognized and considered by the den2st. objective: to assess, in databases, the presence of congenital hypertrichosis associated with gene2c syndromes and condi2ons affec2ng the head and neck. methods: the search was conducted in pubmed-ncbi databases; gene2c condi2ons along with their characteris2cs were available in online mendelian inheritance in man (omim) and in protein®. results: the analysis was performed on 63 ar2cles and all diseases were considered as rare. in the syndromes, prevalence of autosomal dominant inheritance was observed in 48.64%, followed by autosomal recessive in 45.9%, 1 x-linked recessive case, and 1 arising from defec2ve mitochondrial energy genera2on. among the 63 gene2c condi2ons, 44 showed neurodevelopmental changes. besides the craniofacial involvement itself, there is a high prevalence of altera2ons of the oral structures involving 47 of all condi2ons, including mainly dental abnormali2es, palatal and gingival changes. conclusion: the associa2on of hypertrichosis with gene2c components is strongly associated with major craniofacial changes. therefore, the knowledge of the dental surgeon about the condi2ons that can affect the oral cavity is impressive because it is related to a correct treatment and bezer quality of life for the pa2ent. keywords: craniofacial abnormali2es; dermatology; gene2c disorders; hirsu2sm; hypertrichosis cita9on: caixeta, m et al. (2022) hypertrichosis associated with gene9c condi9ons with head and neck altera9ons den9stry 3000. 1:a001 doi:10.5195/d3000.2022.237 received: september 8, 2021 accepted: march 25, 2022 published: june 14, 2022 copyright: ©2022 caixeta, m et al. this is an open access ar9cle licensed under a crea9ve commons awribu9on work 4.0 united states license. email: mirellecaixeta@gmail.com introduction hypertrichosis [#307150 (wmo)] is a rare disorder in which the body hair grows longer than is considered normal when compared to people of the same age, sex, race, and for a particular area of the body [1-3]. it usually occurs in regions where hair follicles are present that are not necessarily androgen-dependent, except for those locations such as the lips, palms of the hands, and soles of the feet that are normally hairless [1,3]. one condition that can occasionally lead to a misdiagnosis of hypertrichosis is hirsutism [4]. this variation affects especially females, resulting in abnormal hair growth and giving them a male phenotype, since in these cases hair growth is androgen-dependent, thus distinguishing it from hypertrichosis [1,4-8]. the clinical features of hypertrichosis vary according to the type of hair and there are three variations that can hypertrichosis associated with gene2c condi2ons with head and neck altera2ons vol 10 no 1 (2022) doi 10.5195/d3000.2022.237 h#p://den*stry3000.pi#.edu eventually manifest themselves [4,5]. lanugo is the hair that covers the fetal skin and is lost in the first weeks of the baby's life, being thin and nonpigmented. the vellus replaces the lanugo and corresponds to most of the postnatal body hair, not being androgendependent, besides being lightly pigmented, thin and short. the terminal hairs, on the other hand, are thicker, more pigmented, and their length varies according to their location due to androgen dependence [1,4,5,9-11]. one of the mechanisms that occurs in congenital hypertrichosis is the conversion of vellus hair to terminal hair in regions where terminal hair would not normally develop, but this change is not well understood [4]. another is based on changes in the hair growth cycle. hair follicles go through three stages anagen, catagen, and telogen [11,12]. a systemic influence will lead to changes in this pattern, so that the follicles persist longer in the anagen phase, the active phase of growth, resulting in the characteristic phenotype [4]. in its idiopathic form, atavism is an assumption that would explain the typical physical appearance. the ancestral gene responsible for hair growth would remain latent during its evolution and by an erroneous reactivation in them, the hypertrichotic [1] profile would result. its classification is based on the etiology and location and may be related to a congenital or acquired form, with a localized or generalized pattern, and according to the type of hair [2,4,13]. for the most part, hypertrichosis is considered an aesthetic problem, but often it may signal an underlying systemic alteration, since the incidence increases when it is associated with genetic syndromes or conditions [1,4,14]. some syndromes with associated hypertrichosis have been mentioned featuring specific molecular defects, such as fontaine progeroid syndrome with involvement of the slc25a24 gene; coffin-siris syndrome affecting the genes of the baf complex and wiedeman-steiner syndrome involving the kmt2a gene [1,15-17]. systemic conditions such as kidney, heart and bone problems may be associated with the presence of congenital hypertrichosis [1]. moreover, craniofacial dysmorphisms with hypertelorism, eyebrow abnormalities, prominent mouth philtrum, dental anomalies with oligodontia, microcephaly, short neck, high and arched palate and gingival hyperplasia are signs commonly found in these carriers [1,18]. the recognition of these alterations in these individuals by the dentist is essential, since it will provide a differential diagnosis and a better clinical outcome for the dental abnormalities [19]. therefore, this paper presents a review of the literature on syndromes and genetic conditions associated with the presence of congenital hypertrichosis with head and neck involvement. material and methods the present study consists of a qualitative exploratory research work. an extensive search of the pubmed-ncbi databases for scientific papers was performed in a limited period from july to september 2020, with no limits set as to the date they were published. the scope of this project is the investigation of genetic conditions that appear concomitantly with the clinical picture of hypertrichosis with craniofacial changes. syndromes and polymorphisms based on these features were available in online mendelian inheritance in man (omim) and protein, which were considered sufficient for this manuscript taking into account the inclusion and exclusion criteria [19,20]. papers that did not associate hypertrichosis as a characteristic were excluded, as well as those considered extremely rare with not very relevant craniofacial features, those associated with acquired hypertrichosis, and those citing isolated hirsutism not associated with hypertrichosis. all cases that presented clinical conditions of hypertrichosis with craniofacial comorbidities and those rarely associated with hypertrichosis, but with relevant head and neck alterations, were included. the hypertrichosis associated with gene2c condi2ons with head and neck altera2ons vol 10 no 1 (2022) doi 10.5195/d3000.2022.237 h#p://den*stry3000.pi#.edu descriptor used for the search was "hypertrichosis" limited to the english language. occasionally, an extension of the search to complement the study was performed by pubmed ncbi [20]. results after a rigorous search in the english language literature, a total of 143 genetic conditions were found, and of these, 63 were included in the research. the studies were analyzed individually and, in all of them, where hypertrichosis appeared associated with some genetic component, a great reflection on the head and neck structures was noticed, as well as a great oral and dental involvement. these genetic disorders were tabulated so that, in summary, they described the most commonly encountered general and craniofacial features (charts 1 and 2), and those conditions that rarely had the hypertrichotic condition but with relevant head and neck changes (chart 3) [19]. discussion the syndromes identified that presented hypertrichosis as a common feature were those in which this cutaneous finding manifested itself in a generalized manner; in these cases, the phenotype was consistent with craniofacial involvement. these include cahmr, cantu, and gingival overgrowth syndrome [21-23]. in addition to the syndromes, hypertrichosis as a consiste feature has been cited in polymorphisms such as hypertrichosis lanuginosa congenita, hypertrichosis congenita generalized, a atp linkage cassette subfamily a member 5 and hypertrichosis congenita generalized, with or without gingival hyperplasia [24-26]. in syndromes associated with hypertrichosis, it is noteworthy that the mode of inheritance commonly found is autosomal dominant (ad), with an involvement of 48.64% (18). in addition, the remainder showed to be autosomal recessive (ar) in 45.94% (17), 1 case arising from defective mitochondrial energy generation (mi) and 1 x-linked recessive [19,20]. an analysis of the general characteristics of the disorders showed a significant prevalence of neurodevelopmental changes, with an involvement in 29 out of 37 of the syndromes identified, and polymorphisms bringing an involvement in 15 out of 26. embryologically, the nervous system and the skin derive from the ectoderm, so the presence of these congenital skin conditions are highly associated with neurological changes [27]. traits such as intellectual and cognitive impairment, delayed psychomotor development, limb ataxia, spasticities, cerebellar atrophy, and some psychiatric behavioral manifestations have been cited. furthermore, it can be seen that all the genetic disorders selected in the study presented head and neck alterations, but some features were more commonly found than others. these included those showing nasal changes (39 of 63), dental abnormalities including changes in shape, size, quantity, and delayed eruption (26 of 63), ear dysmorphisms (24 of 63), with palate changes (19 of 63), gingival involvement (17 of 63), showing microcephaly (16 of 63), and traits of micrognathia (14 of 63). it was observed during the selection of genetic conditions, 01 study in which there was disagreement regarding the classification of the diagnosis of the skin condition, not making clear the distinction between true hypertrichosis or hirsutism, in which the term used in the clinical synopsis of the study did not match the term that appeared described in the text [28]. according to wendelin et al., (2003), there are reports in the literature that hypertrichosis is erroneously referred to as hirsutism [4]. according to mofid a. et al. (2007), a differential diagnosis must be made because these are two different [29] conditions. hirsutism is excessive hair growth in women in androgen-dependent areas including facial beards and moustaches, and body hair in the pubic area and inner thighs, while hypertrichosis is excessive hair growth in androgenindependent areas and gender [4-8]. hypertrichosis associated with gene2c condi2ons with head and neck altera2ons vol 10 no 1 (2022) doi 10.5195/d3000.2022.237 h#p://den*stry3000.pi#.edu in view of the research carried out at omim, we noticed that all syndromes and polymorphisms associated with congenital hypertrichosis are considered rare diseases, since there are few case reports described in the literature [5]. the definition of being rare is based on the prevalence of affected individuals, but this definition varies in different countries, since some consider the total number of affected individuals and not the proportion of the affected population [30,31]. according to mukherjee k. (2019), in the united states a rare condition is defined as one that affects fewer than 200,000 patients, in the european union fewer than 250,000, and in japan fewer than 50,000 patients. according to the world health organizationwho (2016), in which was also adopted by the ministry of health in brazil, define rare diseases those that affect up to 65 people in every 100,000 individuals [31,32]. it is evident that systemic capillary pathology associated with a genetic component is accompanied by several changes in the head and neck, both in its morphology and physiology, as shown in the researched literature [1]. it is possible to observe some of these morphophysiological changes in coffin-siris syndrome 3, which include microcephaly, visual and hearing impairment, anteverted nostrils, and trichomegaly, as described in charts 1. dysmorphisms of the facial bones have been widely reported, including micrognathia, prognathism and hypoplasia of the malar bones as shown in bloom syndrome, autosomal recessive mental retardation 35, congenital disorder of glycosylation type iie, barber-say and progeroid fontaine syndromes. furthermore, oral and dental involvement is quite prevalent, and in certain genetic diseases accompanied by hypertrichosis, dental involvement is quite characteristic of the systemic condition carried as shown in congenital erythropoietic porphyria (chart 2), since erythrodoontia is the most typical dental alteration. according to gomes (2020), calcified tissues show high susceptibility in pigment deposition and porphyrins have a higher affinity for phosphate and calcium, explaining a greater deposition of them during osteogenesis and odontogenesis, thus conferring a reddish coloration both in bones and teeth [33]. the research carried out shows significant gingival involvement in those with congenital hypertrichosis, highlighting hyperplasias, hypoplasias, hypertrophies, and fibromatoses. gingival fibromatosis is described as an excessive and benign enlargement of the free and attached gingiva [34] and, according to chacon-camacho, its etiological basis may be isolated or as part of a genetic condition. it is considered rare, but its incidence increases when it is associated with a genetic disease, commonly those characterized by hypertrichosis, and gingival fibromatosis is largely considered an important diagnostic component of these diseases [35]. hypertrichosis as characteristics of genetic syndromes involve a series of head and neck alterations, as mentioned before, and must be taken into consideration by health professionals, especially dentists. in view of the results, it can be stated that hypertrichosis, when associated with a genetic component, whether a disease or a syndrome, reflects major changes in the head and neck region, besides the face itself, secondarily affecting the oral structures, including changes in the quantity and morphology of the teeth, gingival structures, tongue and palate. the relevance of knowledge of congenital hypertrichosis accompanied by craniofacial dysmorphisms by oral health professionals can be seen, since the dental surgeon routinely practices clinical evaluation of the maxillomandibular complex, not limited only to the evaluation of the oral cavity, but also being made a careful observation of the head and neck region [19,20]. although rare, it is advisable that these professionals have an understanding of this genetic picture since it will influence the clinical conduct of the patient. in cases of suspicion, medical follow-up should be done and, when associated with dental interventions, will result in better oral clinical outcomes. hypertrichosis associated with gene2c condi2ons with head and neck altera2ons vol 10 no 1 (2022) doi 10.5195/d3000.2022.237 h#p://den*stry3000.pi#.edu therefore, this paper provides a study that will help to contribute to the expansion of knowledge by oral health professionals about these conditions with head and neck involvement, helping in the differential diagnosis and multiprofessional work. hypertrichosis associated with gene2c condi2ons with head and neck altera2ons vol 10 no 1 (2022) doi 10.5195/d3000.2022.237 h#p://den*stry3000.pi#.edu chart 1: genetic syndromes reported with hypertrichosis: features syndromes moi chromo some omim features cahmr ar n/a 211770 general: congenital lamellar cataract, generalized hypertrichosis, and mental retardation. h&n: highly arched palate, microdontia, and depressed nasal bridge. ramon ar n/a 266270 general: low weight, kyphosis, scoliosis, hypertrichosis, mental retardation, and seizures. h&n: kerubism, gum fibromatosis, papillomatosis, and delayed tooth eruption. barber-say ad 2q37 #209885 general: generalized hypertrichosis and mental retardation. h&n: mandibular prognathism, micrognathia, dysmorphic ears, hypertelorism, macrostomia, high arched palate, and delayed eruption. progeroid fontaine ad 1p36 #612289 general: hair abnormalities, aged appearance, and hypertrichosis. h&n: microcephaly, progeroid appearance, micrognathia, retrognathia, dysmorphic ears, hypertelorism, microstomia, high arched palate, and oligodontia. coffin siris 1 ad 6q25 #135900 general: intellectual disability, hypertrichosis, poor growth. h&n: facial hypertrichosis, dysmorphic ears, strabismus, long eyelashes, wide nasal tip, macrostomia, and delayed dentition. histiocytosis lymphadenopathy plus ar 10q22 #602782 general: short stature, splenomegaly, camptodactyly, hypogonadism and hypertrichosis. h&n: submandibular lymphadenopathy, orbital and nasal mass due to histiocytosis, exophthalmos, retropharyngeal lymphadenopathy, and cervical lymphadenopathy. zimmermann laband 3 ad 1q21 #618658 general: hypoplasia or aplasia of phalanges, hypertrichosis, joint hyperextensibility and hepatosplenomegaly. h&n: facial hypertrichosis, thick eyebrows, long eyelashes, triangular nostrils, gingival hyperplasia, high and arched palate, and a bifid uvula. wiedemann-steiner ad 11q23 #605130 general: hypertrichosis cubitalis, intellectual disability and short stature. h&n: dysmorphic ears, hypertelorism, strabismus, thick eyebrows, long eyelashes, high arched palate, and abnormal dentition. cantu ad 12p12 #239850 general: generalized congenital hypertrichosis, osteochondrodysplasia, cardiomegaly and lymphedema. h&n: macrocephaly, coarse facies, prominent forehead, long eyelashes, wide and flat nasal bridge, gingival hypertrophy, and short neck. hypertrichosis associated with gene2c condi2ons with head and neck altera2ons vol 10 no 1 (2022) doi 10.5195/d3000.2022.237 h#p://den*stry3000.pi#.edu zimmermannlaband 1 ad 1q32 #135500 general: hypertrichosis, joint hyperextensibility, and intellectual disability. h&n: prognathism, dysmorphic ears, thick eyebrows, gingival fibromatosis, gingival hyperplasia, high arched palate, and delayed tooth eruption. schinzel-giedion midface retraction ad 18q12 #269150 general: hypertrichosis, mental retardation, skeletal abnormalities, genitourinary, renal and cardiac malformations. h&n: coarse facies, midface hypoplasia, facial hemangioma, shallow orbits, proptosis, hypertelorism, macroglossia, and short neck. leigh ar, mi n/a #256000 general: hypertrichosis, psychomotor retardation, hypotonia, ataxia, and dysphagia. h&n: ophthalmoplegia, optic atrophy, nystagmus, strabismus, ptosis, and pigmentary retinopathy. seckel syndrome 9 ar 3p21 #616777 general: growth retardation, microcephaly, mental retardation and hypertrichosis. h&n: microcephaly, scaphocephaly, micrognathia, and pointy nose. lymphedema hypoparathyroidism ar n/a 247410 general: lung disease, nephropathy, brachydactyly, and hypoparathyroidism. h&n: cataracts, ptosis, telecanthus, wide nasal bridge, and hypertrichosis. zimmermannlaband 2 ad 8p21 #616455 general: hypertrichosis, joint hyperextensibility, hepatosplenomegaly, and intellectual disability. h&n: sensorineural deafness, thick eyebrows, wide and bifid nasal tip, gingival hyperplasia, macroglossia, and short neck. coffin-siris 3 ad 22q11 #614608a general: intellectual disability, hypertrichosis, and poor growth. h&n: microcephaly, coarse facies, thick eyebrows, long eyelashes, anteverted nostrils, macroglossia, and delayed dentition. donohue ar 19p13 #246200 general: cystic ovaries, delayed bone age, acanthosis nigricans, pachyderma, hypertrichosis, and precocious puberty. h&n: elven facies, small face, large and low set ears, prominent eyes, gingival hyperplasia and macrostomia. warburg micro 1 ar 2q21 #600118 general: short stature, severe mental retardation, and hypogonadism. h&n: microcephaly, micrognathia, large ears, microphthalmia, optic atrophy, ptosis, deep eyes, and facial hypertrichosis. trichohepatoneurode velopmental ar 17q23 #618268 general: woolly hair, hypotonia, developmental delay, and hypertrichosis. h&n: microcephaly, ptosis, hypertelorism, visual impairment, hyperopia, nasal bone hypoplasia, high arched palate, macroglossia, severe diastemas, crowding of teeth, microdontia, and prognathism. coffin-siris 4 ad 19p13 #614609 general: intellectual disability, hypertrichosis and poor growth. h&n: microcephaly, hearing and visual impairment, long eyelashes, macrostomia, macroglossia, and delayed teething. hypertrichosis associated with gene2c condi2ons with head and neck altera2ons vol 10 no 1 (2022) doi 10.5195/d3000.2022.237 h#p://den*stry3000.pi#.edu tenorio ad 18q12 #616260 general: intellectual disability, hypoglycemia, inflammatory diseases similar to sjögren's syndrome, and hypertrichosis. h&n: macrocephaly, telecanthus, dry keratoconjunctivitis, thick eyebrows, recurrent stomatitis, macroglossia, prognathism, and delayed dentition. warburg micro 3 ar 10p12 #614222a general: severe mental retardation, hypogonadism and hypertrichosis. h&n: microcephaly, brachycephaly, optic atrophy, nystagmus, blepharophimosis, down-turned corners of mouth, and prominent secondary alveolar ridges. sandestig-stefanova ar 9q34 #618804 general: trigonocephaly, camptodactyly, delayed myelination and hypertrichosis. h&n: microcephaly, trigonocephaly, retrognathia, arched eyebrows, epicanthus, cleft lip and palate, high arched palate, and short neck. coffin-siris 2 ad 1p36 #614607 general: intellectual disability, poor growth and hypertrichosis. h&n: gross facies, thick eyebrows, long eyelashes, anteverted nostrils, macrostomia, macroglossia, and delayed dentition. marshall-smith ad 19p13 #602535 general: accelerated skeletal maturation, mental retardation, and hypertrichosis. h&n: micrognathia, retrognathia, prominent eyes, bluish sclera, thick eyebrows, glossoptosis, gingival hypertrophy, and irregular dentition. bloom ar 15q26 #210900 general: growth deficiency, telangiectatic skin and hypertrichosis. h&n: dolicocephaly, microcephaly, malar hypoplasia, prominent nose and absence of upper lateral incisors. ambras ad 8q22 % 145701 general: generalized hypertrichosis, and hexadactyly. h&n: facial roughness, wide intercanthal distance, anteverted nostrils, flat mentholabial groove, and dental abnormality. rabson-mendenhall ar 19p13 # 262190 general: insulin resistant diabetes mellitus with pineal hyperplasia, skin abnormalities and hypertrichosis. h&n: prognathism, macroglossia, fissured tongue, gingival hypoplasia, highly arched palate, premature eruption of teeth, and dental anomalies. hunter xlr xq28 # 309900 general: skeletal deformities, developmental delay, hyperactivity, mental retardation, dementia, and hypertrichosis. h&n: macrocephaly, papilledema, retinal pigmentation, ptosis, macroglossia, delayed tooth eruption, widely spaced teeth, and short neck. berardinelli-seip ar 11q12 # 269700 general: adipose tissue shortage, hepatic steatosis, and hypertrichosis. h&n: prognathism, triangular facies, acromegaloid appearance and large ears. gingival overgrowth syndrome ad 11q13 # 618381 general: delayed motor and intellectual development, and generalized hypertrichosis. h&n: hypotonic facies, micrognathia, gingival growth, prominent lip vermilion and everted upper lip. hypertrichosis associated with gene2c condi2ons with head and neck altera2ons vol 10 no 1 (2022) doi 10.5195/d3000.2022.237 h#p://den*stry3000.pi#.edu chart 2: polymorphisms with hypertrichosis: features disease omim characteristics hypertrichosis lanuginosa congenita 145700 general: universal hypertrichosis, skeletal disorders and mental retardation. h&n: dental deformity, double eyebrows, and glaucoma. congenital generalized hypertrichosis, with or without gingival hyperplasia 135400 general: generalized hypertrichosis. h&n: hirsutism, epicanthal folds, and gingival fibromatosis. generalized congenital hypertrichosis 307150 general: scoliosis and congenital generalized hypertrichosis in males. h&n: deformed and badly positioned teeth and hirsutism. potassium channel, subfamily k, member 4 605720 general: hypertrichosis, epilepsy, intellectual and developmental delay. h&n: facial dysmorphism and gingival growth syndrome. mandibulofacial dysostosis with macroblephon and macrostomia 602562 general: n/a. h&n: micrognathia, retrognathism, hypertelorism, hypertrichosis of the eyebrows, anteverted nostrils, macrostomia, malpositioned teeth, and oligodontia. congenital glycosylation disorder, type iaa 617082 general: hypertrichosis, hypotonia, epilepsy, spasticity, and delayed psychomotor development. h&n: microcephaly, hearing and visual impairment, retinal pigmentary spotting and macular lesions. congenital glycosylation disorder, type iq 612379 general: ichthyosiform dermatitis, hyperkeratosis, hypertrichosis, and mental retardation. h&n: brachycephaly, coloboma, hypertelorism, and visual loss. mental retardation, x-linked 99, syndromic, female restricted 300968 general: scoliosis, hypertrichosis, delayed psychomotor development, and muscular hypotomia. h&n: brachycephaly, facial asymmetry, dysmorphic ears, eye abnormalities, and dental abnormalities. torg ar 16q12.2 259600 general: severe multicentric osteolysis and hypertrichosis. h&n: corneal opacities, hyperpigmented skin patches, and gingival hypertrophy. okamoto n/a n/a 604916 general: congenital hydronephrosis, severe mental retardation and growth deficiency. h&n: cleft palate, hypoplasia of the mid-face, hypertrichosis, long eyelashes, prominent eyes, epicanthus, dysmorphic ears, short snub nose, and winged neck. coffinsiris 8 ad 12q13 618362 general: intellectual disability and hypoplastic or absent fifth finger phalanges. h&n: rough facial features, hypertrichosis, thick eyebrows, long eyelashes, ptosis, anteverted nostrils, ophthalmologic abnormalities, and a snub nose. hypertrichosis associated with gene2c condi2ons with head and neck altera2ons vol 10 no 1 (2022) doi 10.5195/d3000.2022.237 h#p://den*stry3000.pi#.edu mental retardation, autosomal dominant 57 618050 general: scoliosis, hypertrichosis, neurological alterations, and muscular hypotonia. h&n: microcephaly, asymmetrical face, blepharophimosis, telecanthus, epicanthal folds, microstomia, and deep palate. congenital erythropoietic porphyria 263700 general: short stature, photosensitivities, hypertrichosis, and alopecia. h&n: conjunctivitis, corneal scarring, red stained teeth (erythrodontia). dental anomalies and short stature 601216 general: short stature, delayed bone age, scoliosis, and hypertrichosis. h&n: maxillary hypoplasia, oligodontia, amelogenesis imperfecta, microdontia, yellow teeth, severe diastemas, taurodontia, anadontia of permanent teeth. alpha b lysosomal mannosidosis 248500 general: hypertrichosis, mental retardation, spasticity, and nystagmus. h&n: macrocephaly, frontal bossing, prognathism, epicanthic folds, macroglossia, gingival hypertrophy and severe diastemas. mental retardation, autosomal recessive 35 % 615162 general: clinodactyly, global developmental delay, and hypertrichosis. h&n: malar hypoplasia, dysmorphic ears, hypertelorism, upturned lower lip, and micrognathia. mullerian derivatives, persistence of, with lymphangiectasia and postaxial polydactyly % 235255 general: hypertrichosis, and lymphedema. h&n: micrognathia, hypertelorism, hypertrophied alveolar ridge, highly arched palate, cleft palate and short neck. agenesis of the corpus callosum, cardiac, ocular and genital 618929 general: pectus excavatum, global developmental delay, and intellectual disability. h&n: macrocephaly, frontal bossing, micrognathia, facial hypertrichosis, dysmorphic ears, hypertelorism, atresic palate, and winged neck. congenital disorder of glycosylation, type iie 608779 general: hypertrichosis, global developmental delay, and muscular hypotonia. h&n: microcephaly, micrognathia, retrognathia, flat malar region, protruding tongue, thick gums, and short neck. gm1-gangliosidosis type i 230500 general: dwarfism, joint stiffness, dermal melanocytosis, hypertrichosis, and mental retardation. h&n: coarse facies, hypertelorism, gingival hyperplasia and short neck. neurodevelopmental disorder with progressive microcephaly, spasticity and brain anomalies 617527 general: pectus carinatum, hirsutism, muscular hypotonia, and mental retardation. h&n: microcephaly, micrognathia, dysmorphic ears, nystagmus, optic atrophy, and highly arched palate. hypertrichosis associated with gene2c condi2ons with head and neck altera2ons vol 10 no 1 (2022) doi 10.5195/d3000.2022.237 h#p://den*stry3000.pi#.edu atp link cassette a, subfamily a, member 5 612503 general: generalized hypertrichosis and epilepsy. h&n: severe gingival hyperplasia. trichomegaly 190330 general: hereditary spherocytosis. h&n: hypertrichosis of the cheeks, forehead and eyebrows, and long eyelashes. facial hypertrichosis 134000 general: n/a. h&n: hypertrichosis of the face. gingival fibromatosis with hypertrichosis and mental retardation 605400 general: mental retardation, epilepsy and brachymetacarpalia. h&n: hirsutism, dysmorphic ears, and gingival hypertrophy. intellectual developmental disorder with cardiac defects and dysmorphic facies 618316 general: variable congenital heart disease and global developmental delay. h&n: triangular face, deep eyes, hypertelorism, dysmorphic ears, very arched eyebrows and hypertrichosis. chart 3: syndrome and polymorphisms with rare hypertrichoses and with relevant h&n involvement: features conditions and syndromes chromosome omim moi features chromosome 17q12 deletion syndrome. 17q12 # 614527 ad general: short stature, mental retardation, and autistic features. h&n: dolicocephaly, micrognathia, retrognathia, hypertrichosis of the upper lip, and high palate. philippi syndrome 2q13 # 272440 ar general: short stature, syndactyly, and intellectual disability. h&n: microcephaly, hairy forehead, microdontia, dental anomaly, hypodontia, and serrated incisors. autosomal recessive spinocerebellar ataxia 20 616354 general: psychomotor problems, and intellectual disability. h&n: macrocephaly, epicanthal folds, highly arched palate, macroglossia, delayed tooth eruption, and tooth crowding. pontocerebellar hypoplasia, type 10 615803 general: psychomotor problems, convulsions, and brain atrophy. h&n: microcephaly, prominent eyes, long eyelashes, strabismus, nystagmus, and well arched palate. spinal muscular atrophy with congenital bone fractures 1 616866 general: delayed psychomotor development, and congenital bone fractures. h&n: microretrognathia, hypertelorism, microstomia, highly arched palate. adducted thumbs syndrome n/a %201550 ar general: generalized hypotonia, respiratory insufficiency and hypertrichosis. h&n: myopathic rigid facies, open mouth, arched high palate, cleft palate, microcephaly and dysphagia. h&n (head and neck); n/a (not available); ad (autosomal dominant); ar (autosomal recessive); omim (online mendelian inheritance in man); moi (mode of inheritance); mi (mitochondrial) hypertrichosis associated with gene2c condi2ons with head and neck altera2ons vol 10 no 1 (2022) doi 10.5195/d3000.2022.237 h#p://den*stry3000.pi#.edu references 1. congenital generalized hypertrichosis: the skin as a clue to complex malformajon syndromes. pavone p, prajcò ad, falsaperla r et al. ital j pediatr. 2015; 41:1-8. pmid: 26242548 2. single case localized hypertrichosis with traumajc panniculijs: a case report and literature review. ploydaeng m, rojhirunsakool s, suchonwanit p. case rep dermatol 2019; 11:180-186. pmid: 31320866 3. first japanese case of congenital generalized hypertrichosis with a copy number variajon on chromosome 17q24. hayashi r, yoshida k, abe r, niizeki h, shimomura y. j dermatol sci. 2017; 85:63-65. pmid: 27780627 4. hypertrichosis. wendelin ds, pope dn, mallory sb. j am acad dermatol. 2003; 48:161-182. pmid: 12582385 5. hypertrichosis. saleh dahlia, yarrarapu siva naga s, cook christopher. statpearls. 2020; jul 08. available from: hnps://www.ncbi.nlm.nih.gov/b ooks/nbk534854/ 6. endocrinology of hirsujsm: from androgens to androgen excess disorders. yilmaz b, yildiz bo. front horm res. 2019; 53:108119. pmid: 31499500 7. ejological diagnosis of hirsujsm and implicajons for the treatment. spritzer pm. rev bras ginecol obs. 2009; 31:41-47. pmid: 19347228 8. hirsujsm. hafsi w, badri t. statpearls. 2020. available from: hnps://www.ncbi.nlm.nih.gov/b ooks/nbk470417/. 9. the involvement of androgens in human hair growth. alwaleedi sa. am j biomed sci. 2015; 7:105-124. available from: hnps://www.nwpii.com/ajbms/ papers/ajbms_2015_2_05.pdf 10. physiology, hair. hoover e, krishnamurthy k. statpearls publishing. 2018. available from: hnp://www.ncbi.nlm.nih.gov/pu bmed/29763123. 11. the human hair: from anatomy to physiology. buffoli b, rinaldi f, labanca m et al. int j dermatol. 2014; 53:331-341. pmid: 24372228 12. promojon of anagen, increased hair density and reducjon of hair fall in a clinical seqng following idenjficajon of fgf5inhibijng compounds via a novel 2-stage process. burg d, yamamoto m, namekata m, haklani j, koike k, halasz m. clin cosmet invesjg dermatol. 2017; 10:71-85. pmid: 28280377 13. hypertrichosis. trüeb rm. hautarzt. 2008; 59:325-338. available from hnps://link.springer.com/arjcle /10.1007/s00105-008-1489-z 14. foxn1 duplicajon and congenital hypertrichosis. gilhooley e, gormally s, irvine a, lynch sa, collins s. pediatr dermatol. 2017; 34:77-79. pmid: 28297140 15. a rare male pajent with fontaine progeroid syndrome caused by the new p.r217h mutajon in slc25a24. rodríguez-garcía me, cotrinavinagre fj, cruz-rojo j et al. am j med genet part a. 2018; 176:2479-2486. available from hnps://pubmed.ncbi.nlm.nih.go v/30329211/ 16. genejc abnormalijes in a large cohort of coffin-siris syndrome pajents. sekiguchi f, tsurusaki y, okamoto n et al. j hum genet. 2019; 64:1173-1186. pmid: 31530938 17. wiedemann-steiner syndrome in two pajents from portugal. grangeia a, leão m, moura cp. am j med genet part a. 2020; 182:25-28. pmid: 31710778 18. hypertrichose universelle congénitale. maza a, gaudymarqueste c, collet-vilene am, joubert f, richard ma, grob jj. ann dermatol venereol. 2009; 136:300-302. pmid: 19328322 19. a review of syndromes associated with blue sclera, with inclusion of malformajons of the head and neck. brooks jk. oral surg oral med oral pathol oral radiol. 2018; 126:252-263. pmid: 29980417 20. cafe-au-lait spots as a clinical sign of syndromes. carvalho aa, martelli d, barbosa g et al. clinical pediatrics. 2020; 07:1-23. 21. cataract, hypertrichosis and mental retardajon (cahmr): a new autosomal recessive syndrome. temtamy sa, sinbawy ahh. am. j. med. genet. 1991; 41:432-433. pmid: 1776632 22. cloning of the beta cell highaffinity sulfonylurea receptor: a regulator of insulin secrejon. aguilar bryan l, nichols cg, wechsler sw et al. science. 1995; 268:423426. pmid: 7716547 23. mutajons in kcnk4 that affect gajng cause a recognizable neurodevelopmental syndrome. bauer ck, calligari p, radio fc et al. am j hum genet. 2018; 103:621-630. pmid: 30290154 24. congenital hypertrichosis lanuginosa. beighton p. arch dermatol. 1970; 101:669-672. pmid: 5424483 25. characterizajon of the human abc superfamily: isolajon and mapping of 21 new genes using the expressed sequence tags database. allikmets r, gerrard b, hutchinson a, dean m. hum mol genet. 1996; 5:1649-1655. pmid: 8894702 26. hereditary gingival fibromatosis. anderson j, cunliffe wj, roberts df, close h. br med j. 1969; 3: 218-219. pmid: 5792612 27. intellectual disability: when the hypertrichosis associated with gene2c condi2ons with head and neck altera2ons vol 10 no 1 (2022) doi 10.5195/d3000.2022.237 h#p://den*stry3000.pi#.edu hypertrichosis is a clue. pezzani l, milani d, tadini g. j pediatr genet. 2015; 04:154-8. pmid: 27617126 28. neurodevelopmental disorder with progressive microcephaly, spasjcity, and brain anomalies in china caused by novel mutajons of plaa. dai c, zeng s, tan z et al. clin genet. 2019; 96: 380-381. pmid: 31322726 29. hirsujsm. mofid a, seyyed alinaghi sa, zandieh s, yazdani t. int j clin pract. 2007; 62: 43343. pmid: 18081798 30. global view on rare diseases: a mini review. blanca k, michael s, marjn v, kamil k. curr med chem. 2017; 24: 31533158. pmid: 28494745. 31. care for rare: spotlight on rare diseases. mukherjee k. trends pharmacol sci. 2019; 40: 227-8. pmid: 30905358 32. rare diseases debated in the senate. anon. n.d.. retrieved february 17, 2021. available from: hnps://www.conass.org.br/doen cas-raras-em-debate-nosenado/. 33. denjnogenesis imperfecta: differenjal diagnosis and clinical treatment. gomes mc. porto. 2020. available from: hnps://repositorioaberto.up.pt/bitstream/10216/1 27966/2/409881.pdf 34. hereditary gingival fibromatosis: idenjficajon and treatment. serra mc, falabella mev, tinoco emb, ribeiro msm, silva dg, maior jms. rev. cir. traumatol. buco-maxillofac. v.7, n.3, p. 15 22. 2007. available from: hnps://www.revistacirurgiabmf. com/2007/v7n3/2.pdf 35. expanded gingival fibromatosis syndrome phenotype, mental retardajon and hypertrichosis (zimmermann-laband). chacon camacho of, vázquez j, zenteno jc. am j med genet a. 2011; 7: 1716-20. pmid: 21626675 physicochemical characteristics and discolouration potentials of pulpine mineral® and pulpine ne® vol 12 no 1 (2024) doi 10.5195/d3000.2024.533 http://dentistry3000.pitt.edu physicochemical characteristics and discolouration potentials of pulpine mineral® and pulpine ne® mai fakhr-eldeen m1*, ashraf m. abu-seida2,3, maram f. obeid4, ahmed a. hashem4,5 1 department of endodontics, faculty of dentistry, king salman international university. el tor city, egypt. 2 faculty of dentistry, galala university. suez, egypt. 3 department of surgery, anaesthesiology and radiology, faculty of veterinary medicine, cairo university. giza, po: 12211, egypt. 4 department of endodontics, faculty of dentistry, ain shams university. cairo, egypt. 5 director of research at cleveland dental institute. ohio, usa. abstract aim: to compare the physicochemical properties (solubility, ph, radiopacity and crown discoloration) of pulpine mineral (pmin) and pulpine ne (pne) with the conventional material, mineral trioxide aggregate (mta). methodology: specimens of the tested materials were prepared according to the manufacturer’s instructions using split teflon ring molds. solubility was evaluated by the percentage of material mass loss over 24 h and one week. the alkalinity was measured after each evaluation period using a ph meter. other specimens were digitally radiographed on a size 2 sensor plate along with an aluminum step wedge to analyze the radiopacity by the image j software. finally, crown discoloration was assessed after applying the tested materials in the pulp chamber of sound human premolars using spectrophotometer. all data were statistically analyzed. results: compared to mta, both materials had significantly higher solubility and lower radiopacity (p<0.05). the alkalinity of pmin was higher than that of mta and pne. unlike pmin, pne and mta caused crown discoloration. conclusions: pmin exhibits promising results related to high alkalinity and adequate color stability but it needs modifications for radiopacity and solubility. keywords: crown discoloration; mta; pulpine ne; pulpine mineral; radiopacity; solubility. citation: mai fakhr-eldeen m., et al. (2024) physicochemical characteristics and discolouration potentials of pulpine mineral® and pulpine ne® dentistry 3000. 1:a001 doi:10.5195/d3000.2024.533 received: july 13, 2023 accepted: july 14, 2023 published: may 9, 2024 copyright: ©2024 mai fakhr-eldeen m., et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: maifakhreldeeen@gmail.com introduction vital pulp therapy (vpt) is indicated whenever the exposed pulp shows signs of reversible pulpitis [1]. it is mostly dependent on correct diagnosis and proper selection of an optimum capping material with adequate physicochemical and biological properties to stimulate dentinogenesis. [2]. because of its outstanding biocompatibility and capacity to build dentin bridge with minimal inflammation, mta is the gold standard material applied for vpt [2]. unfortunately, it showed poor handling characteristics, long setting time and crown discoloration [3]. hofmann dental manufacture (hofmann dental manufaktur, hdm, berlin, germany) lately released pulpine mineral (pmin) and pulpine ne (pne) materials as alternatives to mta in direct and indirect pulp capping [ 4,5]. both used propolis (ps) as the principal constituent in their liquid which is a natural product that has been identified as a valuable material as it contains high levels of flavonoids, aromatic acids, and esters, providing antibacterial and anti-inflammatory activities [6, 7]. the powder in pmin composes 70% hydroxyapatite (hap) implanted in a calcium hydroxide [4, 5]. hap is a calcium phosphate biomaterial that was previously employed as a pulp capping material due to its biocompatibility and osteoconductivity [8]. on the other hand, the powder of pne contains zinc compounds, and calcium compounds [4, 5], the latter combines with the tissue's carbon dioxide to produce calcite crystals, so http://dentistry3000.pitt.edu/ physicochemical characteristics and discolouration potentials of pulpine mineral® and pulpine ne® vol 12 no 1 (2024) doi 10.5195/d3000.2024.533 http://dentistry3000.pitt.edu commencing the calcification process [9]. the physicochemical features of vpt materials are highly critical to their therapeutic relevance [10]. these features include solubility, alkalinity, and radiopacity, for example. the decrease in solubility is critical for long-term sealing capabilities of any dental cement [11], and the solubility in distilled water shouldn’t be greater than 3% according to american national standard/american dental association (ansi/ada 2000) specification no. 57 [12]. adequate radiopacity is another significant physical property of dental materials, as it aids in the differentiation of cements and surrounding tissues and improves the radiographic examination of root fillings [13]. endodontic sealing materials must have a radiopacity of at least 3 mm of aluminium, according to american national standard/american dental association (ansi/ada 2000) specification no. 57 [12]. furthermore, the long-term ph maintenance is mandatory to create an apatite-like layer on the surface of the capping materials when it contacts the phosphate-containing fluids[14], thus alkalinity is another important chemical property that guaranteed dental tissue healing and remineralization. crown discoloration, on the other hand, is a serious clinical issue that has been documented as an undesirable consequence related to the application of various medications in the pulp chamber [15]. thus, the purpose of this study was to compare the new hoffmann's pmin and pne to mta in terms of solubility, alkalinity, radiopacity, and crown discoloration. materials and methods ethical approval the research proposal was approved by the ethical committee at faculty of dentistry; ain shams university, egypt (end 16-18d/7-2017). materials table 1 shows the composition, manufacturers, and batch number of all tested materials. solubility the solubility test was carried out in accordance with previous studies [1618]. for all tested materials, six specimens were prepared with split teflon ring molds (1.5±0.1 mm height and 7.75±0.1 mm internal diameter). specimens were left to set for three times longer than the manufacturer’s recommended (i.e. 15 min for mta, 30 min for pmin, and 45 min for pne). to determine the net weight of each specimen with mold (w0), a high precision scale with 1 x10-4 g accuracy (precision electrical weighing balance, sartorius max 220 g, germany) was used. the specimens were immersed in 60 ml deionized water then stored in an incubator for 24 hours and one week (at 37o c and 95% relative humidity). after that, the specimens were partially dried using absorbent papers then placed in a silica-containing desiccator for 24 hours. specimens with molds were then reweighed (w1). the specimens were unmolded, and the weight of the molds was reported in order to obtain the exact weight of the specimens without molds (w2). for each specimen, the percentage of solubility was computed as follows: % wight loss = (w0-w2) -(w1w2)/(w0-w2) x100. measurement of the ph the ph was measured according to bernardi et al [17]. in brief, newly mixed samples (n=6) were made in the same teflon mold. in addition; 2 split teflon mold without materials were utilized, to act as a base line of measurement, then placed in 60 ml of distilled water and incubated for 24 hours, and 7 days. after each interval, the ph was measured using a ph meter (orion 3 star; thermo scientific, singapore). radiopacity following oliveira et al. [18], two specimens for each material were prepared using the same teflon mold, stored at 37°c and 100% humidity for 48h then digitally radiographed on a size 2 sensor plate (fona, sri viag, galilei, italy) along with an aluminum step wedge (artinis cd dent 1.0, netherlands) (1 to 5 mm thickness), using a vario-2200 x-ray machine operating at 70 kv, 3.5 ma, 0.2 exposure time and focus sensor distance of 30 cm. image j software (national institutes of health, bethesda, md) was used to analyse the images. the radiopacity was calculated using a radiographic http://dentistry3000.pitt.edu/ physicochemical characteristics and discolouration potentials of pulpine mineral® and pulpine ne® vol 12 no 1 (2024) doi 10.5195/d3000.2024.533 http://dentistry3000.pitt.edu density tool and a region of interest (roi). crown discoloration forty single rooted mandibular sound premolars (free of restorations and cracks, extracted for orthodontic or periodontal issues) were collected. the specimens (n=10) were then randomly assigned to four groups: three experimental groups (mta, pmin, and pne) and a control group (without filling). the teeth were sectioned horizontally 2 mm apical to the cement enamel junction then accessed apically. chemo-mechanical preparation was made with h files from (30-80) and 10 ml of 2.5% sodium hypochlorite (naocl) and finally flushed with 5 ml saline solution (19). materials were prepared according to the manufacturers’ instructions and placed into the pulp chamber, then sealed with resin modified glass ionomer cement (riva light cure, sdi limited, australia). all specimens were immersed in test tubes containing 10 ml deionized water at room temperature and under light condition. color was evaluated by vita easy shade spectrophotometer (easyshade advance 4.0, vita zahanfabrik, germany) at 3 time points: l0, a0, b0: baseline (after preparation but before insertion of the materials), l1, a1, b1: (immediately after insertion of the filling material), l2, a2, b2: (after 6 weeks of storage). changes in the commission internationale de l’eclairage (cie l*a*b*) were represented for each experimental group and the corresponding δe values were calculated using the following formula: δe= [(δl) 2+(δa)2+(δb)2]1/2 as mentioned by chen et al. [20]. statistical analysis the statistical package for the social sciences was used for the statistical analysis (spss 25, ibm, usa). the normal distribution of data was assessed using kolmogorov-smirnov and shapiro-wilk tests. parametric data were accomplished using anova test to compare different groups, followed by a tukey's post hoc test to determine the significant one. a dependent t-test was applied for intragroup comparisons. the significance level was set at p < 0.05. table 1: tested materials and their composition according to the manufacturers results solubility values (percentage of weight loss) the data are collected in table (2). at 24 hours, the pmin group had the largest value of solubility (-2.5), followed by pne (-2.4) while mta had the least value (-1.2) with no statistically significant difference between pmin and pne but a statistically significant difference between mta and the other groups (p=0.027). at one week, the pmin group exhibited the largest value (12.8), followed by the pne group (3.4), dislikes) x100. while mta group showed the least mean percentage value (-1.6) with a statistically significant difference between the three groups (p<0.000). material composition manufacturer batch no mta -calcium oxide 55%–60% -silicon dioxide 17%–20% -aluminum oxide 2%–4% -bismuth oxide 18%–22% angelus, londrina, pr, brazil 41840 pulpine mineral (pmin) 5g powder containing calcium and hydroxyapatite compounds -10 ml liquid containing ethanol and propolis. hoffmann’s germany company 84078 84077 pulpine ne (pne) -10g powder containing calcium and zinc compounds -10 ml liquid containing ethanol and propolis. hoffmann’s germany company 8456 8457 http://dentistry3000.pitt.edu/ physicochemical characteristics and discolouration potentials of pulpine mineral® and pulpine ne® vol 12 no 1 (2024) doi 10.5195/d3000.2024.533 http://dentistry3000.pitt.edu moreover, mta and pne groups showed a slight increase in the mean weight percentage loss from 24 h to one week without statistically significant difference (p=0.093, 0.301 respectively) while pmin group had a high increase in the mean weight percentage loss from 24 h to one week with a significant difference (p<0.001) . ph values the results of ph values are collected in table (3). at 24 hours and one week, the greatest ph value was recorded in pmin group , followed by mta group, while the least value was recorded in pne group with no statistically significant difference between the mta and pmin groups, but a statistically significant difference was found between the pne group and other groups (p<0.05). furthermore, the mean ph value slightly decreased in mta and pne groups but increased in pmin group without statistically significant difference within each group. radiopacity values the values are collected in table (4) and figure (1). the greatest mean radiopacity value was recorded in mta group (equivalent to 5 aluminum step-wedge), followed by pne group (equivalent to 3 aluminum step-wedge, and the least value was recorded in pmin (equivalent to less than number one aluminum stepwedge). there was statistically significant difference between different groups (p<0.05). tooth discoloration values mta group showed a slight increase in luminance l*; while other groups showed a slight decrease with a statistically significant difference between them after each evaluation period (p<0.05). additionally, all groups showed a decrease in a* and b* values except the control group that showed a slight increase with a statistically significant difference between them after each evaluation period (p<0.05). still, all groups revealed clinically noticeable discoloration with δe > 3.3, however, the control group showed the least change in discoloration (δe= 3.9) followed by the pmin group (δe =7.66) and mta group (δe =17.71), while the highest value was recorded in the pne group (22.14 ( as shown in table (5) figure (2). statistically, there was no significant difference between mta and pne groups nor between pmin and control groups (p>0.05). however, there were significant differences between mta and both pmin and control groups (p<0.05). times mta pmin pne p-value 24 hours -1.2±0.251aa -2.5±0.305 ab -2.4±0.556 ab 0.027* one week -1.6±0.200 aa -12.8±0.200 bb -3.4±0.556 ac 0.000* p-value 0.093 0.001* 0.301 times mta pmin pne p-value 24 hours 11.5±0.100aa 11.6±0.152 aa 8.1±0.208 ab 0.000* one week 11.2±0.568aa 11.9±0.100 aa 7.2±0.550 ab 0.000* p-value 0.547 0.250 0.05 table 2: mean± (sd) values of solubility (percent of weight loss) among the different tested materials at different time intervals means with different capital superscript letters in the same column are statistically significant different. means with different small letters in the same row are statistically significant difference (p<0.05). table 3: mean± (sd) values of ph among the different tested materials at different time intervals. means with different capital superscript letters in the same column are statistically significant different. means with different small letters in the same row are statistically significant difference (p<0.05). http://dentistry3000.pitt.edu/ physicochemical characteristics and discolouration potentials of pulpine mineral® and pulpine ne® vol 12 no 1 (2024) doi 10.5195/d3000.2024.533 http://dentistry3000.pitt.edu also, there were significant differences between the pne and both pmin and control groups (p<0.05). discussion because the selection of an acceptable capping material with adequate physicochemical qualities is required to stimulate dentinogenesis in vpt procedures, this study compared the solubility, ph, radiopacity, and crown discoloration of pmin, and pne to mta. the results revealed that none of the evaluated materials had optimal attributes in this investigation. although pmin showed promising results related to high alkalinity and adequate color stability but it needs modifications for radiopacity and solubility. in the current study, split teflon molds with smaller dimensions than ansi/ada standards 57 were used to support the materials during the experiment, notably with pmin, which became crumble when stressed during mold removal, resulting in cracks and disintegration of the samples. the solubility of mta recorded here was 1.6% in one week that is like the results of previous studies [17,21]. this low value could be explained by leaching calcium and hydroxide ions at the initial setting [22]. conversely, after one week, pmin showed an increase in solubility (12.8%) which exceeds the ansi/ada recommendation. this can be attributed to its chemical composition that includes ps which in an aqueous solution creates phenoxide ions and raised the solubility as found by ganapathi mr et al [23]. additionally, hesperidin, which is the most important flavonoids present in ps, increased the solubility in distilled water after 28 days [24]. on the other hand, pne showed lower solubility in comparison to pmin which slightly exceeds the maximum recommendation. this could be explained by the addition of zinc compound in its powder as this resulted in a lower solubility [25]. groups mean±sd minimum maximum median p-value mta 165.18±2.40a 163.72 167.95 163.86 0.000* pmin 38.47±1.90 b 36.38 40.09 38.94 0.000* pne 124.64±1.44c 123.30 126.16 124.46 0.000* groups δl δa δb δe (mean±sd) p-value mta 2.0250 -6.9250 -16.1250 17.713±3.646a 0.000* pmin -4.6000 -3.7000 -3.7750 7.6615±2.216b 0.000* pne -2.9250 -8.6250 -20.1500 22.142±2.111a 0.000* control -3.30 -1.05 1.7 3.9695±0.159b 0.000* table 4: mean± (sd), minimum, maximum and median values of radiopacity among the different tested materials. means with different superscript letters in the same column are statistically significant different (p<0.05). table 5: δl,δa,δb and mean±(sd) of δe among the different tested materials means with different superscript letters in the same column are statistically significant different (p<0.05) http://dentistry3000.pitt.edu/ physicochemical characteristics and discolouration potentials of pulpine mineral® and pulpine ne® vol 12 no 1 (2024) doi 10.5195/d3000.2024.533 http://dentistry3000.pitt.edu alkalinity of the environment plays a crucial role in the process of hard tissue formation and is usually assessed by the ph meter [26]. in the present study, mta revealed a significant increase in ph value after 24h and slight decrease after one week and this agrees with the results of previous research [3]. on the contrary, another study demonstrated a lower ph after one week [27] which could be attributed to the difference in the preparation and evaluation methods used. on the other hand, pne group showed ph of 8.1 after 24 h and reached 7.2 after one week, this is totally related to the chemical properties of propolis, as the flavonoids present in propolis are acidic [24]. on contrary, pmin group showed high ph value (11.6) at 24h that increased to 11.9 after one week and properly, this could be explained by the addition of hap to its powder which are responsible for releasing hydroxyl ions that elevates the ph value. the radiopacity was also evaluated as it is an important feature that should be considered when evaluating material for vpt. radiopacity of the capping materials facilitate the observation of gaps or material deficiency in dental radiographs [28]. this was done in accordance with ansi/ada specification #57 which suggested that endodontic material should develop radiopacity correspondent to at least 3 mm aluminum steps [29]. in the present study, three different regions were evaluated (roi) within the same material to balance out the effect of localized irregularities not observed by the naked eye or air bubbles incorporated [29]. mta demonstrated greater radiopacity values than pne and pmin among studied materials due to the presence of bismuth oxide in its component, which possesses appropriate radiopacity features [28] and this was like earlier studies [30,31]. pmin group had the lowest radiopacity value which was lower than the no.1 aluminium step wedge, thus, further research involving the addition of radiopacifiers to pmin is highly advised. pne group demonstrated the minimum aluminium thickness suggested by ansi/ada specification n.57 (3mm), this is because zn compound is present in its powder [32]. the spectrophotometric and cie l*a*b* systems were chosen to evaluate the color change (δe) because they can detect minute variations in color and have several advantages such as repeatability, objectivity, and sensitivity [33]. the current results showed that pne had the most discoloration, followed by mta, and the pmin group had the least. the presence of ps and ethanol in pne liquid may explain this discoloration [34] while adding hap to pmin may explain the little change in tooth color as it is used for whitening purposes [35]. this is a preliminary study that analyzed pmin and pne materials' physicochemical qualities; more research on more physical, mechanical, and biological features of both products is required to emphasize the overall characteristics of these innovative materials. figure 1: the different radiopacity of the mta (a), pmin (b) and pne (c) inside the split teflon mold along with aluminum step wedge. figure 2: the different tooth discoloration of the pne (a), pmin (b), mta (c) and control groups (d) after 6 weeks immersed in deionized water and under light condition. http://dentistry3000.pitt.edu/ physicochemical characteristics and discolouration potentials of pulpine mineral® and pulpine ne® vol 12 no 1 (2024) doi 10.5195/d3000.2024.533 http://dentistry3000.pitt.edu conclusion compared to mta, both pmin and pne had significantly higher solubility and lower radiopacity. pmin showed significant color stability, while pne didn’t. the alkalinity of pmin was higher than that of mta while that of pne wasn’t. clinical relevance pmin showed promising results related to high alkalinity and adequate color stability but it needs modifications for radiopacity and solubility. conflict of interest the authors declare that they have no conflict of interest. acknowledgments not applicable references 1. mesenchymal stem cells promote hard-tissue repair after direct pulp capping. obeid m, saber sel d, ismael ael d, hassanien e. j endod. 2013 may;39(5):626-31. pmid: 23611380. 2. outcome of pulpotomy in permanent teeth with irreversible pulpitis: a systematic review and meta-analysis. ather a, patel b, gelfond jal, ruparel nb. sci rep. 2022 nov 16;12(1):19664. pmid: 36385132. 3. physicochemical properties of a bioceramic repair material biomta. coaguila-llerena h, ochoa-rodriguez vm, castronúñez gm, faria g, guerreirotanomaru jm, tanomaru-filho m. braz dent j. 2020 sepoct;31(5):511-515. pmid: 33146335. 4. https://hoffmanndental.com/produkte/?lang=en. 5. histopathological and immunohistochemical profiles of pulp tissues in immature dogs' teeth to two recently introduced pulpotomy materials. mohamed m, hashem aar, obeid mf, abuseida a. clin oral investig. 2023 jun;27(6):3095-3103. pmid: 36781475. 6. evaluation of antibacterial activity of propolis on regenerative potential of necrotic immature permanent teeth in dogs. el-tayeb mm, abu-seida am, el ashry sh, el-hady sa. bmc oral health. 2019 aug 6;19(1):174. pmid: 31387578. 7. biological evaluation of hesperidin for direct pulp capping in dogs' teeth. abo el-mal eo, abu-seida am, el ashry sh. int j exp pathol. 2021 feb;102(1):3244. pmid: 33405328. 8. hydroxyapatite applied as direct pulp capping medicine substitutes for osteodentin. hayashi y, imai m, yanagiguchi k, viloria il, ikeda t. j endod. 1999 apr;25(4):225-9. pmid: 10425944. 9. evaluation of the tissue reaction to fast endodontic cement (cer) and angelus mta. gomes-filho je, rodrigues g, watanabe s, estrada bernabé pf, lodi cs, gomes ac, faria md, domingos dos santos a, silos moraes jc. j endod. 2009 oct;35(10):1377-80. pmid: 19801233. 10. physiochemical properties of experimental nano-hybrid mta. akhavan zanjani v, tabari k, sheikh-al-eslamian sm, abrandabadi an. j med life. 2017 jul-sep;10(3):182-187. pmid: 29075348. 11. marginal adaptation, solubility and biocompatibility of theracal lc compared with mtaangelus and biodentine as a furcation perforation repair material. alazrag ma, abu-seida am, el-batouty km, el ashry sh. bmc oral health. 2020 oct 29;20(1):298. pmid: 33121465. 12. ansi/ada (2000) specification no 57 endodontic sealing material. chicago, usa: ada publishing. 13. densitometric measurement of radiopacity of gutta-percha cones and root dentin. katz a, kaffe i, littner m, tagger m, tamse a. j endod. 1990 may;16(5):211-3. pmid: 2074412. 14. bioceramic materials in endodontics. endodontic topics. wang z. 2015;32:3-0. 15. a review of tooth discoloration after regenerative endodontic therapy. kahler b, rossi-fedele g. j endod. 2016;42(4):563-569. pmid: 26852148. 16. solubility and dimensional change after setting of root canal sealers: a proposal for smaller dimensions of test samples. carvalho-junior jr, correr-sobrinho l, correr ab, sinhoreti ma, consani s, sousa-neto md. j endod. 2007;33(9):1110-1116. pmid: 17931945. 17. effects of the addition of nanoparticulate calcium carbonate on setting time, dimensional change, compressive strength, http://dentistry3000.pitt.edu/ https://hoffmann-dental.com/produkte/?lang=en https://hoffmann-dental.com/produkte/?lang=en physicochemical characteristics and discolouration potentials of pulpine mineral® and pulpine ne® vol 12 no 1 (2024) doi 10.5195/d3000.2024.533 http://dentistry3000.pitt.edu solubility and ph of mta. bernardi a, bortoluzzi ea, felippe wt, felippe mc, wan ws, teixeira cs. int endod j. 2017;50(1):97105. pmid: 26659859. 18. a laboratory evaluation of cell viability, radiopacity and tooth discoloration induced by regenerative endodontic materials. oliveira lv, da silva gr, souza gl, magalhães tea, barbosa glr, turrioni ap. int endod j. 2020; 53:1140-1152. pmid: 32299123. 19. color stability of teeth restored with biodentine: a 6month in vitro study. vallés m, roig m, duran-sindreu f, martínez s, mercadé m. j endod. 2015; 41:1157-1160. pmid: 25937179. 20. spectrophotometric analysis of coronal tooth discoloration induced by tricalcium silicate cements in the presence of blood. chen sj, karabucak b, steffen jj, yu yh, kohli mr. j endod. 2020;46(12):1913–9. pmid : 32949559. 21. solubility of root-end-filling materials: a comparative study. poggio c, lombardini m, alessandro c, simonetta r. j endod. 2007;33(9):1094–7. pmid: 17931941. 22. changes in the surface of four calcium silicate-containing endodontic materials and an epoxy resin-based sealer after a solubility test. borges rp, sousa-neto md, versiani ma, rached-júnior fa, dedeus g, miranda ces. int endod j. 2012; 45:419-428. pmid: 22150403. 23. nucleophilic effects on the deprotonation of phenol radical cations. chem phys lett. 2001; 337:335-340. 24. a comparative study of the physicochemical properties of hesperidin, mta-angelus and calcium hydroxide as pulp capping materials. abo el-mal eo, abu-seida am, el ashry sh. saudi dent j. 2019; 31: 219-227. pmid: 30983832. 25. antimicrobial activity and ph of calcium hydroxide and zinc oxide nanoparticles intracanal medication and association with chlorhexidine. aguiar as, guerreiro-tanomaru jm, faria g, leonardo rt, tanomaru-filho m. j contemp dent pract. 2015; 16:624629. pmid: 26423497. 26. physical evaluation of a new pulp capping material developed from portland cement. negm a, hassanien e, abu-seida am, nagy m. j clin exper dent. 2016; 8, e278– e283. pmid: 27398178. 27. influence of powder-towater ratio on radiopacity, setting time, ph, calcium ion release and micro-ct volumetric solubility of white mineral trioxide aggregate. cavenago bc, pereira tc, duarte mah. int endod j. 2014; 47:120126. pmid: 23647286. 28. radiopacity of portland cement associated with different radiopacifying agents. húngaro duarte ma, de oliveira el kadre gd ar, vivan rr, guerreiro tanomaru jm, filho mt, de moraes ig. j endod. 2009; 35:737-740. pmid: 19410095. 29. radiopacity of root filling materials using digital radiography. carvalho-junior jr, correr-sobrinho l, correr ab, sinhoreti mac, consani s, sousa-neto md. int endod j. 2007; 40 :514-520. pmid: 17511790. 30. mineral trioxide aggregate enriched with iron disulfide nanostructures: an evaluation of their physical and biological properties. argueta-figueroa l, delgado-garcía jj, garcía-contreras r. eur j oral sci. 2018; 126:234-243. pmid: 29442393. 31. comparison of the physical and mechanical properties of mta and portland cement. islam i, kheng chng h, jin yap au. j endod. 2006; 32:193-197. pmid: 16500224. 32. effect of zirconium oxide and zinc oxide nanoparticles on physicochemical properties and antibiofilm activity of a calcium silicate-based material. guerreirotanomaru jm, trindade-junior a, costa bc. sci world j. 2014: article id 975213. pmid: 25431798. 33. color stability of white mineral trioxide aggregate. vallés m, mercadé m, duran-sindreu f, bourdelande jl, roig m. clin oral investig. 2013; 17:1155-1159. pmid: 22814761. 34. does modification of amalgomer with propolis alter its physicomechanical properties? an in vitro study. abdallah abdallah rm, abdelghany am, aref ns. int j biomater. 2020;2020:3180879. pmid: 32454828. 35. whitening effect and morphological evaluation of hydroxyapatite materials. dabanoglu a, wood c, garcíagodoy f, kunzelmann kh. am j dent. 2009; 22:23-29. pmid: 19281109. http://dentistry3000.pitt.edu/ microsoft word 281 2022.docx evalua&on of the efficacy of 3d-guided piezosurgery in accelera&ng mandibular orthodon&c teeth alignment: a randomized controlled trial in adults. vol 10 no 1 (2022) doi 10.5195/d3000.2022.281 h#p://den*stry3000.pi#.edu evaluation of the efficacy of 3d-guided piezosurgery in accelerating mandibular orthodontic teeth alignment: a randomized controlled trial in adults. omar gibreal1, yasser al-modallal1, maher al-assaf2 1department of oral and maxillofacial surgery, faculty of dentistry, damascus university, damascus, syria 2department of oral histology and pathology, faculty of dentistry, damascus university, damascus, syria abstract objective: the aim of this study was to inves&gate the effects of 3d guided piezocision (a minimally invasive surgical technique) in accelera&ng orthodon&c tooth movements (otm) since the literature does not provide high quality evidence to confirm that piezocision results in significant otm accelera&on. furthermore, no randomized controlled trial (rct) has u&lized 3d piezo-surgical guides to accelerate severe orthodon&c de-crowding. study design: a parallel-group randomized controlled trial was conducted on pa&ents with severe mandibular teeth irregularity. sample size was 34 pa&ents (19 females and 15 males), then subjects were divided into two parallel groups using minitab® version 17 sowware. first group received conven&onal orthodon&c treatment, whereas the second group received 3d-guided piezo-assisted orthodon&c one. liyle irregularity index (lii) changes were studied using dental cast in four &me points before inser&ng the first archwire (t0), awer 1 month of treatment onset (t1), awer 2 months (t2), and at the end of the alignment stage (t3) and the overall alignment &me (oat) required to complete anterior alignment of the mandibular arch was also measured. results: ninety-five severe dental crowding pa&ents were evaluated for eligibility, 46 of them fulfilled the inclusion criteria. thirty-four par&cipants were allocated to the treatment groups randomly. there were no withdrawals during the trial phases. accordingly, the results of 34 pa&ents were sta&s&cally analyzed. oat was reduced by 48% in the experimental group compared to the control group, with a sta&s&cally significant difference between the two groups. likewise, the &me difference between all arch changes was significantly lower when 3d guided piezocision was performed, except for the first arch (t0) and the last arches (t3). conclusion: minimally invasive 3d guided piezocision seems to be a very effec&ve and easy technique in accelera&ng orthodon&c tooth movement keywords: 3d surgical guide; piezosurgery; accelerated tooth movement; minimally invasive surgery cita9on: gibreal, o et al. (2022) evalua9on of the efficacy of 3d-guided piezosurgery in accelera9ng mandibular orthodon9c teeth alignment: a randomized controlled trial in adults den9stry 3000. 1:a001 doi:10.5195/d3000.2022.281 received: december 18, 2021 accepted: april 12, 2022 published: august 11, 2022 copyright: ©2022 gibreal, o et al. this is an open access ar9cle licensed under a crea9ve commons auribu9on work 4.0 united states license. email: dr.maher.assaf1990@gmail.com introduction reduction in the duration of orthodontic treatment is considered one of the major challenges facing orthodontists today. moreover, prolonged orthodontic treatment times have complications such as periodontal disease, caries and root resorptions [1]. extraction-based orthodontic cases that could last up to 35 months are particularly vulnerable [2]. therefore, accelerating tooth movement and shortening the treatment time have evalua&on of the efficacy of 3d-guided piezosurgery in accelera&ng mandibular orthodon&c teeth alignment: a randomized controlled trial in adults. vol 10 no 1 (2022) doi 10.5195/d3000.2022.281 h#p://den*stry3000.pi#.edu become the common goals of both orthodontists and patients [3, 4]. recent studies have shown that corticotomy-accelerated orthodontic treatment is associated with regional acceleration phenomenon (rap) [5, 6, 7]. although these corticotomies have proven their effectiveness, they are considered to be aggressive therapies [8]. therefore, minimally invasive flapless techniques, as an alternative to traditional corticotomy, have been introduced into the orthopedic field [9-11]. piezocision appears to be an effective, safe and minimally invasive procedure in terms of accelerating orthodontic tooth movement [12]. however, no randomized controlled trial (rct) has employed 3d surgical guided techniques in piezocision planning. this technique could permit the engineering of an easy, accurate, predictable and safe localized alveolar decortication through a minor incision. thus, without raising a flap, no harm would occur to any nearby anatomical structures. so, the aim of this randomized controlled clinical trial was to investigate the effect of piezocision with a pre-customized 3d surgical guide in orthodontic treatment. material and methods study design the study was designed as a twoarmed, parallel group rct to evaluate the influence of 3d-guided piezocision-assisted orthodontic treatment (test group) compared to the traditional orthodontic treatment alone (control group). participants were recruited from the departments of orthodontics at damascus university dental school between august 2019 and may 2021. the local research ethics committee approval was obtained (udds-365616,032,033/src-5671). this trial was funded by the university of damascus dental school postgraduate research budget (ref no: 83054205788den). sample size estimation sample size was calculated using the g*power 3.1.7 software presuming that a reduction of 30 per cent in total treatment duration could be evidenced with a power of 80 per cent at the 5 per cent significance level. the smallest clinically significant difference in the time needed for leveling and alignment of severely crowded incisors was assumed to be 52.8 days depending on a previous report showing that crowding relief of anterior teeth took a mean of 132 days with a standard deviation 39 days [3]. therefore, a sample of 32 patients was required for both groups. to account for possible withdrawal, the final sample size for the study was set at 17 patients per group, yielding a total of 34 patients. patient selection, recruitment, and follow-up patients were selected from the department of orthodontics at university of damascus dental school. the treatment plan of 95 severe dental crowding patients was reviewed, but the number of patients who met the inclusion criteria and agreed to participate in this study after the acquaintance with the information sheet was 46. thirty-four subjects were equally and randomly assigned to the two groups; figure 1 represents the consort flow diagram. information sheets were distributed to all patients and informed consents were obtained. inclusion criteria ü adult healthy pabents from both sexes within an age range 17–24 years. ü absence of previous orthodonbc treatment. ü class ii division i pabents requiring first upper premolars extracbon. ü severe crowding (tooth-size– arch-length discrepancy >5 mm) in the anterior mandible with lifle’s irregularity index (lii). ü complebon permanent denbbon (except of third molars). ü absence of medicabons intake that interfere with pain percepbon for at least evalua&on of the efficacy of 3d-guided piezosurgery in accelera&ng mandibular orthodon&c teeth alignment: a randomized controlled trial in adults. vol 10 no 1 (2022) doi 10.5195/d3000.2022.281 h#p://den*stry3000.pi#.edu one week before the beginning of the treatment. exclusion criteria ü medical condibons that would affect tooth movement (corbcosteroid treatments, nsaids consumpbon, bisphosphonates, hyperparathyroidism, osteoporosis, uncontrolled diabetes). ü contraindicabon to oral surgery (medical–social– psychological). ü presence of primary teeth in the mandibular arch. ü missing permanent mandibular teeth (except for third molars). ü previous orthodonbc treatments. ü history of periodonbbs with a loss of alveolar support more than 10 percent. randomization and allocation concealment patients were assigned to the experimental group or the control group with an allocation ratio of 1:1 using a software-generated list of random numbers. allocation sequence was concealed using sequentially numbered, opaque, sealed envelopes which were opened only after the completion of premolars extraction. first group received 3d-guided piezocisionassisted orthodontic treatment, whereas the second group received conventional orthodontic treatment (figure 1). the generation of random allocation sequence, participants’ enrollment and assignment to the two groups were performed by one of the academic staff not involved in this research. orthodontic procedures all subjects underwent conventional orthodontic treatment with fixed appliances. fixed orthodontic appliances with an mbt prescription and 0.022-in. slot height (master series®, american orthodontics™, sheboygan, wi usa) were bonded four days following first-premolar extraction. these brackets were conventionally ligated. then, a 0.014inch niti archwire (american orthodontics, sheboygan, wisconsin, usa) was inserted and tied to each bracket using 0.010-inch stainless steel ligature wires. for both groups, the following sequence of archwires was adopted: 0.014-in. niti followed by 0.016-in., 0.016 x0.022-in. and 0.017 x 0.025-in. niti, and finally 0.019 x 0.025-in. stainless steel [11]. patients were asked to return every 2 weeks, so that change in archwires was performed when it was felt that an improvement had occurred in teeth positions and there was a possibility of inserting the next archwire without exerting excessive figure 1. consort flow diagram of patients’ recruitment and follow-up. evalua&on of the efficacy of 3d-guided piezosurgery in accelera&ng mandibular orthodon&c teeth alignment: a randomized controlled trial in adults. vol 10 no 1 (2022) doi 10.5195/d3000.2022.281 h#p://den*stry3000.pi#.edu force on the engaged teeth. treatment was considered finished when lii was less than 1 mm, indicating complete alignment of the teeth and the feasibility of inserting the final archwire passively into all brackets [13]. 3d piezocision surgical procedure in the test group, piezocision surgery was performed on the same premolars extraction day to minimize the number of anesthesia depending sessions in terms of patients` comfort and satisfaction. after local infiltration was injected (lidocaine hydrochloride 2% with epinephrine 1:80,000), a pre-customized 3d surgical guide was placed to perform vertical interproximal micro-incisions (varying between 5 and 8 mm) below each interdental papilla (figure 2). the incisions began 4 mm below the papilla to prevent any further gingival recessions, then corticotomies (5 mm long and 3 mm deep) guided by the same 3d guide were made using a piezoelectric device(implant center™ 2, satelec, france) with a bs1 cutting tip and irrigation solution pump 80 ml/m (figure 3). all of the above-mentioned aspects were pre-planned and applied by cbct imaging and transferred to the casts in order to produce a reliable and precise surgical 3d guide (figure 4). no subsequent sutures were performed, and the surgical side was covered by a piece of iodoform gauze. careful tooth brushing and the use of a mouthwash (chlorhexidine gluconate 0.12%) were prescribed for 7 days; ice packs for the first 12 h following surgery; and patients were instructed to take one or two tables of panadol® (acetaminophen; 500 mg) when they suffer from moderate/severe pain. experimental patients check-ups were scheduled a day after the procedure ensuring absence of postoperative complications, four days post procedure to continue their orthodontic treatment and were followed up every two weeks for orthodontic treatment sequence. outcome measures the primary outcome measure was the overall alignment time (oat) required to complete anterior alignment of the dental arch. followup of this trial was considered finished when the lii was less than 1 mm [13]. the secondary outcome measure was little’s irregularity index (lii) measured on study models taken at the following assessment times: before inserting the first archwire (t0), after 1 month of treatment onset (t1), after 2 months (t2), and at the end of the alignment stage which was accomplished when lii was less than 1 mm (t3). measurements were made according to methodology mentioned by little (13). lii was measured using a fine tip digital caliper with an accuracy of 0.1 mm (mitutoyo, zhejiang, china). statistical analysis parametric tests were used since anderson-darling normality tests showed normal distributions of the collected data. two-sample t-tests were used to detect significant differences between the two groups at each assessment time. single blinding was employed in this trial figure 2. 3d piezo-surgical guide. figure 3. the guide is in place to perform the incisions and piezocisions. figure 4. cbct pre-planning. evalua&on of the efficacy of 3d-guided piezosurgery in accelera&ng mandibular orthodon&c teeth alignment: a randomized controlled trial in adults. vol 10 no 1 (2022) doi 10.5195/d3000.2022.281 h#p://den*stry3000.pi#.edu regarding outcome measure assessment and data analysis. minitab® program version 17.0 (minitab inc., pennsylvania, usa) was used to perform descriptive and inferential statistics. patients were given serial numbers and patients’ allocation was concealed from the principal investigator through the different stages of the trial and at the statistical analysis phase to ensure blindness of the assessor and to avoid detection bias. results 34 patients were enrolled in this study as presented in the basic sample characteristics table (table no. 1), while descriptive statistics of the evaluated variables are given in table no. 2. no withdrawals were registered through the whole trial stages. a statistical significance was found between the two groups regarding the oat. the experimental group required less mean treatment time (i.e. 61.9 ± 12.5 days) in the leveling and alignment stage compared to the control group (i.e. 129.1 ± 35.5 days; p <0.0001) with a 48% decrease in the oat. the results regarding changes of the lii according to assessed time points are given in (table no. 3). no statistically significant difference between the two groups was found at t0 and t3 (p = 0.511, p=0.832, respectively), whereas statistically significant differences were observed at t1 and t2 (p <0.001, p=0.003, respectively). discussion this is the first published rct having the objective of employing 3d guiding techniques in accelerating severe orthodontic teeth de-crowding utilizing piezocision. it was found that 3d guided piezocision accelerated leveling and alignment and reduced the overall time by about 48%. this could be explained by the regional acceleratory phenomenon (rap) following the intentional bone injury [7, 14]. this result was in accordance with some other recent clinical trials studying the effectiveness of the flapless piezocision technique in accelerating teeth leveling and alignment. in comparable recent studies [3, 5, 15, 16], similar acceleration rates in the piezocision groups were found. none of them used 3d guiding techniques to achieve safe and accurate piezoelectric cortectomies. however, the trial that had performed guided piezocorticision utilized mild non extraction crowding cases. group gender n (%) p-value* mean age (sd) min. age max. age p-value† control male 7 (43.75%) 0.531 21.27 (1.87) 18 24 0.092 female 9 (56.25%) experimental male 8 (44.4%) 20.86 (1.98) 17 23 female 10 (55.6%) all sample 34 (100%) 21.03 (1.96) 17 24 table 1: basic sample characteristics. *: employing chi-square test, †: employing two-sample t test, min: minimum, max: maximum evalua&on of the efficacy of 3d-guided piezosurgery in accelera&ng mandibular orthodon&c teeth alignment: a randomized controlled trial in adults. vol 10 no 1 (2022) doi 10.5195/d3000.2022.281 h#p://den*stry3000.pi#.edu in contrast, uribe et al reported no significant difference in the alignment time between the conventional and the piezocision groups [17]. the difference between our findings and theirs is probably related to the fact that they applied just three cortical incisions in the labial cortical plate (4mm length and 1-mm depth of cortical bone) between the mandibular central incisors, and lateral incisors and canines, while in the current study, five cortical incisions in the labial cortical plate (5 to 8-mm length and 3-mm depth of cortical bone) between the six anterior teeth. additionally, patients in the current study underwent extraction of first premolars which may have enhanced the effect of the rap [8, 18]. other rcts carried out in the past few years regarding the efficacy of piezocision in accelerating other orthodontic treatments such as canine retraction [19-21] and en masse retraction [22]. alfawal et al. in a rct [19] evaluated the effectiveness of piezocision in the variable group mean sd se min q1 median q3 max liit0 control 11.66 1.17 0.24 10.00 10.88 11.00 12.00 13.00 exp 11.59 0.69 0.32 10.00 10.84 11.50 12.25 14.00 liit1 control 8.90 1.14 0.25 6.50 7.25 8.50 9.00 10.00 exp 4.35 1.67 0.42 4.00 4.75 5.00 5.25 6.00 liit2 control 5.75 1.12 0.28 4.50 5.00 5.50 6.25 7.80 exp 2.91 0.34 0.21 2.00 2.57 2.75 2.89 3.50 liit3 control 0.32 0.12 0.02 0.10 0.25 0.30 0.40 0.50 exp 0.24 0.10 0.02 0.10 0.20 0.20 0.30 0.40 ott control 129.11 35.53 7.55 98.00 101.50 121.00 130.50 179.00 exp 61.90 12.52 4.33 49.00 51.00 58.00 62.50 81.00 little's index of irregularity group mean sd mean difference (%) 95% ci of the difference p-value significance of differences lower bound upper bound before control 11.66 1.17 -0.226 -1.004 0.510 0.671 ns experimental 11.59 0.69 at one month control 8.90 1.14 3.2899 3.192 5.488 <0.0001 ** experimental 5.25 1.67 at two months control 5.66 1.12 1.8099 1.074 4.544 0.002 * experimental 2.95 0.34 at three months control 0.32 0.12 0.0824 0.0050 0.1597 0. 545 ns experimental 0.24 0.10 table 2: descriptive statistics of the little’s index of irregularity (mm) and the overall alignment time (days) in the two groups (n = 17 for each group). liit0: little’s irregularity index before inserting the first archwire; liit1: after 1 month of orthodontic treatment onset; liit2: after 2 months; liit3: at the end of the alignment stage; exp.: experimental group; sd: standard deviation; se: standard error; min: minimum; q1: first quartile; q3: third quartile, max: maximum. table 3: the results of significance tests of the observed lii changes (in mm); (n=17 in each group)†. †: two sample t test, *: significant at p<0.05, **: significant at p<0.001, ns: not significant. evalua&on of the efficacy of 3d-guided piezosurgery in accelera&ng mandibular orthodon&c teeth alignment: a randomized controlled trial in adults. vol 10 no 1 (2022) doi 10.5195/d3000.2022.281 h#p://den*stry3000.pi#.edu acceleration of upper canine retraction after first premolar extraction. this showed that the rate of upper canine retraction was significantly higher in the experimental side compared to the control side, by two-fold the first month and 1.5-fold in the second month. these findings are comparable with the studies of abbas et al [21] and aksakalli et al [20] that reported an acceleration of the rate of canine retraction by 1.5–2 times during the first 3 months of tooth movement when piezocision was used. to the contrary, tunçer et al [22] concluded that piezo surgery was ineffective in accelerating en masse retraction for upper anterior teeth following premolar extraction in class ii division 1 patients. the lack of significant results may be explained by underpowered sample size calculation of that study, intending to detect at least 50 per cent difference in the rate of tooth movement instead of 30 per cent as in our study. nevertheless, the direct comparison between our findings and those studies is not straightforward since these rcts have been based on canine or en-masse retraction and not crowding relief corrections. no significant difference at t0 in lii was found, which assured that the groups were almost similar in the initial crowding. furthermore, after a month lii had a mean of 4.25 mm (± 1.67) in the experimental group and a mean of 8.9 mm (± 1.14) for the control group. these results indicated a 51% higher leveling rate for the experimental group than the control one. this percentage slightly decreased to 49% after 2 months; but further decreased to 25% at the end of the alignment stage (t3). this decrease could be explained by the gradual decrease of the rap over time which was documented by wilcko et al., however they report an onset after 1–2 months following bone injury and found that the effects lasted from 2 to 4 months [23]. conclusion minimally invasive 3d guided piezocision seems to be a very effective and easy technique in accelerating orthodontic tooth movement. references 1. adverse effects of orthodontic treatment: a clinical perspective. talic nf. saudi dent j. 2011 apr;23(2):55-59. doi: 10.1016/j.sdentj.2011.01.003. pmid: 24151415. 2. corticotomy facilitated orthodontics: review of a technique. alghamdi as. saudi dent j. 2010 jan;22(1):1-5. doi: 10.1016/j.sdentj.2009.12.008. pmid: 23960473. 3. efficacy of piezocision-based flapless corticotomy in the orthodontic correction of severely crowded lower anterior teeth: a randomized controlled trial. gibreal o, hajeer my, brad b. eur j orthod. 2019 mar 29;41(2):188-195. doi: 10.1093/ejo/cjy042. pmid: 29931294. 4. acceleration of tooth movement during orthodontic treatment--a frontier in orthodontics. nimeri g, kau ch, abou-kheir ns, corona r. prog orthod. 2013 oct 29;14:42. doi: 10.1186/21961042-14-42. pmid: 24326040. 5. localized piezoelectric alveolar decortication for orthodontic treatment in adults: a randomized controlled trial. charavet c, lecloux g, bruwier a, rompen e, maes n, limme m, lambert f. j dent res. 2016 aug;95(9):1003-1009. doi: 10.1177/0022034516645066. pmid: 27129491. 6. piezocision™-assisted orthodontics: past, present, and future. dibart s, keser e, nelson d. semin orthod. 2015;21(3):170-175. doi: 10.1053/j.sodo.2015.06.003. 7. the regional acceleratory phenomenon: a review. frost hm. henry ford hosp med j. 1983;31(1):3-9. pmid: 6345475. 8. mechanism of action and morphologic changes in the alveolar bone in response to selective alveolar decorticationfacilitated tooth movement. baloul ss, gerstenfeld lc, morgan ef, carvalho rs, van dyke te, kantarci a. am j orthod dentofacial orthop. 2011 apr;139(4 suppl):s83-101. doi: 10.1016/j.ajodo.2010.09.026. pmid: 21435543. 9. effects of corticision on paradental remodeling in orthodontic tooth movement. kim sj, park yg, kang sg. angle orthod. 2009 mar;79(2):284291. doi: 10.2319/020308-60.1. pmid: 19216591. 10. effect of corticision and different force magnitudes on orthodontic tooth movement in a rat model. murphy ca, chandhoke t, kalajzic z, flynn r, utreja a, wadhwa s, nanda r, uribe f. evalua&on of the efficacy of 3d-guided piezosurgery in accelera&ng mandibular orthodon&c teeth alignment: a randomized controlled trial in adults. vol 10 no 1 (2022) doi 10.5195/d3000.2022.281 h#p://den*stry3000.pi#.edu am j orthod dentofacial orthop. 2014 jul;146(1):55-66. doi: 10.1016/j.ajodo.2014.03.024. pmid: 24974999. 11. evaluation of the levels of pain and discomfort of piezocisionassisted flapless corticotomy when treating severely crowded lower anterior teeth: a singlecenter, randomized controlled clinical trial. gibreal o, hajeer my, brad b. bmc oral health. 2019 apr 16;19(1):57. doi: 10.1186/s12903-019-0758-9. pmid: 30991984. 12. piezocision™: accelerating orthodontic tooth movement while correcting hard and soft tissue deficiencies. dibart s. front oral biol. 2016;18:102108. doi: 10.1159/000351903. pmid: 26599123. 13. the irregularity index: a quantitative score of mandibular anterior alignment. little rm. am j orthod. 1975 nov;68(5):554-563. doi: 10.1016/0002-9416(75)90086-x. pmid: 1059332. 14. orthodontic method and device. wilcko mt, wilcko wm. united states patent us 6,109,916. 2000 aug. 15. piezocision-assisted orthodontic treatment using cad/cam customized orthodontic appliances: a randomized controlled trial in adults. charavet c, lecloux g, jackers n, albert a, lambert f. eur j orthod. 2019 sep;41(5):495-501. doi: 10.1093/ejo/cjy082. pmid: 30649257. 16. piezocorticision-assisted orthodontics: efficiency, safety, and long-term evaluation of the inflammatory process. strippoli j, durand r, schmittbuhl m, rompré p, voyer r, chandad f, nishio c. am j orthod dentofacial orthop. 2019 may;155(5):662-669. doi: 10.1016/j.ajodo.2018.06.013. pmid: 31053282. 17. efficiency of piezotomecorticision assisted orthodontics in alleviating mandibular anterior crowding-a randomized clinical trial. uribe f, davoody l, mehr r, jayaratne ysn, almas k, sobue t, allareddy v, nanda r. eur j orthod. 2017 nov 30;39(6):595-600. doi: 10.1093/ejo/cjw091. pmid: 28371882. 18. cytokine expression and accelerated tooth movement. teixeira cc, khoo e, tran j, chartres i, liu y, thant lm, khabensky i, gart lp, cisneros g, alikhani m. j dent res. 2010 oct;89(10):1135-1141. doi: 10.1177/0022034510373764. pmid: 20639508. 19. evaluation of piezocision and laser-assisted flapless corticotomy in the acceleration of canine retraction: a randomized controlled trial. alfawal amh, hajeer my, ajaj ma, hamadah o, brad b. head face med. 2018 feb;14(1):4. doi: 10.1186/s13005-018-0161-9. pmid: 29454369. 20. accelerated tooth movement with piezocision and its periodontal-transversal effects in patients with class ii malocclusion. aksakalli s, calik b, kara b, ezirganli s. angle orthod. 2016 jan;86(1):59-65. doi: 10.2319/012215-49.1. pmid: 25989211. 21. evaluation of corticotomyfacilitated orthodontics and piezocision in rapid canine retraction. abbas nh, sabet ne, hassan it. am j orthod dentofacial orthop. 2016 apr;149(4):473-480. doi: 10.1016/j.ajodo.2015.09.029. pmid: 27021451. 22. efficiency of piezosurgery technique in miniscrew supported en-masse retraction: a single-centre, randomized controlled trial. tunçer ni, arman-özçirpici a, oduncuoglu bf, göçmen js, kantarci a. eur j orthod. 2017 nov;39(6):586594. doi: 10.1093/ejo/cjx015. pmid: 28402521. 23. accelerated osteogenic orthodontics technique: a 1stage surgically facilitated rapid orthodontic technique with alveolar augmentation. wilcko mt, wilcko wm, pulver jj, bissada nf, bouquot je. j oral maxillofac surg. 2009 oct;67(10):2149-2159. doi: 10.1016/j.joms.2009.04.095. pmid: 19761908. microsoft word 449 2023.docx effect of vocal anesthesia on adult dental pa5ents during covid-19 pandemic period vol 11 no 1 (2023) doi 10.5195/d3000.2023.449 h#p://den*stry3000.pi#.edu effect of vocal anesthesia on adult dental patients during covid-19 pandemic period mudher mb. alsunbuli1, marwa a. a3yia2, amer hassan3, hasanain faisal ghazi4 1bds, fibms, oral and maxillofacial surgery department, college of dentistry. al-bayan university. baghdad, iraq 2bds, fibms, oral and maxillofacial surgery unit, al-yarmouk teaching hospital, baghdad, iraq 3mbchb, cabs, college of dentistry, al-bayan university, baghdad, iraq 4mbchb, phd (public health), college of nursing, al-bayan university, baghdad, iraq abstract background: pain is as a major concern with dental pa5ents. the den5st-pa5ent interac5on can reveal the presence of anxiety and fear, which may result in rise of pain. this study aimed to assess vocabulary communica5on as support to dental anesthesia on the adult dental pa5ent. materials and methods: this is a prospec5ve, randomized clinical study, conducted from february 2020 through march 2021. two hundred individuals (112 males, 88 females) of the age range (16 to 84 years) were separated into two groups for tes5ng the level of pain during dental local anesthesia, with vocal communica5on and without vocal communica5on. results: while the control group showed no difference between males and females, there was a difference between the sexes in the experimental group with past dental visits, vas and injec5on type having less anxiety (p=0.0001) while with educa5on/vas having (p£0.01) as appear in sex to age (p£0.05). there was no difference when data were compared by age, or type of anesthesia (inferior alveolar nerve block or infiltra5on technique). conclusion: dental anxiety can be alleviated by talking to the pa5ents before the procedure, which could reduce the amount of pain. keywords: anesthesia; local; dental anxiety; vocabulary; communica5on cita9on: alsunbuli, mmb et al. (2023) effect of vocal anesthesia on adult dental pa9ents during covid-19 pandemic period den9stry 3000. 1:a001 doi:10.5195/d3000.2023.449 received: february 18, 2023 accepted: february 19, 2023 published: march 31, 2023 copyright: ©2023 alsunbuli, mmb et al. this is an open access ar9cle licensed under a crea9ve commons avribu9on work 4.0 united states license. email: dr.mudhar@albayan.edu.iq introduction most dental procedures require pain control [1]. pain in its traditional sense can be considered as an indicator or alarm for potential tissue damage. pain response varies based on psychological state, social stratum, age, and other factors. hence, one can say that pain loses some efficiency, and no longer is a reliable indicator of tissue damage [2]. the site of the dental needle injection, the thickness of the tissue to be pierced by a dental needle, and the degree of tissue adherent to deeper structures are strongly related to the patient’s pain sensitivity and anxiety [3]. the interaction between the dentist and the patient can reveal the presence of anxiety and fear [4]. it is not uncommon that dental injection is used as intimidation for children [5]. patients with generalized anxiety disorder exposed to changing in talk used as treatment ended with significantly different levels of response according to the severity of the case [19]. to demonstrate that vocal anesthesia can improve analgesia in dental procedures this study aimed to assess vocabulary effect of vocal anesthesia on adult dental pa5ents during covid-19 pandemic period vol 11 no 1 (2023) doi 10.5195/d3000.2023.449 h#p://den*stry3000.pi#.edu communication as support to dental anesthesia on adult dental patients. material and methods this is a prospective, single-blinded, randomized observational prognostic controlled clinical study. the study was conducted through the covid-19 pandemic, from february 2020 through march 2021, at al-bayan university, college of dentistry, in bagdad, iraq. approval by the local ethics committee according to the declaration of helsinki guidelines was obtained. two hundred (112 males, 88 females) of the age range (16 to 84 years) participated in the study. all participants had a covid-19 infection and recovered from the disease. the individuals were separated into two groups for testing the level of pain due to dental local anesthesia. written consent was obtained from patients, explaining that information asked was only for research purposes. the treatment modality (with vocal communication vs. without vocal communication) was decided by tossing a coin, where 'face' was the experimental side and 'back' for control. the study was performed using the ''single injection method'' to remove acquiescence bias. this is where only one injection of local anesthesia was performed on the patient after a vocabulary communication with the patient to explain the pain pattern before injection (study group, n=94), and direct injection of local anesthetic agent to patient without any verbal communication (control group, n=106). patients were numbered from 1 to 200. excluded patients from the study were the ones that had uncontrolled systemic diseases or a clinically significant medical history, or if they regularly used analgesic medications or antiinflammatory medication in the last 24 hours prior the day of the experiment. we also excluded pregnant women or women breastfeeding. tobacco smokers using ten or more cigarettes per day were also excluded. all areas that were injected were free from infection, inflammation, and the patient did not seek emergency treatment because of pain. all patients were injected with 2% lidocaine with 1:80000 adrenalin dental local anesthetic solution through dental needle gauge 27. six different dentists were operating in this study with different days and times through the day and different degrees of experience from general practitioners, orthodontists, prosthodontists, and oral surgeons. ibm spss statistics v.26 statistical analysis software program was used to evaluate the experiment through t test using p-value ≤0.05 as statistically significant for rejection of the null hypothesis. every patient experience was recorded using a questionnaire. pain was assessed by a 10 mm visual analog scale (vas) with the two ends assigned as no pain and worst pain ever. when the pain was present, the patient was asked to select a category from 1 to 10. then, the score was recorded in the case sheet used in this study (appendix 1). appendix 1. study instrument. effect of vocal anesthesia on adult dental pa5ents during covid-19 pandemic period vol 11 no 1 (2023) doi 10.5195/d3000.2023.449 h#p://den*stry3000.pi#.edu pain sensitivity differences between sexes was assessed. patient age was tested for compliance to dental work and dental-injection despite anxiety. type of injection was recorded. infiltration of almost all the maxillary teeth and some of the mandibular injections or block anesthesia mainly for the inferior alveolar nerve block were used for treating the study participants. past dental procedures and patient regular visits to the dentist were recorded as null, little, frequent, and usual. frequency of dental visits was recorded as first-time the patient seeking a dental office, once in a year, once a month, and if he the patient went to the dentist on a monthly basis or even less. education level, procedures, and marital status were also recorded. results table 1 summarizes the 200 study participants. table 2 describes the distribution of the sex of the participants by ethnicity. differences were found when age was compared to the sex of the participants. samples were tested according to age and vas score, as well as the distribution of samples according to age and marital status and vas score, appeared statistically significant (p≤0.05) (tables 2, 3 and 5). several differences were detected when looking at the distribution of the sample by sex and vas score, education and vas score, past dental office visits and vas score. other comparisons included distribution of the sample by injection of anesthesia type and vas, which appeared to be statistically significant different (tables 4, 6, 7, 8 and 9). variable n chi-square (p-value) male 112 2.88 (0.09) female 88 young adults 41 51.6 (0.0001) middle age 114 elderly 45 single 116 5.12 (0.02) married 84 low 15 71.06 (0.0001) mid 111 high 74 null 4 186.72 (0.0001) little 130 frequent 44 usual 22 iadb 67 21.78 (0.0001) infiltration 133 control 106 0.49 (0.48) experiment 94 no pain 22 95.4 (0.0001) mild 61 moderate 79 severe 37 worst pain 1 table 1. distribution of variables in the sample studied. effect of vocal anesthesia on adult dental pa5ents during covid-19 pandemic period vol 11 no 1 (2023) doi 10.5195/d3000.2023.449 h#p://den*stry3000.pi#.edu age group 15-25 26-35 36-45 46-55 56-65 66-75 76-85 totals m (no.) 23 10 17 20 16 12 14 112 f (no.) 18 12 17 10 12 12 7 88 total 41 22 34 30 28 24 21 200 chi-square -χ2 (pvalue) 5.29 * (0.05) * (p≤0.05). age 15-25 26-35 36-45 46-55 56-65 66-75 76-85 totals vas e c e c e c e c e c e c e c e c never 6 0 4 0 4 0 4 0 2 0 1 0 1 0 22 0 mild 11 0 7 0 7 0 10 1 10 1 8 0 6 0 59 2 moderate 2 13 2 6 3 12 1 9 1 6 2 9 2 11 13 66 sever 0 8 0 3 0 8 0 5 0 8 0 4 0 2 0 38 worst 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 total 20 22 13 9 14 20 15 13 13 15 11 13 8 12 95 106 percent % 21.1 20. 7 13. 7 8.5 14. 7 18. 9 15. 8 12. 3 13. 7 14. 2 11. 6 12. 3 8. 4 11.3 100% chi-square -χ2 (p-value) 6.38* (0.02) -- * (p≤0.05). sex male female totals vas e c e c e c table 2. distribution of sample according to age and sex. table 3. distribution of sample according to age and vas/e&c. table 4. distribution of sample according to sex and vas/e&c. effect of vocal anesthesia on adult dental pa5ents during covid-19 pandemic period vol 11 no 1 (2023) doi 10.5195/d3000.2023.449 h#p://den*stry3000.pi#.edu never 4 0 18 0 12 0 mild 22 2 37 0 59 2 moderate 1 53 12 13 13 66 sever 0 30 0 7 0 37 worst 0 0 0 1 0 1 total 27 85 67 21 94 106 percent % chi-square -χ2 (p-value) 34.69 ** (0.0001) -- marital status single married totals vas e c e c e c never 8 0 14 0 12 0 mild 39 0 20 0 59 0 moderate 6 38 7 22 13 60 sever 0 24 0 21 0 45 worst 0 1 0 0 0 1 total 53 63 41 43 94 106 percent % 56.38% 59.43% 43.72% 40.57 % 100% chi-square -χ2 (p-value) 5.87* (0.0277) -- table 5. distribution of sample according to marital status and vas/e&c. effect of vocal anesthesia on adult dental pa5ents during covid-19 pandemic period vol 11 no 1 (2023) doi 10.5195/d3000.2023.449 h#p://den*stry3000.pi#.edu education pre-school primaryhigh school university higher totals vas e c e c e c e c e c never 2 0 13 0 6 0 1 0 22 0 mild 2 0 30 0 21 2 6 0 59 0 moderate 1 7 8 38 4 18 0 3 13 66 sever 0 3 0 21 0 9 0 4 37 0 worst 0 0 0 1 0 0 0 0 0 1 total 5 10 51 60 31 29 7 7 94 106 chi-square -χ2 (p-value) 35.02 (p=0.0001). -- (p≤0.01). past dental. procedures null little frequent usual totals vas e c e c e c e c e c never 0 0 14 7 0 1 0 22 0 mild 1 34 1 16 1 8 0 59 2 moderate 0 2 8 46 4 10 1 8 13 66 sever 0 0 0 27 0 6 0 4 0 37 worst 0 1 0 0 0 0 0 0 0 1 total 1 3 56 74 27 17 10 12 94 106 table 6. distribution of sample according to education/vas/e&c. table 7. distribution of sample according to past dental visits. effect of vocal anesthesia on adult dental pa5ents during covid-19 pandemic period vol 11 no 1 (2023) doi 10.5195/d3000.2023.449 h#p://den*stry3000.pi#.edu percent % 1.06% 2.83% 59.57% 69.81% 28.72% 16.04% 10.64% 11.32% 100% chi-square -χ2 (p-value) 39.205 ** (0.0001) -- vas group never mild moderate sever worst totals control 0 2 66 37 1 106 experiment 22 59 13 0 0 94 total 22 61 79 37 1 200 chi-square -χ2 (p-value) 95.400 ** (0.0001) -- ianb infiltration totals (mean) vas e c e c e c never 7 0 15 0 22 0 mild 16 1 44 0 60 1 moderate 2 24 11 34 13 58 sever 0 16 0 29 0 45 worst 0 1 0 0 0 1 total 25 42 70 63 95 105 chi-square -χ2 (p-value) 23.483 ** (0.0001) -- discussion age, sex, and the surroundings are just a few of the many factors that might have an impact on how people experience pain [20]. the results of the current study showed that injection with the aid of vocabulary anesthesia caused statistically less pain in comparison with the conventional method in males and females. in comparison to men, women reported more pain and had table 8. distribution of sample according to vas/e&c. table 9. distribution of sample according to injection type/vas/e&c. effect of vocal anesthesia on adult dental pa5ents during covid-19 pandemic period vol 11 no 1 (2023) doi 10.5195/d3000.2023.449 h#p://den*stry3000.pi#.edu a lower pain threshold. this difference was not seen for the other variables. the mechanism underlying this disparity may be due to that neuro-perception processes of pain and pain inhibition correlates with sex differences (20,23). although needle phobia is a common phobic condition and over 20% of adults aged 20–40 years old have fear of needles [24,25]. age differences in pain perception were less consistent in our study and the results were insignificant. some studies implied that pain sensitivity is higher in older adults, whereas others suggest a decrease in sensitivity with age [26]. pain is commonly under recognized and undertreated in older adults compared to younger adults [27]. there was a significant difference in the experience of pain between the single and married patients who received vocal anesthesia and the effect of marital status on pain. there is a report describing the relation of marriage and chronic pain (29), which stated that marital status was not associated with immediate painrelated unpleasantness. in contrast to a previous study from the united states [30], which stated that adults with higher levels of education generally report less pain our results were did not show differences. kaufman et al. [31] reported that the inferior alveolar nerve block was more painful than local infiltration or another dental nerve block. however, differences in our study results were insignificant. another study [32, 33] also found no difference between injection techniques. previous dental experience likely prepares patients for pain associated with injections [33]. the majority of patients in this study had previous dental procedures and hence injections. pain perception during the past dental injection is associated with anxiety for the next injections. this result is consistent with other studies that reported the presence of an association between the amount of pain felt during injection and the progressive level of anxiety for injection within subsequent dental visits [34-36]. conclusion dental anxiety and phobia can be alleviated by simply talking to the patients before the procedure. which could reduce the amount of pain felt by the patient. the dentist should take the time needed to communicate with the patient before the dental procedure. even before explaining the procedure needed for treatment, to reduce anxiety before interventions requires the identification of their fears and any social conflict or previous bad experiences. all successful treatment will rest on dentist-patient acceptance, and thus a relaxed patient will result in a less stressful environment for the dental team and lastly treatment outcomes. references 1. pain, anxiety, and quality of life related to the oral health of patients treated in a dental emergency clinic. queiroz mf, verli fd, marinho sa, paiva pcp, santos smc, soares ja. cien saude colet. 2019;24(4):127786. 2. evolution, emotions, and emotional disorders. am psychol.nesse rm, ellsworth pc. 2009;64(2):129-39. 3. pain perception and dental anxiety during periodontal probing in patients visiting community oral health programme: a cross sectional study. singh a, shrestha a, bhagat t. bmc oral health. 2021;21(1):82. 4. dental fear in children: the role of previous negative dental experiences. mendoza-mendoza a, perea mb, yanez-vico rm, iglesias-linares a. clin oral investig. 2015;19(3):745-51. 5. what sensitivities matter in dental anxiety? investigating sensitivity to anxiety, pain, and disgust. witcraft sm, wickenhauser me, maack dj, dixon lj. psychol health med. 2021;26(3):313-21. 6. pain and anxiety management for pediatric dental procedures using various combinations of sedative drugs: a review. gazal g, fareed wm, zafar ms, alsamadani kh. saudi pharm j. 2016;24(4):379-85. 7. use of virtual reality for the management of anxiety and pain in dental treatments: systematic review and metaanalysis. lopez-valverde n, muriel fernandez j, lopezeffect of vocal anesthesia on adult dental pa5ents during covid-19 pandemic period vol 11 no 1 (2023) doi 10.5195/d3000.2023.449 h#p://den*stry3000.pi#.edu valverde a, valero juan lf, ramirez jm, flores fraile j, et al. j clin med. 2020;9(4). 8. effects of pretreatment exposure to dental practice using a smartphone dental simulation game on children's pain and anxiety: a preliminary double-blind randomized clinical trial. meshki r, basir l, alidadi f, behbudi a, rakhshan v. j dent (tehran). 2018;15(4):250-8. 9. comparative evaluation of virtual reality distraction and counter-stimulation on dental anxiety and pain perception in children. nunna m, dasaraju rk, kamatham r, mallineni sk, nuvvula s. j dent anesth pain med. 2019;19(5):277-88. 10. association between anxiety and pain in dental treatment: a systematic review and metaanalysis. lin cs, wu sy, yi ca. j dent res. 2017;96(2):153-62. 11. associations between dental anxiety and postoperative pain following extraction of horizontally impacted wisdom teeth: a prospective observational study wang tf, wu yt, tseng cf, chou c. medicine (baltimore). 2017;96(47):e8665. 12. does preoperative dental anxiety play a role in postoperative pain perception after third molar surgery?. onwuka ci, udeabor se, alhunaif am, al-shehri wak, alsahman la. ann afr med. 2020;19(4):269-73. 13. state anxiety and depression as factors modulating and influencing postoperative pain in dental implant surgery. a prospective clinical survey. gomez-de diego r, cutandosoriano a, montero-martin j, prados-frutos jc, lopezvalverde a. med oral patol oral cir bucal. 2014;19(6):e592-7. 14. assessment of the impact of stress and anxiety on pain perception in patients undergoing surgery for placement of their first dental implant. pani sc, algarni b, alzain lm, alqahtani ns. oral health dent manag. 2014;13(2):464-8. 15. association between dental anxiety and intraoperative pain during root canal treatment: a cross-sectional study. murillobenitez m, martin-gonzalez j, jimenez-sanchez mc, cabanillasbalsera d, velasco-ortega e, segura-egea jj. int endod j. 2020;53(4):447-54. 16. the prevalence of dental anxiety and its association with pain and other variables among adult patients with irreversible pulpitis. dou l, vanschaayk mm, zhang y, fu x, ji p, yang d. bmc oral health. 2018;18(1):101. 17. atraumatic restorative treatment in relation to pain, discomfort and dental treatment anxiety. frencken je, flohil ka, de baat c. . ned tijdschr tandheelkd. 2014;121(7-8):38893. 18. comparative study of minimally invasive technique and traditional decaying method for dental caries and the impact of dental treatment on pain and anxiety during pregnancy. tang h, huang q. shanghai kou qiang yi xue. 2019;28(3):297-300. 19. change talk and sustain talk in treatment of generalized anxiety disorder: a secondary analysis of cognitive behavioral therapy and metacognitive therapy in adult outpatients. isak joramo, stian solem, bendik romundstad, hans m. nordahl. journal of behavior therapy and experimental psychiatry. 2021:72, 101650. issn 00057916, 20. the perception of pain in others: how gender, race, and age influence pain expectations. wandner ld, scipio cd, hirsh at, torres ca, robinson me. j pain. 2012;13(3):220-7. 21. pain management during needle insertion with low level laser. ghaderi f, ghaderi r, davarmanesh m, bayani m, arabzade moghadam s. eur j paediatr dent. 2016;17(2):151-4. 22. pain and anxiety reduction using a manual stimulation distraction device when administering local analgesia oro-dental injections: a multi-center clinical investigation. touyz lz, lamontagne p, smith be. j clin dent. 2004;15(3):88-92. 23. sex differences in pain and analgesia: the role of gonadal hormones. craft rm, mogil js, aloisi am. eur j pain. 2004;8(5):397-411. 24. the fear of needles: a systematic review and metaanalysis. mclenon j, rogers mam. journal of advanced nursing. 2019;75(1):30-42. 25. author correction: comparison between the analgesic effectiveness and patients' preference for virtual reality vs. topical anesthesia gel during the administration of local anesthesia in adult dental patients: a randomized clinical study. almugait m, abumostafa a. scientific reports. 2022;12(1):805. 26. age effects on pain thresholds, temporal summation and spatial summation of heat and pressure pain. lautenbacher s, kunz m, strate p, nielsen j, arendtnielsen l. pain. j. 2005;115(3):410-8. effect of vocal anesthesia on adult dental pa5ents during covid-19 pandemic period vol 11 no 1 (2023) doi 10.5195/d3000.2023.449 h#p://den*stry3000.pi#.edu 27. pain assessment and management in persons with dementia. horgas al, elliott af. nurs clin north am. 2004;39(3):593-606. 28. an emotional component analysis of chronic pain. wade jb, price dd, hamer rm, schwartz sm, hart rp. pain. j. 1990;40(3):303-10. 29. the relationship between marital status and psychological resilience in chronic pain wade jb, hart rp, wade jh, bajaj js, price dd.. pain res treat. 2013;2013:928473. 30. the relationship between education and pain among adults aged 30-49 in the united states. zajacova a, rogers rg, grodsky e, grol-prokopczyk h. j pain. 2020;21(11-12):1270-80. 31. a survey of pain, pressure, and discomfort induced by commonly used oral local anesthesia injections. kaufman e, epstein jb, naveh e, gorsky m, gross a, cohen g. anesth prog. 2005;52(4):122-7. 32. patient's pain perception during mandibular molar extraction with articaine: a comparison study between infiltration and inferior alveolar nerve block. bataineh ab, alwarafi ma. clin oral investig. 2016;20(8):224150. 33. articaine infiltration for anesthesia of mandibular first molars. corbett ip, kanaa md, whitworth jm, meechan jg. journal of endodontics. 2008;34(5):5148. 34. a randomised, double-blind, placebo-controlled, comparative study of topical skin analgesics and the anxiety and discomfort associated with venous cannulation. speirs af, taylor kh, joanes dn, girdler nm. br dent j. 2001;190(8):444-9. 35. highly anxious dental patients report more pain during dental injections. van wijk aj, makkes pc. br dent j. 2008;205(3):e7; discussion 142-3. 36. strategies to manage patients with dental anxiety and dental phobia: literature review. appukuttan dp. clin cosmet investig dent. 2016;8:35-50. microsoft word 258 2022.docx the influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model vol 10 no 1 (2022) doi 10.5195/d3000.2022.258 h#p://den*stry3000.pi#.edu the influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model maria johanna jacoba heezen1, weibo zhang2, sara rudolph2, x. frank walboomers1, pamela c. yelick2 1radboud university medical center, radboud institute for molecular life sciences, department of dentistry – regenerative biomaterials, philips van leydenlaan 25, 6525ex nijmegen, the netherlands 2division of craniofacial and molecular genetics, department of orthodontics, tufts university school of dental medicine, boston, massachusetts 02111, united states of america abstract aim: tooth decay and associated periodontal disease remain the most common chronic diseases in current society. in the future, individually tailored, more effec@ve therapeu@c treatment op@ons could be provided through the applica@on of @ssue engineering and regenera@ve medicine and den@stry (termd) approaches. a relevant finding is the poten@al of both dental mesenchymal and dental epithelial cells to regenerate mineralized den@n and enamel @ssues, respec@vely. materials and methods: in the current study, a mul@-layered, bioengineered tooth bud model was assembled by combining human dental mesenchymal (hdm) and porcine dental epithelial cells (pde). the hdm cells were seeded onto poly (epsiloncaprolactone)/poly (lac@de-co-glycolide) (pcl/plga) wet electro-spun scaffolds overlayed with confluent pde cell sheet harvested from thermo-reversible @ssue culture plates. the so-formed mul@-layered bioengineered tooth bud was then used to study the mineraliza@on poten@al of the dental cells in in vitro culture. it was hypothesized that the addi@on of the in media soluble factors insulin-like growth factor 1 (igf-i) and the extracellular matrix derivate emdogain (emd) would result in enhanced differen@a@on and mineralized dental @ssue forma@on. results: scanning electron morphological observa@on was used to characterize scaffolds porosity. histological and immunofluorescent analyses confirmed the localiza@on of hdm cells inside the scaffold, an intact pde cell sheet, and the presence of beta-integrin 1posi@ve cell-cell junc@ons connec@ng the two. scanning electron microscopy showed that emd, in par@cular, enhanced the mineraliza@on poten@al of pde cells. qrt-pcr analyses showed that both emd and igf-1 significantly enhanced the expression of ameloblas@n (ambn), reflec@ng pde cell differen@a@on. conclusion: in conclusion, these results proved the hypothesis that both emd and igf-1 should be considered for their u@lity in preclinical dental @ssue engineering approaches. keywords: tissue engineering; dental epithelial/mesenchymal cells; scaffold; igf-1; emdogain cita9on: heezen, m et al. (2022) the influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model den9stry 3000. 1:a001 doi:10.5195/d3000.2022.258 received: october 21, 2021 accepted: april 23, 2022 published: july 8, 2022 copyright: ©2022 heezen, m et al. this is an open access ar9cle licensed under a crea9ve commons ayribu9on work 4.0 united states license. email: pamela.yelick@tubs.edu frank.walboomers@radboudumc.nl introduction tooth decay remains the most common chronic disease in modernday society. according to a recent systematic analysis for the global burden of disease (including data from 195 countries worldwide), over 3.5 billion people are affected by untreated oral diseases [1] that often lead to complete or partial tooth loss as well as systemic diseases. the influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model vol 10 no 1 (2022) doi 10.5195/d3000.2022.258 h#p://den*stry3000.pi#.edu prevention and tooth replacement therapies are the current solutions for these problems [2], for example using proper oral hygiene and partial or complete dentures, bridges, crowns and dental implants. tissue engineering and regenerative medicine and dentistry (termd) approaches, used as future therapeutic options to treat oral diseases, could provide the means for living dental tissue regeneration, thus providing individuals with restored comfort and function of their masticatory system adapted to their specific needs [3]. prior studies in dental tissue engineering have shown that interactions between dental mesenchymal (i.e. the dentin forming odontoblasts) and ectodermal (i.e. the enamel forming ameloblasts) cells are a prerequisite to initiating both dental cell differentiation and mineralization processes. especially, epithelial cells were proven not to grow and differentiate without the presence of supporting mesenchymal cells. previously, such interactions were facilitated by seeding dental mesenchymal cells into poly(epsiloncaprolactone)/ poly (lactide-coglycolide) (pcl/plga) wet electro spun scaffolds, and simultaneously or subsequently seeding dental epithelial cells on the surface in absence of the addition of growth factors [4]. these scaffolds provided a three-dimensional environment to support dental cell attachment, migration, proliferation and/or differentiation [5, 6]. moreover, the use of an appropriate scaffold could ensure the morphologically correct shape of the bioengineered tooth, which remains unattainable using scaffold-free methods. however, the current main challenge in any in vitro approach for tooth engineering is how to regenerate an adequate epithelial cell layer and sufficient quantities of enamel-like tissue. while odontoblasts remain viable during the life of a tooth, ameloblasts undergo apoptosis prior to tooth eruption [7]. furthermore, enamel is not only very highly mineralized (>96% of hydroxyapatite), but also consists of a highly complex, hexagonal honeycomb like, crystalline lattice organization, which is extremely strong and able to withstand the significant and variable oral forces of chewing [8]. therefore, in vitro formed enamel required proper dental epithelial cell proliferation and differentiation, and the ability to form an elaborate mineralized enamel matrix. many studies have focused on how best to increase the mineralization potential of (stem)cells. for instance, a recent study by fujioka-kobayaski et al. demonstrated a significant increase in the total mineralized tissue volume of calvarial defects after the addition of emdogain, an enamel matrix derivative (emd) originating from unerupted porcine tooth buds [9] [10]. another investigation characterized the functional mineralization potential of three bio inductive materials for vital pulp therapies – emdogain, mta (mineral troxide aggregate) and biodentine. in this study, emdogain showed highest cell viability, osteo and odonto-induction capacity [11]. although these and many more studies showed an enhanced mineralization potential of emd in relation to bone and or pulp regeneration therapy, emd has not yet been used in termd approaches that focus on the mineralization potential of dental epithelial cells. another potential approach to enhance enamel formation could be the addition of insulin-like growth factor i (igf-i). igf-i is present during the differentiation stages of ameloblasts, most abundantly during the secretory stage, and igf-1 expression is not detectable in maturation stage ameloblasts [12]. a three-year japanese follow up study of epidemiological data by kouda et al. indicated that higher serum levels of igf-1 resulted in higher bone mineral acquisition in children [13]. extrapolating this data, it could be assumed that igf-1 might influence the mineralization potential in tooth engineering in a similar way. therefore, the purpose of the current study was to investigate the effect of emd and igf-i on the mineralization potential of dental epithelial cells in a three-dimensional pcl/plga wet electro spun scaffold model. we hypothesized that bioengineered three-dimensional mineralized enamel formation could be achieved the influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model vol 10 no 1 (2022) doi 10.5195/d3000.2022.258 h#p://den*stry3000.pi#.edu by seeding porcine dental epithelial (pde) cells onto these scaffolds, and that the addition of igf-i and emdogain would result in superior osteogenic – odontogenic and ameloblast differentiation. material and methods electrospun scaffolds the scaffolds were chosen based on earlier experiment with the same cells [4]. the electrospinning solution consisted of pcl (75%, 0.12g/ml) and plga 85/31(25%, 0.12 g/ml), both dissolved in liquid 222-trifluorethanol (tfe, sigma aldrich, zwijndrecht, the netherlands). the polymers were fully dissolved by magnetic stirring overnight. the syringe, filled with solution, was vertically installed (together with the blunt-end 18g nozzle) in the electrospinning machine (es-2000s, esprayer, tokyo, japan). the fibers forming the threedimensional scaffolds were collected in a 96% ethanol bath (surface covered in aluminum foil). the distance between the nozzle and the ethanol was adjusted to 20 cm. the voltage was set at 25.0 kv and a 25 µl/min feeding rate was used. the scaffolds were collected from the ethanol bath after 5 minutes and stored at -80°c for 60 minutes before freeze-drying (virtis benchtop pro, sp scientific, pennsylvania, usa) overnight. thereafter, scaffolds were cut with a 6mm disposable biopsy punch (kai medical, solingen, germany) and stored at -80°c until use. before use, scaffolds were sterilized and made hydrophilic by radio frequent glow discharge treatment (rfgd-t; harrick plasma cleaner/ sterilizer, harrick plasma, ithaca, usa)[14]. cell culture cryopreserved pde [4] cells were recovered (passage 1) and cultured with epithelial cell media [lhc-8 #12678-017 (gibco, bleiswijk, the netherlands) containing 10% fbs, 1% psa (penicillin/streptomycin/amphoterici n), and 0.5 μg/ml epinephrine]. the medium was refreshed every 2-3 days. once ≥ 95% confluency was reached, pde cells were seeded into 12-well upcell thermo-responsive plates (thermo scientific, netherlands) at a cell seeding density of 1.6 x 106 cells per 12-well upcell (=3.5cm2)[15]. non-seeded cells were collected to serve as a day 0 value for rna isolation ( ̴20 million total). the pde cells were grown in upcell thermo-responsive wells for ̴7 days until confluent. human dental pulp stem cells (hdpscs, p3) [4] were retrieved from cryopreservation approximately one week after the pde cells were thawed to ensure that both types of cells would reach confluency at the same time. human cells were used from discarded m3 teeth, following national guidelines for working with human materials, and did not require further ethical committee approval. upon reaching confluency, hdpscs were statically seeded onto scaffolds at 2.0x105 cells per scaffold, in hdm cell media [advanced dmem/f12 #12634 (gibco) containing 10% fbs, 25 μg/ml ascorbic acid, 1% glutamax, 1% psa], and refreshed every 2-3 days. both pde and hdm cells were cultured at 37°c in a humidified 5% co2 atmosphere. construct preparation after 7 days in vitro culture, hdmseeded scaffolds and pde cell sheets (pde-cs) were combined as follows. first, media was removed from the hdpsc seeded scaffolds to facilitate the attachment between the scaffolds and pde-cs. the hdm seeded scaffolds were then transferred to upcell 12-well plates containing pde cell sheets. four scaffolds were transferred to each well. then 160 µl (40 µl/ scaffold) of specific, group assigned, medium (see below) was added per well to prevent drying. after a 30 minutes’ incubation period to ensure attachment between the hdm seeded scaffold and pde cell sheet, the medium was supplemented to 250 µl, and finally after 7 days in vitro culture to 500 µl. 250 µl of the medium was refreshed daily. three groups were examined and all groups were a mixture of dm medium and de medium (1:1): 1dm/de normal medium (nm; 1:1, control group) 2dm/de medium (1:1) with 100 µg/ml igf-1 3dm/de medium (1:1) with 200 ng/ml emd the influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model vol 10 no 1 (2022) doi 10.5195/d3000.2022.258 h#p://den*stry3000.pi#.edu samples were harvested and fixed at days 3, 14 and 21. for cs detachment, media was aspirated and 50 µl/well of fresh media was added to prevent dehydration during the detachment procedure (according to manufacturer’s instructions) [16]. the samples were placed at room temperature (24°c) for ±30 minutes until detachment from the upcell plates was visually observed. the cell sheet edges were loosened with a needle and split in 4 with a scalpel under the dissecting microscope (zeiss stereo discovery. v8, pleasanton, usa). each scaffold was then wrapped with an epithelial cs and transferred on top of filter paper in a new 12 well plate, positioning them so that the dental epithelial cs was positioned on top of the hdpsc seeded scaffold. after the appointed time points in culture, samples were harvested, fixed in formalin on at 40c and stored in pbs until further processing. histological staining (h&e) fixed constructs were embedded in oct, serially sectioned at six microns (leica rm 2135, leica, bannockbur/illinois, usa), mounted using a premiere tissue floating bath xh-1001 and slide warmer (premiere xh-2001, c&a scientific, virginia, usa) set to 45°c for the first 60 minutes, and then increased to 54°c overnight. samples were rehydrated in xylene and ethanol series, stained with hematoxylin for 1 minute, rinsed in water and diluted hydrochloric acid water, ammonia water, and stained with eosin for 20 seconds. samples were dehydrated in ethanol series and xylene, and mounted using permount medium (fisher scientific, illinois, usa). micrographs were made with a zeiss axio imager/ z1 equipped with digital zeiss axiocam hrc camera (zeiss, pleasanton, usa). immunofluorescent (if) analyses if staining was performed to visualize hdm and pde cell-cell interactions. the primary antibodies included: antivimentin (vm, mouse, 1:50), antiecad (rabbit, 1:50) and anti-βintegrin 1 (mouse, 1:25). secondary antibodies included goat anti-mouse (excitation: 568m, cy5), goat antimouse (excitation: 488nm, green), goat anti-rabbit (excitation: 652nm, cy5, with 4ʹ,6-diamidino-2phenylindole (dapi, blue) counterstain. porcine tooth bud sections were used as positive controls for primary antibodies ecad and vm, and human dental pulp was used as positive control for integrin. samples lacking the primary antibody were included as negative controls. if staining color’s intensity fluctuates for the different groups yet is positively present in all. scanning electron microscope (sem) analysis sem was executed to visualize the status of mineralization and to monitor cell growth in harvested 3, 14 and 21 day samples. samples were washed in pbs, fixed with 2.5% glutaraldehyde (ph 7.31), post fixed with 1% osmium tetroxide for 1 hour on ice, dehydrated in a series of ethanol, and air dried in tetramethylsilane. samples were sputter coated twice with 10nm chromium (q150t s, quorum technologies, lewes, uk). images were taken in a zeiss gemini sigma 300 scanning electron microscope (zeiss, oberkochen, germany). rna isolation and quantitative realtime polymerase chain reaction (qrt-pcr) analyses qrt-pcr was performed to monitor cell differentiation marker expression in d0 and d21 construct samples. four scaffolds per group were pooled to ensure that sufficient amounts of rna were obtained. control 2d cultured hdm and pde cells were trypsinized, lysed in βmercaptoethanol cell suspension (trizol), and preserved at -80°c until processing. the scaffolds were dissociated into small pieces using rnase treated scissors, and the rna extraction was performed using the rneasy mini kit (qiagen sciences, baltimore, usa). the rna yield was confirmed with a spectrophotometer (nanodrop 1000, thermo scientific). reverse transcription was carried out with the superscript first-strand synthesis system (invitrogen, thermo scientific), and cdna yield was the influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model vol 10 no 1 (2022) doi 10.5195/d3000.2022.258 h#p://den*stry3000.pi#.edu determined using a spectrophotometer. the qrt-pcr was executed using the protocol for real time pcr with the stratagene mx3000p (stratagene, san diego, california). the primers, as displayed in table 1, (1 μl each) used were hdspp, gapdh (control primer), hbglap and pambn (qiagen sciences, baltimore, usa). all three media conditions were tested with all primers in triplicate. statistical analysis all data were presented as mean ± standard error. statistically significant values were defined as p≤0.05 based on one-way analysis of variance (anova) and post hoc tukey testing. results histological staining dental cell-distribution throughout the scaffolds was investigated using histological analysis. at day 3 (figure 1a-c), all scaffolds showed the presence of hdm cells dispersedly evenly throughout the scaffolds, and covered by a pdecs. the 3 day pde-cs constructs showed evidence of fragmentation likely due to handling. on days 14 (figure 1 d-f) and 21 (figure 1 g-i) the interstitium between the scaffold fibers appeared to be filled with hdm cells, and the cell sheets were in close contact with the scaffolds and had remained largely intact. the cell sheet thickened over time, by visual inspection most evidently the igf-i group. the epithelial polygonal shaped cells multiplied in the days following the experiment while remaining on top of the scaffolds, not migrating inwards. the mesenchymal cells multiplied as well. day 3 full image 10x detailed polarized image 40x nm igf emd primer (1 μl): catalog number: hdspp pph57747e-200 gapdh (control primer) pph00150f-200 hbglap pph01898a-200 pambn pps00441a-200 table 1: overview of the four used primers for the qrt-pcr analyses and their origin/catalog numbers. a b c the influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model vol 10 no 1 (2022) doi 10.5195/d3000.2022.258 h#p://den*stry3000.pi#.edu day 14 nm igf emd day 21 nm igf emd d e f g h i figure 1. cell seeding procedure assessed with histological staining. h&e stained sectioned scaffolds cultured with hdm cells and pde cells for 3 (a-c), 14 (d-f) and 21 (g-i) days in normal medium (a, d & g), medium supplemented with igf-i (b, e & h), and emd (c, f & i) enriched medium. for each sample, low magnification (10x, scale bar = 500μm) image provides the overview to monitor cell-growth and visualize the cell-distribution within the scaffold. the high magnification polarized images (40x; inserts) demonstrate the presence of both dental epithelial as mesenchymal cells, as well as mineralized tissue ecm polarized structures particularly evident in nm d14 (d) and nm d21 (g)(arrows) the influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model vol 10 no 1 (2022) doi 10.5195/d3000.2022.258 h#p://den*stry3000.pi#.edu immuno-fluorescent staining (if) to visualize the distribution of cells (figure 2), epithelial cells were stained for ecad, and hdpscs for vimentin. hdpscs were seen all through the sample. the if images corroborated the h&e staining, with pde cells maintaining the cultured cell sheet shape, whereas the hdm cells were evenly distributed throughout the sample. to visualize hdm-pde cell contacts, if imaging for b-integrin 1 was performed (figure 3). if results showed the formation of hdm-pde cell contacts at all time points, in all three types of media. vm&eca d (1:50, 40x) nm igf-1 emd day 3 day 14 day 21 b c a d g e h f i figure 2. locakon and interackons of dental cells as assessed by immunofluorescent staining. immunofluorescent staining at days 3 (a-c), 14 (d-f) and 21 (g-i) in nm (a, d & g), igf-1 (b, e, h) and emd (c, f & i). dapi (blue) was used to counterstain all nuclei. vimenwn (green) labels hdm cells, while ecad (red) labels pde-css. very close contact between the different layers was observed in all constructs (scale bar = 50μm). diswnguished polarized structures are clearly visible in the d21 samples (arrows) the influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model vol 10 no 1 (2022) doi 10.5195/d3000.2022.258 h#p://den*stry3000.pi#.edu scanning electron microscopy (sem) the scaffold, hdm-de cell morphology, mineralized dental matrix formation were assessed using sem. in the nm group (figure 4a, d & g) the pde-cs was clearly observed, with no obvious differences in pde cell morphology over time. a similar result was observed in the igf group (figure 4b, e & h), where a pde-cs covering the hdm cell seeded scaffold was clearly observed, with no obvious difference over time in culture. in contrast, the emd group (figure 4c, f & i) showed a markedly different appearance, in that the pde-cs transparency decreased over time, and the hdm seeded scaffold surface structure exhibited a complex, honeycomb like, crystalline lattice organization at d21 (figure 4i). integrin (1:25, 40x) nm igf-1 emd day 3 day 14 day 21 a d g b e h c f i figure 3. locakon and interackons of dental cells as assessed by immunofluorescent staining. immunofluorescent staining for anw-integrin 1 (red) at days 3 (a-c), 14 (d-f) and 21 (g-i) in constructs cultured in nm (a, d & g), igf-1 (b, e, h) and emd media (c, f & i). note the abundant cell-cell contact between pde and hdm cells throughout several of the constructs (arrows)(scale bar = 50μm) the influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model vol 10 no 1 (2022) doi 10.5195/d3000.2022.258 h#p://den*stry3000.pi#.edu nm 1000x igf 1000x emd 1000x day 3 day 14 day 21 a d g b e h c f i figure 4. sem imaging of dental epithelial cell sheets. sem imaging of in vitro cultured bioengineered tooth bud constructs at days 3 (a-c), 14 (d-f) and 21 (g-i), in the nm (a, d & g), igf (b, e & h) and emd (c, f & i) culture condijons (1000x magnificajon). all images show a clearly visible de cell sheet (arrows). the arrows in the sem images are poinjng at a small piece of the visible de cell sheet, to evidence that the sheet obviously spreads across most of the image (apart from small cracks, most likely due to fragmentajon/ human handling). the surface structure of the emd treated group portrays a complex honeycomb (hexagonal crystallized (clear deposijon of minerals) structures, depicjng the external likes of a beehive) like crystalline lauce organizajon at d21(i) as compared to d3(c) the influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model vol 10 no 1 (2022) doi 10.5195/d3000.2022.258 h#p://den*stry3000.pi#.edu quantitative real-time polymerase chain reaction (qrt-pcr) qrt-pcr analyses of dental cell differentiation marker expression were performed (figure 5). the expression of the ameloblast differentiation marker ambn was significantly higher in the igf-1 and emdogain groups as compared to the nm group (p<0.05). no significant differences in the expression of dspp and oc were observed. discussion dental tissue damage remains a considerable health concern in our current society. the ability to regenerate organic, vital tooth material would be highly desirable to enable future therapeutic options. moreover, cultured mineralized tooth-like tissue could serve as an ex vivo screening tool for novel pharmaceutical or regenerative medicinal approaches. this study showed the construction of a multidimensional scaffold-dental epithelial cell sheet-based model, and the utility of this model to demonstrate the positive effects on mineralization potential of igf-1 and emdogain. in hindsight, the 21-day period of time is not long enough for considerate in vitro mineralized tissue formation. future research could incorporate the use of alizarin red s staining or eds sem analysis to determine ca2+ deposition on the scaffolds. the pcl/plga scaffolds were assumed not to exhibit parameters such as an increased cytotoxicity and/or genotoxicity potential due to scaffold degradation. the material is believed to be biocompatible based on multiple experiments [17, 18] that used pcl/plga in tissue engineering approaches on this matter. of note, several technical issues were encountered. one concerned the static seeding method used to seed hdpscs onto the electrospun scaffold. based on literature, it might be argued that a dynamic cell seeding method would be a better method [19], as rotational seeding leads to a more even distribution of cells, especially in the deep interstitium of porous carrier materials. on the other hand, rotational seeding approaches might also damage or present undesirable mechanical cues to the cells. our results indicated that static seeding of hdm cells on the surface, directly adjacent to where the cell sheet would be placed, apparently was sufficient for the current scaffold dimensions used in this study. furthermore, the results showed an adequate enough amount of cell 0 0.0002 0.0004 0.0006 0.0008 0.001 0.0012 nm igf emd o c ex pr es sio n oc 0 0.05 0.1 0.15 0.2 0.25 nm igf* emd* am bn e xp re ss io n ambn 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 nm igf emd ds pp e xp re ss io n dsppa c figure 5. qpcr analyses of dental cell differenkakon marker expression. expression of (a) dspp (b) ambn(c) bglap/oc as normalized to d0 dental cell cultures. the expression of the ameloblast differenwawon marker ambn was significantly higher in the igf-1 and emdogain treated groups as compared to the nm group (p<0.05). no significant differences in the expression of dspp and oc were observed b the influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model vol 10 no 1 (2022) doi 10.5195/d3000.2022.258 h#p://den*stry3000.pi#.edu distribution for the researched to be executed. another technical aspect was the choice of how best to combine the hdm seeded scaffold with the epithelial cells, after these epithelial cells had been grown separately as confluent sheets. this approach was based on the assumption that de cells have the tendency to form coherent monolayers, as shown before in a study performed by seo et al. [20] and by our group [15]. our results indeed confirmed the assumption that pde cells formed polarized monolayers, and that the cell sheet technology was accommodating for the purpose of making layered constructs with distinct cellular phenotypes. still, a certain amount of fragmentation of the epithelial cell sheets occurred, shown in the histological images of day 3 constructs, raising a concern towards the applied model, although such fragmentation likely occurred as a result of manual handling during the harvesting. the day 3 samples proved quite challenging to collect due to their extremely fragile nature after culture for such a short period of time. although no damage was visible by eye during the construct harvest, it was clearly observed in harvested and sectioned specimens examined via light microscopy. at later timepoints, the constructs appeared more robust, as more rigid sheets could be formed, and no fragmentation occurred anymore. future research could explore the different methods for adapting the scaffold structure to make it less susceptible to fragmentation in early stages. a study performed by hong et al. describes different biomaterials that could be considered [21] to possibly enhance the handling efficiency and minimize fragmentation. although our results clearly showed that igf-1 and emd exhibited positive and significant effects on dental cell differentiation, in most of the constructs analyzed as evidenced by q-pcr, the expression levels of dspp and oc were not affected. this may be due to the relatively low cell seeding density used, and/or in combination with the limited in vitro culture time frame. at later timepoints, increased amounts of mineralized dental matrix were observed via light and sem microscopical analyses. as such, a longer experimental time frame would be advisable for future research when regarding specifically the expression of mineralized tissue markers. when comparing these results to the published literature, certain discrepancies are noteworthy. for instance, grandin et al. recently reviewed the in vitro cellular effects of emdogain, showing that in periodontal ligament and osteoblastic cells, emdogain enhanced cell proliferation, gene expression, protein production, and differentiation [22]. also, angiogenesis was shown to be stimulated by emd in this study [22]. however, for epithelial cells specifically, a cytostatic tendency was described. this is in contrast with our results which showed normal unhindered growth and enhanced effect on the mineralization potential of dental epithelial cells. our results are supported by published literature including that of qu et al., who investigated the effects of emdogain on the proliferation and migration of human oral epithelial cells [23]. this study concluded that the addition of emd resulted in significant enhancement of the proliferation and viability of the epithelial cells [18]. however, this study used a 2d model and did not assess the mineralization of treated epithelial cells. likewise, a recent study by li et al. showed that igf-1 facilitated odontogenesis of cultured periodontal ligament stem cells [24], which again corroborated the data for our differentiated epithelial and dental mesenchymal cell co-cultured constructs. in summary, our hypotheses were validated in this study, which demonstrated that both emdogain and igf-i are important for the enhancement of dental cell differentiation and proliferation in our system. conclusion based on the results of this study, we conclude that a multi-layered threedimensional tooth bud model was successfully created by combining hdpsc-seeded scaffolds with dental epithelial cell sheets. this multithe influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model vol 10 no 1 (2022) doi 10.5195/d3000.2022.258 h#p://den*stry3000.pi#.edu layered bioengineered 3d tooth bud model exhibited abundant hdm-pde cell-cell contact and was sufficient to support pde and hdpsc cell proliferation and differentiation. in particular, the addition of igf-1 and emd increased the mineralization potential of the pde cells. future research is therefore justified and warranted to further verify these conclusions in a preclinical in vivo model. acknowledgements radboud honours programme medical sciences (radboud honours academy, nijmegen), associatie nederlandse tandartsen (ant), radboud umcn student grant, nih/nibib r01 de026731 (pcy). disclosure of potential conflict of interest the authors designated no potential conflict of interest. references 1. global, regional, and national prevalence, incidence, and disability-adjusted life years for oral conditions for 195 countries, 1990-2015: a systematic analysis for the global burden of diseases, injuries, and risk factors. kassebaum nj, smith agc, bernabe e, fleming td, reynolds ae, vos t, et al. j dent res. 2017;96(4):380-7. 2. caries management science and clinical practice. in: effenberger ds, editor. hendrik meyer-leuckel sp, kim r. ekstrand. stuttgart new york: thieme; 2013. p. 8,9. 3. the replacement of missing teeth. canadian family physician medecin de famille canadien. zarb ga. 1988;34:1435-40. 4. influence of highly porous electrospun plga/pcl/nha fibrous scaffolds on the differentiation of tooth bud cells in vitro. journal of biomedical materials research part a. cai x, ten hoopen s, zhang w, yi c, yang w, yang f, et al. 2017;105(9):2597-607. 5. electrospinning approaches toward scaffold engineering--a brief overview. artificial organs. boudriot u, dersch r, greiner a, wendorff jh. 2006;30(10):78592. 6. the influence of electrospun fibre scaffold orientation and nano-hydroxyapatite content on the development of tooth bud stem cells in vitro. odontology. van manen eh, zhang w, walboomers xf, vazquez b, yang f, ji w, et al. 2014;102(1):14-21. 7. dental tissue regeneration a mini-review. gerontology. yen ah, yelick pc. 2011;57(1):85-94. 8. advances and perspectives in tooth tissue engineering. journal of tissue engineering and regenerative medicine. monteiro n, yelick pc. 2017;11(9):2443-61. 9. straumann.com [available from: https://www.straumann.com/en /dental-professionals/productsandsolutions/biomaterials/biologics. html. 10. effect of enamel matrix derivative liquid in combination with a natural bone mineral on new bone formation in a rabbit gbr model. clin oral implants res. kobayashi e, fujiokakobayashi m, saulacic n, schaller b, sculean a, miron rj. 2019;30(6):542-9. 11. response of stem cells from human exfoliated deciduous teeth (shed) to three bioinductive materials an in vitro experimental study. dahake pt, panpaliya np, kale yj, dadpe mv, kendre sb, bogar c. saudi dent j. 2020;32(1):4351. 12. expression and regulation of insulin-like growth factor-i in the rat incisor. growth factors. joseph bk, savage nw, young wg, gupta gs, breier bh, waters mj. 1993;8(4):267-75. 13. associations between serum levels of insulin-like growth factor-i and bone mineral acquisition in pubertal children: a 3-year follow-up study in hamamatsu, japan. j physiol anthropol. kouda k, iki m, ohara k, nakamura h, fujita y, nishiyama t. 2019;38(1):16. 14. [effect of radio frequency discharge plasma on surface properties and biocompatibility of polycaprolactone matrices]. biomed khim. bolbasov en, antonova lv, matveeva vg, novikov va, shesterikov ev, bogomolova nl, et al. 2016;62(1):56-63. 15. dental cell sheet biomimetic tooth bud model. biomaterials. monteiro n, smith ee, angstadt s, zhang w, khademhosseini a, yelick pc. 2016;106:167-79. 16. scientific tf. [available from: https://assets.thermofisher.com /tfsassets/lsg/manuals/d17402.pd e. 17. design, synthesis, characterization, and cytotoxicity of pcl/plga scaffolds through plasma treatment in the presence of pyrrole for possible use in urethral tissue engineering. j biomater appl. sánchez-pech jc, the influence of igf-i and emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model vol 10 no 1 (2022) doi 10.5195/d3000.2022.258 h#p://den*stry3000.pi#.edu rosales-ibáñes r, cauichrodriguez jv, carrillo-escalante hj, rodríguez-navarrete a, avilaortega a, et al. 2020;34(6):84050. 18. biocompatibility of pcl/plgabcp porous scaffold for bone tissue engineering applications. j biomater sci polym ed. thi hiep n, chan khon h, dai hai n, byong-taek l, van toi v, thanh hung l. 2017;28(9):864-78. 19. static versus vacuum cell seeding on high and low porosity ceramic scaffolds. journal of biomaterials applications. buizer at, veldhuizen ag, bulstra sk, kuijer r. 2014;29(1):3-13. 20. epithelial monolayer culture system for real-time single-cell analyses. physiological reports. seo jb, moody m, koh ds. 2014;2(4):e12002. 21. cell-electrospinning and its application for tissue engineering. int j mol sci. hong j, yeo m, yang gh, kim g. 2019;20(24). 22. enamel matrix derivative: a review of cellular effects in vitro and a model of molecular arrangement and functioning. tissue eng part b rev. grandin hm, gemperli ac, dard m. 2012;18(3):181-202. 23. effect of emdogain on proliferation and migration of different periodontal tissueassociated cells. oral surg oral med oral pathol oral radiol endod. qu z, laky m, ulm c, matejka m, dard m, andrukhov o, et al. 2010;109(6):924-31. 24. odontogenesis and neuronal differentiation characteristics of periodontal ligament stem cells from beagle dog. j cell mol med. li x, liao d, sun g, chu h. 2020;24(9):5146-51. compara've evalua'on of indocyanine green and 810nm diode laser assisted an'microbial photodynamic therapy with 810nm diode laser assisted new acachment procedure as adjuncts to scaling and root planning in suppor've periodontal therapy: a randomized controlled split-mouth clinical trial vol 12 no 1 (2024) doi 10.5195/d3000.2024.392 h#p://den*stry3000.pi#.edu comparative evaluation of indocyanine green and 810nm diode laser assisted antimicrobial photodynamic therapy with 810nm diode laser assisted new attachment procedure as adjuncts to scaling and root planning in supportive periodontal therapy: a randomized controlled split-mouth clinical trial sruthima nvs go.umukkala1, sathya valli veluri2, gautami subhadra penmetsa3, ramesh ksv4, mohan kumar pasupule=5, dinesh gera2, sravanthi vundavalli2 1professor, department of periodontics and implantology, vishnu dental college, bhimavaram 2post-graduate student, department of periodontics and implantology, vishnu dental college, bhimavaram 3professor and head, department of periodontics and implantology, vishnu dental college, bhimavaram 4associate professor, department of periodontics and implantology, vishnu dental college, bhimavaram 5reader, department of periodontics and implantology, vishnu dental college, bhimavaram abstract background: minimally invasive nonsurgical management of recurrent periodontal pockets remains the most viable op'on during suppor've periodontal therapy (spt), but with inconclusive results. the purpose of this study is to evaluate and compare the adjunc've use of indocyanine green (icg) and 810nm diode laser assisted photodynamic therapy (ig-pdt) with 810nm diode laser assisted new acachment procedure (lanap) in the management of persistent pockets in spt. materials and methods: this single blinded randomized controlled split mouth clinical trial included 20 par'cipants with at least two adjacent teeth with ppd ≥5mm and clinical acachment level (cal) ≥3mm in the contralateral quadrants. ig-pdt group received applica'on of 5mg/ml solu'on of icg for one minute followed by 810±10nm diode laser with 1w power density. lanap group received 810±10nm diode laser applica'on at 3w power density with 100ms and 650ms pulse dura'on at two consecu've passes. clinical parameters i.e., plaque index (pi), gingival index (gi), ppd, cal gain were recorded at baseline, 3 and 6 months. landry’s healing index was assessed on the 7th day and 1 month postopera'vely. pa'ent comfort was assessed using visual analog scale (vas) immediate post-opera'vely. results: student t test and repeated measures of anova were used for sta's'cal analysis. both site level and pa'ent level assessments of clinical parameters showed improvement in clinical parameters in both groups. however, ig-pdt group showed significantly greater improvements in ppd (<0.001) and cal (<0.001) at 3 and 6 months evalua'on. ig-pdt showed becer healing (p-0.03) at 7th post-opera've day. however, lanap showed less discomfort (2.40±0.50) compared to ig-pdt group (3.20±0.61). conclusion: ig-pdt is shown to provide becer clinical outcomes compared to lanap in management of pa'ents under spt. keywords: indocyanine green; laser; photodynamic therapy; periodon''s; periodontal acachment loss cita9on: go=umukkala, snvs et al. (2024) compara9ve evalua9on of indocyanine green and 810nm diode laser assisted an9microbial photodynamic therapy with 810nm diode laser assisted new a=achment procedure as adjuncts to scaling and root planning in suppor9ve periodontal therapy: a randomized controlled splitmouth clinical trial den9stry 3000. 1:a001 doi:10.5195/d3000.2024.392 received: october 6, 2022 accepted: april 4, 2023 published: february 13, 2024 copyright: ©2024 go=umukkala, snvs et al. this is an open access ar9cle licensed under a crea9ve commons a=ribu9on work 4.0 united states license. email: sruthima@gmail.com http://dentistry3000.pitt.edu/ compara've evalua'on of indocyanine green and 810nm diode laser assisted an'microbial photodynamic therapy with 810nm diode laser assisted new acachment procedure as adjuncts to scaling and root planning in suppor've periodontal therapy: a randomized controlled splitmouth clinical trial vol 12 no 1 (2024) doi 10.5195/d3000.2024.392 h#p://den*stry3000.pi#.edu introduction periodontitis is the most common and prevalent oral chronic inflammatory disease caused by a dysbiosis of the host response and oral microorganisms, resulting in periodontal attachment and bone loss [1]. comprehensive periodontal therapy, which combines non-surgical and surgical periodontal therapy, has been shown to be effective in achieving adequate attachment gain [2]. periodontal maintenance, also known as supportive periodontal therapy (spt), is a phase of periodontal therapy in which the periodontal condition is monitored and etiological factors are reduced or eliminated following periodontal treatment [3,4,5]. following active periodontal therapy, maintenance consists of three components: patient personal care, preventive procedures performed by dental health care professionals, and supportive periodontal therapy interventions addressing disease recurrence due to re-emergence of subgingival biofilm and pocket formation [6]. scaling and root planning (srp) with disruption of bacterial biofilm, as well as adjunctive use of various nonsurgical treatment procedures, such as systemic and local delivery of antimicrobial agents, have been tried for pocket reduction during spt [711]. however, there are inconsistent results and insufficient evidence to determine the superiority of different protocols or adjunctive strategies to improve tooth maintenance during spt when performed alone or in combination with srp [11]. lasers have been proposed in the treatment of periodontal disease as a more efficient and atraumatic technique that improves periodontal healing. various dental laser wavelengths have been used by clinicians in the treatment of periodontitis over the last decade, most notably diode lasers (809-980 nm), nd:yag (1064 nm), er:yag and er,cr:ysgg (2940 nm and 2780 nm, respectively), and co2 (10,600 nm) [12]. lasers are thought to improve decontamination during treatment, provide antiseptic action on nonvascularized tissues, overcome antibiotic resistance in subgingival biofilms, and stimulate fibroblast and osteoblast activity [13-15]. gregg and mccarthy pioneered the use of the lanap protocol, claiming it could regenerate bone growth and stimulate new attachment [16]. the laser-assisted new attachment procedure (lanap) aims to reduce pocket depth by promoting the growth of new bone, cementum, and periodontal ligament on previously damaged root surfaces [17-19]. the nd:yag laser with a shorter wavelength of 1,064nm was initially considered for use in lanap protocols due to its affinity for melanin or dark pigmentation, as opposed to longer wavelengths that are highly absorbed in water and would have a shallow depth of penetration [20]. diode lasers are also known for this specific ingestion in pigmented tissues and deeper penetration into biological tissues, as well as producing a relatively thick coagulation layer on the lased softtissue surface [20]. the shortcomings of conventional treatments for the management of spt patients have led to the proposal of adjunctive antimicrobial photodynamic therapy (pdt) as an alternative treatment [21,22]. when a non-toxic photosensitizing chemical is stimulated by light of a specific wavelength, reactive oxygen species (ros) are produced. ros can cause dna and cell membrane damage, which can lead to microbial killing [23]. pdt works by activating photosensitizers in the presence of oxygen when they are exposed to light. one consideration in periodontal disease is the lack of oxygen in the subgingival area, which may render the antimicrobial pdt ineffective when combined with traditional photosensitizers. indocyanine green (icg), a commonly used photosensitizer, has been widely used in clinical settings in recent years for diagnostic procedures such as angiography in ophthalmology, monitoring liver function and splanchnic perfusion, perfusionrelated examination of tissues and organs, sentinel lymph node biopsy, and rheumatic disorder diagnosis [2325]. icg is used in pdt in addition to http://dentistry3000.pitt.edu/ compara've evalua'on of indocyanine green and 810nm diode laser assisted an'microbial photodynamic therapy with 810nm diode laser assisted new acachment procedure as adjuncts to scaling and root planning in suppor've periodontal therapy: a randomized controlled splitmouth clinical trial vol 12 no 1 (2024) doi 10.5195/d3000.2024.392 h#p://den*stry3000.pi#.edu diagnostic applications because of its ability to absorb light at a wavelength of 810 nm, which is closer to the absorption wavelength of diode lasers [26]. because anaerobic subgingival pdt does not require oxygen, free radicals and singlet oxygen are known to be produced during the activation process of icgmediated pdt(ig-pdt). given the benefits listed above, it was hypothesised that ig-pdt, like the well-established lanap protocol, could be a promising tool for periodontal disease management. as a result, the current split mouth randomised controlled clinical trial sought to evaluate and compare the efficacy of icg-mediated pdt and lanap as adjuvants to srp in the management of recurrent periodontal pockets during spt. material and methods study design the present study was a single blinded randomized controlled splitmouth clinical trial to evaluate the efficacy of ig-pdt over lanap as an adjunct to scaling and root planning in the treatment of periodontitis. a single calibrated (with intra-examiner correlation coefficient k-0.82) and blinded examiner performed the clinical assessments throughout the study period. the study was conducted in the department of periodontology at vishnu dental college, bhimavaram from march 2021 to june 2022. ethical aspects institutional ethical committee provided the ethical clearance (iec vdc/2021/pg01/pi/ivv/49) and the study was registered under clinical trials registry of india (ctri/2021/08/035658). in accordance with the 2003 revision of the declaration of helsinki, the patients signed a written statement of informed consent. sample size using g*power software (version3.1.9.4), the required sample size was calculated to be 18 patients in both groups with a statistical power of 0.80, an alpha of 0.05, and effect sizes of 0.32 (anova repeated measures within-between interactions). the sample size calculation was done for patient level assessment and was rounded to 20 patients considering 10% loss of follow-up. selection criteria inclusion criteria were participants in the age range of 30-55 years, previous surgical treatment of periodontitis stage ii and stage iii, grade a and b (according to 2017 classification of periodontal diseases by world workshop of periodontal and peri-implant diseases) at least 6 months to 3 years earlier, systemically healthy individuals and participants not being administered antibiotics or not having undergone periodontal care in the past six months [27]. exclusion criteria were participants with systemic diseases that would influence the periodontium and effect healing, pregnant and lactating women, smokers, and usage of other kinds of tobacco products, allergy history known to exist to any type of pigmented chemical use, teeth with persistent angular defects and bony deformities requiring osseous surgery, grade ii and grade iii mobility, non-compliant patients, poor oral hygiene maintenance, presence of occlusal discrepancies and mal-aligned teeth. patient selection the patients who satisfied all the inclusion and exclusion criteria were enrolled in the study consort guidelines were followed (figure 1). http://dentistry3000.pitt.edu/ compara've evalua'on of indocyanine green and 810nm diode laser assisted an'microbial photodynamic therapy with 810nm diode laser assisted new acachment procedure as adjuncts to scaling and root planning in suppor've periodontal therapy: a randomized controlled splitmouth clinical trial vol 12 no 1 (2024) doi 10.5195/d3000.2024.392 h#p://den*stry3000.pi#.edu randomization, allocation, and blinding a total of 26 participants (16 females and 10 males) with previous history of surgical periodontal therapy at least 6 months to 3 years before inclusion in the study were selected. all the participants received a complete periodontal, clinical, and radiographic examination and oral prophylaxis. participants were recalled after 3 weeks of root planning and sites with persistent probing pocket depths (ppd) of ≥5mm in contralateral sites with minimum of 2 teeth in each quadrant were selected for the study. the ppd and clinical attachment loss (cal) were calculated using unc-15 probe (hu-friedy®). the sites were randomly allocated into both the groups i.e., ig-pdt and lanap. allocation concealment was done by using computer generated random numbering system which is sealed in opaque envelopes. clinical parameters clinical parameters assessed in the study were evaluated at baseline, 3 months and 6 months after the treatment in both the groups. the parameters recorded include full mouth plaque index (pi), full mouth gingival index (gi), bleeding index (bi), ppd, and cal gain at baseline, 3 figure 1. consort flow diagram http://dentistry3000.pitt.edu/ compara've evalua'on of indocyanine green and 810nm diode laser assisted an'microbial photodynamic therapy with 810nm diode laser assisted new acachment procedure as adjuncts to scaling and root planning in suppor've periodontal therapy: a randomized controlled splitmouth clinical trial vol 12 no 1 (2024) doi 10.5195/d3000.2024.392 h#p://den*stry3000.pi#.edu months and 6 months [28]. healing was assessed on the 7-post operative day and 1 month postoperatively by using landry et al healing index [29, 30]. healing was assessed as an ordinal value on a scale of 1-5, where score 1 indicates very poor and score 5 excellent healing. the healing is based on tissue colour, response to palpation, presence or absence of bleeding epithelisation of gingival margin and presence or absence of suppuration [29, 30]. patient comfort was assessed immediate postoperatively using vas scale. the vas scale is a numeric scale assessed as an ordinal value from 0 to 10 with 0 indicating ‘no discomfort’ and 10 indicating ‘greatest discomfort’ [31]. preparation of indocyanine green solution icg aqueous solutions should be used within 24 hours in sterile circumstances due to their instability. therefore, a fresh 5 mg/ml solution was created as needed as follows: to create a 5 mg/ml icg solution, icg (aurogreen, aurolabs®, madurai, india) was dissolved in 5 ml of sterile water and gently shaken for 1 min according to manufacturer instructions, resulting in a concentration of 0.1 mg/ml icg [32]. treatment procedure ig-pdt group the icg photosensitizer system was combined with a calibrated class 4 laser device developed for dental use with multi-functional soft tissue applications (denlase®, pune, maharashtra) at the ig-pdt sites. the laser light is transmitted via an optical fibre connected to a hand piece to photosensitize icg dye within a wavelength of 810±10 nm. the active solution of prepared icg was then transferred via disposable 2ml syringe to the test sites and injected with a thin canula to the deep periodontal pockets until excess fluid was visible around the teeth, followed by resting for 1 to 2 min (figure 2a, 2b, 2c). irradiation of icg-loaded sites was performed using a 400 µm laser fibre tip in a pulsed wave mode and a pulse repetition rate of 2 khz with a mean output power of 100 mw, and a power density of 1 w yielding an applied radiation energy of 1414 j/cm2 per site for 60s(figure 2d) [32]. lanap group the first pass with an 810±10 nm diode laser and a 400µm fibre diameter, laser settings of 3.0 w and energy density of 1,965 mj/mm2, 100-ms pulse duration, and 20 hz were applied from the gingival margin to the base of the pocket parallel to the root surface and moved laterally and apically to remove the diseased pocket epithelium. with piezo ultrasonic instrumentation, the teeth were then completely scaled and root planed. a second pass was performed from the apical extent of the bone defect to the gingival margin using a 400µm fibre diameter, laser settings of 3.0 w and energy density of 1,965 mj/mm2, 650ms pulse duration, and 20 hz (figure 3a, 3b) [19, 33]. statistical analysis a sample size of 20 participants each having two contralateral sites was included under 5% alpha error and 80% of power of the test to detect the significant difference. all data collected were entered into microsoft excel (ms office version 2010) and tabulated. statistical analysis of the results was done using spss 26.0 (ibm inc. chicago, il, usa). the intergroup comparison of the data was done using the student t test and figure 2. ig-pdt group; a. presence of persistent probing pocket depth; b. intrapocket delivery of icg solution; c. icg dye injected into the sulcus; d. diode laser tip inserted into the sulcus. figure 3. lanap group; a. presence of persistent probing pocket depth; b. diode laser tip inserted into the sulcus and moved circumferentially http://dentistry3000.pitt.edu/ compara've evalua'on of indocyanine green and 810nm diode laser assisted an'microbial photodynamic therapy with 810nm diode laser assisted new acachment procedure as adjuncts to scaling and root planning in suppor've periodontal therapy: a randomized controlled splitmouth clinical trial vol 12 no 1 (2024) doi 10.5195/d3000.2024.392 h#p://den*stry3000.pi#.edu the intra group comparisons were performed using repeated measures of anova. non para-metric test mannwhitney u test was performed. results a total of 22 participants were allocated to the intervention. however, two were lost to follow up which led to inclusion of 20 participants (including 12 females and 8 males) in the final analysis. both patient level and site level analysis was done for both the groups. the mean age of the participants was 42.20±5.90. in both the treatment groups healing took place uneventfully. a total of 240 sites were treated and evaluated in the ig-pdt and 234 sites in the lanap group. the clinical parameters like ppd, cal, pi, gi and bi were assessed in all the sites at 3 and 6 month postoperatively. the patient level analysis was also done for all the parameters. the mean baseline pi, gi and bi scores were 1.66±.07, 1.61±.09 and 2.40±0.14 respectively which reduced significantly (p=<0.001) to 0.42±0.07, 0.41±0.06 and 0.19±0.02 at 6 months. ppd and cal reduction the baseline site level mean ppd were 5.19±0.78 and 5.22±0.73 in igpdt and lanap groups respectively with no statistically significant difference (p=0.64). a significant reduction in ppd was observed from baseline to 3 and 6 months in both the groups (p=<.001). however, intergroup analysis at both 3and 6months interval showed highly significant reduction of ppd in both groups (p<0.001) with ig-pdt showing less ppd (3.21±0.46) compared to lanap (3.52±0.50) (table 1). the patient level analysis also showed similar ppd reduction scores with lesser ppd in ig-pdt group (3.21±0.10) compared to lanap group (3.50±0.50) at 6 months (table 2) (figure 4). the baseline site level mean cal values were 5.15±0.61, 5.22± 0.62 in ig-pdt and lanap groups respectively showing no significant difference (p-0.67). at 3 and 6 months postoperatively a significant difference (p<0.001) in the mean cal were observed between the two groups with ig-pdt group showing greater cal gain. the site level mean cal values at 6 months were 2.03±0.54 and 3.22±0.62 in ig-pdt and lanap groups respectively (table 1). the intergroup patient level assessment of cal values also showed similar reduction at 6 months i.e., 2.01±0.64 in ig-pdt and time interval ig-pdt lanap ig-pdt vs lanap mean ±sd p-value mean±sd p-value ppd baseline 5.19±0.78 <0.001** 5.22±0.73 <0.001** 0.641 3months 3.97±0.65 4.24±0.69 <0.001** 6months 3.21±0.46 3.52±0.50 <0.001** cal baseline 5.15±0.61 <0.001** 5.22± 0.62 <0.001** 0.20 3months 4.02± 0.54 4.22±0.62 <0.001** 6months 2.03±0.54 3.22±0.62 <0.001** table 1: intra and intergroup site level assessment of clinical parameters ** p<0.001highly statistically significant ppdprobing pocket depth; calclinical attachment loss figure 4. ig-pdt group; a. baseline ppd of 5mm; b. 3mm ppd at 3-month ppd reevaluation; c. 3mm ppd at 6-month reevaluation http://dentistry3000.pitt.edu/ compara've evalua'on of indocyanine green and 810nm diode laser assisted an'microbial photodynamic therapy with 810nm diode laser assisted new acachment procedure as adjuncts to scaling and root planning in suppor've periodontal therapy: a randomized controlled splitmouth clinical trial vol 12 no 1 (2024) doi 10.5195/d3000.2024.392 h#p://den*stry3000.pi#.edu 3.02±0.72 in lanap groups (table 2, figure 5). healing index and patient discomfort (vas) the landry et al., healing index was used to assess the healing at 7 days and 1 month postoperatively. the mean healing index score at 7 days in ig-pdt and lanap group were 4.95±0.22 and 4.70±0.47 respectively and the difference in healing index scores was statistically significant (p=0.03). at 1month postoperatively mean healing index scores were 4.95±0.22 and 4.55±0.51 for ig-pdt and lanap groups respectively and the difference was highly significant (p-value =0.00) (table 3). patient discomfort was assessed immediately after surgery by using vas scale. the mean vas scores postoperatively were 3.20±0.61 and 2.40±0.50 in ig-pdt and lanap groups respectively with ig-pdt group showing greater discomfort(p=<0.001) (table 3). discussion amongst the various adjunctive nonsurgical treatment procedures lanap has been widely accepted for routine clinical care. however, there is a lack of evidence to support the use of lanap in supportive periodontal care, and no previous trials compared the effects of pdt with a well-established lanap protocol in recurrent periodontal pockets during supportive periodontal care. as a result, the purpose of this in vivo study was to examine the effect of ig-pdt and lanap as adjuncts to srp in the treatment of periodontal pockets in supportive periodontal therapy. the current study's methodology for treating periodontal pockets in both groups is well established. to our knowledge, no study has compared the efficacy of pdt and lanap in supportive periodontal therapy. furthermore, icg has been shown to be a potential alternative to the conventional photosensitizers used in time interval ig-pdt lanap ig-pdt vs lanap mean ±sd p-value mean±sd p-value ppd baseline 5.17±0.27 <0.001** 5.22±0.73 <0.001** 0.641 3months 4.12±0.25 4.21±0.29 0.143 6months 3.21±0.46 3.52±0.50 <0.001** cal baseline 5.25±0.24 <0.001** 5.38± 0.25 <0.001** 0.20 3months 1.05± 0.12 0.95±0.16 <0.001** 6months 1.80±0.54 1.63±0.62 <0.001** parameter time interval groups mean ±sd p-value healing index 7 days ig-pdt 4.95±0.22 0.03* lanap 4.70±0 .47 1 month ig-pdt 4.85± 0.36 0.68 lanap 4.80± 0.41 patient discomfort(vas) after the procedure ig-pdt 3.20±0.61 <0.001** lanap 2.40±0.50 figure 5. lanap group; a. baseline ppd of 6mm; b. 5mm ppd at 3-month re-evaluation; c. 5mm ppd at 6 month re-evaluation. table 2: intra and intergroup patient level analysis of clinical parameters ** p<0.001highly statistically significant ppdprobing pocket depth; calclinical attachment loss table 3: intra and intergroup analysis of healing index and patient discomfort (vas) **p<0.001highly statistically significant *p<0.05statistically significant http://dentistry3000.pitt.edu/ compara've evalua'on of indocyanine green and 810nm diode laser assisted an'microbial photodynamic therapy with 810nm diode laser assisted new acachment procedure as adjuncts to scaling and root planning in suppor've periodontal therapy: a randomized controlled splitmouth clinical trial vol 12 no 1 (2024) doi 10.5195/d3000.2024.392 h#p://den*stry3000.pi#.edu the majority of the studies [32, 3439]. in the current study no detrimental/ adverse effects were reported in either of the groups showing good tissue acceptability of both the procedures which is in accordance with earlier studies [40,41]. the shorter wavelengths of deep-tissue penetrating diode lasers do not interact with the periodontal tissues within the pocket or tooth crown, and should not cause any thermal changes within the gingival tissues and root surfaces, or destruction of the intact attachment apparatus at the base of pockets [42,43]. to avoid confounders, the full mouth pi, gi and bi scores were assessed at specific time intervals to assess the patient's overall oral hygiene status throughout the study period. the full mouth plaque and bleeding scores reduced significantly after both the treatments. reduction in the plaque scores dictates the adequate maintenance of oral hygiene and good patient compliance. also, the full mouth bleeding scores reduced significantly and maintained throughout the study period. this is in accordance with study done by christodoulides n et al who reported a significantly higher reduction in bleeding scores in pdt group compared to scaling and root planning alone [34]. the lower bleeding scores can be attributed to the use of laser light in both groups, which has been shown to reduce the formation of abnormal blood vessels and bleeding, thereby preserving gingival health. the results of the present study illustrated statistically significant positive treatment outcomes in terms of cal gain and reduction in ppd at 3 and 6 months follow up in both the groups however significantly greater reduction in ppd and cal were observed in the ig-pdt group. recent studies reported a significant improvement in periodontal parameters using ig-pdt compared to scaling and root planning [32,35,39,44]. ig-pdt may convey anti-inflammatory properties and is effective in eliminating periodontal pathogens. the enhanced treatment outcomes shown with adjunctive igpdt may be explained by the toxic effect on the microbial organisms responsible for the disease, along with anti-inflammatory abilities [26]. ig-pdt downregulates inflammatory mediators such as tumor necrosis factor-α, nitric oxide, and 5lipoxygenase which play a significant role in the pathogenesis of periodontal disease. mccawley et al., conducted a clinical trial on the immediate effects of lanap procedure on human periodontal microbiota, and reported that 85% of the patients treated with lanap showed negative culture tests immediately for the red and orange complex microbes [36]. marc nevins et al conducted a study to evaluate the clinical response to the lanap in a 9-month re-evaluation and the results demonstrated a statistically significant improvement in majority of the sites treated showing clinical improvement in terms of decreased ppd and increased cal which is in accordance with our study [45]. two histologic studies on humans have been done so far using lanap technique. yukna et al examined healing in teeth extracted three months following treatment in the first study and reported that laserassisted new attachment procedure showed the formation of new cementum and connective tissue attachment, as well as new bone in four of the six specimens. in contrast, five of the six teeth that underwent the control procedure healed by a long junctional epithelium [18]. a similar histological study by nevins et al. also reported similar results with greater new attachment gain in laserassisted new attachment procedure treated teeth [17]. till date no clinical trial compared the outcomes of pdt with lanap procedure. alsarhan ma et al reported a significant greater reduction in probing pocket depth and bleeding on probing in ig-pdt compared to the control group where only scaling and root planning were performed in a three-month clinical trial [32]. the above study reported a reduction in mean probing pocket depths in the ig-pdt group from 5.22± 0.924mm to http://dentistry3000.pitt.edu/ compara've evalua'on of indocyanine green and 810nm diode laser assisted an'microbial photodynamic therapy with 810nm diode laser assisted new acachment procedure as adjuncts to scaling and root planning in suppor've periodontal therapy: a randomized controlled splitmouth clinical trial vol 12 no 1 (2024) doi 10.5195/d3000.2024.392 h#p://den*stry3000.pi#.edu 2.27± 0.749mm. in the current study, the mean ppd decreased from 5.17±0.27 to 3.21±0.46mm in the igpdt group. this difference in residual ppd at the end of study period could be attributed to al sarhan ma et al’s protocol of multiple applications of pdt at 3 intervals one week apart [32]. in contrary, another split mouth trial by muller campanile vs et al evaluating the multiple application protocol of pdt did not show any significant difference in ppd reduction in single (2.8±1.1 mm) or twice (2.9±1.1 mm) application protocols. however, no sites in twice application and two sites in single application protocol had persistent ppd >4mm [37]. a recent systematic review and meta-analysis by bashir et al., aimed to evaluate the efficacy of ig-pdt as an adjunct to nspt in the management of chronic periodontitis. the results of the rcts included showed positive treatment outcomes in terms of reduced ppd and increase in cal gain at 3 and 6 months postoperatively and hence concluded that ig-pdt can be used as an adjunct to nspt [38]. post operative healing was assessed at 7th day and one month post operatively to assess early healing of the sites after both the protocols [30]. the current study showed a significant improvement in healing in both the groups from one week to one month post-operatively with igpdt group showing significantly better healing at one week. there are hardly any clinical trials evaluating the healing after pdt. however, laser biostimulation is shown to increase the healing in various clinical trials suggesting that low level lasers increases the motility of epithelial cells and proliferation of fibroblasts suggesting an enhanced wound healing [46]. also laser biostimulation is thought to increase the revascularization process [47]. the results show a significant (p=0.03) difference with better healing in the ig-pdt group at the seventh post-operative day, with no difference at the one-month interval, indicating faster healing in the ig-pdt group, which can be attributed to the photosensitizer used in the ig-pdt group in conjunction with laser irradiation. laser irradiation with deep penetrating lasers such as diode laser was shown to produce a thicker coagulation layer due to greater tissue denaturation compared to superficially absorbed lasers resulting in slower healing. photobiomodualtion with use of icg causes biostimulation thus promoting cell proliferation and differentiation, as well as anti-inflammatory effects, which may positively modulate wound healing [48,49]. the findings of the current study are at odds with previous reviews that indicated pdt to be only marginally clinically effective in the management of periodontitis [50]. but this can be accounted for by the fact that the systematic reviews were conducted before icg was approved as a photosensitizer for periodontitis, and as a result, their conclusions were based on the outcomes seen with less-than-ideal photosensitizers such toluidine blue and methylene blue. there is currently very little evidence for the use of pdt in supportive periodontal care. previously published clinical trials showed contradictory results for the use of pdt as adjuncts in supportive periodontal care [23,51,52]. a single episode of pdt as an adjunct to scaling and root planning did not result in an additional improvement in terms of ppd reduction and cal gain, but it did result in a significantly higher reduction in bleeding scores than scaling and root planning alone [50]. repeated (five times) application of pdt as adjunct to debridement yielded improved clinical outcomes in residual pockets in maintenance patients [51]. mongardinii et al. reported that adjunctive photodynamic treatment with light-emitting diode (led) may improve short-term clinical and microbiological outcomes in periodontitis patients undergoing spt [53]. limitations and future research the current study, like most previous studies, is a short-term (6-month) clinical trial with a small cohort (n=20). a single concentration of icg was also used, along with specific laser properties and protocols based http://dentistry3000.pitt.edu/ compara've evalua'on of indocyanine green and 810nm diode laser assisted an'microbial photodynamic therapy with 810nm diode laser assisted new acachment procedure as adjuncts to scaling and root planning in suppor've periodontal therapy: a randomized controlled splitmouth clinical trial vol 12 no 1 (2024) doi 10.5195/d3000.2024.392 h#p://den*stry3000.pi#.edu on previous research. despite having similar properties, using a diode laser instead of the well-established nd:yag laser assisted lanap protocol may be a limitation. further research with larger sample size, different protocols and multiple applications at different time intervals could provide more insight into developing a defined protocol for routine management of patients receiving supportive periodontal care. conclusion ig-pdt outperformed lanap in terms of probing depth reduction, clinical attachment gain, and faster healing within the limitations of the current study. the promising impact of igpdt as an additional therapeutic procedure necessitates additional research, including long-term clinical trials, to validate its clinical use as a treatment strategy in supportive periodontal therapy. references 1. könönen e, gursoy m, gursoy uk. periodontts: a multfaceted disease of tooth-supportng tissues. j clin med. 8 (8) (2019) 1135, h_ps:// doi.org/ 10.3390/jcm8081135. 2. heitz-mayfield lj, trombelli l, heitz f, needleman i, moles d. a systemasc review of the effect of surgical debridement vs non-surgical debridement for the treatment of chronic periodonsss. j clin periodontol. 29 (3) (2002) 92102, hups://doi.org/ 10.1034/j.1600051x.29.s3.5.x. 3. hirata t, fuchida s, yamamoto t, kudo c, minabe m. predicsve factors for tooth loss during supporsve periodontal therapy in pasents with severe periodonsss: a japanese mulscenter study. bmc oral health. 19 (19) (2019), hups://doi.org/10.1186/s129 03-019-0712-x. 4. atarbashi-moghadam f, talebi m, mohammadi f, sijanivandi s. recurrence of periodonsss and associated factors in previously treated periodonsss pasents without maintenance follow-up. j adv periodontol implant dent. 12 (2) (2020) 79-83, hups://doi.org/10.34172/japi d.2020.010. 5. sanz-marwn i, cha jk, yoon sw, sanz-sánchez i, jung uw. long-term assessment of periodontal disease progression ayer surgical or non-surgical treatment: a systemasc review. j periodontal implant sci. 49 (2) (2019) 60-75, hups://doi.org/10.5051/jpis.2 019.49.2.60. 6. mombelli a. maintenance therapy for teeth and implants. periodontol 2000. 79 (1) (2019) 190-199, hups://doi.org/10.1111/prd. 12255. 7. mikami r, mizutani k, sasaki y, iwata t, aoki a. pasentreported outcomes of laserassisted pain control following non-surgical and surgical periodontal therapy: a systemasc review and meta-analysis. plos one. 15 (9) (2020) e0238659, hups://doi.org/10.1371/jour nal.pone.0238659. 8. suprith s, seuy s, thakur s. diode laser as an adjuncsve to nonsurgical periodontal therapy during maintenance phase: a randomized controlled clinical trial. int j laser dent 6 (1) (2016) 1217, hups://doi.org/10.5005/jpjournals-10022-1080. 9. ratka-krüger p, mahl d, deimling d, mönsng js, jachmann i, al-machot e, sculean a, berakdar m, jervøe-storm pm, braun a. er:yag laser treatment in supporsve periodontal therapy. j clin periodontol. 39 (5) (2012) 483-489, hups//doi.org/10.1111/j.160 0-051x.2012.01857.x. 10. choi e, um hs, chang bs, lee sy, lee jk. clinical and microbiological effects of adjuncsve local delivery of minocycline (periocline®) in pasents receiving supporsve periodontal therapy: a pilot study. j periodontal implant sci. 51 (1) (2021) 53-62, hups://doi.org/10.5051/jpis.2 002720136. 11. manresa c, sanz-miralles ec, twigg j, bravo m. supporsve periodontal therapy (spt) for maintaining the densson in adults treated for periodonsss. cochrane database syst rev. 1 (1) http://dentistry3000.pitt.edu/ compara've evalua'on of indocyanine green and 810nm diode laser assisted an'microbial photodynamic therapy with 810nm diode laser assisted new acachment procedure as adjuncts to scaling and root planning in suppor've periodontal therapy: a randomized controlled splitmouth clinical trial vol 12 no 1 (2024) doi 10.5195/d3000.2024.392 h#p://den*stry3000.pi#.edu (2018) cd009376, hups://doi.org/10.1002/1465 1858.cd009376. 12. cobb cm, low sb, coluzzi dj. lasers and the treatment of chronic periodonsss. dent clin north am. 54 (1) (2010) 35-53, hups://doi.org/10.1016/j.cde n.2009.08.007. 13. noguchi t, sanaoka a, fukuda m, suzuki s, aoki t. combined effects of nd:yag laser irradiason with local ansbiosc applicason into periodontal pockets. j int acad periodontol. 7 (1) (2005) 8-15, pmid: 15736890. 14. schwarz f, aoki a, sculean a, becker j. the impact of laser applicason on periodontal and peri-implant wound healing. periodontol 2000. 51 (2009) 79-108, htpps://doi.org/10.1111/j.16 00-0757.2009.00301.x. 15. kim i s, cho t h, kim k, weber f e and hwang s j. high power pulsed nd:yag laser as a new ssmulus to induce bmp-2 expression in mc3t3–e1 osteoblasts lasers. surg. med. 42 (2010) 510–518. 16. harris dm, gregg rh, mccarthy dk, colby le, tilt lv. laser-assisted new auachment procedure in private pracsce. general densstry. 52 (5) (2004) 396403. 17. nevins ml, camelo m, schupbach p, kim sw, kim dm, nevins m. human clinical and histologic evaluason of laser-assisted new auachment procedure. int j periodonscs restorasve dent,32 (2012) 497–507. pmid: 22754897 18. yukna ra, carr rl, evans gh. histologic evaluason of an nd:yag laser-assisted new auachment procedure in humans. int j periodonscs restorasve dent. 27 (6) (2007) 577-587, pmid: 18092452. 19. jha a, gupta v, adinarayan r. lanap, periodonscs and beyond: a review. j lasers med sci. 9 (2) (2018) 7681, hups://doi.org/10.15171/jlm s.2018.16. 20. niemz mh. laser-sssue interacson. fundamentals and applicasons. berlin: springer-verlag, (1996) 5887, hups://doi.org/10.1007/9783-662-03193-3. 21. kolbe mf, ribeiro fv, luchesi vh, casarin rc, sallum ea, nocis fh jr, ambrosano gm, cirano fr, pimentel sp, casas mz. photodynamic therapy during supporsve periodontal care: clinical, microbiologic, immunoinflammatory, and pasent-centered performance in a split-mouth randomized clinical trial. j periodontol. 85 (8) (2014) e277-286, hups://doi.org/ 10.1902/jop.2014.130559. 22. park, d., choi, e. j., weon, k.y., lee, w., lee, s. h., choi, j.s., … choi, j. w. non-invasive photodynamic therapy against -periodonsss-causing bacteria. sciensfic reports, 9 (1) (2019), hups://doi.org/10.1038/s415 98-019-44498-4. 23. fan z, zong j, lau wy, zhang y. indocyaninee green and its nanosynthesc parscles for the diagnosis and treatment of hepatocellular carcinoma. am j transl res.12 (6) (2020) 2344-2352. pmid: 32655776; pmcid: pmc7344064. 24. alander jt, kaarsnen i, laakso a, päslä t, spillmann t, tuchin vv, venermo m, välisuo p. a review of indocyaninee green fluorescent imaging in surgery. int j biomed imaging. (2012) 940585, hups://doi.org/10.1155/2012 /940585. 25. sevieri m, silva f, bonizzi a, sisa l, truffi m, mazzucchelli s, corsi f. indocyaninee green nanoparscles: are they compelling for cancer treatment? front chem. 8 (2020) 535, hups://doi.org/10.3389/fche m.2020.00535. 26. raut cp, sethi ks, kohale br, mamajiwala a, warang a. indocyaninee greenmediated photothermal therapy in treatment of chronic periodonsss: a clinico-microbiological study. j indian soc periodontol. 22 (2018) 221-227, hups://doi.org/10.4103/jisp.ji sp_128_18. 27. papapanou pn, sanz m, buduneli n, dietrich t, feres m, fine dh, et al. periodonsss: consensus report of workgroup 2 of the 2017 world workshop on the http://dentistry3000.pitt.edu/ compara've evalua'on of indocyanine green and 810nm diode laser assisted an'microbial photodynamic therapy with 810nm diode laser assisted new acachment procedure as adjuncts to scaling and root planning in suppor've periodontal therapy: a randomized controlled splitmouth clinical trial vol 12 no 1 (2024) doi 10.5195/d3000.2024.392 h#p://den*stry3000.pi#.edu classificason of periodontal and peri-implant diseases and condisons. j periodontol. 89 (1) (2018) 173-182, hups://doi.org/10.1002/jper. 17-0721. 28. löe h. the gingival index, the plaque index and the retenson index systems. j periodontol. 38 (6) (1967) 610-616, hups://doi.org/10.1902/jop.1 967.38.6.610. 29. lingamaneni s, mandadi lr, pathakota kr. assessment of healing following low-level laser irradiason ayer gingivectomy operasons using a novel soy sssue healing index: a randomized, double-blind, splitmouth clinical pilot study. j indian soc periodontol. 23 (2019) 53-57, hups://doi.org/10.4103/jisp.ji sp_226_18. 30. masse jf, landry rg, rocheue c, dufour l, morency r, d'aoust p. effecsveness of soy laser treatment in periodontal surgery. int dent j. 43 (2) (1993) 121-127, pmid: 8320005. 31. koppolu p, mishra a, swapna la, butchibabu k, bagalkokar a, baroudi k. comparison of efficacy among various topical anesthescs: an approach towards painless injecsons in periodontal surgery. saudi j anaesth. 10 (1) (2016) 55-7, hups://doi.org/10.4103/1658 -354x.169476. 32. alsarhan ma, altammami ma, alaqeely rs, alebdi a, jasser ra, otaibi da, et al. short-term improvement of clinical parameters and microbial diversity in periodonsss pasents following indocyaninee green-based ansmicrobial photodynamic therapy: a randomized single-blind splitmouth cohort. photodiagnosis photodyn ther. 35 (2021) 102349, hups://doi.org/10.1016/j.pdp dt.2021.102349. 33. kirpalani t, dym h. role of piezo surgery and lasers in the oral surgery office. dent clin north am. 64 (2) (2020) 351-363, hups://doi.org/10.1016/j.cde n.2019.12.007. 34. christodoulides n, nikolidakis d, chondros p, becker j, schwarz f, rössler r, sculean a. photodynamic therapy as an adjunct to non-surgical periodontal treatment: a randomized, controlled clinical trial. j periodontol. 79 (9) (2008) 1638-1644, hups://doi.org/10.1902/jop.2 008.070652. 35. joshi k, baiju cs, khashu h, bansal s. clinical effecsveness of indocyaninee green mediated ansmicrobial photodynamic therapy as an adjunct to scaling root planing in treatment of chronic periodonsssa randomized controlled clinical trial. photodiagnosis photodyn ther.29 (2020) 101591. hups://doi.org/10.1016/j.pdp dt.2019.101591. 36. mccawley tk, mccawley mn, rams te. immediate effect of nd:yag laser monotherapy on subgingival periodontal pathogens: a pilot clinical study. j periodontal implant sci. 52 (1) (2020) 77-87, hups://doi.org//10.5051/jpis. 2100900045. 37. müller campanile vs, giannopoulou c, campanile g, cancela ja, mombelli a. single or repeated ansmicrobial photodynamic therapy as adjunct to ultrasonic debridement in residual periodontal pockets: clinical, microbiological, and local biological effects. lasers med sci. 30 (1) (2015) 27-34, hups://doi.org/10.1007/s101 03-013-1337-y. 38. bashir nz, singh ha, virdee ss. indocyanine greenmediated ansmicrobial photodynamic therapy as an adjunct to periodontal therapy: a systemasc review and meta-analysis. clin oral invessg. 25 (10) (2021) 56995710, hups://doi.org/10.1007/s007 84-021-03871-2. 39. sufaru ig, martu ma, luchian i, stoleriu s, diaconu-popa d, martu c, teslaru s, pasarin l, solomon sm. the effects of 810 nm diode laser and indocyanine green on periodontal parameters and hba1c in pasents with periodonsss and type ii diabetes mellitus: a randomized controlled http://dentistry3000.pitt.edu/ https://doi.org/10.4103/jisp.jisp_226_18 https://doi.org/10.4103/jisp.jisp_226_18 https://doi.org/10.1016/j.pdpdt.2021.102349 https://doi.org/10.1016/j.pdpdt.2021.102349 https://doi.org/10.5051/jpis.2100900045 https://doi.org/10.5051/jpis.2100900045 compara've evalua'on of indocyanine green and 810nm diode laser assisted an'microbial photodynamic therapy with 810nm diode laser assisted new acachment procedure as adjuncts to scaling and root planning in suppor've periodontal therapy: a randomized controlled splitmouth clinical trial vol 12 no 1 (2024) doi 10.5195/d3000.2024.392 h#p://den*stry3000.pi#.edu study. diagnosscs (basel). 12 (7) (2022) 1614, hups://doi.org/10.3390/diag nosscs12071614. 40. parker s. the use of diffuse laser photonic energy and indocyanine green photosensiszer as an adjunct to periodontal therapy. br dent j. 215 (2013) 167–171, hups://doi.org/10.1038/sj.bd j.2013.790. 41. benson rc, kues ha. fluorescence properses of indocyanine green as related to angiography. phys med biol. 23 (1978) 159–163, hups://doi.org/10.1088/0031 -9155/23/1/017. 42. takasaki aa, aoki a, mizutani k, et al. applicason of ansmicrobial photodynamic therapy in periodontal and peri-implant diseases. periodontol 2000. 51 (2009) 109–140, hups://doi.org/10.1111/j.160 0-0757.2009.00302.x. 43. topaloglu n, gulsoy m, yuksel s. ansmicrobial photodynamic therapy of resistant bacterial strains by indocyanine green and 809nm diode laser. photomed laser surg. 31 (2013) 155– 162, hups://doi.org/10.1089/pho. 2012.3430. 44. dalvi s, khetal n, ansari s, benedicens s, hanna r. uslizason of 810 nm diode laser treatment in periodonsss as an alternasve to surgical debridement approach. photochem photobiol. 97 (3) (2021) 566-573, hups://doi.org/10.1111/php. 13417. 45. nevins m, kim sw, camelo m, marsn is, kim d, nevins m. a prospecsve 9-month human clinical evaluason of laser-assisted new auachment procedure (lanap) therapy. int j periodonscs restorasve dent. 34 (1) (2014) 21-7, hups://doi.org/10.11607/prd .1848. 46. tuby h, maltz l, oron u. modulasons of vegf and inos in the rat heart by low level laser therapy are associated with cardioprotecson and enhanced angiogenesis. lasers surg med. 38 (7) (2006) 682-688, hups://doi.org/10.1002/lsm. 20377. 47. camachoa ad, paredesb ac, aldana rl. an in vitro study of the reacson of periodontal and gingival fibroblasts to low-level laser irradiason: a pilot study. j oral laser appl. 8 (2008) 235-244. 48. aoki a, mizutani k, schwarz f, sculean a, yukna ra, takasaki aa, romanos ge, taniguchi y, sasaki km, zeredo jl, koshy g, coluzzi dj, white jm, abiko y, ishikawa i, izumi y. periodontal and peri-implant wound healing following laser therapy. periodontol 2000. 68 (1) (2015) 217-69, hups://doi.org/10.1111/prd. 12080. 49. srikanth k, chandra rv, reddy aa, reddy bh, reddy c, naveen a. effect of a single session of ansmicrobial photodynamic therapy using indocyanine green in the treatment of chronic periodonsss: a randomized controlled pilot trial. quintessence int. 46 (5) (2015) 391-400, hups://doi.org/10.3290/j.qi.a 33532. 50. aseh ma. photodynamic therapy as an adjuncsve treatment for chronic periodonsss: a metaanalysis. lasers med sci. 25 (4) (2010) 605-613, hups://doi.org/10.1007/s101 03-009-0744-6. 51. chondros p, nikolidakis d, christodoulides n, rössler r, gutknecht n, sculean a. photodynamic therapy as adjunct to non-surgical periodontal treatment in pasents on periodontal maintenance: a randomized controlled clinical trial. lasers med sci.24 (5) (2009) 681698, hups://doi.org/10.1007/s101 03-008-0565-z. 52. lulic m, leiggenergörög i, salvi ge, ramseier ca, mauheos n, lang np. oneyear outcomes of repeated adjuncsve photodynamic therapy during periodontal maintenance: a proof-ofprinciple randomizedcontrolled clinical trial. j clin periodontol. 36 (8) (2009) 661-666, hups://doi.org/10.1111/j.160 0-051x.2009.01432.x. http://dentistry3000.pitt.edu/ https://doi.org/10.3390/diagnostics12071614 https://doi.org/10.3390/diagnostics12071614 https://doi.org/10.1089/pho.2012.3430 https://doi.org/10.1089/pho.2012.3430 https://doi.org/10.1111/php.13417 https://doi.org/10.1111/php.13417 compara've evalua'on of indocyanine green and 810nm diode laser assisted an'microbial photodynamic therapy with 810nm diode laser assisted new acachment procedure as adjuncts to scaling and root planning in suppor've periodontal therapy: a randomized controlled splitmouth clinical trial vol 12 no 1 (2024) doi 10.5195/d3000.2024.392 h#p://den*stry3000.pi#.edu 53. mongardini c, di tanna gl, pilloni a. light-acsvated disinfecson using a lightemi�ng diode lamp in the red spectrum: clinical and microbiological short-term findings on periodonsss pasents in maintenance. a randomized controlled splitmouth clinical trial. lasers med sci. 29 (1) (2014) 1-8, hups://doi.org/10.1007/s101 03-012-1225-x. http://dentistry3000.pitt.edu/ microsoft word 410 2023.docx monitoring of the inverted rela2on of gingival pads in a non-syndromic infant vol 11 no 1 (2023) doi 10.5195/d3000.2023.410 h#p://den*stry3000.pi#.edu monitoring of the inverted relation of gingival pads in a non-syndromic infant silvia josé chedid1 1clinical instructor of pediatric dentistry, school of dentistry, são paulo association of dental surgeons, and private practitioner, são paulo, sp, brazil abstract background: monitoring the occlusal rela2onship of the infant’s gum pads is not a common prac2ce in pediatric den2stry. in general, pediatric den2sts are used to observe the conforma2on of the arches and clinical findings that may be outside of the infant’s normal oral anatomy. here we tested the hypothesis that interven2on before erup2on of the primary den22on will minimize anterior crossbite in the primary den22on. to address our hypothesis, we used one case to demonstrate the importance of monitoring the rela2onship of the gum pads and primary teeth erup2on to avoid possible devia2ons in the pagern of orofacial development and prevent future malocclusions. materials and methods: aier proper consen2ng and anamnesis, an inverted rela2onship of the gum pads was observed in a 7-month-old baby while sleeping and at occlusal rest. given that the pa2ent was exclusively breasleeding, adjustments of the catch were made, such as a greater opening towards the areola and nipple, and adjustments in the posi2on and posture of the baby. results: during the erup2on phase of the anterior teeth, an anterior crossbite was observed. caregivers were prescribed exercises that were to be performed in the maxillary and mandibular arches to s2mulate maxillary growth and guide the posture of the mandible. the exercises were intensified during the erup2on phase of the lateral incisors and molars and the occlusion was stabilized with bilateral mas2catory guidance and different textures to be offered during food transi2on. conclusion: based on the results observed for this case, the interven2ons allowed for a more sa2sfactory occlusion. keywords: gingival rounding; prognathism; child; infant; angle class iii cita9on: chedid, sj (2023) monitoring of the inverted rela9on of gingival pads in a non-syndromic infant den9stry 3000. 1:a001 doi:10.5195/d3000.2022.410 received: december 16, 2022 accepted: december 20, 2022 published: february 10, 2023 copyright: ©2023 chedid, sj this is an open access ar9cle licensed under a crea9ve commons avribu9on work 4.0 united states license. email: chedidsj@gmail.com introduction angle class iii malocclusion in the permanent dentition has a multifactorial etiology [1]. among the various genetic and epigenetic factors, we can highlight inadequate postural habits of the mandible and inadequate posture of the tongue [2,3]. we can also include a history of prolonged sucking habits, alterations in the establishment of mastication, atypical swallowing, obstruction of the nasal airways, mouth breathing, size of the tongue or tonsils and the presence of adenoids, among other factors. most of these factors can be observed by the pediatric dentist, therefore, a timely intervention may prevent prognathism in the permanent dentition [2,3]. transdisciplinary care in the first years of facial development will aid in the accurate diagnosis and timely interventions for cases of mouth breathing, airway obstruction, swallowing problems, and lip and tongue positioning, providing for the esther kwon monitoring of the inverted rela2on of gingival pads in a non-syndromic infant vol 11 no 1 (2023) doi 10.5195/d3000.2023.410 h#p://den*stry3000.pi#.edu opportunity to help establishing a healthy and functional primary occlusion. these early interventions can minimize and prevent future vertical, transverse, and anteroposterior problems in the deciduous and/or permanent dentitions [4]. a device used in infants born with down syndrome aiming to sensory motor stimulation [5], in addition to sensorimotor manipulation techniques for the tongue, lips and mandible posture, have shown effective results in tongue posture and lip sealing in these patients. other devices to reposition the mandible have also been suggested [6-9]. some of these devices involve the use of a wooden or plastic spatula (tongue depressor) behind the upper anterior teeth and in front of the lower anterior teeth as an inclined plane to position the mandible distally and depress the tongue, along with postural exercises for the mandible in cases of postural mandibular protrusion. these techniques are suggested for syndromic or non-syndromic patients [6-10]. preventive interventions in infants require attention to distinct parameters that are considered in the primary and mixed dentitions. given the relevance of this theme, we tested the hypothesis that intervention before eruption of the primary dentition will minimize anterior crossbite in the primary dentition. to address our hypothesis, we used one case to demonstrate the importance of monitoring the relationship of the gum pads and primary teeth eruption to avoid possible deviations in the pattern of orofacial development and prevent future malocclusions. for that purpose, one case that received preventive interventions performed in a 7-month-old, with a pattern of mandibular protrusion and on exclusive breastfeeding. muscle relaxation techniques and digital compression exercises were used. study case the mother of a 7-month-old female, residing in the city of são paulo, state of são paulo, brazil, sought dental care on october 19, 2020, for her first visit to the pediatric dentist. in this first evaluation, extra and intraoral exams were performed [11,12]. regarding the relationship of the gum pads [13-15], an inverted relationship between them was observed [16] (figure 1). this relationship was determined with the infant at occlusal rest while sleeping [17,18]. in the anamnesis, the mother reported normal delivery, exclusive breastfeeding, and no other cases of prognathism in the family. there was no relevant medical history. the child's diet was still exclusively breast milk. nasal breathing, absence of habits, and no ankyloglosia or short lip frenulum were found. the observation of the feeding [11] was carried out and, in this phase, the only intervention was the adjustment of the catch and the position of the baby (figure 2). the infant was monitored monthly to observe feeding and latching, as well as the evolution of mandibular posture with reference to the relationship between occlusal arches. in the eruption phase of the lower incisors, which happened around 9 months of age, mandibular protrusion was still observed. the evaluations figure 1. the infant had gingival rounds in inverted relation. photograph was taken with the patient asleep. figure 2. mother was instructed to allow for a greater mouth opening and catch on of the areola. monitoring of the inverted rela2on of gingival pads in a non-syndromic infant vol 11 no 1 (2023) doi 10.5195/d3000.2023.410 h#p://den*stry3000.pi#.edu were performed with the child asleep (figure 3). at follow-up visits, it was observed while the child asleep the eruption of the upper incisors, and the establishment of an inverse relationship (figure 4). at this time, transition feeding had already started. at this stage, preventive interventions were suggested to stimulate the growth of the premaxilla and to improve mandibular posture. an intervention of functional digital compression exercises was implemented [10]. all procedures were demonstrated to the mother in the first consultation and the exercises were repeated three times in the presence of the author until they were properly performed. all exercises were performed at home 2 or 3 times a day for 3 to 5 minutes according to the child's cooperation. the exercises were monitored once a week by the professional [10] (figures 5 and 6). the occlusal relationship was constantly monitored and adapted according to the child's collaboration. there was a change in the mandibular posture and the anteroposterior relationship was re-established after performing the exercises (figure 7). the occlusion was stabilized with the eruption of the first deciduous molars, and subsequent better masticatory function (figure 8). discussion the extra and intra-oral observation of an infant regarding the relationship of the gum pads is important from the point of view of monitoring the orofacial development and eruption of the deciduous teeth. it is important that the pediatric dentist is attentive to the child’s figure 3. evaluation of the relationship of the gingival edges during the eruption of the lower primary central incisors. figure 5. intervention where the index finger is placed under the palatine papilla with slight pressure (up to a small ischemia) [10]. figure 6. mandibular posture was guided with the index finger in the cervical region of the lower incisors or chin region, guiding the mandible in a more backward position. figure 7. anterior overlap and reestablishment of the anteroposterior relationship after performing the exercises. figure 8. stabilized occlusion after eruption of primary first molars. figure 4. observation of the relationship between the arches and the sleeping baby, in occlusion. monitoring of the inverted rela2on of gingival pads in a non-syndromic infant vol 11 no 1 (2023) doi 10.5195/d3000.2023.410 h#p://den*stry3000.pi#.edu harmonious development of the arches and functional occlusion. the correction of facial disharmonies of dental, functional, or skeletal origin in early childhood is still not a common practice, as it involves several limitations. one of them is the adherence of the parents to the attention and carrying out of the preventive conducts at home with the due collaboration and conditioning of the child. for these practices to be viable, it is important that the professionals involved are qualified and specialized. when parents observe these changes and seek professional help, their adherence to carry out the proposed treatment at home is facilitated [10]. muscle relaxation massages and postural guidance and digital compression exercises performed in defined areas and at the right time can provide quick and effective results for establishing a harmonic relationship between the arches. although these exercises have already been proposed for patients with disabilities [5], their practice in non-syndromic patients is still not common [5,6,19,20]. there is little evidence regarding the effectiveness of these techniques. more studies are needed, especially in the first months of the infant's life. we know that functional or skeletal problems that are not treated in early childhood can be perpetuated in the mixed and permanent dentitions4. waiting to intervene can lead to more invasive, complex, long, and expensive orthodontic treatments that can negatively affect the patient's quality of life [21]. in addition, the concepts of aesthetics and self-esteem also influence the quality of life of young patients, hence the need for preventive intervention as soon as any change is identified [21]. proper guidance from dental prenatal care can positively influence mothers towards effective practices in health and caries prevention [22]. the observation of facial images on morphological ultrasound can influence positive health behaviours, thus, they can also be important tools for promoting oral health during pregnancy [22-24]. parents must be willing to cooperate, and this is the key to successful results. acting as early as possible, even during the eruption phase of the deciduous teeth, can be effective as this is still a time when functional, postural, dental, or skeletal problems have not been established. performing digital compression exercises can be even more effective if started from the first years of the infant's life, before the eruption of the first deciduous molars, preventing vicious postures of the mandible or tongue [3,4]. most therapies proposed in the literature are designed for later, when patients with complete deciduous dentition [25,26]. future clinical studies should be encouraged aiming at early interventions to prevent malocclusions. additional investigations are needed with larger samples, well-defined diagnostic criteria, rigorous scientific methodologies, and long-term control [27]. conclusion this paper reports an early intervention where constant monitoring of orofacial development and proper guidance to parents were carried. the goal was establishing a harmonious incisor guidance and a favourable anteroposterior relationship of the arches during the eruption phase of the first deciduous teeth. the hope is that these early interventions will decrease severity of malocclusions. references 1. early orthodon.c treatment for class iii malocclusion: a systema.c review and metaanalysis. woon sc, thiruvenkatachari b. am j orthod dentofacial orthop 2017;151(1):28-52. 2. skeletal and dental effects of class iii orthopaedic treatment: a systema.c review and metaanalysis. rongo r, d'antò v, bucci r, polito i, mar.na r, michelow a. j oral rehabil 2017;44(7):545-62. 3. orthodon.c treatment need and .ming: assessment of evolu.ve malocclusion condi.ons and associated risk factors. grippaudo mm, quinzi v, manai a, paolantonio eg, valente f, la torre g, marzo g. eur j paediatr dent 2020;21(3):203-8. 4. deciduous-den..on malocclusion predicts orthodon.c treatment needs monitoring of the inverted rela2on of gingival pads in a non-syndromic infant vol 11 no 1 (2023) doi 10.5195/d3000.2023.410 h#p://den*stry3000.pi#.edu later: findings from a popula.on-based birth cohort study. peres kg, peres ma, thomson wm, broadbent j, hallal pc, menezes ab. am j orthod dentofacial orthop 2015;147(4):492-8. 5. possibilieà di tratamento della patologia orofaciale nelle sindrome di down com la placa di regolalazione motoria. ca.llomorales r, avalle c, schimdt r. rev pediatr preven.va sociale 1984;34(1):1-4. 6. programa preven.vo de maloclusiones en los bebés. camargo mcf. in: feller c, gonçalves ean. actualización en clinica dental. 1st ed. são paulo:artes médicas; 1998:40542. 7. orientações básicas e propostas de intervenção na den.ção decídua. roulet cp, chedid sj, correa fnp, correa msnp. in: chedid sj. ortopedia e ortodon.a para a den.ção decídua. atendimento integral ao desenvolvimento infan.l. 1st ed. são paulo: ed. santos; 2013:223-41. 8. proposta de intervenção preven.va – massagens e exercícios de compressão digital para casos de retrogna.smo e progna.smo. chedid sj, roulet pc, silva mo. in: chedid sj. prevenção de maloclusão no bebê. monitoramento do desenvolvimento crânio facial desde a gestação. 1st ed. são paulo:editora napoleão/quintessence; 2022:388-404. 9. anterior crossbite correc.on in the early deciduous den..on. vadiakas g, viazis ad. am j orthod dentofacial orthop 1992;102(2):160-2. 10. interven.on proposal for infant prognathism: a case report in a non-syndromic pediatric pa.ent. chedid sj, húngaro r. int j innov res med sci 2021;6(11):1270. 11. proposta de ficha clínica para a avaliação extraoral do bebê com ênfase na prevenção de oclusopa.as. chedid sj, garcia e. in: chedid sj. prevenção de maloclusão no bebê. monitoramento do desenvolvimento cranio facial desde a gestação. 1st ed. são paulo:editora napoleão/quintessence; 2022:220-7. 12. proposta de ficha clínica para a avaliação intraoral do bebê com ênfase na prevenção de oclusopa.as. in: chedid sj. chedid sj, leite esg, roulet pc. prevenção de maloclusão no bebê. monitoramento do desenvolvimento cranio facial desde a gestação. são paulo:editora napoleão/quintessence; 2022:275-83. 13. varia.ons in the mutual rela.onships of the maxillary and mandibular gum pads in the newborn child. clinch l. int j orthod dent child 1934;20(4):359-74. 14. rela.onship of maxillary and mandibular gum pads in the newborn infant. sillman jh. am j orthod oral surg 1938;24(5):409-24. 15. gum pad rela.onships of infants at birth. simpson wj, cheung dk. j can dent assoc (toronto) 1973;39(3):182-8. 16. assessment of intraoral findings of neonates, born in and around meerut city. niranjan mm, srivastava n, rana v, chandna p int j clin pediatr dent 2020;13(1):48-52. 17. aspectos epidemiológicos do desenvolvimento da oclusão no período pré-dentário [dissertação de mestrado pelo curso de pós -graduação em ortodon.a]. brandão amr. campinas:universidade são leopoldo mandic; 2003:85p. 18. avaliação da oclusão no período pré-dentário em prematuros nascidos na fundação santa casa de misericórdia do pará. almeida mnf, siqueira to, ribeiro smm. rev paraense med 2007;2(4):31-6. 19. es.mulação sensoriomotora intra e extra-oral em neonatos prematuros: revisão bibliográfica. lemes ef, silva thmm, correr ama, almeida eoc, luchesi kf. rev cefac 2015;17(3):945-55. 20. brasil. ministério da saúde. guia de atenção a saúde bucal da pessoa com deficiência. 2019:78-82. 21. the impact of orthodon.c treatment on the quality of life a systema.c review. zhou y, wang y, wang x, volière g, hu r. bmc oral health 2014;14:66. 22. interven.ons with pregnant women, new mothers and other primary caregivers for preven.ng early childhood caries. riggs e, kilpatrick n, slack-smith l, chadwick b, yelland j, muthu ms, gomersall jc. cochrane database syst rev 2019;2019(11):cd012155. 23. an overview for clinical applica.on of gesta.onal ultrasonography in prenatal dental care. a literature review. chedid sj. int j paediatr dent abstracts iapd 2021:1534. 24. three-dimensional ultrasound and maternal bonding, a third trimester study and a review. de jong-pleij eap, ribbert lsm, pistorius lr, tromp e, mulder ejh, bilardo cm. prenatal diag 2012;33(1):81-8. 25. reabilitação neuro-oclusal. 2nd ed. planas p. rio de janeiro:medisi; 1997. monitoring of the inverted rela2on of gingival pads in a non-syndromic infant vol 11 no 1 (2023) doi 10.5195/d3000.2023.410 h#p://den*stry3000.pi#.edu 26. the effec.veness of early interven.ons on malocclusion and its impact on craniofacial growth: a systema.c review. valerio p, poklepovic-pericic t, rossi a, grippaudo c, campos jst, borges do nascimento ij. contemp pediatr dent 2021;2(2):72-89. 27. predic.on of post-treatment outcome a~er combined treatment with maxillary protrac.on and chincap appliances. yoshida i, yamaguchi n, mizoguchi i. eur j orthod 2006;28(1):89–96. 822 final evalua&on on microhardness of self-adhesive pediatric filling vol 13 no 1 (2025) doi 10.5195/d3000.2025.822 h#p://den*stry3000.pi#.edu evaluation of microhardness of self-adhesive pediatric filling sohaib qais alwan, rusal saad ahmed, ali saad ahmed, saif saad kamil tikrit university, tikrit, iraq abstract objec&ve: the purpose of this study was to evaluate how beverages affected the self-adhesive hybrid pediatric cement's microhardness. materials and methods: specimens measuring 5 mm in diameter and 2 mm in thickness were fabricated from surefil one self-adhesive hybrid gic and subjected to exposure by acidic beverages. the study involved three groups (arhficial saliva as control, cola, sparkling water with lemon). samples for vickers hardness were immersed for 15 minutes daily for 28 days. then, samples were tested using a vickers microhardness tester. stahshcal analysis involved the use of one-way anova and post hoc tucky test at the level of significance of 0.05. results: when surefil one samples were submerged in simulated saliva, their vickers microhardness significantly decreased. samples submerged in cola showed the largest decline, indicahng a negahve impact on material integrity. conclusions: beverages affected the self-adhesive hybrid pediatric cement's microhardness. keywords: fluoride release; glass ionomer cement; microhardness; surefil one. cita9on: alwan sq, et al. (2025) evalua9on of microhardness of self-adhesive pediatric filling. den9stry 3000. 1:a001 doi:10.5195/d3000.2025.822 received: january 7, 2025 accepted: january 14, 2025 published: february 17, 2025 copyright: ©2025 alwan sq, et al. this is an open access ar9cle licensed under a crea9ve commons ayribu9on work 4.0 united states license. email: ali.s.ahmed@tu.edu.iq introduction because of its exceptional qualities, which include fluoride release, biocompatibility, and adhesion to dentin and enamel, glass-ionomer cements have been used in both restorative and preventative dentistry [1,2]. glass ionomer cements are a crucial and essential component of several restorative, pedodontic, and preventative dental procedures. this is because these materials may closely mimic the structure of teeth and allow for remineralization at the toothmaterial contact. gics stand out as practical and clinically essential among the wide range of dental materials that are accessible, and they are a mainstay of modern dentistry treatment [3]. recent developments in dental material science have sparked a great deal of interest in learning more about how different environmental conditions affect gic characteristics. one area of interest is how various drinks and beverages affect some of these restorations' mechanical, chemical, and physical characteristics [4]. for both academics and clinicians, knowing how popular beverages affect glass ionomer materials is crucial because it provides valuable information on how long dental restorations will last in real-world situations [5]. depending on the beverage's composition and ph levels, different drinks may interact differently with glass-ionomer cements. acidic drinks, including carbonated drinks and citrus juices, pose a risk to gics because they cause acidic erosion and filling material deterioration [6,7]. the integrity and qualities of the cement matrix may be compromised by the breakdown of glass particles brought on by the acidic environment. furthermore, extended exposure to acidic drinks might hasten the gic's fluoride release, decreasing its effectiveness and encouraging remineralization [8]. in a previous study, conventional and resinmodified glass ionomer cements evalua&on on microhardness of self-adhesive pediatric filling vol 13 no 1 (2025) doi 10.5195/d3000.2025.822 h#p://den*stry3000.pi#.edu revealed qualitative surface changes after immersion in different drinks (coca-cola, lemonade, and fuzetea), characterized by the presence of unevenly distributed micro-cavities on the surface [9]. furthermore, research suggests that such drinks can have erosive effects on gics, with different erosion patterns observed. the depth of erosion was found to be greater at the margin than at the body of the restoration [10]. fluoride release from dental restorative materials is an important aspect of their effectiveness in avoiding tooth decay and supporting oral health [11]. several research have been undertaken over time to explore the fluoride release qualities of different dental repair materials [12,13]. however, these studies have frequently produced significantly variable results, which can be due to variances in methodology, specimen size, storage conditions, and measuring procedures [14]. in a previous study, it was found that glass carbomer exhibited the highest fluoride release into deionized water compared to conventional gic (chemfil rock) and resinmodified gic (fuji ii lc). however, the fluoride uptake by glass carbomer did not lead to increased fluoride release, unlike the conventional and resin-modified gics, where fluoride uptake enhanced subsequent fluoride release. this suggests that while glass carbomer may be highly effective in initial fluoride release, its recharging mechanism differs from other gics [15,16]. material and methods the materials and its composition used in the study are listed in (table 1). table 1. materials used. material composition surefil one self-adhesive composite hybrid powder: silanated aluminum-phosphorus-strontium-sodiumfluoro-silicate glass, dispersed silicon dioxide, ytterbium fluoride, and pigments liquid: acrylic acid, polycarboxylic acid, bifunctional acrylate, self-cure initiator, camphorquinone, and stabilizer cola carbonated water high fructose corn syrup (or sucrose) caramel color (e150d) phosphoric acid natural flavors caffeine citric acid sodium citrate evalua&on on microhardness of self-adhesive pediatric filling vol 13 no 1 (2025) doi 10.5195/d3000.2025.822 h#p://den*stry3000.pi#.edu sparkling water with lemon sparkling water fresh lemon juice lemon slices study design the purpose of this prospective in vitro study was to investigate the effects of acidic drinks on surefil one's vickers microhardness. three groups—group a, group b, and group c—were formed according to the immersion solutions that were used: sparkling water, cola, and artificial saliva, respectively. there were ten samples in each group, for a total of thirty samples per test condition. samples preparation samples were fabricated using molds crafted from clear acrylic plates (perspex cell cast acrylic, clairvaux-les-lacs, france). for the microhardness test, disks were standardized to dimensions of 5 mm in height and 2 mm in thickness [11,17,18]. the molds were put on the surface of a thick glass plates, then celluloid transparent strips (stripmat, polydentia, ch-6805 mezzovico, switzerland) were interposed between them to ensure a even surface and reduce the risk of air bubbles. after filling the molds with surefil one material, excess material was removed, and another transparent strip was applied followed by a second glass plate. samples were polymerized for 20 seconds using an led curing light source (optilight ld max, gnatus, ribeirão preto, sp. brasil). the tip of the light cure 8mm in diameter and the intensity of the light of 1200 mw as measured with a radiometer (denshine, china). after curing, the samples were cautiously taken out of the molds and finished by only trimming any excess with sheets of 1200 grit silicon carbide. immersion procedure three groupings of samples were created: group a: as a control, disks were submerged in fake saliva for the length of the trial. group b: for 28 days, samples were submerged in the acidic beverage (cola) for 15 minutes every day. group c: similarly to group b, samples were submerged in the freshly made acidic beverage (sparkling water with lemon) for 15 minutes every day for 28 days, and the ph was measured with a ph meter (milwaukee, usa). when not submerged in liquids, samples in groups b and c were maintained in artificial saliva. vickers microhardness ten disc-shaped samples per group, each measuring 5 mm in height and 2 mm in diameter, were prepared for vickers microhardness testing. after removal from the molds, the samples were incubated for 24 hours at 37°c. surface microhardness was evaluated by a digital tester (laryee hvs5manufacturing limited, beijing, china) as shown in (figure 1). three indentations were made within 15 seconds of dwell time with a load of 100 g and a magnification of 20×. the mean surface microhardness value for each sample was recorded in vickers hardness numbers (vhn). figure 1. vickers microhardness test. evalua&on on microhardness of self-adhesive pediatric filling vol 13 no 1 (2025) doi 10.5195/d3000.2025.822 h#p://den*stry3000.pi#.edu statistical analysis the statistical analysis for this study involved descriptive statistics such as mean, standard deviation, minimum, and maximum values. analysis of variance (anova) was used to compare the mean values of compressive strength and vickers microhardness among the different groups. independent t test was used for fluoride release and uptake tests. finally, post-hoc analysis such as donnette’s test and tuckey’s hsd, was conducted to determine which specific groups differed significantly from each other results vickers microhardness the highest recorded value was observed in group a, followed by group c, and then group b. tuckey post hoc test revealed highly significant differences between all the groups (table 2). table 2. descriptive statistics for vickers microhardness test. group min max mean ±sd f p-value groups p-value group a 76.50 79.50 78.1000 0.87000 102.345 0.000 a b 0.000 group b 71.50 73.50 72.8000 0.85000 a c 0.000 group c 73.50 76.50 75.0000 0.89000 b c 0.000 levene’s statistics = 3.045, p-value = 0.085 [ns] discussion the observed decrease vickers microhardness of surefil one following immersion in acidic beverages such as cola and sparkling water with lemon underscores the intricate interplay of chemical and physical processes inherent in the composition and behavior of the material. surefil one, a self-adhesive composite hybrid, comprises a unique blend of components designed to impart superior mechanical properties and ease of application in dental restorations. the material's main component, high molecular weight polyacrylic acid functionalized with polymerizable groups (referred to as mopos), forms the backbone of its structural matrix. this polyacrylic acid bears similarities to vitrebond copolymer, present in vitremer and ketac nano. moreover, surefil one contains monomers with two photopolymerizable ends (badep), introduced to the polyacrylic acid chains as cross-linkers, enhancing the material's mechanical strength [19,20]. cola and sparkling water with lemon are both acidic. cola contains phosphoric acid and carbonic acid, sparkling water with lemon contains organic acids such as malic and citric acids. upon exposure to such acidic beverages, the chemical constituents of surefil one are subjected to degradation and alteration. the acidic environment catalyzes hydrolytic degradation of the resin matrix, facilitated by the presence evalua&on on microhardness of self-adhesive pediatric filling vol 13 no 1 (2025) doi 10.5195/d3000.2025.822 h#p://den*stry3000.pi#.edu of phosphoric acid in cola and organic acids in the sparkling water juice. acid-catalyzed hydrolysis and ion exchange processes lead to the leaching of fillers from the resin matrix, resulting in a depletion of reinforcing components and a consequent decline in compressive strength [21,22,23]. in addition, the acids in such beverages can erode the surface layer of surefil one. the acrylic and polycarboxylic acids of surefil one may react with these external acids which leads to breakdown of polymer network. such erosion can expose the underlying filler particles and decrease the surface resistance to wear and indentation [24]. furthermore, the acidic constituents of the beverages can interact with the chemical constituents of surefil one, such as the modified polyacid mopos and cross-linker badep, precipitating complex chemical transformations that compromise the intermolecular bonding within the material [25.] acid-base reactions between the acidic components of the material and those of the beverages result in the formation of new chemical species, altering the material's microstructure and contributing to a reduction in vickers microhardness [26,27]. in addition, the presence of water in such beverages enables the entrance of water inside the structure of surefil one increasing water absorption and causes hydrolysis of the ester bonds in the bifunctional acrylate, which can induce swelling and plasticization of the material, further weakening the mechanical properties and aggravating the decrease in compressive strength and microhardness [28-30]. conclusion almost all glass ionomer cements are affected by beverages, and this study showed that surefil one is also affected by the exposure to cola and sparkling water with lemon, that’s why clinicians should pay attention to such factors to maintain the long-term performance of the restoration and ensure the satisfaction of patients. conflicts of interest the authors declare no competing interest. references 1. park ey, kang s. current aspects and prospects of glass ionomer cements for clinical denmstry. yeungnam univ j med. 2020 jul;37(3):169. doi: 10.5152/yujm.2020.01. [doi not available] 2. hill r. glass ionomer polyalkenoate cements and related materials: past, present and future. br dent j. 2022 may 13;232(9):6537. doi: 10.1038/s41415022-3796-5. 3. pratap b, gupta rk, bhardwaj b, nag m. resin based restoramve dental materials: characterismcs and future perspecmves. jpn dent sci rev. 2019 nov 1;55(1):126-38. doi: 10.1016/j.jdsr.2019.08.001 . 4. ipikhar s, jahanzeb n, saleem m, ur rehman s, mamnlinna jp, khan as. the trends of dental biomaterials research and future direcmons: a mapping review. saudi dent j. 2021 jul 1;33(5):229-38. doi: 10.1016/j.sdentj.2021.04.0 05. 5. sikka n, brizuela m. glass ionomer cement. statpearls. 2024 mar 4. [doi not available] 6. jaiswal s, pusarapu k, alla rk, guduri v, av r, mc ss. solubility of glass ionomer cement in various acidic beverages at different mme intervals: an in vitro study. evalua&on on microhardness of self-adhesive pediatric filling vol 13 no 1 (2025) doi 10.5195/d3000.2025.822 h#p://den*stry3000.pi#.edu int j dent mater. 2022 dec 31;4(3):78-81. doi: 10.1097/idm.0000000000 000211. 7. ganesh s, ganesh sb, jayalakshmi s. effect of carbonated beverages on flexural strength property of restoramve glass ionomer cement. j adv pharm technol res. 2022 nov 1;13(suppl 1). doi: 10.4103/japtr.japtr_238_2 2. 8. nica i, stoleriu s, iovan a, tărăboanță i, pancu g, tofan n, brânzan r, andrian s. convenmonal and resinmodified glass ionomer cement surface characterismcs aper acidic challenges. biomedicines. 2022 jul 21;10(7):1755. doi: 10.3390/biomedicines1007 1755. 9. hübel s, mejàre i. convenmonal versus resinmodified glass-ionomer cement for class ii restoramons in primary molars. a 3-year clinical study. int j paediatr dent. 2003 jan;13(1):2-8. doi: 10.1046/j.09607439.2003.00420.x. 10. wan bakar wz, abdullah a, hussien a. erosion effect of acidic drinks on two types of glass ionomer cement. malays dent j. 2011 jul 1;33(2). [doi not available] 11. ahmed, ali saad; kamil, saif saad; arab, luma nasrat. assessment of peel bond strength between two facial prosthemc materials aper surface treatment methods: in vitro study. journal of internamonal oral health, 2024, 16.1: 63-68. 12. wiegand a, buchalla w, a{n t. review on fluoridereleasing restoramve materials—fluoride release and uptake characterismcs, anmbacterial acmvity and influence on caries formamon. dent mater. 2007 mar 1;23(3):343-62. doi: 10.1016/j.dental.2006.02.0 09. 13. ahmed, ali saad, et al. influence of some plant extracts on anmfungal propermes, hardness, and peel bond strength of heatcured denture sop liner. diyala journal of medicine, 2024, 26.2: 14-28. 14. vandever c. introducmon to research stamsmcal analysis: an overview of the basics. hca healthcare j med. 2020;1(2):71. [doi not available] 15. hasan am, sidhu sk, nicholson jw. fluoride release and uptake in enhanced bioacmvity glass ionomer cement (“glass carbomer™”) compared with convenmonal and resin-modified glass ionomer cements. j appl oral sci. 2019 feb 21;27. doi: 10.1590/1678-77572018-0230. 16. hasan am, sidhu sk, nicholson jw. fluoride release and uptake in enhanced bioacmvity glass ionomer cement (“glass carbomer™”) compared with convenmonal and resin-modified glass ionomer cements. j appl oral sci. 2019 feb 21;27. doi: 10.1590/1678-77572018-0230. 17. ahmed, ali saad; ahmed, rusal saad; arab, luma nasrat. the anmfungal potenmal of cinnamon oil incorporated into a heatpolymerized sop liner. journal of dental materials & techniques, 2024, 13.3. 18. altamimi a, majeed ma. development and evalua&on on microhardness of self-adhesive pediatric filling vol 13 no 1 (2025) doi 10.5195/d3000.2025.822 h#p://den*stry3000.pi#.edu assessment of new bioacmve glass fiber post. part ii: ion’s release. indian j forensic med toxicol. 2021 jul 1;15(3). doi: 10.37506/ijfmt.v15i3.1453 0. 19. iso 9917-1:2007 denmstry—water-based cements—part 1: powder/liquid acid-base cements; iso: geneva, switzerland, 2017. [doi not applicable] 20. chesterman j, jowe� a, gallacher a, nixon pj. bulkfill resin-based composite restoramve materials: a review. br dent j. 2017 mar 10;222(5):337-44. doi: 10.1038/sj.bdj.2017.263. 21. kamil, saif saad; khaudhair, aseel taha; hamid, hind thyab. associamon of dental caries with different abo blood groups. den$stry 3000, 2024, 12.1. 22. valinom ac, neves bg, silva em, maia lc. surface degradamon of composite resins by acidic medicines and ph-cycling. j appl oral sci. 2008;16:257-65. doi: 10.1590/s167877572008000300003. 23. münchow ea, ferreira ac, machado rm, ramos ts, rodrigues-junior sa, zanchi ch. effect of acidic solumons on the surface degradamon of a microhybrid composite resin. braz dent j. 2014;25:321-6. doi: 10.1590/01036440201300035003. 24. zhao q, gao y, jin x, han f, chen k, chen c. influence of acidic environment on hydrolymc stability of mdpca salts with nanolayered and amorphous structures. int j nanomedicine. 2022 apr 13;17:1695-709. doi: 10.2147/ijn.s334292. 25. facio rc, vieira-junior w, basmng rt, turssi cp, do amaral fl, frança fm. influence of fluoridereleasing materials in the inhibimon of enamel and denmn demineralizamon around restoramons. braz dent sci. 2022 dec 19;25(4). doi: 10.5935/16784391.20220052. 26. kukia�rakoon b, hengtrakool c, kedjaruneleggat u. effect of acidic agents on surface roughness of dental ceramics. dent res j (isfahan). 2011;8(1):6. doi: 10.4103/1735-3327.78697. 27. bitencourt sb, catanoze ia, da silva ev, dos santos ph, dos santos dm, turcio kh, guio{ am. effect of acidic beverages on surface roughness and color stability of armficial teeth and acrylic resin. j adv prosthodont. 2020 apr;12(2):55. doi: 10.4047/jap.2020.12.2.55. 28. zimmer s, kirchner g, bizhang m, benedix m. influence of various acidic beverages on tooth erosion. evaluamon by a new method. plos one. 2015 jun 2;10(6). doi: 10.1371/journal.pone.0129 462. 29. almudaihesh f, holford k, pullin r, eaton m. the influence of water absorpmon on unidirecmonal and 2d woven cfrp composites and their mechanical performance. compos part b eng. 2020 feb 1;182:107626. doi: 10.1016/j.compositesb.201 9.107626. 30. abuzenada bm, souror yr, waly as, khlifa y. mutans streptococci growth on evalua&on on microhardness of self-adhesive pediatric filling vol 13 no 1 (2025) doi 10.5195/d3000.2025.822 h#p://den*stry3000.pi#.edu glass ionomer incorporated with chlorhexidine: in-vivo study. braz dent sci. 2020;23(2):1-7. doi: 10.5935/16784391.20200013. 704 2024 maximum mouth opening measurement in rela2on to maximum bite force among children vol 13 no 2 (2024) doi 10.5195/d3000.2024.704 h#p://den*stry3000.pi#.edu maximum mouth opening measurement in relation to maximum bite force among children dalya rafie mohammed, aseel haidar m j al haidar college of dentistry, university of baghdad, iraq abstract objec&ve: this research aimed to study the rela1on between maximal bite force (mbf) and maximum mouth opening among 12-year-old school children. methods: in this cross-sec1onal study, a total of 400 children aged 12 years (194 boys and 206 girls) were examined. the mbf for the right and lei side, as well as the anterior region, were evaluated for all children. the mmo was measured using an electronic digital caliper. to analyze the data path analysis method was used. results: boys showed a higher mmo of 48.797 ± 6.500 than girls (46.710 ± 5.926 mm). the mmo increased with increasing mbf, with significant differences between females and males. conclusion: the mmo was higher in boys than in girls. gender plays a significant role in mbf strength. keywords: bite force; children; digital caliper; human; mouth opening; sensor. cita:on: mohammed dr, et al. (2024) maximum mouth opening measurement in rela:on to maximum bite force among children. den:stry 3000. 2:a001 doi:10.5195/d3000.2024.704 received: august 14, 2024 accepted: august 24, 2024 published: september 25, 2024 copyright: ©2024 dalya r, et al. this is an open access ar:cle licensed under a crea:ve commons asribu:on work 4.0 united states license. email: dr.dalya_1993@yahoo.com introduction bite force (bf) refers to "the force exerted by the masticatory muscles during teeth contact, and it is a key factor in assessing the performance and efficiency of the masticatory system". various factors, including age, gender, craniofacial morphology, body size, masseter muscle thickness, number of erupted teeth, and dental occlusion, influence human bite force (1,2). it is closely associated with the health of the masticatory system (3). tooth eruption indicates overall body growth. also delayed tooth eruption can limit overall body growth (4). teeth play an essential part in the masticatory apparatus as they form the occlusal area responsible for the breakdown of food particles. thus, the total occlusal surface and the number of teeth will determine the grinding force (5). maximum mouth opening can be defined as "the greatest distance, when the mouth is open as widely as possible, between the incisal edges of the mandibular and maxillary central incisors at the midline" (6). both dental disease and the therapy itself can really have a detrimental effect on the quality of life related to oral health (7). various diseases and conditions, including trauma, inflammation, temporomandibular joint problems, neurological disorders, hyperplasia of the coronoid process, rheumatoid arthritis, adverse effects of medicines, tumors, and results of head and neck cancer radiotherapy, can restrict mouth opening (5). in addition, the inability to open the mouth can sometimes lead to legal issues, and pediatric dentists who work with a child's oral cavity encounter maximum mouth opening measurement in rela2on to maximum bite force among children vol 13 no 2 (2024) doi 10.5195/d3000.2024.704 h#p://den*stry3000.pi#.edu different levels of difficulties when there are limitations in mouth opening (8). the present study aimed to examine the possible relationship between the mmo with gender and mbf. material and methods design and sample the study was approved by the scientific and the ethical committee of baghdad university, college of dentistry (731322) and was carried out in agreement with the principles of the declaration of helsinki. all the selected students were in healthy mental and physical conditions. all were examined by the author to reduce bias during the collection of data. a clinical assessment was performed following the receipt of a signed informed consent from the appropriate school authorities and the parents of each child involved in the study. the inclusion criteria for the students in this study were as follows: absence of any preceding orthodontic treatment and/or orthognathic surgery, absence of an anterior or posterior crossbite or an open bite, no history of any systemic disease, no dysfunction of the temporomandibular joint and no congenital disability (cleft lip and palate) or deformed teeth. children with incompletely filled consent forms, facial abnormalities or open bites, fractured central incisors, or any prosthetic device were not included in the study. a total of 400 students (206 girls and 194 boys) who were 12 years old, from various public schools in baghdad were included in this study, which was conducted from december 2022 until may 2023. the participants were separated into two groups, one consisting of 206 girls and the other of 194 boys. ethical considerations the study proposal was submitted to the ethical and scientific committee at the department of pediatrics and preventive dentistry at the college of dentistry at baghdad university in iraq, and a formal registration number was obtained (project no. 731322). the study's objective was clarified to the parents, and explicit agreement was acquired from the parents of the children participating in the study. mbf measurement bite forces were assessed via a portable biting force sensor manufactured by loadstar sensor company, based in the united states. the device had a maximum capacity of 100 kg, it had a diameter of 13 mm and a total vertical height of 7 mm. a computer was utilized to receive, process, and display the information using a specialized software program (9). each student sat upright, with the head in a natural posture without head support and the students were instructed to bite the sensor (that was covered with sterile disposable latex) as hard as they could. bf measurements were taken between the central incisors and the first permanent molars (both right and left). this method was performed three times per student on each side, with a 10second rest period between each bite. the student's mbf was determined by taking the highest of the three bite force readings (9). the latex finger cots were changed after each recording, and 70% isopropyl alcohol was used to clean the device. mmo measurement mmo was evaluated by instructing the students to open their mouth to the maximum possible extent. the examiner recorded the maximum distance at the midline between the incisal edges of the mandibular and maxillary central incisors. in order to measure the mmo, an electronic digital caliper was utilized while the students maximum mouth opening measurement in rela2on to maximum bite force among children vol 13 no 2 (2024) doi 10.5195/d3000.2024.704 h#p://den*stry3000.pi#.edu were in an upright posture, with their heads resting on a solid wall surface (6). statistical analysis spss (version 22, chicago, illinois) was used to examine data. a significant criterion of p < 0.05 was established. frequency of mbf, including the means and standard deviations were calculated. the findings were analyzed using independent t and chi-square tests. regression analysis was used for determining the best predictors of mmo. results table 1 illustrates the distribution of study sample based on gender regarding mmo. a statistical significant difference was found, where the boys had higher mean of mmo than that of girls (48.797 ± 6.500, 46.710 ± 5.926 mm, respectively). according to the results presented in table 2, a significant linear relationship, but a weak positive correlation, was found between the maximal mouth opening and mbf for the left, right, and anterior areas and the total mbf. *: significant. variable maximum mouth opening gender boys girls number % 194 (48.5 %) 206 (51.5 %) minimum 31.05 29.39 maximum 62.08 59.40 mean± sd 48.797 ± 6.500 46.710 ± 5.926 t-test 3.358 p value 0.001* max. mouth opening gender mbfr mbfl mbfa mbf r p r p r p r p boys 0.197 0.006* 0.136 0.058 0.090 0.213 0.176 0.014* girls 0.151 0.030* 0.102 0.144 0.067 0.338 0.130 0.063 total 0.202 0.000* 0.144 0.004* 0.102 0.042* 0.181 0.000* table 1. distribution of study sample based on gender regarding mmo. table 2. pearson's correlation coefficient between maximal mouth opening and maximum bite force. maximum mouth opening measurement in rela2on to maximum bite force among children vol 13 no 2 (2024) doi 10.5195/d3000.2024.704 h#p://den*stry3000.pi#.edu discussion the mmo measurement has clinical and social implications. the inter-incisal distance plus overbite measurement represents the vertical distance travelled by the mandible (6). nevertheless, mezitis et al. (10) emphasized that the functional act of opening the mouth holds more importance since it directly affects both chewing and dental procedures. measurements of the individual bite force have been conducted to assess the therapeutic effectiveness of the prosthetic device and understand the mechanics of chewing. this information is essential for the process of treatment planning, especially when creating the prosthesis (11). the novelty of the present study was to investigate maximal mouth opening in healthy children and mbf in 12-year-old iraqi school children. the current crosssectional study can potentially establish a reference value for biting force that can be used in future investigations. hence, the bite force sensor is suitable for pediatric use according to the present study. the device is userfriendly, noninvasive, and portable. the device has a digital screen and compact load cells that are sufficient for measuring bite force in clinical settings. in addition, it provides standard values and details on the variables that affect their variability. the results revealed a statistically significant difference between mmo and gender, like the results of other studies (5,12). in the present study, the maximal mouth opening was observed to be larger in boys than in girls, which is in line with the previous studies that reported higher mmo among male subjects (6,13,14). however, the present results disagreed with some studies that did not show the effect of gender in mmo (15– 18). on the contrary, some studies showed that girls had higher mmo than boys (13,19,20). this study utilized a specialized bite force sensor to get accurate and comfortable measure to the maximal force exerted while biting. the pearson correlation value indicated a slight positive association between mbf and mmo in permanent teeth. this finding coincides with the results reported by su et al. (21). this study's findings differ from previous research conducted in 2003 by the team of su et al., which focused on children aged nine to twelve. the jawbone and masticatory muscles of young preschoolers still in the process of developing. in contrast, regression analysis revealed a positive connection between bf and maximal mouth opening on both the left and right sides. these findings indicated that the bite force increases as the size of the mouth aperture increases. fields et al. did a study that discovered a similar association among adults (22). according to the findings of the current inquiry, there is a direct relation between mbf and mmo. the study potential limitations may include the study's restricted period. the study utilized a crosssectional design rather than a longitudinal design. additional research is required to be conducted using a larger sample size while also comparing various bf instruments and assessing biting force across different people, as well as other anthropological characteristics and variables. conclusion the study's results indicated that there was a substantial variation in maximum mouth opening across genders among children with permanent dentition, where maximal mouth opening was higher in boys than in girls. the mmo showed favorable associations with mbf. a weak but maximum mouth opening measurement in rela2on to maximum bite force among children vol 13 no 2 (2024) doi 10.5195/d3000.2024.704 h#p://den*stry3000.pi#.edu positive correlation has existed between maximal biting force and mmo. acknowledgements we appreciate all personnel at college of dentistry, university of baghdad, iraq for their support and assistance during the study. authors’ contribution conceptualization, investigation, methodology and writing original draft: dalya rafie. supervision: aseel haidar. review & editing: aseel haidar. source of funding none. conflicts of interest the authors claim no conflicts of interest. references 1. sun kt, chen sc, li yf, chiang hh, tsai hh, li cy, et al. bite-force difference among obese adolescents in central taiwan. j formos med assoc. 2016;115(6):404–10. 2. al-saadi dk, al-mulla aa. digitalized measurement of maximum bite force in iraqi adult sample aged 18–25 years with different malocclusion groups. j bagh coll dent. 2011;23. 3. braun s, bantleon hp, hnat wp, freudenthaler jw, marcotte mr, johnson be. a study of bite force, part 1: relationship to various physical characteristics. angle orthod. 1995;65(5):367–72. 4. salih st, diab bs. breastfeeding effect on primary teeth emergence in relation to craniofacial growth among iraqi infants. med j babylon. 2023;20(1):101–7. 5. medhat ah, al haidar ah. maximum bite force in relation to maximum mouth opening among primary school children. j baghdad coll dent. 2019;31(4). 6. moosa zh, slihem ag, junaidallah aa, alshathri aa, al samh aka, kandil mm. maximum mouth opening and its association with gender, age, height, weight, body mass index, and systemic disease in adult saudi population: a crosssectional study. j int oral heal. 2020;12(2):173–81. 7. ali ra, radeef sm, mohammed nb, diab bs. oral health–related quality of life among dental implant patients in relation to temporomandibular joint function. med j babylon. 2022;19(4):609–14. 8. kumar a, dutta s, singh j, mehta r, hooda a, namdev r. clinical measurement of maximal mouth opening in children: a pioneer method. j clin pediatr dent. 2012;37(2):171–6. 9. khalaf ms, ban ali bds, alsahaf n. maximum bite force among a group of iraqi children in relation to mandibular growth rotation. j ortho, pedo prev dent. 2011;23(1):125–30. 10. mezitis m, rallis g, zachariades n. the normal range of mouth opening. j oral maxillofac surg. 1989;47(10):1028–9. 11. mutt nh, george mkm, nallanchakrava s, charlet r v, alampally hs, animireddy d. estimation of maximum occlusal bite force of school-going children in different dentition: a crosssectional study. int j clin pediatr dent. 2023;16(6):804. 12. komiyama o, arai m, kawara m, kobayashi k, de laat a. pain patterns and mandibular dysfunction following experimental trapezius muscle pain. j orofac pain. 2005;19(2). maximum mouth opening measurement in rela2on to maximum bite force among children vol 13 no 2 (2024) doi 10.5195/d3000.2024.704 h#p://den*stry3000.pi#.edu 13. pullinger ag, liu s, low g, tay d. differences between sexes in maximum jaw opening when corrected to body size. j oral rehabil. 1987;14(3):291–9. 14. patel sm, patel nh, khaitan gga, thanvi rs, patel p, joshi rn. evaluation of maximal mouth opening for healthy indian children: percentiles and impact of age, gender, and height. natl j maxillofac surg. 2016;7(1):33–8. 15. agerberg gör. maximal mandibular movements in children. acta odontol scand. 1974;32(3):147–59. 16. vanderas ap. mandibular movements and their relationship to age and body height in children with or without clinical signs of craniomandibular dysfunction: part iv. a comparative study. asdc j dent child. 1992;59(5):338– 41. 17. sun kt. the changes of bite force and associated influencing factors of the chung hsiao elementary school students in the mixed dentition stage. thesis, china medical college, taichung; 2003. 18. kiliaridis s, kjellberg h, wenneberg b, engström c. the relationship between maximal bite force, bite force endurance, and facial morphology during growth: a cross-sectional study. acta odontol scand. 1993;51(5):323–31. 19. ferrario vf, sforza c, miani a, d’addona a, tartaglia g. statistical evaluation of some mandibular reference positions in normal young people. int j prosthodont. 1992;5(2). 20. rahmania a, tanti i, gita f. the association of normal mouth opening with gender and height. j int dent med res. 2017;10:406–9. 21. su cm, yang yh, hsieh ty. relationship between oral status and maximum bite force in preschool children. j dent sci. 2009;4(1):32–9. 22. fields hw, proffit wr, case jc, vig kwl. variables affecting measurements of vertical occlusal force. j dent res. 1986;65(2):135–8. 703 2024 clinical efficiency of diode lasers (810+980nm) in exposure of maxillary impacted canines vol 13 no 2 (2024) doi 10.5195/d3000.2024.703 h#p://den*stry3000.pi#.edu clinical efficiency of diode lasers (810+980nm) in exposure of maxillary impacted canines alaa faiz abdullah alashbal 1, balsam saadi abdul hameed 2, soudad salman ahmed 3 1 health ministry, baghdad, iraq 2 imam al-kazemin medical city, baghdad, iraq 3 university of baghdad, baghdad, iraq abstract objec&ve: an impacted canine has no erup1on following the comple1on of the normal growth pa6ern. to expose an impacted canine, a laser can be used as an addi1onal tool in addi1on to a scalpel and electrocautery. to compare the effec1veness of a dual diode laser with a tradi1onal scalpel procedure for exposing impacted canines, with the parameters of bleeding and pain as main outcomes. methods: thirty pa1ents were chosen for this inves1ga1on. each of them needed an opera1on for canine impac1on, and they were divided into two groups at random: the study group, which were treated with a dual diode laser (980+810nm), and included fiieen pa1ents, and the control group, which were treated with a scalpel. data obtained prior to, during, and following surgery were compared. there were follow-ups aier two hours, on the second, and third days aier surgery. the student's t and the one-way anova tests were used for all comparisons. results: thirteen males and seventeen females were examined in this study, and their mean age was 23 years. intraopera1ve bleeding score showed a significantly decrease in laser group versus the surgical blade group (0.4666 vs. 0.29333, p=0.001). the postopera1ve pain score was significantly decreased in the laser group compared to the surgical blade group, at two hours (4.6 vs 8.233, p = 0.001), two days (2.067 vs 4.833, p=0.001), and three days (1.066 vs 2.01, p=0.001). there were significant differences in the pain and bleeding scores (p≤ 0.05). conclusion: when using a dual diode laser (810+910nm) as opposed to a scalpel, hemostasis can be achieved with minimal pain. keywords: impacted canine; dual diode laser; 810+980nm; scalpel. cita:on: alashbal afa, et al. (2024) clinical efficiency of diode lasers (810+980nm) in exposure of maxillary impacted canines. den:stry 3000. 2:a001 doi:10.5195/d3000.2024.703 received: august 13, 2024 accepted: august 24, 2024 published: september 25, 2024 copyright: ©2024 alashbal afa, et al. this is an open access ar:cle licensed under a crea:ve commons ayribu:on work 4.0 united states license. email: aalaa.faez2102m@ilps.uobaghdad.edu.iq introduction a tooth that does not erupt after the regular development pattern is finished is known as an impacted tooth [1]. after the third molar teeth, maxillary canines are the teeth that are impacted most frequently. pediatric, orthodontic, or general dentists frequently identify tooth impaction during routine dental examinations. results that are both aesthetically pleasing and practical can be achieved with early discovery, prompt management, and appropriate surgical and orthodontic intervention. impacted canines may result in undesirable complications such as cyst formation and neighboring incisor resorption [2]. radiographs such as opg (orthopantomogram) or cbct scan (cone beam computed tomography) assist in identifying impacted canines in the anteroposterior position regarding the adjacent teeth [3]. classification of maxillary canine impaction includes type iii (distally tipped canine with an overlapped canine crown and the root of the first premolar); type i (canine submerged between lateral incisor and first premolar); type ii (crown is mesially tipped overlapping, pressing lateral incisor tooth to provide a distal tipping of the lateral); canine long axis orientations are type iv/v (horizontally oriented); type vi (canine crown directed upward into orbital fossae); and type vii (horizontally oriented canine clinical efficiency of diode lasers (810+980nm) in exposure of maxillary impacted canines vol 13 no 2 (2024) doi 10.5195/d3000.2024.703 h#p://den*stry3000.pi#.edu long axis with crown situated buccally or interchanging with adjacent teeth) (figure 1) [4]. to develop and carry out the appropriate flap design, as well as orthodontic devices, it is essential that oral surgeons and orthodontists understand the location and angulation of the maxillary impacted canine. it is suggested that only the tissue above the crown of an impacted canine tooth is removed using a laser or conventional surgery for types i, ii, and iii impacted canine teeth. if bone is present, it can be removed with a round handpiece and normal saline irrigation to avoid the bone from heating up. there are three methods for exposing impacted canines: laser, electrosurgery, and scalpel. the most popular technique for canine exposure is still the conventional method, which uses a scalpel. this method has the advantage of being less expensive and having a more durable instrument, but it may be difficult to achieve hemostasis [5–9]. by simultaneously closing blood vessels and cutting, electrocautery can produce excellent hemostasis; nevertheless, excessive heat generation can result in thermal damage, which can cause healing to be delayed relative to scalpel surgery [9,10]. in contrast to patients exposed with a scalpel in a typical manner, those exposed with a laser exhibited fewer local anesthetics during surgery, less discomfort following surgery (requiring fewer analgesics), and no post-operative symptoms, such as pain, oedema and bleeding [10-13]. the fiber-optic tip of the diode laser device cuts, ablates, and reshapes the oral soft tissues more easily than a scalpel can [14]. there is no or minimal discomfort, bleeding, or suturing required when using the laser [15-18]. it provides several advantages for the treatment of soft tissue diseases that conventional surgery does not [19]. this is mostly because the deep penetration of the laser into the surrounding tissues, which promotes the growth of new tissue and cell growth without altering the temperature of the surrounding tissues permanently (photobiomodulation) [17]. since diode lasers are easily absorbed by pigments such as hemoglobin and melanin, they are becoming more and more popular in periodontal surgery. this is because they are safe to use near dental hard tissues due of their poor absorption by water and hydroxyapatite [20]. figure 1. yamamoto et al. [4] classification of canine maxillary impaction. first premolar, canine, and lateral incisor are represented by teeth 2, 3, and 4, respectively. methods for this investigation, thirty patients older than 16 years were chosen. they were separated into two groups at random: the control group, which included fifteen patients treated with a scalpel, and the study group, which included fifteen patients treated with a dual diode laser (980+810nm). all of them needed surgical treatment for maxillary canine impaction, either buccal or palatal. all patients did not have concomitant medical issues. patients who were pregnant or otherwise physically compromised were not included in this study. patients that were treated at the department of surgery, al-kadhimiya teaching hospital were treated with dual diode laser applied at wavelength 810+980nm, 2.5w power, continuous wave mode with a 400 μm tip diameter. whereas the patients treated al amriya specialized dental center were treated with the scalpel method. prior to surgery, all patients had medical history, dental history, clinical examination (extraoral and intraoral examinations), opg (orthopantomogram), or cbct scan (cone beam computed tomography) done to determine the location of the tooth and see whether there was bone covering the impacted tooth. pain, edema, clinical efficiency of diode lasers (810+980nm) in exposure of maxillary impacted canines vol 13 no 2 (2024) doi 10.5195/d3000.2024.703 h#p://den*stry3000.pi#.edu and healing were recorded using follow-up scores on the first-, second-, and fifth-days postsurgery as well as the firstand second week following surgery. the research received ethical approval on september 21, 2023, with decision 435 from the laser institute for postgraduate studies' research ethics committee at the university of baghdad. following the exposure of the impacted canines, an orthodontist attached a bracket. exposure of impacted canine by scalpel the treatment was performed under local anesthetic. cone-beam computed tomography systems (cbct) were used to identify the location of the impacted tooth and whether it was covered by bone. after that, a surgical tool (a blade) was used to create a window and expose the impacted tooth. if bone was present, it was removed surgically using a round bur and continuously irrigation with saline to prevent the bone from heating up. then, irrigation of surgical site by normal saline was done (figure 2) [21]. figure 2. exposure of impacted canine by conventional surgery (scalpel). a cbct, b. before exposure, c. after exposure. exposure of impacted canine by dual diode laser 810+980nm there were fifteen patients in the laser canine exposure study group. the process was carried out with a high-intensity laser called quicklase810+980 nm (canterbury, united kingdom). before the fiber end has been carbonized and initialized, the new laser fiber tip will not cut. using a piece of back carbon paper to start lasing is the simplest way to accomplish this. articulating paper was used to initiate the fiber tip. the patient, the assistant, and the operator were all wearing safety glasses. avoiding instruments with highly reflective or mirrored surfaces was advised due to the possibility of laser beam reflection. care was taken to ensure that the laser was not used close to explosive gases. local infiltration was used to achieve anesthesia (2 percent lidocaine with 1:100:000 epinephrine). laser dots were used to define the surgical area. the procedure began after determining the location of the tooth and if covered by bone or not by cone-beam computed tomography (cbct). then, the impacted tooth was exposed using dual diode laser (wavelength 810+980nm), power 2.5w, a continuous mode energy output of 60mj, and an optical fiber diameter of 400 µm. fiber tip in contact mode and sweeping brushstroke motions were used to cut tissue to make a window in the place of the impacted canine tooth, and then bone coverage if present was removed by surgical round bur with irrigation by normal saline to avoid heating the bone after that, the orthodontist placed a brace on the canine tooth until it pulls it to its natural place .the surgical site was irrigated with normal saline (figure 3). clinical efficiency of diode lasers (810+980nm) in exposure of maxillary impacted canines vol 13 no 2 (2024) doi 10.5195/d3000.2024.703 h#p://den*stry3000.pi#.edu figure 3. exposure of impacted canine by laser. a. cbct, b. before exposure, c. after exposure. statistical analysis spss, version 21.0, france, was used to analyze the data. to compare numerical variables, one way anova and student's t-test were employed. p≤ 0.05 was used as the significant level. results bleeding score (who bleeding scale) [22] table 1. statistical analysis of bleeding score after exposure of impacted canines. intraoperative bleeding score showed significantly lower scores in laser group versus the surgical blade group (0.4666 vs. 0.2.9333, p=0.001) (table 1). pain score (visual analogue scale, vas) [23] when comparing pain scores between laser and surgical groups, laser had lower scores two hours after operation, two days after, and three days after (table 2). table 2. pain scores at two hours, two days, and three days after operation. post-operative bleeding score n means sd se confidence interval (ci) 95% p -value diode laser 15 0.4666 0.639 0.1652 0.1122 -0.8210 <.000 scalpel 15 2.9333 0.258 0.0666 2.7903-3.07631 post-operative pain exposure canine surgery p value scalpel diode laser mean±sd mean±sd 2 hours 8.233±0.903 4.6±1.055 <.0001 2nd day 4.833±0.227 2.067±0.22 <.0001 3rd day 2.01±0.188 1.066 ±0.188 0.0016 clinical efficiency of diode lasers (810+980nm) in exposure of maxillary impacted canines vol 13 no 2 (2024) doi 10.5195/d3000.2024.703 h#p://den*stry3000.pi#.edu discussion the intraoperative and postoperative benefits of dental lasers have resulted in its application in oral soft tissue surgery [24]. the purpose of this study was to determine if treatment exposure of an impacted canine based on two parameters—pain and bleeding—allows the dual diode laser to be more effective than the conventional scalpel procedure. each case treated by the diode laser had less post-operative bleeding and pain at all times assessed in addition to excellent hemostasis and a clear view of the surgical field. hemostasis is enhanced by the contraction of the irradiated tissue against the proximal vasculature and the shrinkage of collagen in blood vessel walls. increased platelet aggregation was seen in erythrocytes damaged by lasers, which promotes intraluminal thrombosis and reduces blood loss [10]. melanin and hemoglobin greatly absorb diode lasers. this enables deep penetration, precise cutting of soft tissue with great homeostasis, and laser treatment of all cases without bleeding [8,25]. these results use the same bleeding scores and are consistent with previous reported data [8]. all cases treated with the diode laser had considerably lower post-operative pain compared to conventional surgery, which was also consistent with previous data [8]. conclusion in conclusion, when compared to scalpel surgery, dual diode laser 810nm+980nm surgery demonstrated efficacy as an alternate approach in the exposure of impacted canines with excellent hemostasis and decreased postoperative pain. the advantages of using lasers include improved patient compliance, less discomfort, no infection or need of post-operatory medication, bloodless and sterile field, faster healing, swift operation, fewer appointments and treatment duration. nonetheless, is desirable that clinicians understand the physics of lasers, their safety, and how they interact with tissue. conflicts of interest the authors declare no competing interests. references 1. lazim ai. the prevalence of impacted maxillary canine among iraqi patients of al-basrah city. journal of the college of dentistry, university of baghdad. 2016 mar;325(3154):1-5. [1] 2. rahman vf, fatah aa. localization of maxillary impacted canine using cone beam computed tomography for assessment of angulation, distance from occlusal plane, alveolar width and proximity to adjacent teeth. journal of baghdad college of dentistry. 2017 mar 13;29(1):70-5. [2] 3. warford jr. jh, grandhi rk, tira de. prediction of maxillary canine impaction using sectors and angular measurement. american journal of orthodontics and dentofacial orthopedics 2003;124(6):651-655. 4. yamamoto g, ohta y, tsuda y, tanaka a, nishikawa m, inoda h. a new classification of impacted canines and second premolars using orthopantomography. asian j oral maxillofac surg. 2003;15:31-7. [3] 5. fornaini c, rocca jp, bertrand mf, merigo e, nammour s, vescovi p. nd: yag and diode laser in the surgical management of soft tissues related to orthodontic treatment. photomedicine and laser surgery 2007;25(5), 381-392. [4] 6. sobouti f, rakhshan v, chiniforush n, khatami m. effects of laser-assisted cosmetic smile lift gingivectomy on postoperative bleeding and pain in fixed clinical efficiency of diode lasers (810+980nm) in exposure of maxillary impacted canines vol 13 no 2 (2024) doi 10.5195/d3000.2024.703 h#p://den*stry3000.pi#.edu orthodontic patients: a controlled clinical trial. progress in orthodontics 2014;15(1), 1-5. 7. matthews dc. seeing the light-the truth about soft tissue lasers and nonsurgical periodontal therapy. journal of the canadian dental association 2010;76(2). 8. ize-iyamu in, saheeb bd, edetanlen be. comparing the 810nm diode laser with conventional surgery in orthodontic soft tissue procedures. ghana medical journal 2013; 47(3), 107-111. 9. amaral mbf, de ávila jms, abreu mhg, mesquita ra. diode laser surgery versus scalpel surgery in the treatment of fibrous hyperplasia: a randomized clinical trial. international journal of oral and maxillofacial surgery 2015; 44(11), 1383-1389. 10. kumar p, rattan v, rai s. comparative evaluation of healing after gingivectomy with electrocautery and laser. journal of oral biology and craniofacial research 2015; 5(2), 69-74. 11. qassab ah, hamad sa. comparison between diode laser and scalpel for lip lengthening in patients with gummy smile. journal of baghdad college of dentistry 2019 mar 15;31(1):60-4. 12. al-karadaghi ts, gutknecht n, jawad ha, vanweersch l, franzen r. evaluation of temperature elevation during root canal treatment with dual wavelength laser: 2780 nm er, cr: ysgg and 940 nm diode. photomedicine and laser surgery 2015 sep 1;33(9):4606. 13. al-karadaghi ts, franzen r, jawad ha, gutknecht n. investigations of radicular dentin permeability and ultrastructural changes after irradiation with er, cr: ysgg laser and dual wavelength (2780 and 940 nm) laser. lasers in medical science 2015 nov;30:2115-21. 14. jawad ha, hamdi sa. nonanesthetic second stage implant surgery by 970nm diode laser. iosr j dent med sci ver i. 2015;14(6):2279-861. 15. migliario m, rizzi m, lucchina ag, renò f. diode laser clinical efficacy and miniinvasivity in surgical exposure of impacted teeth. j. craniofac. surg. 2016, 27, e779–e784. 16. borzabadi-farahani a. the adjunctive soft-tissue diode laser in orthodontics. compend. contin. educ. dent. 2017, 38, e18–e31. 17. aoki a, mizutani k, schwarz f, sculean a, yukna ra, takasaki aa, romanos ge, taniguchi y, sasaki km, zeredo jl, et al. periodontal and periimplant wound healing following laser therapy. periodontol. 2000 2015, 68, 217–269. 18. borzabadi-farahani a, cronshaw m. lasers in orthodontics. in lasers in dentistry—current concepts. textbooks in contemporary dentistry; coluzzi d, parker s, eds.; springer: cham, switzerland, 2017. 19. tawi sr, ahmed ss, algarak bs. histopathological evaluation of two diode lasers 450 and 980 nm in excision biopsies of oral benign lesion. lasers in dental science. 2023 dec;7(4):251-8. 20. al ghazali sr, ahmed ss, abdulhameed bs. the clinical evaluation of oral fibroma lesion removal utilizing two types of diode lasers 980 nm and 450 nm. iraqi journal of laser. 2024 jun 15;23(1):80-9. 21. björksved m, arnrup k, lindsten r. magnusson a, sundell al, gustafsson a, bazargani f. closed vs open surgical exposure of palatally displaced canines: surgery time, postoperative complications, and patients’ perceptions: a multicentre, randomized, controlled trial. european journal of orthodontics, 2018;40(6), 626635. 22. webert ke, cook rj, sigouin cs, et al. the risk of clinical efficiency of diode lasers (810+980nm) in exposure of maxillary impacted canines vol 13 no 2 (2024) doi 10.5195/d3000.2024.703 h#p://den*stry3000.pi#.edu bleeding in thrombocytopaenic patients with acute myeloid leukaemia. haematologica. 20 06;91:1530–1537. 23. sung y-t, wu j-s. the visual analogue scale for rating, ranking and pairedcomparison (vas-rrp): a new technique for psychological measurement. behav res methods 2018; 50(4):1694– 1715. 24. akram hm, ali oh, omran nk, ali ao. diode laser versus scalpel gingivectomy. biomedical and pharmacology journal 2017 dec 21;10(4):1799-804. 25. musaa fe, awazli lg, alhamdani f. gingival enlargement management using diode laser 940 nm and conventional scalpel technique (a comparative study). iraqi journal of laser 2017;16(b):19. a potential natural alternative to chlorhexidine mouthwashes: an in vivo study in rats vol 11 no 1 (2023) doi 10.5195/d3000.2023.503 http://dentistry3000.pitt.edu a potential natural alternative to chlorhexidine mouthwashes: an in vivo study in rats mariana bezamat1,2, alice spitz1*, daniela sales alviano3, dayanne lopes da silva1, vicente telles1,2, marcia grillo cabral4, ana maria bolognese1 1 department of pediatric dentistry and orthodontics, federal university of rio de janeiro. 2 department of oral and craniofacial sciences, university of pittsburgh school of dental medicine. 3 department of microbiology, federal university of rio de janeiro. 4 department of oral pathology, federal university of rio de janeiro. abstract background: in the brazilian northeast region, the leaves of a plant scientifically known as aristolochia cymbifera (a. cymbifera) are therapeutically used to treat different conditions, including fevers, ulcers and stomach disorders. objective: to explore its effects in the oral cavity, this study aimed to evaluate the presence of morphological, cellular, and tissue modifications in the oral mucosa of rats treated with different a. cymbifera solutions. materials and methods: the rats were divided into 6 groups: group 1 was treated with 0.3 ml of vehicle (dmso and water); group 2 treated with 0.3 ml of a. cymbifera-ethanolic extract 4mg/ml; group 3 treated with 0.3 ml of a. cymbifera-hexane extract 4mg/ml; group 4 treated with 0.3 ml of a. cymbiferaethanolic extract 10mg/ml; group 5 treated with 0.3 ml of a. cymbifera-hexane extract 10mg/ml; and group 6 treated with 0.3 ml of chlorhexidine gluconate 0.12%. all groups were treated twice daily by applying the solutions into the oral cavity, over a total period of 24 days. to analyze cytotoxicity on cells, the micronucleus test was performed, and an additional histopathologic analysis was conducted in order to evaluate tissue changes such as inflammatory infiltration, desquamation, and degeneration of cells. results: results showed that the cytotoxicity of the a cymbifera -based solutions was similar to those of chlorhexidine, and all groups showed morphological characteristics of normal mucosa after treatments. conclusion: it is concluded that no cytotoxicity was present after treatment with extracts of aristolochia cymbifera, showing its potential for the development of future clinical trials. keywords: alternative medicine, cytotoxicity, plant extract, aristolochia cymbifera, oral medicine. citation: bezamat, m et al. (2023) a potential natural alternative to chlorhexidine mouthwashes: an in vivo study in rats. dentistry 3000. 1:a001 doi:10.5195/d3000.2023.503 received: may 1, 2023 accepted: may 13, 2023 published: october 20, 2023 copyright: ©2023 bezamat m, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: mbl29@pitt.edu introduction the daily use of toothbrushes, toothpaste and dental floss is essential to control plaque, avoiding the occurrence of dental caries and periodontal disease [1]. nevertheless, in periods of higher cariogenic risk, when quantitative and qualitative changes of oral microbiota occur [2], the use of mouthwashes is highly recommended. the anti-plaque action of these mouthwashes consists of the reduction of bacterial adhesion to the teeth surfaces, inhibition of growth and proliferation of microorganisms and plaque’s intercellular matrix formation [3]. furthermore, mouthwashes have also shown to modify bacterial biochemistry, leading to a less pathogenic biofilm overall [4]. http://dentistry3000.pitt.edu/ a potential natural alternative to chlorhexidine mouthwashes: an in vivo study in rats vol 11 no 1 (2023) doi 10.5195/d3000.2023.503 http://dentistry3000.pitt.edu for everyday plaque control in chronic situations of cariogenic challenge, such as in patients subjected to orthodontic appliances, regular mouthwash with fluoride and triclosan are commonly indicated [5]. however, the incidence of white spot lesions around brackets is still extremely high, highlighting the need for new therapies to prevent this outcome [6]. for short periods of increased cariogenic challenge, such as during treatment of periodontal disease and pericoronitis, the gold standard is the use of chlorhexidine rinses for 14 days. chlorhexidine mouthwashes, although extremely effective, have significant side effects such as alteration of taste perception and teeth staining that limit their long term use [7]. because of the side effects of these mouthwashes commonly used in dentistry, there is a need for safe, effective and affordable alternatives [8]. in the pharmaceutical industry, rinses derived from essential oils and plant extracts have been explored due to their ability to inhibit growth of fungi and bacteria and the effectiveness in reducing plaque and changing the composition of biofilm [9]. for everyday use and for long periods, these natural rinses might be preferable in comparison to chlorhexidine in controlling plaque and mild to moderate gingivitis because they may present less sideeffects. among the essential oils, an in vitro study performed by our group with the alcoholic extract of aristolochia cymbifera (a. cymbifera) showed high effectiveness, reducing the microbial population by about 94.2% after one-hour in vitro tests with the reproduction of biofilms of human saliva [10]. aiming to translate this research into future clinical application of the a. cymbifera extract, the objective of this study was to assess the cytotoxicity of this substance in the oral mucosa of rats, as the experimental animal model. materials and methods experimental design all procedures have been conducted according to the guidelines described by the ethics committee of the federal university of rio de janeiro (ceua-ufrj). the sample consisted of 30 wistar rats, approximately 8 weeks old and weighing approximately 250g. the animals were kept under controlled temperature (22º ± 2ºc), humidity (50 ± 10%) and cycle of 12 hours light/ dark. food and water were available ad libitum. the rats were divided into 6 groups: group 1 (negative control) was treated with 0.3 ml of vehicle (dmso and water); group 2 treated with 0.3ml of a. cymbifera ethanolic extract 4mg/ml; group 3 treated with 0.3 ml of a. cymbifera -hexane extract 4mg/ml; group 4 treated with 0.3 ml of a. cymbifera ethanolic extract 10mg/ml; group 5 treated with 0.3 ml of a. cymbifera -hexane extract 10mg/ml; and group 6 treated with 0.3 ml of chlorhexidine gluconate 0.12% (positive control). all groups were treated twice daily with the solutions applied into the oral cavity by syringes. the experiment was carried out for 24 days. collection of cells occurred three times, with 8 days interval between them: t1 – after 8 days, t2 – after 16 days, and t3 after 24 days. cells were collected from the right and left buccal mucosa and hard palate by scraping http://dentistry3000.pitt.edu/ a potential natural alternative to chlorhexidine mouthwashes: an in vivo study in rats vol 11 no 1 (2023) doi 10.5195/d3000.2023.503 http://dentistry3000.pitt.edu these areas using sterile spatulas. the collection was done in two different ways: a) a smear directly on the slide, wetted with pbs; b) cells collected from each animal were placed in an eppendorf with 0.5 ml of pbs. for fixation of the cells to the eppendorf, 0.5 ml of methanol: acetic acid (3:1 v / v) was added. thereafter, the resulting solution of each collection was distributed into two slides (0.5 ml in each slide). the slides were dried for 24 hours at room temperature before being stained. at the end of the experimental period all animals were sacrificed, and tissue samples were collected from the right and left buccal mucosa, palate, and tongue for histopathology. cytology and imaging we performed an analysis of micronucleus formation since its appearance has been shown to be a reliable marker for cytogenetic damage [11, 12]. the feulgen-fast green method was used for staining. initially the slides were dipped in 1n hcl solution for 15 minutes for acid hydrolysis of nitrogenous bases purine and pyrimidine, allowing subsequent staining of the nuclear material. the slides were then washed with distilled water, where they remained for 15 minutes. subsequently they were immersed in schiff reagent (merck, darmstadt, hessen, germany) for 90 minutes, which is a specific dye for the nuclear chromosomal material exposed by acid hydrolysis. after removal of the schiff reagent, the slides were dipped in 0.5% fast green for cytoplasmic staining (merck, darmstadt, hessen, germany) for 60 seconds and washed with distilled water again. the slides were then subjected to four batteries of absolute alcohol in a row for 2 minutes each, and after dehydration in alcohol, the slides were subjected to three batteries of xylol for 5 minutes each. final preparation of the slides with entellan was then performed (merck, darmstadt, hessen, germany), covering them with coverslips to avoid bubble formation. the slides dried for 24 hours at room temperature. finally, the cells were evaluated with the nikon eclipse e600 microscope (nikon instruments, melville, new york, usa) at a 400x magnification, to determine the frequency of micronucleated cells indicating mutagenicity. figure 1 illustrates possible nuclear changes that indicate genotoxicity and cytotoxicity. data were recorded in tables and analyses of variance with post hoc tukey (intergroup) and the student t test (intragroup) were performed. histopathology the tissue samples collected from right and left buccal mucosa, palate, and tongue were processed, embedded with paraffin, cut into 5μm sections and adhered to glass slides. finally, the slides were stained by hematoxylin and eosin and analyzed by light microscopy. histopathology analysis evaluated the presence of tissue changes such as inflammatory infiltration, desquamation, degeneration of cells and other injuries. photomicrographs were taken using a microscope coupled to a leica dm500 digital camera of 5 mega pixels icc50. http://dentistry3000.pitt.edu/ a potential natural alternative to chlorhexidine mouthwashes: an in vivo study in rats vol 11 no 1 (2023) doi 10.5195/d3000.2023.503 http://dentistry3000.pitt.edu figure 1. schematic of potential nuclear changes. results in the cytologic analysis we were able to observe the presence of different nuclear changes, such as cells without nucleus and with or without granules, illustrated in figure 2. figure 2. nuclear changes (100x): a) control cell, b) pyknosis, c) cell without nucleus and without granules, d) karyolysis, e) cell without nucleus and with granules. exposure genotoxicity chromosomal breakage classic micronucleous spindle’s interference apoptosis pyknosis, chromatin condensation, cariorrexis cytotoxicity cytotoxicity necrosis keratinization pyknosis, chromatin condensation, cariorrexis, karyolysis http://dentistry3000.pitt.edu/ a potential natural alternative to chlorhexidine mouthwashes: an in vivo study in rats vol 11 no 1 (2023) doi 10.5195/d3000.2023.503 http://dentistry3000.pitt.edu the a. cymbifera -hexane extract 10mg/ml (group 5) showed a significant increase in the count of cells without nucleus with time. however, no other treatment groups showed the same trend (table 1). on the other hand, when the count of cells without nucleus and without granules at different intervals was analyzed, group 2 treated with 4mg/ml of the a. cymbifera ethanolic extract showed a significant increase count from day 8 to day 16 and a significant decrease from day 16 to day 24 (table 2). the cytotoxicity capacity of the a. cymbifera based solutions was similar to those of chlorhexidine and no significant differences intergroups were observed (tables 1 and 2). table 1. count of cells without nucleus at intervals of 8, 16, and 24 days. group 8 days (t8) 16 days (t16) 24 days (t24) average (sd) intergroup comparison* average (sd) intergroup comparison* average (sd) intergroup comparison* group 1 59.50 (62.93)a a 31.50 (30.40)a a 54.50 (19.09)a a group 2 60.50 (33.23)a a 59.50 (21.92)a a 40.50 (9.19)a a group 3 69.50 (24.74)a a 42.50 (0.70)a a 51.00 (19.79)a a group 4 38.00 (18.38)a a 32.00 (5.65)a a 41.00 (16.97)a a group 5 36.00 (11.31)a a 45.50 (10.60)b a 62.00 (14.14)b a group 6 66.50 (27.57)a a 80.50 (57.27)a a 44.50 (7.77)a a a, b, c, different lowercase letters mean that there is statistically significant difference (p <0.05) between different time in the same group (same row). *different uppercase letters mean that there is a statistically significant difference (p <0.05) between groups at each time (same column). sd – standard deviation. http://dentistry3000.pitt.edu/ a potential natural alternative to chlorhexidine mouthwashes: an in vivo study in rats vol 11 no 1 (2023) doi 10.5195/d3000.2023.503 http://dentistry3000.pitt.edu table 2. count of cells without nucleus and without granules at intervals of 8, 16, and 24 days group 8 days (t8) 16 days (t16) 24 days (t24) average (sd) intergroup comparison* average (sd) intergroup comparison* average (sd) intergroup comparison* group 1 16.50 (3.53)a a 15.50 (0.70)a a 23.50 (6.36)a a group 2 9.50 (6.36)a a 29.00 (7.07)b a 21.00 (16.97)ab a group 3 12.50 (3.53)a a 18.50 (16.26)a a 24.50 (20.50)a a group 4 11.00 (1.41)a a 13.00 (1.41)a a 10.00 (7.07)a a group 5 7.00 (1.41)a a 13.50 (4.94)a a 23.00 (2.82)a a group 6 12.50 (12.02)a a 24.00 (7.07)a a 10.50 (9.19)a a a, b, c, different lowercase letters mean that there is statistically significant difference (p <0.05) between different time in the same group (same row). *different uppercase letters mean that there is a statistically significant difference (p <0.05) between groups at each time (same column). sd – standard deviation. http://dentistry3000.pitt.edu/ a potential natural alternative to chlorhexidine mouthwashes: an in vivo study in rats vol 11 no 1 (2023) doi 10.5195/d3000.2023.503 http://dentistry3000.pitt.edu upon examination of the tissues previously submitted to treatments, all groups showed morphological characteristics of normal mucosa in the histopathologic analysis. figure 3 shows the most representative photomicrographs of each group. discussion in the northeast of brazil, leaves of a. cymbifera are used to treat liver and stomach disorders, snake bites, fever and ulcer [13]. the root of a. cymbifera is believed to have therapeutic properties [13]. this study aimed to evaluate the presence of morphological, cellular, and tissue changes in the oral mucosa of 30 wistar rats after topical application of different a. cymbifera extract solutions. our results showed that cytologically, most of the extract concentrations tested did not show significant nuclear changes that would indicate cytotoxicity. histologically, all tissue samples were also considered normal. both the efficacy and the absence of cytotoxicity of this plant extract suggests the possibility of a natural alternative for chlorhexidine mouthwashes. a disadvantage of chlorhexidine is the alteration of taste perception that it causes after a few days of use. chlorhexidine is amphiphilic, which means it’s both lipophilic and hydrophilic, a characteristic suggested to be the main cause of the taste alteration as side effect [7]. on the other hand, a. cymbifera has a strong bitter taste that we have tried to overcome with different dilutions. even though natural and herbal substances a b c d e f g h i j figure 3. selection of tissue structures in different treatment groups: a) tongue (group 4 – 4x), b) tongue (group 3 – 40x); c) tongue (group 1 – 4x), d) palate (group 4 – 40x), e) palate (group 5 – 40x), f) palate (group 2 – 40x); g) palate (group 3 -10x), h) buccal mucosa (group 4 – 10x), i) buccal mucosa (group 3 – 10x), j) buccal mucosa (group 5 -10x). http://dentistry3000.pitt.edu/ a potential natural alternative to chlorhexidine mouthwashes: an in vivo study in rats vol 11 no 1 (2023) doi 10.5195/d3000.2023.503 http://dentistry3000.pitt.edu popularly used for nutritional purposes, medications and even mouthwashes are taken considerably well by users, additional experiments are warranted in order to provide the extract with a more pleasant taste before commercialization. additional tests to determine whether a. cymbifera would cause any teeth staining would also be desirable as the next step. using the kill-kinetics assay, the a. cymbifera alcoholic extract has previously demonstrated favorable results in microorganism’s growth inhibition after 1 hour of in vitro exposure [10]. in conclusion, although the extract of a. cymbifera showed no cytotoxicity, no abnormal tissue characteristics, there is a need for clinical trials to confirm the usability and efficacy of this substance in humans before it is applied clinically or commercialized. disclosure of interest the authors declare that they have no competing interests. authors' contributions these authors contributed equally to this work. references 1. madan, c., et al., a knowledge, attitude, and practices study regarding dental floss among dentists in india. j indian soc periodontol, 2014. 18(3): p. 361-8. 2. lucchese, a., et al., changes in oral microbiota due to orthodontic appliances: a systematic review. j oral microbiol, 2018. 10(1): p. 1476645. 3. chen, f. and d. wang, novel technologies for the prevention and treatment of dental caries: a patent survey. expert opin ther pat, 2010. 20(5): p. 681-94. 4. de oliveira, j.r., et al., mouthwashes: an in vitro study of their action on microbial biofilms and cytotoxicity to gingival fibroblasts. gen dent, 2018. 66(2): p. 28-34. 5. haas, a.n., et al., mouthwashes for the control of supragingival biofilm and gingivitis in orthodontic patients: evidence-based recommendations for clinicians. braz oral res, 2014. 28(spe): p. 1-8. 6. benson, p.e., et al., fluorides, orthodontics and demineralization: a systematic review. j orthod, 2005. 32(2): p. 102-14. 7. helms, j.a., et al., effects of chlorhexidine on human taste perception. arch oral biol, 1995. 40(10): p. 91320. 8. mulani, m.s., et al., emerging strategies to combat eskape pathogens in the era of antimicrobial resistance: a review. front microbiol, 2019. 10: p. 539. 9. khameneh, b., et al., review on plant antimicrobials: a mechanistic viewpoint. antimicrob resist infect control, 2019. 8: p. 118. 10. alviano, w.s., et al., in vitro antioxidant potential of medicinal plant extracts and their activities against oral bacteria based on brazilian folk medicine. arch oral biol, 2008. 53(6): p. 545-52. 11. erdemir, e.o., a. sengun, and m. ulker, cytotoxicity of mouthrinses on epithelial cells by micronucleus test. eur j dent, 2007. 1(2): p. 80-5. 12. tolbert, p.e., c.m. shy, and j.w. allen, micronuclei and other nuclear anomalies in buccal smears: a field test in http://dentistry3000.pitt.edu/ a potential natural alternative to chlorhexidine mouthwashes: an in vivo study in rats vol 11 no 1 (2023) doi 10.5195/d3000.2023.503 http://dentistry3000.pitt.edu snuff users. am j epidemiol, 1991. 134(8): p. 840-50. 13. giorgetti, m., g. negri, and e. rodrigues, brazilian plants with possible action on the central nervous system: a study of historical sources from the 16th to 19th century. j ethnopharmacol, 2007. 109(2): p. 338-47. http://dentistry3000.pitt.edu/ 784 2025 phytochemical analysis and an1bacterial ac1vity of stevia rebaudiana bertoni leaves extract against streptococcus sanguis (a primary inhabitant of dental plaque): in vitro study vol 13 no 1 (2025) doi 10.5195/d3000.2025.784 h#p://den*stry3000.pi#.edu phytochemical analysis and antibacterial activity of stevia rebaudiana bertoni leaves extract against streptococcus sanguis (a primary inhabitant of dental plaque): in vitro study manar ibrahim ahmed1, safa ali hamad2, maha abdulsalam2 1 al-bayan university college of dentistry, baghdad, iraq 2 al-hikma college university, department of dentistry, baghdad, iraq abstract objec&ve: dental plaque is considered the primary causa4ve agent in developing periodontal diseases. early colonizers of dental plaque, such as streptococcus sanguis (s.sanguis), are crucial in the succession steps of biofilm forma4on. as an alterna4ve to the commonly used chlorohexidine (chx), it is of interest to find naturally occurring an4microbial substances from plants. materials and methods: volunteers were asked to provide plaque samples. microscopic examina4on, gram stain, optochin test, catalase test and polymerase chain reac4on were used to ensure the iden4fica4on of s. sanguis. stevia rebaudiana bertoni leaves extracted by 70% ethanol alcohol. four experiments have been done in this study: the suscep4bility of s. sanguis to stevia extract, the minimum inhibitory (mic) and the minimum bactericidal (mbc) concentra4ons, and explora4on of the extract effec4ve cons4tuents by using hplc. results: stevia extract had good an4bacterial ac4vity with varying inhibi4on zone diameters that were concentra4on dependent, but 0.2% chx showed bever ac4vity with a sta4s4cally significant difference (p < 0.05). both mic and mbc were at 16 mg/ml. hplc analysis confirmed the presence of an4bacterial cons4tuents: narigenin 25.76 ppm, catechin 30.25 ppm, coumarin 25.47 ppm, and kaempferol 4.59 ppm. conclusions: the an4microbial ac4vity of the alcoholic stevia rebaudiana bertoni leaf extract was sa4sfactory. the study extract exhibited lower an4bacterial ac4vity at 512 mg/ml of stevia extract, while 0.2% chx had superior ac4vity overall. hplc showed that the alcoholic leaves extract of stevia rebaudiana bertoni contains several ac4ve an4bacterial components: narigenin, catechin, coumarin and kaempferol. keywords: streptococcus sanguis, stevia rebaudiana (bertoni), an4bacterial ac4vity, high performance liquid chromatography. cita;on: ahmed mi, et al. (2025) phytochemical analysis and an;bacterial ac;vity of stevia rebauduana bertoni leaves extract against streptococcus sanguis (a primary inhabitant of dental place): in vitro study. den;stry 3000. 2.a001 doi:10.5195/d3000.2025.784 received: november 29, 2024 accepted: december 18, 2024 published: february 5. 2025 copyright: ©2025 ahmed mi, et al. this is an open access ar;cle licensed under a crea;ve commons axribu;on work 4.0 united states license. email: manar.i@albayan.edu.iq introduction many kinds of microbes call the mouth and gums their home [1]. the mouth supports distinct microbial communities, with each site containing an average of 50 species (a subset of 1,000 species capable of oral colonization) [2]. oral surfaces are most often colonized by streptococci and actinomyces species, interbacterial streptococci binding has major implications for periodontal disease development in that several co-adhering organisms are periodontal pathogens. decreased oxygen tensions favor population shifts, allowing strict anaerobes like bacteroidaceae spp. and spirochaetes to flourish [3,4]. biofilm in the form of supragingival and subgingival plaque causes periodontal disease [5]. tamura et al. (2008) [6] postulated that some kinds of bacteria found above the gum line would entice free-living pathogens to the subgingival plaque, hence exacerbating periodontal disorders. ximenez et al. (2000a,b) [7,8] discovered that phytochemical analysis and an1bacterial ac1vity of stevia rebaudiana bertoni leaves extract against streptococcus sanguis (a primary inhabitant of dental plaque): in vitro study vol 13 no 1 (2025) doi 10.5195/d3000.2025.784 h#p://den*stry3000.pi#.edu improving periodontal condition and reducing pathogenic bacterial species, including p. gingivalis, may be achieved by eliminating the supragingival plaque from periodontal patients. biofilm bacteria produce a thin extracellular matrix that encloses and protects microbial populations from the environment, including chemotherapy attacks [9]. mechanical methods are required to remove the dental biofilm on a regular basis, and antiseptics such as mouth rinse can aid in biofilm control. medicinal plants are becoming increasingly popular around the globe to treat a wide range of illnesses [10]. stevia rebaudiana (bertoni) "sweet herb of paraguay" is a zero-calorie sweetener native to south america, where it was initially ingested over 200 years ago by native people [11]. stevia rebaudiana leaves contain over 100 phytochemicals, which are secondary metabolites that plants accumulate to defend themselves against microbial infections. they are active ingredients with therapeutic properties used to make medicines and drugs [12]. stevia has anti-microbial, antihypertensive, anti-cariogenic, diuretic, anti-viral, anti-diarrheal, immunomodulatory, antimutagenesis, anti-teratogenesis and anti-allergic properties [13,14]. as far as we are aware, the potential antimicrobial properties of stevia extract against many microorganisms have been investigated, but no one proved the presence of an antibacterial effect against s. sanguis. material and methods the research ethics review board of the college of dentistry medical ethical committee at baghdad university approved the protocol of this study. stevia leaves were collected and extracted at university of baghdad research unit of aromatic and medical plants. by maceration according to seidel, 2012 [15]; stevia leaves powder (100gm) was extracted with 1 liter of 70% ethanol alcohol. using a laboratory spray dryer, the filtered ethanolic extract was spray dried to yield powder. prior to taking plaque samples, consents were obtained. patients were required to refrain from using antibacterial mouthwash for a minimum of one month before the research. brain heart infusion broth, 1.5 ml, was added to sterile collection tubes as soon as samples were taken [16]. samples were cultured on mitis salivarious blood agar aerobically for 24 hours at 37°c. gram stain [17], hemolysis in blood agar [18], sensitivity to optochin [19], catalase reaction [20], and polymerase chain reaction [21] were all used to identify and characterize the isolated colonies. the abiopuretm genomic dna protocol was used to extract the dna from bacterial growth. the concentration of dna sample (20 ng/μl) was measured using a quantus fluorometer device. macrogen company provided the primers in lyophilized state: s. sanguis-f 5`ggatagtggctcagggcagccagt t-3`, s. sanguis-r 5`gaacagttgctggacttgcttgtc3`, annealing temperature 70˚c and product size (pb) 313 [21]. to obtain the stock solution, a concentration of 100 pmol/µl was achieved by dispersing lyophilized primers in 300µl of nuclease-free water. by mixing 10μl of stock primers with 90μl of nuclease-free water, a solution with a concentration of 10 pmol/μl was prepared. following the manufacturer's instructions, a final solution of 20 μl was created by mixing 10 μl of master mix with 1 μl each of forward primer, reward primer, 6 μl of nuclease-free water, and 2 μl of the sample dna. phytochemical analysis and an1bacterial ac1vity of stevia rebaudiana bertoni leaves extract against streptococcus sanguis (a primary inhabitant of dental plaque): in vitro study vol 13 no 1 (2025) doi 10.5195/d3000.2025.784 h#p://den*stry3000.pi#.edu the thermal cycling protocol was followed as directed by the manufacturer, with a reaction volume per run of 20 and a total of 30 pcr cycles. the pcr device was used to amplify the bacterial dna samples with the following settings (table 1). table 1. pcr program. stages temperature (°f) time (min.) rounds preliminary denaturation 203 5 1 denaturation 203 0.5 30 annealing 158 0.5 elongation 161.6 1 definitive extension 161.6 7 1 hold 50 10 to verify pcr amplification, an agarose gel electrophoresis was performed. after sealing the edges of the gel tray with cellophane tapes, the agarose was poured into the tray and allowed to solidify at room temperature for 30 minutes. then, the tray was filled with 1x tae-electrophoresis buffer until the buffer reached a depth of 3-5 mm over the gel's surface. we loaded the pcr products immediately. wells were immediately filled with 5μl of pcr product. after 60 minutes, the electricity was switched on at 100 volts per milliampere. the dna molecule cycles between the cathode and anode ends. gel imaging system was used to visualize the bands stained in ethidium bromide. according to cavalieri et al, (2005) [19], a suspension of s. oralis was prepared by using “direct colony suspension method”, confined colonies were picked from the agar media and grown in 1 ml muellerhinton broth (mhb). the suspension was vortexed and adapted to a 0.600 absorbance at 600 nm, which corresponds to 0.5 mcfarland using an absorbance plate reader [22]. method for dispersing agar drops, as described by cavalieri et al, 2005 [22] was used to test the activity of the study extract against s. sanguis. using a pasteur pipette under aseptic conditions, 6 mm wells of similar depth were created in each mueller hinton agar plate. 50 μl from each of stevia extract concentrations (512mg/ml, 256mg/ml, 128mg/ml, 64mg/ml, 32mg/ml and16mg/ml) were added to each well. the positive control was 0.2 % chlorhexidine gluconate (non-alcoholic), and the negative control was sterile distilled water. for 24 hours, the plates were maintained in an aerobic atmosphere at 37°c. each well's inhibition zone was measured in (mm) using a ruler. in order to determine the minimum inhibitory concentration (mic), the experiment used twofold serial broth micro-dilutions [19,22]. between wells 1 and 9, 100 μl of brain-heart infusion broth (bhi) was added to two rows of a 96-well microplate. there was a two-fold serial dilution process starting from well 1 and continuing until well 9, after which 100 μl of the 512 mg/ml alcoholic stevia extract was added to each well in the appropriate row. well 10 in the first row had 100 μl of broth and 100 μl of 0.2% chx, whereas wells 11 and 12 had 100 μl of mhb alone; the whole first row was regarded as plain, devoid of bacteria. one hundred microliters of the test organism were added to each well in the second row. well 10 in the second row served as the positive control, with 100 μl of 0.2% chx in broth and 100 μl of bacterial suspension; in contrast, wells 11 and 12 in the negative control group contained 100 μl of mhb and 100 μl of bacterial suspension, respectively. overnight, the microtiter plate was incubated at 37 ˚c in an aerobic environment. an "absorbance micro plate reader" calibrated to a wavelength of 600 nm was used to assess the turbidity of the wells after incubation. minimum bactericidal concentration (mbc) was established by placing 50 μl phytochemical analysis and an1bacterial ac1vity of stevia rebaudiana bertoni leaves extract against streptococcus sanguis (a primary inhabitant of dental plaque): in vitro study vol 13 no 1 (2025) doi 10.5195/d3000.2025.784 h#p://den*stry3000.pi#.edu portions from the minimum inhibitory concentration (mic) well and the well immediately above it onto brain-heart agar plates and leaving them to incubate at 37 °c for 24 hours. the appearance of bacteria was then determined by looking at the agar plates [23]. an hplc analysis was used to determine the concentrations of the active ingredients in an alcohol-based stevia rebaudiana leaf extract [24]. this test was carried out at the ministry of science and technology/ material's research department. to detect and quantify narigenin, catechin, coumarin and kaempferol, a stevia alcoholic extract was prepared for hplc testing. blend 1 gram of stevia extract with 5 milliliters of hplcgrade acetonitrile in a glass vial; cover with paraffin wax; vortex well; and subject to an ultrasonic bath set at 35 degrees celsius for 15 minutes. the standards concentration was set to 5 part per million (ppm). after filtering the solution with a 0.45 micro filter, 20 l of the solution was injected into an hplc binary pump machine. to calculate the sample's concentration, the following formula was used: “conc. of sample = (conc.of standard ×area of sample)/(area of standard) ×dilution factor” the data were summarized by maximum, minimum, standard deviation (sd), standard error (se), and mean values. levene test, one way analysis of variance (anova), and shapiro wilk test were used. these tools were part of the statistical package for the social sciences (spss version -22, chicago, illinois, usa). p>0.05 was considered not significant, whereas p<0.05 was considered significant. results identification of s. sanguis: on msa, s. sanguis colonies were small spherical colonies, blue in color with slightly raised surfaces and greatly adhere to the agar surface. on 40x magnification, cells of s. sanguis appeared as spherical purple cocci (grampositive) and they were arranged in medium to long chains. results from biochemical tests confirmed that the study organism is an alpha hemolytic, gram positive, catalase negative and optochin resistant streptococci. the pcr results validated the diagnosis of s. sanguis, the identification of s. sanguis was clarified by pcr findings (figure1). figure 1. s. sanguis dna amplification results. m stands for 100bp ladder marker, and bp stands for base pair. the zones of inhibition for s. sanguis grew in diameter with increasing concentrations of the study extract; the greatest one was in chlorohexidine, and this difference was statistically significant (p > 0.05). also, when comparing each group to each other and to chlorohexidine, the results demonstrated a significant difference using multiple comparisons among groups by tukey's hsd (p more than 0.05). tables 2, 3, and 4 show the results. according to an “absorbance microplate reader”, mic and mbc of the study extract against s. sanguis both were 16 mg/ml. bacteriostatic activity has been observed with 0.2% chx (0.1 percent in broth). phytochemical analysis and an1bacterial ac1vity of stevia rebaudiana bertoni leaves extract against streptococcus sanguis (a primary inhabitant of dental plaque): in vitro study vol 13 no 1 (2025) doi 10.5195/d3000.2025.784 h#p://den*stry3000.pi#.edu table 2. descriptive statistics for the diameter of the s. sanguis inhibition zone in different groups (levene test= 0.829, p value= 0.558). groups/ (mg/ml) mean (±sd) (±se) min. max. 16 12.4 0.418 0.187 12.000 13.000 32 15.1 0.418 0.187 14.500 15.500 64 17.3 0.447 0.200 17.000 18.000 128 19.8 0.570 0.255 19.000 20.500 256 22.6 0.548 0.245 22.000 23.000 512 26.3 0.274 0.122 26.000 26.500 chx 28 0.500 0.224 27.500 28.500 table 3. a one-way analysis of variance (anova) was used to compare the groups' inhibitory zone diameters for s. sanguis. a p-value less than 0.05 is considered statistically significant. anova sum of squares df mean square f p value between groups 996.143 6 166.024 774.778 0.000 sig. within groups 6.000 28 0.214 total 1002.143 34 table 4. tukey hsd analysis of multiple comparisons of s. sanguis inhibition zone diameter between groups. groups/ (mg\ml) ( mean differenc e) (pvalue) 16 32 -2.700 0.00000 significant 64 -4.900 0.00000 128 -7.400 0.00000 256 -10.200 0.00000 512 -13.900 0.00000 chx -15.600 0.00000 32 64 -2.200 0.00000 128 -4.700 0.00000 256 -7.500 0.00000 512 -11.200 0.00000 chx -12.900 0.00000 64 128 -2.500 0.00000 256 -5.300 0.00000 512 -9.000 0.00000 chx -10.700 0.00000 128 256 -2.800 0.00000 512 -6.500 0.00000 chx -8.200 0.00000 256 512 -3.700 0.00000 chx -5.400 0.00000 512 chx -1.700 0.00006 using high-performance liquid chromatography, the presence of effective antibacterial phytochemical compounds was encountered at the following concentrations: narigenin (25.76 ppm), catechin (30.25 ppm), coumarin (25.47 ppm), and kaempferol (4.59 ppm) (figure 2). figure 2. narigenin, catechin, coumarin, and kaempferol were found in the study extract using hplc. rt: retention time. discussion more and more studies are being conducted on plant extracts and the active ingredients in them from various parts of the world for their antibacterial activity [25]. bacteria phytochemical analysis and an1bacterial ac1vity of stevia rebaudiana bertoni leaves extract against streptococcus sanguis (a primary inhabitant of dental plaque): in vitro study vol 13 no 1 (2025) doi 10.5195/d3000.2025.784 h#p://den*stry3000.pi#.edu cannot develop resistance to plant extracts because they are chemicaly complicated in structure, and any of its constituents may have an antimicrobial role [26]. according to studies, stevia rebaudiana (bertoni) is a possible drug candidate with numerous nutritional and medicinal properties. it has been found to have anti-inflammatory, antigingivitis, anti-plaque, anticariogenic and health-promoting characteristics [27, 28]. the current study has been conducted and may be regarded the first report because of the previously stated characteristics and because stevia has no proven antibacterial activity on s. sanguis. the antibacterial effect of the study extract against s. sanguis was found to be concentration dependent, as the concentration of the extract rose, the inhibition zone widened, suggesting that the quantity of soluble active chemicals in the extract was also rising. after comparing the advantages of stevia with the disadvantages of chx, the research extract showed impressive results, with an average inhibitory zone width of 26.3 mm against s. sanguis at 512 mg/ml, which is comparable to the 28 mm seen at 0.2% chx, we might come up with a satisfactory result. the extract's mic and mbc were both found to be 16 mg/ml, showing that it exhibits bactericidal activity. levison, (2004), as cited by mogana et al., (2020) (29), said that if the mbc/mic ratio is less than 4, the agent is bactericidal. the similar outcome was reached by parvekar et al., 2020 [30], who investigated the mic / mbc for silver nanoparticles on staph. aureus. the exact mechanism of stevia's antibacterial action is unknown, although it could be linked to the existence of antimicrobial compounds such as: dihydrodeoxy-streptomycin, diterpene glycosides, tannins, flavonoids, polyphenols and saponins [31-35]; and the extract's phytochemical components' synergism. due to the interaction of multiple distinct chemicals in plant extracts, developing microbial resistance through genetic alterations driven by external stimuli is more challenging [36]. many active compounds were found in alcoholic stevia extract by hplc analysis, including narigenin, catechin, coumarin, and kaempferol. narigenin is a bioactive flavonoid with good antibacterial properties, its concentration in the study extract was (25.76 ppm). studies have reported its inhibitory action against: escherichia coli, staphylococcus aureus, lactobacillus rhamnosus and salmonella typhimurium [37, 38]. after being exposed to narigenin, by altering the ratio of certain fatty acids in their membranes, e. coli and staphylococcus aureus cells demonstrated an increase in membrane fluidity. narigenin also significantly down-regulated genes in bacteria involved in fatty acid production at higher doses [39]. catechin is a polyphenol found in high concentration )30.25 ppm) in the study extract, it has antibacterial activity [40]. the mechanism of catechin action propose that the catechins influence the microbial membrane, altering its fluidity and lowering thiourea and cycloleucine flux [41]. the presence of the negatively charged lipopolysaccharide may explain why it is more effective against gram positive bacteria than gram negative bacteria, since it alters the structure of the membrane and damages the lipid bilayer [42,43]. coumarin concentration in the study extract was (25.47ppm). coumarin is a naturally occurring benzopyrone which is a potent inhibitor of dnagyrase, its derivatives exhibit antibacterial effect against both gram negative phytochemical analysis and an1bacterial ac1vity of stevia rebaudiana bertoni leaves extract against streptococcus sanguis (a primary inhabitant of dental plaque): in vitro study vol 13 no 1 (2025) doi 10.5195/d3000.2025.784 h#p://den*stry3000.pi#.edu and methicillin-resistant staphylococcus aureus (mrsa), novobiocin, vancomycin, and teicoplanin-resistant enterococci species are among the grampositive bacteria that are most often seen [44,45]. the concentration of kaempferol in the study extract was (4.59 ppm). kaempferol separated from camellia oleifera demonstrated broad antibacterial activity against staphylococcus aureus, e. coli, salmonella enteriditis, aspergillus niger, bacillus thuringiensis and rhizopus nigricans [46]. conclusion the results of this study demonstrate that the extract has an antibacterial effect and contains abundant antibacterial components; this might pave the way for further studies into its potential usage in oral hygiene products. additional studies examining its toxicity and antibacterial effects on periodontal diseases are necessary to validate its use as a preventive intervention for oral health. references 1abusleme, l., dupuy, a. k., dutzan, n., silva, n., burleson, j. a., strausbaugh, l. d., gamonal, j., & diaz, p. i. (2013). the subgingival microbiome in health and periodontitis and its relationship with community biomass and inflammation. the isme journal, 7(5), 1016–1025. 2griffen, a. l., beall, c. j., campbell, j. h., firestone, n. d., kumar, p. s., yang, z. k., podar, m., & leys, e. j. (2012). distinct and complex bacterial profiles in human periodontitis and health revealed by 16s pyrosequencing. the isme journal, 6(6), 1176–1185. 3aas, j. a., paster, b. j., stokes, l. n., olsen, i., & dewhirst, f. e. (2005). defining the normal bacterial flora of the oral cavity. journal of clinical microbiology, 43(11), 5721–5732. 4dewhirst, f. e., chen, t., izard, j., paster, b. j., tanner, a. c., yu, w. h., lakshmanan, a., & wade, w. g. (2010). the human oral microbiome. journal of bacteriology, 192(19), 5002– 5017. 5haffajee, a. d., dibart, s., kent, r. l., jr, & socransky, s. s. (1995). factors associated with different responses to periodontal therapy. journal of clinical periodontology, 22(8), 628–636. 6tamura, a., ara, t., imamura, y., fujii, t. & wang, p. (2008). the effects of antibiotics on in vitro biofilm model of periodontal disease. eur j med res, 13, 439445. 7ximénez-fyvie, l. a., haffajee, a. d. & socransky, s. s. (2000a). comparison of the microbiota of supraand subgingival plaque in health and periodontitis. j clin periodontol, 27, 648-57. 8ximénez-fyvie, l. a., haffajee, a. d., som, s., thompson, m., torresyap, g. & socransky, s. s. (2000b). the effect of repeated professional supragingival plaque removal on the composition of the supraand subgingival microbiota. j clin periodontol, 27, 637-47. 9saini, r., saini, s. & sharma, s. (2011). biofilm: a dental microbial infection. journal of natural science, biology, and medicine, 2, 71-75. 10rafiq, k., sherajee, s. j., sufiun, m., mostofa, m., alam, a. & barman, b. (2011). comparative efficacy of stevia leaf (stevia rebaudiana bertoni), methi seeds (trigonella foenumgraecum) and glimepiride in streptozotocin induced rats. international journal, 2229, 7472. phytochemical analysis and an1bacterial ac1vity of stevia rebaudiana bertoni leaves extract against streptococcus sanguis (a primary inhabitant of dental plaque): in vitro study vol 13 no 1 (2025) doi 10.5195/d3000.2025.784 h#p://den*stry3000.pi#.edu 11ashwell, m.)2015(. stevia, nature’s zero-calorie sustainable sweetener: a new player in the fight against obesity. nutrition today, 50, 129. 12shakya, a. k. (2016). medicinal plants: future source of new drugs. international journal of herbal medicine, 4, 59-64. 13abou-arab, a. e., abouarab, a. a. & abu-salem, m. f. (2010). physico-chemical assessment of natural sweeteners steviosides produced from stevia rebaudiana bertoni plant. african journal of food science, 4, 269-281. 14 yildiz-ozturk, e., nalbantsoy, a., tag, o. & yesilceliktas, o. (2015). a comparative study on extraction processes of stevia rebaudiana leaves with emphasis on antioxidant, cytotoxic and nitric oxide inhibition activities. industrial crops and products, 77, 961971. 15 seidel, v. (2012). initial and bulk extraction of natural products isolation. natural products isolation, 27-41. 16 abd noor, h.j, 2020. potential effect of gold nanoparticles against streptococcus mitis and streptococcus oralis (primary periodontal colonizeres) (doctoral dissertation, university of baghdad). 17koneman e, schreckenberge p, allens s, et al. diagnostic microbiology. 4th edn. j.b. lippincott co.usa. 1992. 18buxton r. blood agar plates and hemolysis protocols. asm micro library 2013. 19cavalieri, s., harbeck, r., mccarter, y., ortez, j., rankin, i., sautter, r., sharp, s. & spiegel, c. (2005). manual of antimicrobial susceptibility testing. american society for microbiology. pan american health organization: washington, dc, usa. 20reiner k. catalases test protocol. asm micro library 2013. 21hoshino, t., kawaguchi, m., shimizu, n., hoshino, n., ooshima, t. & fujiwara, t. (2004). pcr detection and identification of oral streptococci in saliva samples using gtf genes. diagn microbiol infect dis, 48, 195-9. 22abdulbaqia, h. r., himratulaznita, w. h. & baharuddin, n. a. (2016). anti-plaque effect of a synergistic combination of green tea and salvadora persica l. against primary colonizers of dental plaque. archives of oral biology, 70, 117-124. 23parvekar, p., palaskar, j., metgud, s., maria, r. & dutta, s. 2020. the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) of silver nanoparticles against staphylococcus aureus. biomaterial investigations in dentistry, 7, 105-109. 24gini, t. g. & jeya jothi, g. (2018). column chromatography and hplc analysis of phenolic compounds in the fractions of salvinia molesta mitchell. egyptian journal of basic and applied sciences, 5, 197-203. 25nayan, r.b. and shukla, v.j. 2011. antibacterial and antifungal activities from leaf extracts of cassia fistulal: an ethnomedicinal plant. j adv pharm technol res.2(2): 104– 109 26tariq, m., gore, m. & aruna, k. (2014). antibacterial and synergistic activity of phytochemical analysis and an1bacterial ac1vity of stevia rebaudiana bertoni leaves extract against streptococcus sanguis (a primary inhabitant of dental plaque): in vitro study vol 13 no 1 (2025) doi 10.5195/d3000.2025.784 h#p://den*stry3000.pi#.edu ethanolic ajwain (trachyspermum ammi) extract on esbl and mbl producing uropathogens. int j pharm pharm sci, 6, 278-84. 27de slavutzky, s. m. b. (2010). stevia and sucrose effect on plaque formation. journal für verbraucherschutz und lebensmittelsicherheit, 5, 213216. 28contreras, m. 2013. anticariogenic properties and effects on periodontal structures of stevia rebaudiana bertoni. narrative review. journal of oral research, 2, 158166. 29mogana, r., adhikari, a., tzar, m. n., ramliza, r. & wiart, c. 2020. antibacterial activities of the extracts, fractions and isolated compounds from canarium patentinervium miq. against bacterial clinical isolates. bmc complementary medicine and therapies, 20, 55. 30parvekar, p., palaskar, j., metgud, s., maria, r. & dutta, s. 2020. the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) of silver nanoparticles against staphylococcus aureus. biomaterial investigations in dentistry, 7, 105-109. 31brambilla, e., cagetti, m. g., ionescu, a., campus, g. & lingström, p. (2014). an in vitro and in vivo comparison of the effect of stevia rebaudiana extracts on different cariesrelated variables: a randomized controlled trial pilot study. caries research, 48, 19-23. 32fasiha a, shahid b, faiz-ulhassan s. nutritional and medicinal properties of stevia rebaudiana. curre res diabetes & obes j (2020); 13(4): 555867. 33shakya, a. k. (2016). medicinal plants: future source of new drugs. international journal of herbal medicine, 4, 59-64. 34raut, d. & aruna, k. (2017). antimicrobial activity of stevia rebaudiana against antibiotic resistant esbl producing uropathogens and evaluation of its antioxidant activity. int. j. adv. res. biol. sci, 4, 110-118. 35lemus-mondaca, r., vegagálvez, a., rojas, p., stucken, k., delporte, c., valenzuela-barra, g., jagus, r. j., agüero, m. v. & pasten, a. (2018). antioxidant, antimicrobial and antiinflammatory potential of stevia rebaudiana leaves: effect of different drying methods. journal of applied research on medicinal and aromatic plants, 11, 37-46. 36moussaoui, f. & alaoui, t. (2016). evaluation of antibacterial activity and synergistic effect between antibiotic and the essential oils of some medicinal plants. asian pacific journal of tropical biomedicine, 6, 32-37. 37parkar, s. g., stevenson, d. e., & skinner, m. a. (2008). the potential influence of fruit polyphenols on colonic microflora and human gut health. international journal of food microbiology, 124(3), 295– 298. 38zhang, y., wang, j. f., dong, j., wei, j. y., wang, y. n., dai, x. h., & deng, x. m. (2013). inhibition of α-toxin production by subinhibitory concentrations of naringenin controls staphylococcus aureus pneumonia. fitoterapia, 86, 9299. 39wang, l. h., zeng, x. a., wang, m. s., brennan, c. s., & gong, d. (2018). modification of membrane properties and fatty acids biosynthesis-related genes in escherichia coli and staphylococcus aureus: phytochemical analysis and an1bacterial ac1vity of stevia rebaudiana bertoni leaves extract against streptococcus sanguis (a primary inhabitant of dental plaque): in vitro study vol 13 no 1 (2025) doi 10.5195/d3000.2025.784 h#p://den*stry3000.pi#.edu implications for the antibacterial mechanism of naringenin. biochimica et biophysica acta. biomembranes, 1860(2), 481– 490. 40toda, m., okubo, s., ikigai, h., suzuki, t., suzuki, y., hara, y. and shimamura, t. (1992) microbiol. immunol. 36, 9991001. 41ring, k., ehle, h., foit, b. and schwarz, m. (1977) flavonoids and bioflavonoids, proc. 5th symp. (farkas, l., abor, h. and kally, f., eds.), pp. 427-437. 18 42perrissoud, d., maignan, m.f. and anderset, g. (1981) int. cong. symp. ser.-r. soc. med. 47, 21-25. 43hajime ikigai, taiji nakae, yukihiko hara, tadakatsu shimamura, (1993). bactericidal catechins damage the lipid bilayer, biochimica et biophysica acta (bba) biomembranes, 1147, 132-136. 44thati, b., noble, a., rowan, r., creaven, b. s., walsh, m., mccan, m., egan, d. & kavanagh, k. 2007. mechanism of action of coumarin and silver (i)–coumarin complexes against the pathogenic yeast candida albicans. toxicology in vitro, 21, 801-808. 45qin, h.-l., zhang, z.-w., ravindar, l. & rakesh, k. 2020. antibacterial activities with the structure-activity relationship of coumarin derivatives. european journal of medicinal chemistry, 112832. 46qiu, y., he, d., yang, j., ma, l., zhu, k. & cao, y. 2020. kaempferol separated from camellia oleifera meal by highspeed countercurrent chromatography for antibacterial application. european food research and technology, 246, 2383-2397. microsoft word 483 2023.docx epstein-barr virus predominance and immunological abnormali6es in oral squamous cell carcinoma vol 11 no 1 (2023) doi 10.5195/d3000.2023.483 h#p://den*stry3000.pi#.edu epstein-barr virus predominance and immunological abnormalities in oral squamous cell carcinoma ahmed talib ghadhban1, marwa mohammed ali jassim1, majid mohammed mahmood2 1college of dentistry, al-muthanna university, al-muthanna, iraq 2department of biology, college of science, mustansiriyah university, baghdad, iraq abstract background: both benign and harmful head and neck disorders have been associated with the epstein-barr virus (ebv). many studies have connected ebv to oral squamous cell carcinoma (oscc). oral squamous cell carcinoma is the most prevalent type of cancer. fresh 6ssue samples from pa6ents with oscc were tested for the presence of epstein-barr virus. objective: to determine the frequency of ebv and il10 expression in oscc pa6ents. materials and methods: fily individuals with oscc and 25 with clinically healthy oral mucosa were studied. in situ hybridiza6on was used for the detec6on of ebv. serum il-10 levels were also evaluated in pa6ents and controls using a human il-10 elisa kit. results: ebv was detected in 4 healthy pa6ents, 6 with moderately differen6ated oscc, 10 with poorly differen6ated oscc, and 19 with undifferen6ated oscc. these differences were sta6s6cally significant (p<0.05). il-10 expression was more common in oscc diagnos6c groups than healthy controls, and the difference in blood il-10 levels between pa6ents and controls was sta6s6cally significant (p<0.01). conclusion: the prevalence of ebv in oscc suggests its possible role in oral cavity malignancy. on the other hand, il10 is expressed at higher levels in oscc biopsies; such elevated concentra6ons may promote viral spread. keywords: oral squamous cell carcinoma; epstein-barr virus; in situ hybridiza6on; ebna; il-10 cita:on: ghadhban, at et al. (2023) epstein-barr virus predominance and immunological abnormali:es in oral squamous cell carcinoma den:stry 3000. 1:a001 doi:10.5195/d3000.2023.483 received: march 27, 2023 accepted: april 10, 2023 published: april 27, 2023 copyright: ©2023 ghadhban, at et al. this is an open access ar:cle licensed under a crea:ve commons avribu:on work 4.0 united states license. email: majidmahmood93@yahoo.com introduction oral cancer is increasing worldwide, although the annual death toll is higher in lowand middle-income countries. people are living longer, and there are more factors that might lead to cancer [1]. squamous cell carcinoma (scc) is the most common kind of oral cancer and poses a significant risk to public health across the world. oral cavity scc has a complex etiology; the incidence rate of this cancer varies considerably based on habits or forms of intake, such as alcohol or cigarette use, exposure to other risk factors, viral infection, and individual genetic susceptibility [2]. the most frequent kind of mouth cancer is oral squamous cell carcinoma (oscc). oscc is the most common kind of oral cancer. although it has been observed in young people, it is more frequent in middle-aged and elderly people [3]. oscc accounted for 91% of all instances of cancer diagnosed in iraq between 2000 and 2008, according to the iraqi cancer registry, which epstein-barr virus predominance and immunological abnormali6es in oral squamous cell carcinoma vol 11 no 1 (2023) doi 10.5195/d3000.2023.483 h#p://den*stry3000.pi#.edu records all cases in the country except those in the north [4]. an analysis of 1,425 biopsy reports showed that oscc was found to be frequent in male patients over 50 [5]. frequency of oscc in iraq and saudi arabia varies greatly [6-12]. viruses cause 15% of human cancers worldwide, including head and neck neoplasms [13]. the epstein-barr virus (ebv) is the third most prevalent cause of infections associated with cancer in the united kingdom, and it is present in 90% of nasopharyngeal cancers (npcs)[14] which is widespread around the world [15]. host-tumor interactions, which occur through various genetic and cellular variables inside the tumor microenvironment, are a crucial part of the complicated process of oral cancer progression [16]. interleukin 10 (il10) is a cytokine that helps stop inflammation. it is also called a human cytokine synthesis inhibitory factor (csif). it primarily controls the immunological response, and when it is absent, inflammation is probable. macrophages are the primary source of il-10 production. il10 performs various tasks, including controlling angiogenesis, cytokine synthesis, tcell proliferation, and inflammatory responses. whether il10 has a dynamic function in carcinogenesis and tumor inhibition is debatable. oral malignancies and solid tumors have been shown to have elevated amounts of il-10 production [17,18]. macrophages produce most il-10. tumor tissues, serum, and saliva showed increased il-10 levels in several cancers. moreover, this increased concentration has been recommended as a sign of a bad prognosis [19]. the aim of this work was to define the existence of ebv and il10 expression in oscc patients. material and methods fifty formalin-fixed paraffinembedded archival tissues were used. they included 25 ocss tissues as well as 25 biopsies from apparently normal tissues. according to the manufacturer's protocol, 4-5 mm sections of the studied tissue block were stuck on "positively charged slides" and used for in situ hybridization for the detection of the expression of ebv-ebna (from abcam company, united kingdom). each test included positive and negative controls, and samples were inspected under a microscope at 10 and 40 magnification [20]. following the manufacturer's instructions, a human il-10 elisa kit (biolegend, san diego, usa) was used to measure the concentration of il-10 in the serum. statistical analysis the spss 24 program was utilized for all the analyses. pearson's chi-square or spearman's rho test were used in all comparisons with an alpha of 0.05. results and discussion immunological profile of oscc immunostaining for il-10 antibody was associated with oscc (table 1, p<0.05). mean il10 immune signaling expression levels were 19.3, 20.7, 23.6, and 29.2, moderately, badly, and undifferentiated grades of oscc respectively. in contrast to the findings of the present study, interleukin-10 was not detected in the blood of patients with scc and adenoid cystic carcinoma of the head and neck in a previous study [21]. serum il-10 overproduction was seen in individuals with various solid and hematopoietic tumors [22], suggesting it may be frequent in some cancers. increased blood il-10 levels were seen in patients with more advanced malignancy [23]. salivary il-10 levels in oscc patients were higher than in healthy participants in the current study, which was shown before [24]. our findings suggest that il-10 in saliva likely serves as a physiological purpose but is not a helpful salivary biomarker for oscc [25]. variables in the oral environment, like periodontal disease and oral microbial flora, could cause protein artifacts to be found. this makes it hard to compare people based on their salivary composition. this can be seen as a limitation of this study. acute stress, which was not measured here, elevates serum and salivary epstein-barr virus predominance and immunological abnormali6es in oral squamous cell carcinoma vol 11 no 1 (2023) doi 10.5195/d3000.2023.483 h#p://den*stry3000.pi#.edu il-10 levels, suggesting that psychosocial variables may influence the present results [26]. in situ hybridization detection of the epstein-barr virus ebna gene among studied samples the expression patterns of ebvebna levels in all groups of oss cancer tissues are shown in table 2 and figure 1. this investigation revealed that ebv was more prevalent in oscc tissues. in a previous study, ebv infection was identified in all 36 (100%) osccs [27], indicating the high incidence of ebv infection in oscc samples. a similar finding was unveiled with the use of an ebv genomic microarray. most samples (82.5%) of biopsy tissues from 57 oscc patients were infected with ebv [28]. cytokine diagnosis oscc affected unaffected pearson's chi-square (p-value) il-10 pg/ml well differentiated 19.3±5.12 11.23±2.11 p˂0.01 moderate differentiated 20.7± 9.03 11.23±2.11 poorly differentiated 23.6± 11.23 11.23±2.11 undifferentiated 29.2±5.17 11.23±2.11 study group diagnosis negative ebv-ebna expression pattern pearson chi-square (p-value) oscc well differentiated 0 (0.0%) 4 (10.26%) p=0.04 moderately differentiated 7 (63.64%) 6 (15.38%) poorly differentiated 3 (27.27%) 10 (25.64%) undifferentiated 1 (9.09%) 19 (48.72%) total 11 (100.0%) 39 (100.0%) table 1: immunological detection of il-10 in oscc table 2: ebv prevalence in oscc epstein-barr virus predominance and immunological abnormali6es in oral squamous cell carcinoma vol 11 no 1 (2023) doi 10.5195/d3000.2023.483 h#p://den*stry3000.pi#.edu conclusion this study found more often ebv in oscc tissues in comparison to osccunaffected tissues, suggesting that ebv infection may contribute to oral tissue carcinogenesis. nevertheless, further investigation is required to identify the role of ebv in the development of oscc. in contrast, oscc biopsies had antibodies to il10, whereas normal tissues were immunonegative. references 1. pennacchio+ g, álvarez c, montes r, cárcamo m, sáez r, mar<nez mj. epstein-barr virus in oral cavity squamous cell carcinoma of chilean pahents. int j oral dent health. 2020;6:106. 2. anwar n, pervez s, chundriger q, awan s, moawer t, ali ts. oral cancer: clinicopathological features and associated risk factors in a high risk populahon presenhng to a major terhary care center in pakistan. plos one. 2020;15(8):e0236359. 3. alvarez amézaga j, barbier herrero l, pijoan del barrio ji, mar<n rodríguez jc, romo simón l, genolla subirats j, rios altolaguirre g, ríos ad, arteagoiha calvo i, landa llona s, arruh gonzález ja. diagnoshc efficacy of senhnel node biopsy in oral squamous cell carcinoma: cohort study and meta-analysis. medicina oral, patología oral y cirugía bucal (internet). 2007;12(3):235-43. 4. museedi os, younis wh. oral cancer trends in iraq from 2000 to 2008. the saudi journal for dental research. 2014 ;5(1):417. 5. al-rawi n, talabani n. squamous cell carcinoma of the oral cavity: a case series analysis of clinical presentahon and histological grading of 1425 cases from iraq. clin oral inveshg. 2008;12(1):15–8. 6. al-mahfoud m, alsaimary i, al sa. occurrence of oral and oropharyngeal squamous cell carcinoma among pahents in basrah city. iraqi j biotechnol. 2017;16(3):255–61. 7. khudier h. malignant oral lesions in sulaimania governorate/ a (5 years( retrospechve study. diyala j med. 2012;2(1):13–20. 8. al-niaimi ai. oral malignant lesions in a sample of pahents in the north of iraq (retrospechve study). al–rafidain dent j. 2006; 6(2): 176–180. 9. fuoad sa, mohammad dn, hamied ma, garib bt. oro-facial malignancy in north of iraq: a retrospechve study of biopsied cases. bmc oral health. 2021;21:1-0. 10. mohammed m. the retrospechve survey of oral and maxillofacial malignant neoplasms. mdj. 2014;11(1):129–35. 11. aljazaeri s, al qudsi g, jaber h, al elwi w, haddad s, echrish h. biopsy records to the oral lesions in basrah between 2012– 2017. j oral med oral surg oral pathol oral radiol. 2020;6(2):74–80. 12. basha s, mohamed r, al-thomali y, al sa. the prevalence of oral cancer in saudi arabia—a figure 1. expression of ebna-ebv in diagnostic tumors. epstein-barr virus predominance and immunological abnormali6es in oral squamous cell carcinoma vol 11 no 1 (2023) doi 10.5195/d3000.2023.483 h#p://den*stry3000.pi#.edu systemahc review. ann med health sci res. 2019;9(2):553–7. 13. zaccone r, renzi a, chalfon c, lenzi j, bellei e, marconato l, ros e, rigillo a, be+ni g, faroni e, guerra d. environmental risk factors for the development of oral squamous cell carcinoma in cats. journal of veterinary internal medicine. 2022;36(4):1398-408. 14. bakkalci d, jia y, winter jr, lewis je, taylor gs, stagg hr. risk factors for epstein barr virus-associated cancers: a systemahc review, crihcal appraisal, and mapping of the epidemiological evidence. journal of global health. 2020;10(1). 15. brouwer l, moreni g, wolthers kc, pajkrt d. world-wide prevalence and genotype distribuhon of enteroviruses. viruses. 2021;13(3):434. 16. yu x, he s, shen j, huang q, yang p, huang l, pu d, wang l, li l, liu j, liu z. tumor vessel normalizahon and immunotherapy in gastric cancer. therapeuhc advances in medical oncology. 2022;14:17588359221110176. 17. da silva rr, vasconcelos fd, tavares dd, dos santos pl. associahon between interleukin 10 (il-10) polymorphisms and leishmaniasis progression: a systemahc review and metaanalysis. scienhfic reports. 2022 ; 12(1):11136. 18. caruntu a, scheau c, codrici e, popescu id, calenic b, caruntu c, tanase c. the assessment of serum cytokines in oral squamous cell carcinoma pahents: an observahonal prospechve controlled study. journal of clinical medicine. 2022 ;11(18):5398. 19. xiao l, li x, cao p, fei w, zhou h, tang n, liu y. interleukin-6 mediated inflammasome achvahon promotes oral squamous cell carcinoma progression via jak2/stat3/sox4/nlrp3 signaling pathway. journal of experimental & clinical cancer research. 2022;41(1):166. 20. meng c, zhao x, lao j. a modified immunofluorescence in situ hybridizahon method to detect long non-coding rnas and proteins in frozen spinal cord sechons. experimental and therapeuhc medicine. 2018;15(6):4623-8. 21. hoffmann tk, sonkoly e, homey b, scheckenbach k, gwosdz c, bas m, chaker a, schirlau k, whiteside tl. aberrant cytokine expression in serum of pahents with adenoid cyshc carcinoma and squamous cell carcinoma of the head and neck. head & neck: journal for the sciences and specialhes of the head and neck. 2007;29(5):472-8. 22. gaiolla rd, domingues ma, niéro-melo l, de oliveira de. serum levels of interleukins 6, 10, and 13 before and awer treatment of classic hodgkin lymphoma. archives of pathology & laboratory medicine. 2011;135(4):483-9. 23. ali mn, omar ta, el sheikh sm, swaify ga, fayad ai. expression of interleukin-10 and its value as a potenhal marker in oral squamous cell carcinoma. alexandria dental journal. 2018 ;43(1):11-6. 24. hamzavi m, tadbir aa, rezvani g, ashraf mj, fawahi mj, khademi b, sardari y, jeirudi n. tissue expression, serum and salivary levels of il-10 in pahents with head and neck squamous cell carcinoma. asian pacific journal of cancer prevenhon. 2013;14(3):1681-5. 25. nelson el, wenzel lb, osann k, dogan-ates a, chantana n, reina-pawon a, laust ak, nishimoto kp, chicz-demet a, du pont n, monk bj. stress, immunity, and cervical cancer: biobehavioral outcomes of a randomized clinical trail. clinical cancer research. 2008 ;14(7):2111-8. 26. aziz s, ahmed ss, ali a, khan fa, zulfiqar g, iqbal j, khan aa, shoaib m. salivary immunosuppressive cytokines il-10 and il-13 are significantly elevated in oral squamous cell carcinoma pahents. cancer inveshgahon. 2015;33(7):31828. 27. szabo yz, newton tl, miller jj, lyle kb, fernandez-botran r. acute stress induces increases in salivary il-10 levels. stress. 2016;19(5):499-505. 28. cruz i, van den brule aj, steenbergen rd, snijders pj, meijer cj, walboomers jm, snow gb, van der waal i. prevalence of epstein—barr virus in oral squamous cell carcinomas, premalignant lesions and normal mucosa—a study using the polymerase chain reachon. oral oncology. 1997 ;33(3):182-8. mandible reconstruction with 3d printed models vol 12 no 1 (2024) doi 10.5195/d3000.2024.538 http://dentistry3000.pitt.edu mandible reconstruction with 3d printed models omar bakr hazm1, noor salman nadhum2, alaa mohammed shaheed3 1 al-bayan-university, college of dentistry, al-yarmouk teaching hospital, baghdad, iraq 2 university of baghdad, college of dentistry, baghdad, iraq 3 al-bayan-university, college of dentistry, baghdad, iraq abstract background: when surgeons operate under general anesthesia, length of surgery, not only its outcome, matters. in maxillofacial surgery we can reduce the time in reconstructive surgery by using presurgical bended plates in presurgically designed 3d models. materials and methods: the study measured the mean time to bend the plates preoperatively on a printed three-dimensional model and the capability of the plates to adapt well on the mandible intraoperatively in cases of mandibular reconstruction after partial resection of the mandible due to pathology, such as benign aggressive tumors. results and conclusion: the mean time to bend the plates preoperatively was 17 minutes, with rapid adaptation during surgery. this is important in minimizing the time that the wound is exposed to the environment and minimizes the trauma to the surrounding soft tissues. multiple checks of the plate if bended at time of surgery can increase trauma to the surrounding soft tissues. the study supports the recommendation of the use of the preoperatively corrected and printed 3d models to prebend the plates pre-operatively. this step should be included as a routine workflow in elective cases, since it is not be possible in cases of emergency or urgency. keywords: 3d printed model, maxillofacial reconstruction, reconstruction plates, ameloblastoma, juvenile fibromatosis, keratocyst, osteosarcoma, vascular malformation. citation:hazm, ob et al. (2024) mandible reconstruction with 3d printed models. dentistry 3000. 1:a001 doi: 10.5195/d3000.2024.538 received: july 19, 2023 accepted: july 23, 2023 published: february 13, 2024 copyright: ©2024 hazm, ob et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email:omar.bakr@albayan.edu.iq introduction preoperative virtual planning is considered now essential for good practice in maxillofacial surgery. the reproducible relationship between the part that the surgeons operate on and the surrounding structures makes the treatment plan easier to achieve intraoperatively, reducing the time of operation.1-3 in maxillofacial surgery, a lot of time can be wasted in bending the reconstruction plate during surgery to obtain good adaptation to the resected mandible. printed preoperative three-dimensional (3d) models aim to solve this problem by mirroring the unaffected side to replace the affected side.4 the aim of this study was to answer the question of whether the preoperative bending of the plate on a 3d printed model decreases surgical time. materials and methods ten adult patients treated at the maxillofacial unit from january to july 2022 with asymmetry in the mandible by different causes, managed by http://dentistry3000.pitt.edu/ mailto:omar.bakr@albayan.edu.iq mandible reconstruction with 3d printed models vol 12 no 1 (2024) doi 10.5195/d3000.2024.538 http://dentistry3000.pitt.edu resection and reconstruction by iliac bone graft, were studied. preoperative assessment on clinical examination, all the patients had swelling of the mandible. preoperative screening panoramic radiograph was used for initial assessment and computed tomography (ct) scans were examined to define the nature and extent of the masses. preoperative virtual planning the images obtained from the ct scans were analyzed and processed by the 3d slicer software5 for three-dimensional visualization, as an imageguided therapy. threedimensional models were printed to represent the mandible in diseased and disease-free forms to facilitate the preoperative bending of the reconstruction plate (figure 1). figure 1: preoperative virtual planning. a: ct scans analysis by software for segmental mirroring. b: plate bending on the printed 3d model. surgical procedure under general anesthesia, after skin preparation and draping, the preauricular with neck extension incision was performed to expose the tumor (figure 2a). after en bloc resection of the tumor, a surgical bed was prepared to receive a bone graft from the iliac bone. the well adapted to the recipient site bone graft was then fixed with a prebended reconstruction plate (figure 2b). the surgical field was then checked for any bleeding points and when needed to control them, cauterization or ligation was done, and the flap closed by layers. postoperative care and followup if there were no complications, the patients were discharged on the first postoperative day, keeping them on regular follow-up visits. results the first patient (male, 13 years old) presented with facial asymmetry; left side painless swelling of the mandible, with chronic limitation of the mouth opening of six months duration. the histopathological diagnosis of the excised tumor suggested a juvenile fibromatosis. the second patient (female, 17 years old) presented with left side painless swelling of the mandible of a one-year duration with normal mouth opening. the histopathological http://dentistry3000.pitt.edu/ mandible reconstruction with 3d printed models vol 12 no 1 (2024) doi 10.5195/d3000.2024.538 http://dentistry3000.pitt.edu assessment diagnosed it as an ameloblastoma. the third patient (female, 14 years old) presented with an aneurysmal bone cyst affecting the left side of the mandible. the histological examination revealed multiloculated bloodfilled cystic spaces separated by cellular septae containing fibroblasts, numerous giant cells, woven bone and calcified basophilic material. the fourth patient (male, 34 years old) presented with facial asymmetry and right side mandibular swelling, and normal mouth opening. the histological diagnosis was ameloblastoma. the fifth patient (male, 65 years old) presented with facial asymmetry and mandibular swelling of 1-year history with limited moth opening. the histopathological examination revealed keratocyst. the sixth patient (male, 24 years old) presented with mandibular mass at left side. the pathology revealed an osteosarcoma. in this case, bone graft was postponed, and the reconstruction plate was fixed just to support soft tissues and preserve the facial symmetry. the seventh patient (female, 30 years old) presented with vascular malformation at the left side of the mandible involving body of mandible and part of ramus. the eighth patient (female, 35 years old) presented with mandibular swelling at right side of mandible with facial asymmetry. the histopathological exam result was ameloblastoma. the ninth patient (male, 45 years old) presented with left side keratocyst at right side of body of mandible extending up to ramus. the tenth patient (male, 40 years old) presented with large mass at right side of mandible with limitation of mouth opening. the histopathology revealed an ameloblastoma. the mean time required to bend and adapt the reconstruction plate on the presurgical model was 17 minutes. intraoperatively, all the reconstruction plates adapted well to the mandible without any further need for bending (figure 2b). figure 2: intraoperative view. a: before tumor resection. b: after plate adaptation. http://dentistry3000.pitt.edu/ mandible reconstruction with 3d printed models vol 12 no 1 (2024) doi 10.5195/d3000.2024.538 http://dentistry3000.pitt.edu discussion during traditional surgery, the surgeons try to adapt the plate on the mandible with multiple slippages, inaccuracy or bulging, which may result in weakening of the plate, increased operation time and further injury to the surrounding structures. sometimes the surgeon needs to increase the access to work comfortably, at the expense of increasing the surgical trauma.6, 7 strong et al. (2013) concluded that patient-specific preformed titanium mesh was expensive and required more preoperative time8. although our study reached the same results, it is important to remember that in the case of chronic benign tumors, the time before an operation can be extended a little bit for the best interest of the patients, especially those with concurrent systemic diseases who required less time under general anesthesia. the study concluded that the 3d printed model was preferable in cases of elective surgeries when the operation could be delayed for one week at least, which is the average time to complete the production of the models. the procedure was contraindicated in cases of advanced malignancy, in which the time to start treatment is of essence. the advantages of preoperative plate bending on 3d printed models were reducing the time of the surgery, surgical access, and trauma intraoperatively. disadvantages included that the model production accuracy depended on the efficiency of the programmer, the step is expensive, and the procedure increased the preoperative preparation time. acknowledgment thanks to the programmer humam munir abdulameer. disclosure of interest the authors declare that they have no competing interests. references 1. jeroen liebregts, frank baan, pieter van lierop, martien de koning, stefaan bergé, thomas maal, tong xi. one-year postoperative skeletal stability of 3d planned bimaxillary osteotomies: maxilla-first versus mandible-first surgery. sci rep. 2019 feb 28;9(1):3000. 2. k stokbro, e aagaard, p torkov, r b bell, t thygesen. virtual planning in orthognathic surgery. int j oral maxillofac surg. 2014 aug;43(8):957-65. 3. humberto fernándezolarte, andrés gómezdelgado, juan g gutiérrez-quintero, álvaro rodríguez-sáenz, jaime castro-núñez. the morpho-functional 3d analysis for zygomatic implants: a clinical tool with surgical implications. j craniofac surg. 2020 sep 3. 4. ghada amin khalifa, nahed adly abd el moniem, shadia abdelhameed elsayed, yara qadry. segmental mirroring: does it eliminate the need for intraoperative readjustment of the virtually pre-bent reconstruction plates and is it economically valuable? j oral maxillofac surg. 2016 mar;74(3):621-30. http://dentistry3000.pitt.edu/ mandible reconstruction with 3d printed models vol 12 no 1 (2024) doi 10.5195/d3000.2024.538 http://dentistry3000.pitt.edu 5. fedorov a., beichel r., kalpathy-cramer j., finet j., fillion-robin jc., pujol s., bauer c., jennings d., fennessy f., sonka m., buatti j., aylward s.r., miller j.v., pieper s., kikinis r. 3d slicer as an image computing platform for the quantitative imaging network. magnetic resonance imaging. 2012 nov;30(9):1323-41. pmid: 22770690. 6. azuma m, yanagawa t, ishibashi-kanno n, uchida f, ito t, yamagata k, hasegawa s, sasaki k, adachi k, tabuchi k, sekido m, bukawa h. mandibular reconstruction using plates prebent to fit rapid prototyping 3dimensional printing models ameliorates contour deformity. head face med. 2014 oct 23;10:45. 7. cohen a, laviv a, berman p, nashef r, abu-tair j. mandibular reconstruction using stereolithographic 3dimensional printing modeling technology. oral surg oral med oral pathol oral radiol endod. 2009 nov;108(5):661-6. 8. e bradley strong, scott c fuller, david f wiley, janina zumbansen, m d wilson, marc c metzger. preformed vs intraoperative bending of titanium mesh for orbital reconstruction. otolaryngol head neck surg. 2013 jul;149(1):60-6. http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu use and effectiveness of the failure modes and effects analysis (fmea) for identification of potential errors and failures in the process of root canal treatment mohsen yazdanian1, mostafa alam2, esmail rafiee1,3, elahe tahmasebi1,*, arash ghaffarpasand2, kamyar abbasi4, mohammadkarim bahadori1 1research center for prevention of oral and dental diseases, baqiyatallah university of medical sciences, tehran, iran. 2department of oral and maxillofacial surgery, school of dentistry, shahid beheshti university of medical sciences, tehran, iran 3school of dentistry, baqiyatallah university of medical sciences, tehran, iran. 4department of prosthodontics, school of dentistry, shahid beheshti university of medical sciences, tehran, iran abstract objec�ves: root canal treatment (rct) has high technical sensi�vity, and many human procedural errors can compromise the success of treatment and well-being of pa�ents. the failure modes and effects analysis (fmea) is a risk assessment and management tool that ensures the safety of pa�ent care by a systema�c approach to failures. this study aimed to iden�fy the poten�al errors and failures in the process of rct using the fmea. methods: this descrip�ve study evaluated the failure modes and their effects qualita�vely and quan�ta�vely using the fmea methodology. for this purpose, a fmea team was first established and the steps of rct were described in a flowchart. next, the poten�al errors and failures in each step were iden�fied, and each failure mode was scored from 1 to 10, based on the severity of impact, likelihood of occurrence, and likelihood of detec�on. the three scores were mul�plied to obtain the risk priority number (rpn). correc�ve measures and preven�ve strategies were suggested for high-risk failure modes (rpn≥250). results: the fmea iden�fied 19 steps and 48 poten�al failures in the process of rct. the maximum rpn in the process of rct was assigned to file fracture in the root canal (rpn=324), apical extrusion of irriga�ng solu�on (rpn=320), and inappropriate or no rubber dam isola�on (rpn=315). conclusion: the fmea has high efficacy for detec�on and priori�za�on of improvable points in a complex dental procedure in a busy department such as the endodon�cs department. keywords: den�stry; endodon�cs; failure modes and effects analysis; risk priority number; pa�ent safety; human errors cita�on: yazdanian m, et al. (2022). use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment. den�stry 3000. 1:a001 doi:10.5195/d3000.2022.238 received: september, 13, 2021 accepted: october 21, 2021 published: august 12, 2022 copyright: ©2022 yazdanian m, et al. this is an open access ar�cle licensed under a crea�ve commons atribu�on work 4.0 united states license. email: elahe.tahmasebi.delfan@gmail.com introduction patient safety is a fundamental healthcare principle, which refers to prevention of any harm to patients during provision of healthcare services [1]. in order to ensure patient safety, it is imperative to identify the events that threaten the patient’s health, analyze the process of occurrence of such events, and design and implement corrective strategies to improve the performance of the system [1]. the frequency of medical malpractice lawsuits, as a factor threatening the patient safety, has greatly increased in the recent years. according to the international reports, the frequency of medical malpractice lawsuits has been on the rise despite the great technological advances in diagnostic and http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu treatment procedures. for instance, the percentage of physicians who had been sued at least once in the united states was 3-4% in 1970, 8% in 1972, 20% in 1980, and 25% in 1990 [2]. the dental profession is no exception to this rule and dentists may also be held civilly liable for their practice. in a study the dentists were found guilty in 44.3% of the lawsuit cases researched. endodontics was one of the most frequently involved specialty in the lawsuits analyzed, and was related to a high incidence of damages awarded to settle claims [3]. among different dental specialties, endodontics includes various procedures with high technical sensitivity such as anesthesia, caries removal, working length determination, chemomechanical debridement of the root canal system, and root canal obturation. inaccuracy and errors in any step of the procedures can compromise the patient’s health [4,5]. although errors in provision of dental care are expected to have lower morbidity and mortality rates than the medical errors, their prevention can increase patient satisfaction, decrease costs, increase credibility, and lower the stress level of both patients and dental care providers. part of the errors than may occur in dental practice are related to human errors while another part is related to environmental conditions, quality of dental instruments and equipment, and the dental office setting, that would eventually compromise patient safety [6]. evidence shows that analysis of human errors prior to their occurrence can prevent them to a great extent [7]. moreover, risk assessment and risk management are gaining increasing popularity in many professions and industries in order to prevent or minimize the occurrence of errors and to increase the credibility and efficiency of the system in the current competitive market [8]. implementation of error management techniques is an effective approach for detection and minimization of errors. error management is an organized continuous process for identification, assessment and decision making about the risks and opportunities in a system, which would affect achieving the goals. at present, a great emphasis has been placed on error management as a key factor for ultimate success in achieving the organizational goals [9]. several methods are available for risk assessment and error management. some of them analyze errors prospectively and some retrospectively. the failure modes and effects analysis (fmea), which is a prospective technique, is among the most valid and reliable techniques for risk management and prevention of errors in the healthcare systems according to the national center for patient safety of the united states [5,10]. this technique was first used in the space industry in 1960, and is a qualitative and inductive method, and an organized tool and a completely cognitive preventive measure that operates based on team work. it is used for definition, identification, assessment, prevention, and elimination or control of potential failure modes and effects in a system, process, project or service, before the final product or service is received by the customer or client [11]. the main property of this technique is its ability in prediction of important errors and offering solutions to prevent their occurrence [10]. its modified form is exclusively used for the healthcare systems, and offers a prospective systematic approach for identification and prevention of errors prior to their occurrence in healthcare http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu organizations. the first application of this technique in the healthcare system dates back to 1990 when it was primarily applied for optimization of vital systems, production and development of medications, and prevention of pharmaceutical errors in the hospitals [12]. fmea aims to promote safety by preventing the occurrence of identified errors in a system and minimizing the unwanted consequences. this preventive and prospective approach allows identification and elimination of potential problems in an organization before they could impact on the system, services, and customers/clients [13]. moreover, application of fmea in the healthcare systems establishes a systematic attitude to promote patient safety. evidence shows that fmea can decrease the clinical risks of patients and identify and prioritize the improvable points of a process under implementation [14, 15]. considering the high risk of procedural errors in the endodontics departments and the adverse consequences of such errors, this study sought to identify and prioritize the potential errors and failures in the process of root canal treatment (rct) by the fmea and offer corrective and safety measures for provision of services at the endodontics departments [16,17]. methods this descriptive study evaluated the failure modes and effects qualitatively and quantitatively using the fmea methodology. the logic of this study was inductive, yielding practical results. the fmea is a team-based, systematic and prospective approach to foresee and prevent problems that may arise in the production process or provision of services prior to their occurrence. hypothesis and inclusion criteria this study questioned the possible high-risk errors and failure modes that can occur in the process of root canal treatment; the possibility of identifying and prioritizing risks and their causes, and the feasibility of suggesting and implementing preventive or corrective measures in each step. the study was conducted in the endodontics department of the school of dentistry, shahid beheshti university of medical sciences, tehran, iran, and focused on one main high-risk procedure done in this department, i.e. root canal treatment, between the years of 2019 and 2020. team assembly the fmea team members consisted of 7 individuals that were purposefully chosen to be part of a focus discussion group (fdg) [5]. the team consisted of two endodontists, one assistant from the endodontics department, one head nurse from the department, one clinic supervisor, one infection control personnel, and one quality control personnel of the specialty clinic (table 1). in addition, 23 non-fmea specialized endodontists, were selected as outside examiners to help identifying high risk procedures. http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu table 1. members of the fmea team order organizational ranking educational level work experience 1 head of the department endodontist 7 years 2 attending dental clinician endodontist 8 years 3 assistant bs of nursing 3 years 4 head nurse ms of nursing 5 years 5 quality control personnel bs of nursing 3 years 6 clinic supervisor bs of nursing 2 years 7 quality control personnel ms of healthcare service management 3 years bs: bachelor’s degree; ms: master’s degree inter-examiner agreements the team agreed to identify all root canal treatment steps, errors and failures, as well as implementing opportunities for improvement and correctional changes. team members were allocated adequate time and resources to allow the process to be successful. the team leader (head of the department), who was skilled in team building, played a vital role in facilitating the various steps in the fmea process, controlling the progress of the analysis, as well as assisting the team in applying a structured approach when identifying error causes and outlining necessary actions [6]. data collection data were collected through observation, questionnaires, and discussion group interviews by holding team sessions and meetings. the eindhoven classification model (ecm) was used to find the root causes of errors. in the next step, the fmea team members defined the rct procedural steps in a flowchart using the visio software (flowchart 1). the fmea team members then identified and listed the possible procedural errors which may occur in every step of the process of rct by brain storming, followed by determining the possible impacts of each error, and recorded them in a fmea worksheet. http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu flowchart 1. procedural steps of rct root canal therapy taking the chief complaint and its history can the patient undergo rct? clinical examination including vitality tests, radiographic examination, diagnosis and treatment planning taking medical history noreferral yesmouthwash anesthetic injection has adequate depth of anesthesia been achieved? no yes rubber dam isolation caries removal access cavity preparationdetermination of primary working length by initial file taking a periapical radiograph from initial file filing and irrigation of root canal to master apical file taking a periapical radiograph from master apical file insertion of master apical gutta-percha cone taking a periapical radiograph from master apical cone root canal obturation with accessory cones and sealer cutting the excess length of gutta-percha points taking the final radiograph temporary restoration of the tooth referral to a restorative dentist or prosthodontist does root canal orifice provide adequate access to the entire canal length? yes nowidening of orifice by gates-glidden drills follow-up prioritizing errors and assigning rpn in the fmea, assessment of the sensitivity and significance of a risk is performed by assigning a numerical value to it, known as the risk priority number (rpn) [18]. higher rpn of an error indicates higher risk for the system or its products [19]. accordingly, the identified failure modes in the previous step were prioritized according to their rpn. the rpn is calculated by multiplying the scores assigned to the severity of impact (s), likelihood of occurrence (o), and likelihood of http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu detection (d), which can be scored from 1 to 10 (figure 1). accordingly, considering the range of 1 < rpn < 1000, errors/failures with rpn ≥ 250 were classified as high-risk errors and entered the next level [20] (figures 2 and 3). after prioritizing the errors and calculation of their rpn, the root causes of high-risk errors were identified according to the ecm. in the ecm, the causes of errors are classified into two main categories of latent (technical and organizational errors) and apparent/active errors (human errors and other types). figure 1. severity ra�ng scale of failure modes figure 3. occurrence ra�ng scale of failure modes figure 2. detec�on ra�ng scale of failure modes identifying causes of error modes finally, according to the rpns, possible failure or error causes were discussed and noted, and consequently, corrective and preventive measures were suggested for the high-risk failure modes. these measures, depending on the type of error/failure, focused on decreasing the severity of impact and rate of occurrence of errors/failures, and/or increasing the possibility of their detection. data analysis the data was organized according to the fmea principles and the process of rct was defined stepby-step in a flowchart, and the failure modes and effects and their root causes were determined and listed in the fmea worksheet. the procedures which had higher risks were prioritized using the http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu borda function and the analyzed according to their risk priority number (rpn). ethical measures the patients’ privacy was preserved and their personal information was protected in all steps of the study. informed consent was attained from all patients. results nineteen steps and 48 potential errors/failures were identified and recorded for the rct. next, according to the respective tables for s, o and d, the fmea team members individually scored each potential failure mode in several sessions, and the rpn of each failure mode was determined according to a 1-10 scale by multiplying the scores of s, o and d, and taking into account the ange of 1 < rpn < 1000 (table 2). in this study, rpn > 250 was considered high-risk and unacceptable in rct [21]. next, corrective measures were suggested for high-risk failure modes to prevent their occurrence, increase the likelihood of their detection, or decrease their impact in order to compile the details of process modification by focusing on the aforementioned three strategies. totally, of the defined 19 steps and 48 identified errors, 12 errors were selected and categorized as high-risk, for which, corrective and preventive measures were suggested (table 3). one potential error was found in the process of taking the chief complaint and history of patient. three potential errors were identified in the process of taking a medical history; among which, not asking the patient about the history of systemic diseases acquired the highest score with rpn=63. five errors were identified i the process of clinical examination, radiographic examination, diagnosis and treatment planning; among which, rct of teeth with poor or hopeless periodontal prognosis was classified as a highrisk error (rpn=252). six errors were identified in the process in anesthetic injection; among which, injection of unsafe doses of anesthetic agents containing vasoconstrictor in patients with contraindication (rpn=159), and injection of anesthetic agent in excessive amounts (rpn=158) acquired the maximum scores. two errors were identified in the process of isolation; among which, inappropriate or no rubber dam isolation (rpn=315) was classified as a high-risk error. six potential errors were identified in the process of removal of caries and previous restorations; among which, no reduction of unsupported cusps (rpn=275) was classified as a high-risk error. three errors were identified in the process of access cavity preparation; among which, not finding additional root canals (rpn=271) was classified as a highrisk error. in the process of widening the root canal orifice, careless use of gates-glidden drills (rpn=288) was classified as a highrisk error. one error was identified in the process of primary working length determination by the initial file, and three errors were identified in the process of taking periapical radiograph from the initial file; among which, incorrect determination of root canal working length (rpn=211) acquired the highest score. http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu table 2. primary fmea worksheet for rct process steps modes of errors type of error impact of errors causes of errors rpn taking patient complaint and history incorrect understanding of patient complaint and descriptions human, systematic treating the wrong tooth, incorrect treatment plan not allocating sufficient time by the dentist, crowded clinic, poor verbal communication with patient, poor sympathy with patient 56 taking medical history not asking the patient about important systemic diseases human, systematic occurrence of cardiovascular or other systemic problems not allocating sufficient time by the dentist, crowded clinic, not adhering to the protocol regarding filling out the health questionnaire prior to the onset of treatment, shortage of human resources 63 not asking female patients whether they are pregnant human, systematic injury to the fetus inaccuracy of dentist, not adhering to the protocol regarding filling out the health questionnaire prior to the onset of treatment, shortage of human resources, no option regarding choosing a female dentist for female patients 56 not asking patients about their allergy history human, systematic risk of occurrence of allergic reactions in susceptible patients not allocating sufficient time by the dentist, crowded clinic, inaccuracy of dentist, not adhering to the protocol regarding filling out the health questionnaire prior to the onset of treatment, shortage of human resources 35 clinical and radiographic examination, diagnosis and treatment planning diagnosis and treatment planning merely based on radiographic examination human treating the wrong tooth, incorrect treatment plan not allocating sufficient time by the dentist, inaccuracy of dentist 65 not requesting appropriate radiography human incorrect treatment plan inaccuracy of dentist, poor education and inexperience of dentist 56 excess request of radiography human excessive x-ray exposure, increased risk of cancer poor education and inexperience of dentist 36 root canal treatment of a tooth with poor or hopeless periodontal prognosis human, systematic progression of periodontal disease, mobility, necessitating later extraction not asking for periodontal consultation prior to rct for periodontally compromised or suspected teeth 252 rct of complex root canals by a general dentist human, systematic abscess after rct and treatment failure absence of an organizational treatment protocol 278 local anesthesia administration injection of anesthetic agent containing vasoconstrictor in human development of cardiovascular complications not taking precise medical history, inaccuracy of dentist, poor education and inexperience of dentist 159 http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu excessive amounts in a patient with contraindications not performing aspiration prior to anesthetic injection human entry of anesthetic agent into the bloodstream and subsequent development of cardiovascular problems, signs and symptoms of overdose not allocating sufficient time by the dentist, inaccuracy of dentist, poor education and inexperience of dentist 69 multiple insertions of needle into a highly vascularized area human rupture of vessel wall and development of hematoma inaccuracy of dentist, poor education and inexperience of dentist 93 excessive injection of anesthetic agent without taking into account the patient’s weight human systemic toxicity (overdose) inaccuracy of dentist, poor education and inexperience of dentist 158 sudden change in direction of movement of needle in the tissue, precurving the needle in several points human fracture of needle in the tissue inaccuracy of dentist, poor education and inexperience of dentist 69 induction of long-term anesthesia for a short-duration procedure human development of traumatic ulcer due to soft tissue biting inaccuracy of dentist, poor education and inexperience of dentist 59 isolation of the respective tooth improper or no rubber dam isolation human, systematic aspiration of dental instruments into the respiratory tract, traumatization of soft tissue or adjacent teeth, saliva leakage into the root canal, abscess formation inaccuracy of dentist, poor education and inexperience of dentist, absence of a clear treatment protocol 315 use of latex rubber dam in a patient allergic to latex human, systematic allergy in susceptible patients not asking the patient about history of allergy, inaccuracy of dentist, not adhering to the protocol regarding filling out the health questionnaire prior to the onset of treatment, shortage of medical equipment and instruments 211 removal of caries and previous restoration carelessness in correct use of bur human, systematic traumatization of mucocutaneous surfaces or traumatizing the adjacent teeth with bur inaccuracy of dentist, inexperience of dentist, use of burs without protective guards 222 http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu no irrigation during caries removal systematic thermal damage to enamel and dentin malfunction of medical equipment 196 application of uncontrolled force during caries removal, or excessive removal of tooth structure human irreparability of tooth crown, weakening of tooth structure and increased risk of tooth fracture inexperience of dentist 111 incomplete elimination of caries human development of secondary caries, requiring endodontic retreatment inaccuracy of dentist, inexperience of dentist 96 not reducing unsupported cusps human tooth fracture inexperience of dentist 275 not using face mask when removing old amalgam restoration with highspeed hand-piece human mercury vapor toxicity, increased risk of cancer inexperience of dentist 218 access cavity preparation excessive removal of tooth structure human inability to ideally restore the crown, weakening of tooth structure, increased risk of fracture, tooth perforation inexperience of dentist 86 creating an undersized access cavity human no straight path to the root canals inexperience of dentist 87 not finding additional root canals in molar teeth, particularly the second mesiobuccal canal in the maxillary molars and mid-mesial canal in the mandibular molars and mandibular lateral incisors human, systematic development of abscess after rct, treatment failure rct of complicated cases by a general dentist, absence of an organizational treatment protocol 271 orifice flaring if required careless use of gates-glidden drills human fracture of gates-glidden drills in the root canal, vertical root fracture poor education and inexperience of dentist 288 primary working length determination by initial file incorrect working length determination human, systematic over-filling or under-filling inexperience of dentist, shortage of materials and equipment 211 http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu periapical radiography of initial file incorrect film positioning human repeating radiography, overexposure of patient, increased risk of cancer inexperience of dentist 59 under-exposure or over-exposure of the film human repeating radiography, overexposure of patient, increased risk of cancer inexperience of dentist 68 inappropriate film processing human, systematic repeating radiography, overexposure of patient, increased risk of cancer inexperience of dentist, shortage of materials and equipment 67 filing and root canal irrigation to master apical file, followed by further filing and root canal debridement leakage of root canal irrigating solutions into the oral cavity human mucosal burning, liquid aspiration into the respiratory tract inexperience of dentist 211 inadequate filing and irrigation of root canal system human failure of rct in long-term not allocating sufficient time by the dentist, inexperience of dentist 236 file fracture in the root canal human, systematic abscess formation and failure of rct in long-term inaccuracy of dentist, inexperience of dentist, multiple use of thin files, shortage of materials and equipment 324 apical extrusion of irrigating solution human, systematic hypochlorite accident inexperience of dentist, use of high-concentration hypochlorite, apical extrusion of irrigating solution 320 root canal transportation human apical perforation inexperience of dentist 170 ledge formation human perforation of external root surface inexperience of dentist 159 ignoring the danger zone human strip perforation poor education and inexperience of dentist 245 packing of debris in the root canal and obstruction of the main canal path human not accessing the entire working length and underfilling inexperience of dentist 244 changing the working length human over-filling or under-filling, failure of rct in long-term inexperience of dentist 226 http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu periapical radiography of master apical file improper film positioning human repeating radiography, overexposure of patient, increased risk of cancer inexperience of dentist 59 under-exposure or over-exposure of film human repeating radiography, overexposure of patient, increased risk of cancer inexperience of dentist 68 inappropriate film processing human, systematic repeating radiography, overexposure of patient, increased risk of cancer inexperience of dentist, shortage of dental materials and equipment 67 placement of master apical gutta-percha cone selection of an undersized master apical cone human apical extrusion of guttapercha, inappropriate apical seal, over-filling, failure of rct in long-term poor education and inexperience of dentist 89 selection of an oversized master apical cone human under-filling of root canal, treatment failure in long-term poor education and inexperience of dentist 77 periapical radiography of master apical cone inappropriate film positioning human repeating radiography, overexposure of patient, increased risk of cancer inexperience of dentist 59 under-exposure or over-exposure of film human repeating radiography, overexposure of patient, increased risk of cancer inexperience of dentist 68 inappropriate film processing human, systematic repeating radiography, overexposure of patient, increased risk of cancer inexperience of dentist, shortage of dental materials and equipment 67 root canal obturation by insertion of accessory gutta-percha cones and sealer apical extrusion of sealer or accessory gutta-percha cones human infection, inflammatory reaction inexperience of dentist 236 application of excessive force by the spreader human vertical root fracture inexperience of dentist 288 root canal filling with inadequate density (void formation) human failure of rct in long-term not allocating sufficient time by the dentist, inexperience of dentist 208 cutting excess guttapercha contact of hot endodontic plugger with the skin or mucosa human, systematic burning of skin and mucosa inaccuracy of dentist, inexperience of dentist, shortage of dental materials and equipment 265 http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu falling of the torch human, systematic fire inaccuracy of dentist, inexperience of dentist, unavailability of electronic devices for cutting of excess gutta-percha, shortage of dental materials and equipment 241 final periapical radiography improper film positioning human repeating radiography, overexposure of patient, increased risk of cancer inexperience of dentist 59 under-exposure or over-exposure of film human repeating radiography, overexposure of patient, increased risk of cancer inexperience of dentist 68 inappropriate film processing human, systematic repeating radiography, overexposure of patient, increased risk of cancer inexperience of dentist, shortage of dental materials and equipment 67 temporary restoration carrying the filling material with the wrong instrument human, systematic contamination of filling material, waste of material inexperience of dentist, shortage of dental materials and equipment 56 over-filling of the access cavity with temporary restoration human temporomandibular disorders not allocating sufficient time by the dentist, inexperience of dentist, 69 poor retention of temporary restoration human early loss of temporary restoration, requiring replacement or necessitating endodontic retreatment inexperience of dentist 48 referral to a restorative dentist or prosthodontist for final restoration not showing up for final restoration at an appropriate time human, systematic requiring endodontic retreatment, abscess formation negligence by patient, negligence by assistant, absence of an organizational protocol 252 http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu table 3. suggested measures for high-risk errors in the process of rct high-risk failure modes rpn suggested measures root canal treatment of a tooth with poor or hopeless periodontal prognosis 252 designing an organizational treatment protocol necessitating written periodontal consultation prior to rct for periodontally compromised or suspected teeth holding continuing education courses for dental clinicians inappropriate or no rubber dam isolation 315 designing an organizational treatment protocol necessitating rubber dam isolation for rct use of files with thread holes (at least for patients with poor cooperation) no reduction of unsupported cusps 275 designing an organizational treatment protocol necessitating cusp reduction, and reduction of unsupported walls to prevent tooth fracture (depending on the tooth) holding continuing education courses for dental clinicians not finding additional root canals especially the second mesiobuccal canal in maxillary molars and midmesial canals in mandibular molars and mandibular lateral incisors 271 designing an organizational treatment protocol necessitating treatment of complex root canals exclusively by endodontists careless use of gates-glidden drills 288 holding continuing education courses for dental clinicians file fracture in the root canal 324 holding continuing education courses for dental clinicians provision of high-quality files in the endodontics departments not using thin files for multiple teeth apical extrusion of irrigating solutions 320 holding continuing education courses for dental clinicians designing an organizational treatment protocol necessitating precise determination of the safe concentration of sodium hypochlorite for use in endodontics departments excessive force application by the spreader 288 holding continuing education courses for dental clinicians educating dental clinicians regarding controlled force application in use of spreader contact of hot plugger with the skin or mucosa 265 holding continuing education courses for dental clinicians use of rubber dam until the completion of rct to protect the skin and mucosa not showing up in-time for final restoration of the tooth 252 raising awareness and informing the patients about the problems that may occur in case of not showing up for final restoration in-time, or nursing and following the patient by the assistant http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu a total of 12 errors were identified in the process of taking a periapical radiograph from the master apical file, and continuation of filing and debridement of the root canal; among which, file fracture in the root canal (rpn=324) and apical extrusion of irrigating solutions (rpn=320) were classified as highrisk errors. a total of 8 errors were identified in the process of insertion of master apical cone, taking a periapical radiograph from it, and filling the rest of the root canal with accessory guttapercha points and sealer; among which, excessive force application by the spreader resulting in a bend or deformation, or breakage of the spreader (rpn=288) was considered as a high-risk error. a total of 8 errors were identified in the process of cutting the excess end of gutta-percha points, taking the final radiograph, and temporary restoration of the tooth; among which, contact of hot endodontic plugger with the skin or mucosa (rpn=365) was classified as a high-risk error. in the process of patient referral to a restorative dentist or prosthodontist for final restoration of the tooth, not showing up in-time for final restoration (rpn=252) was classified as a high-risk error. discussion in the recent years, many studies have focused on the human errors in high-risk dental fields due to their significant impact on health, costs, and quality of life of patients [7,22]. in this study, the fmea was employed to identify errors related to rct in the endodontics departments. accordingly, the potential impacts and causes of errors were determined by the team members, and corrective strategies were suggested for high-risk errors. according to the results of this study, file fracture in the root canal, apical extrusion of irrigating solutions, and inappropriate or no rubber dam isolation, acquired the maximum rpns, and were classified as errors with maximum risk in the process of rct. tzanetakis et al. [23] reported the prevalence of file fracture to be 1.83% and discussed that its occurrence had an inverse correlation with the experience level of dental clinicians, such that it had the highest frequency among dental students and lowest frequency among endodontists [23]. according to some studies, file fracture does not decrease the rct prognosis [24,25]. however, some others believe that file fracture increases the risk of rct failure [26,27]. mcguigan et al, [28] in their review study concluded that although file fracture does not increase the rate of endodontic treatment failure in cases without periapical disease, risk of endodontic failure increases following file fracture in teeth with a preexisting periapical lesion. thus, they recommended attempts to remove the broken piece from the root canal. strategies suggested by the fmea team in our study to minimize the occurrence of file fracture and decrease its rpn included continuing education courses for dental clinicians, and provision of high-quality files for the endodontics departments, as well as control over the number of usage of instruments. these strategies have been confirmed by a previous study as well [23]. for instance, evidence shows that higher education and training and greater experience decrease the occurrence of file fracture [23,28]. also, mcguigan et al, [28] in their review study reported that type of alloy used by the manufacturer in the composition of files affects the frequency of file fracture, and use of high-quality files can minimize the occurrence of this adverse event [28]. hypochlorite accident is among the oldest complications of rct [29]. rowland et al. [30] performed a literature review on this topic and concluded that its prevalence is highly variable. leakage of sodium hypochlorite into the periapical tissues can cause hemolysis, prevent the migration of neutrophils, and damage the endothelial cells and fibroblasts [31]. the toxic effects of sodium hypochlorite are due to http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu its alkaline nature and hypertonicity, which can cause oxidation of proteins and lipid membranes [31]. patients often experience excruciating pain, and their clinical examination reveals ecchymosis, hematoma, swelling, and temporary paresthesia (with a lower prevalence rate) [32]. pain and swelling may last for 1-4 months [33,34], and healing of injured mucosa may take up to 2 months [35]. also, hypochlorite accident can even cause irreversible complications such as fibrosis and unesthetic scarring [36,37]. obviously, educating dental clinicians regarding the use of other irrigating solutions such as saline or chlorhexidine, or use of diluted sodium hypochlorite, and delivary of irrigating solutions only in the root canal environment, by use of 30 g needles either with open-ended or side-vented cannulas, while moving inside the root canal with a plunger force of 10–40 n, can decrease the occurrence of hypochlorite accident [19,29,47]. futhermore, the hypochlorite accident may be due to operator malpractice or negligence. over 140 years have passed since the introduction of rubber dam as an effective isolation tool in dentistry [38]. during this time period, its application has been technically simplified, and it is widely used by dental clinicians in restorative dentistry, endodontics, periodontics and pedodontics [39]. according to madarati et al, [18] use of rubber dam alone can serve as an independent factor in prevention of many dental complications, particularly in endodontics [18]. however, evidence shows that still a lot of dental clinicians do not routinely use rubber dam isolation in rct due to a number of reasons such as poor compliance and acceptance by patients, time required for its installation, related costs, and not receiving adequate instruction about its use and its difficult installation [40]. not using rubber dam can cause cross-contamination, prevent ideal irrigation of root canal, adversely affect the treatment results, and increase the risk of swallowing or aspiration of dental materials and instruments [41]. thus, use of rubber dam is imperative as a standard of care, and is an integral part of the organizational treatment protocols in many universities and dental clinics worldwide [42-45]. in addition, during the coronavirus disease (covid-19) outbreak, as aerosols and airborne particles can easily be generated during endodontic treatment, the use of personal protective barriers and rubber dam is recommended at the dental office [48]. the current results well indicated that a large portion of root causes of the most important potential errors in the process of rct are due to inexperience, poor education, and lack of scientific knowledge of dental clinicians. ghasemi et al. [46] reported that the majority of errors performed by post-graduate students of endodontics were procedural errors due to their inexperience, inadequate skills, outdated scientific information, imbalance between the number of patients and attending endodontists, and inefficacy and inadequacy of the protocols and guidelines. accordingly, they suggested the inclusion of a separate course on human errors as part of dental curricula. the fmea identifies and prioritizes the potential errors in a prospective manner. retrospective review of studies regarding the common procedural errors in the process of rct revealed that the potential risks classified as high-risk by fmea were all among the commonly reported, important, and challenging errors in the field of endodontics. this finding highlights the factuality and efficacy of fmea and further confirms its credibility for accurate detection of errors and risks in the process of rct. it appears that adherence to the corrective and preventive measures in a logical time period can decrease the risk of such errors in the respective organizations/institutions. clearly, after the termination of this time period, re-implementation of fmea in the respective organizations/institutions and calculation of rpn of the potential errors can reveal the efficacy of corrective and preventive measures and indicate the quality of their implementation [29]. http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu conclusion according to the current results, identification of potential errors in the process of rct, detection of high-risk (unacceptable) errors, finding their root causes, and offering corrective measures to prevent their occurrence all indicated the high efficacy of fmea for identification, assessment, prioritization, and analysis of errors in endodontics departments. moreover, combining group interviews with fmea, identifying the framework of errors, designing comprehensive tables for the severity of impact, rate of occurrence, and rate of detection of errors, and finding the root causes of high-risk errors by using the rca in this study decreased the effects of some limitations of the fmea such as its time consuming nature and high dependence of the results on team participation of individuals, and consequently increased its efficacy. however, the efficacy of this approach in implementation of corrective measures was not tested in this study, and requires an implementation time period and further studies in this respect. conflicts of interest the authors declare no competing interest. references 1. verklan mt, walden m, forest s. core curriculum for neonatal intensive care nursing ebook: elsevier health sciences; 2020 . .2 yamalik n, perea pérez b. patient safety and dentistry: what do we need to know? fundamentals of patient safety, the safety culture and implementation of patient safety measures in dental practice. international dental journal. 2012;62(4):189-96. pmid: 23017000. .3 zanin aa, herrera lm, melani rf. civil liability: characterization of the demand for lawsuits against dentists. braz oral res. 2016 aug 18;30(1). pmid: 27556556. .4 thusu s, panesar s, bedi r. patient safety in dentistry state of play as revealed by a national database of errors. br dent j. 2012 aug;213(3):e3. pmid: 22878337. .5 wetterneck tb, skibinski ka, roberts tl, et al. using failure mode and effects analysis to plan implementation of smart i.v. pump technology. am j health syst pharm 2006;63(16):1528–1538. pmid: 16896081. .6 bailey e, tickle m, campbell s. patient safety in primary care dentistry: where are we now? british dental journal. 2014;217(7):339-44. pmid: 25303580. .7 chiozza ml, ponzetti c. fmea: a model for reducing medical errors. clinica chimica acta. 2009;404(1):75-8. pmid: 19298799. .8 de wet c, o’donnell c, bowie p. developing a preliminary ‘never event’list for general practice using consensus-building methods. br j gen pract. 2014;64(620):e159-e67. pmid: 24567655. .9 van galen ls, struik pw, driesen be, merten h, ludikhuize j, van der spoel ji, et al. delayed recognition of deterioration of patients in general wards is mostly caused by human related monitoring failures: a root cause analysis of unplanned icu admissions. plos one. 2016;11 .(8). pmid: 27537689. .10 mikulak rj, mcdermott r, beauregard m. the basics of fmea: crc press; 2017 . .11 ramoni rb, walji mf, white j, stewart d, vaderhobli r, simmons d ,et al. from good to better: toward a patient safety initiative in dentistry. the journal of the american dental association. 2012;143(9):956-60. pmid: 22942131. .12 pemberton m. developing patient safety in dentistry. british http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu dental journal. 2014;217(7):335-7. pmid: 25303579. .13 black i, bowie p. patient safety in dentistry: development of a candidate'never event'list for primary care. british dental journal. 2017;222(10):782-8. pmid: 28546608. .14 brunton p. summary of: patient safety in dentistry–state of play as revealed by a national database of errors. british dental journal. 2012;213(3):126-7. pmid: 22878313. .15 renton t, sabbah w. review of never and serious events related to dentistry 2005– 2014. british dental journal. 2016;221(2):71-9. pmid: 27444598. .16 kalenderian e, walji mf, tavares a, ramoni rb. an adverse event trigger tool in dentistry: a new methodology for measuring harm in the dental office. the journal of the american dental association. 2013;144(7):808-14. pmid: 23813262. .17 walji mf, yansane a, hebballi nb, ibarra-noriega am, kookal kk, tungare s, kent k, mcpharlin r, delattre v, obadanudoh e, tokede o, white j, kalenderian e. finding dental harm to patients through electronic health record-based triggers. jdr clin trans res. 2020 jul;5(3):271-277. pmid: 31821766. .18 madarati aa. why dentists don’t use rubber dam during endodontics and how to promote its usage? bmc oral health. 2016;16(1):24. pmid: 26916426. .19 parashos p, messer hh. rotary niti instrument fracture and its consequences. j endod. 2006 nov;32(11):1031-43. pmid: 17055902. .20 kalenderian e, obadanudoh e, yansane a, kent k, hebballi nb, delattre v, et al. feasibility of electronic health record–based triggers in detecting dental adverse events. applied clinical informatics. 2018;9(3):646. pmid: 30134473. .21 tanalp j, güngör t. apical extrusion of debris: a literature review of an inherent occurrence during root canal treatment. int endod j. 2014 mar;47(3):211-21. pmid: 23711187. .22 nahmias y, serota ks, watson jr w. predictable endodontic success: part iimicrostructural replication. oral health. 2003;93(12):36-41. pmid: 12442825. .23 tzanetakis gn, kontakiotis eg, maurikou dv, marzelou mp. prevalence and management of instrument fracture in the postgraduate endodontic program at the dental school of athens: a five-year retrospective clinical study. journal of endodontics. 2008;34(6):675-8. pmid: 18498887. .24 friedman s. expected outcomes in the prevention and treatment of apical periodontitis . essential endodontology: prevention and treatment of apical periodontitis. 2008:408-69. pmid: 21689541 .25 spili p, parashos p, messer hh. the impact of instrument fracture on outcome of endodontic treatment. j endod. 2005;31(12):845-50. pmid: 16306815. .26 kerekes k, tronstad l. long-term results of endodontic treatment performed with a standardized technique. j endod. 1979 mar;5(3):83-90. pmid: 296248. .27 strindberg lz. the dependence of the results of pulp therapy on certain factors-an analytical study based on radiographic and clinical follow-up examination. acta odontol scand. 1956;14:1-175. pmid: 2084204. .28 mcguigan m, louca c, duncan h. the impact of fractured endodontic instruments on treatment outcome. british dental journal. 2013;214(6):285-9. pmid: 23518972. .29 guivarc'h m, ordioni u, ahmed hm, cohen s, catherine jh, bukiet f. sodium hypochlorite accident: a systematic review. j http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu endod. 2017 jan;43(1):16-24. pmid: 27986099. .30 hatton j, walsh s, wilson a. management of the sodium hypochlorite accident: a rare but significant complication of root canal treatment. bmj case rep. 2015 mar 25;2015:bcr2014207480. pmid: 25809429. .31 gernhardt cr, eppendorf k, kozlowski a, brandt m. toxicity of concentrated sodium hypochlorite used as an endodontic irrigant. int endod j. 2004 apr;37(4):272-80. pmid: 15056354. .32 de sermeño rf, da silva la, herrera h, herrera h, silva ra, leonardo mr. tissue damage after sodium hypochlorite extrusion during root canal treatment. oral surg oral med oral pathol oral radiol endod. 2009 jul;108(1):e469. pmid: 19442541. .33 markose g, cotter cj, hislop ws. facial atrophy following accidental subcutaneous extrusion of sodium hypochlorite. british dental journal. 2009;206(5):263-4. pmid: 19287421. .34 pelka m, petschelt a. permanent mimic musculature and nerve damage caused by sodium hypochlorite: a case report. oral surgery, oral medicine, oral pathology, oral radiology ,and endodontology. 2008;106(3):e80-e3. pmid: 18602848. .35 mehra p, clancy c, wu j. formation of a facial hematoma during endodontic therapy. j am dent assoc. 2000 jan;131(1):6771. pmid: 10649874. .36 chaudhry h, wildan tm, popat s, anand r, dhariwal d. before you reach for the bleach. british dental journal. 2011;210(4):157-60. pmid: 21350524. .37 al-sebaei mo, halabi oa, el-hakim ie. sodium hypochlorite accident resulting in lifethreatening airway obstruction during root canal treatment: a case report. clin cosmet investig dent. 2015 mar 4;7:41-4. pmid: 25767406. .38 elderton r. a modern approach to the use of rubber dam--1. the dental practitioner and dental record. 1971;21(6):187. pmid: 5278933. .39 palmer no, ahmed m, grieveson b. an investigation of current endodontic practice and training needs in primary care in the north west of england. br dent j. 2009 jun 13;206(11):e22; discussion 584-5. pmid: 19478812. .40 hill ee, rubel bs. do dental educators need to improve their approach to teaching rubber dam use? j dent educ. 2008 oct;72(10):1177-81. pmid: 18923098. .41 ahmad i. rubber dam usage for endodontic treatment: a review. international endodontic journal. 2009;42(11):963-72. pmid: 19825034. .42 kim sg, malek m, sigurdsson a, lin lm, kahler b. regenerative endodontics: a comprehensive review. int endod j. 2018 dec;51(12):1367-1388. pmid: 29777616. .43 european society of endodontology. quality guidelines for endodontic treatment: consensus report of the european society of endodontology. int endod j. 2006 dec;39(12):921-30. pmid: 17180780. .44 gilbert gh, litaker ms, pihlstrom dj, amundson cw, gordan vv; dpbrn collaborative group. rubber dam use during routine operative dentistry procedures: findings from the dental pbrn. oper dent. 2010 sep-oct;35(5):491-9. pmid: 20945739. .45 american academy on pediatric dentistry clinical affairs committee-pulp therapy subcommittee; american academy on pediatric dentistry council on clinical affairs. guideline on pulp therapy for primary and young permanent http://dentistry3000.pitt.edu/ use and effec�veness of the failure modes and effects analysis (fmea) for iden�fica�on of poten�al errors and failures in the process of root canal treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.238 htp://den�stry3000.pit.edu teeth. pediatr dent. 20082009;30(7 suppl):170-4. pmid: 19216417. .46 ghasemi m, khoshakhlagh ah, mahmudi s, fesharaki mg. identification and assessment of medical errors in the triage area of an educational hospital using the sherpa technique in iran. int j occup saf ergon. 2015;21(3):38290. pmid: 26327157. .47 jäggi m, magni e, eggmann f, elayouti a, connert t, weiger r. apical pressure generated using conventional syringe irrigation in immature teeth—an in vitro study. materials. 2021; 14(10):2580. pmid: 34063549 .48 silva wo, vianna silva macedo rp, nevares g, val rodrigues rc, grossi heleno jf, braga pintor av, almeida bm. recommendations for managing endodontic emergencies during coronavirus disease 2019 outbreak. j endod. 2021 jan;47(1):3-10. pmid: 33045270. http://dentistry3000.pitt.edu/ references .1 verklan mt, walden m, forest s. core curriculum for neonatal intensive care nursing e-book: elsevier health sciences; 2020. 2. yamalik n, perea pérez b. patient safety and dentistry: what do we need to know? fundamentals of patient safety, the safety culture and implementation of patient safety measures in dental practice. international dental journal. 2012;62(4):189-96. p... 3. zanin aa, herrera lm, melani rf. civil liability: characterization of the demand for lawsuits against dentists. braz oral res. 2016 aug 18;30(1). pmid: 27556556. 4. thusu s, panesar s, bedi r. patient safety in dentistry state of play as revealed by a national database of errors. br dent j. 2012 aug;213(3):e3. pmid: 22878337. .5 wetterneck tb, skibinski ka, roberts tl, et al. using failure mode and effects analysis to plan implementation of smart i.v. pump technology. am j health syst pharm 2006;63(16):1528–1538. pmid: 16896081. 6. bailey e, tickle m, campbell s. patient safety in primary care dentistry: where are we now? british dental journal. 2014;217(7):339-44. pmid: 25303580. 7. chiozza ml, ponzetti c. fmea: a model for reducing medical errors. clinica chimica acta. 2009;404(1):75-8. pmid: 19298799. 8. de wet c, o’donnell c, bowie p. developing a preliminary ‘never event’list for general practice using consensus-building methods. br j gen pract. 2014;64(620):e159-e67. pmid: 24567655. 9. van galen ls, struik pw, driesen be, merten h, ludikhuize j, van der spoel ji, et al. delayed recognition of deterioration of patients in general wards is mostly caused by human related monitoring failures: a root cause analysis of unplanned icu ad... 10. mikulak rj, mcdermott r, beauregard m. the basics of fmea: crc press; 2017. 11. ramoni rb, walji mf, white j, stewart d, vaderhobli r, simmons d, et al. from good to better: toward a patient safety initiative in dentistry. the journal of the american dental association. 2012;143(9):956-60. pmid: 22942131. 12. pemberton m. developing patient safety in dentistry. british dental journal. 2014;217(7):335-7. pmid: 25303579. 13. black i, bowie p. patient safety in dentistry: development of a candidate'never event'list for primary care. british dental journal. 2017;222(10):782-8. pmid: 28546608. 14. brunton p. summary of: patient safety in dentistry–state of play as revealed by a national database of errors. british dental journal. 2012;213(3):126-7. pmid: 22878313. 15. renton t, sabbah w. review of never and serious events related to dentistry 2005–2014. british dental journal. 2016;221(2):71-9. pmid: 27444598. 16. kalenderian e, walji mf, tavares a, ramoni rb. an adverse event trigger tool in dentistry: a new methodology for measuring harm in the dental office. the journal of the american dental association. 2013;144(7):808-14. pmid: 23813262. 17. walji mf, yansane a, hebballi nb, ibarra-noriega am, kookal kk, tungare s, kent k, mcpharlin r, delattre v, obadan-udoh e, tokede o, white j, kalenderian e. finding dental harm to patients through electronic health record-based triggers. jdr clin ... 18. madarati aa. why dentists don’t use rubber dam during endodontics and how to promote its usage? bmc oral health. 2016;16(1):24. pmid: 26916426. 19. parashos p, messer hh. rotary niti instrument fracture and its consequences. j endod. 2006 nov;32(11):1031-43. pmid: 17055902. 20. kalenderian e, obadan-udoh e, yansane a, kent k, hebballi nb, delattre v, et al. feasibility of electronic health record–based triggers in detecting dental adverse events. applied clinical informatics. 2018;9(3):646. pmid: 30134473. 21. tanalp j, güngör t. apical extrusion of debris: a literature review of an inherent occurrence during root canal treatment. int endod j. 2014 mar;47(3):211-21. pmid: 23711187. 22. nahmias y, serota ks, watson jr w. predictable endodontic success: part ii-microstructural replication. oral health. 2003;93(12):36-41. pmid: 12442825. 23. tzanetakis gn, kontakiotis eg, maurikou dv, marzelou mp. prevalence and management of instrument fracture in the postgraduate endodontic program at the dental school of athens: a five-year retrospective clinical study. journal of endodontics. 2008... 24. friedman s. expected outcomes in the prevention and treatment of apical periodontitis. essential endodontology: prevention and treatment of apical periodontitis. 2008:408-69. pmid: 21689541 25. spili p, parashos p, messer hh. the impact of instrument fracture on outcome of endodontic treatment. j endod. 2005;31(12):845-50. pmid: 16306815. 26. kerekes k, tronstad l. long-term results of endodontic treatment performed with a standardized technique. j endod. 1979 mar;5(3):83-90. pmid: 296248. 27. strindberg lz. the dependence of the results of pulp therapy on certain factors-an analytical study based on radiographic and clinical follow-up examination. acta odontol scand. 1956;14:1-175. pmid: 2084204. 28. mcguigan m, louca c, duncan h. the impact of fractured endodontic instruments on treatment outcome. british dental journal. 2013;214(6):285-9. pmid: 23518972. 29. guivarc'h m, ordioni u, ahmed hm, cohen s, catherine jh, bukiet f. sodium hypochlorite accident: a systematic review. j endod. 2017 jan;43(1):16-24. pmid: 27986099. 30. hatton j, walsh s, wilson a. management of the sodium hypochlorite accident: a rare but significant complication of root canal treatment. bmj case rep. 2015 mar 25;2015:bcr2014207480. pmid: 25809429. 31. gernhardt cr, eppendorf k, kozlowski a, brandt m. toxicity of concentrated sodium hypochlorite used as an endodontic irrigant. int endod j. 2004 apr;37(4):272-80. pmid: 15056354. 32. de sermeño rf, da silva la, herrera h, herrera h, silva ra, leonardo mr. tissue damage after sodium hypochlorite extrusion during root canal treatment. oral surg oral med oral pathol oral radiol endod. 2009 jul;108(1):e46-9. pmid: 19442541. 33. markose g, cotter cj, hislop ws. facial atrophy following accidental subcutaneous extrusion of sodium hypochlorite. british dental journal. 2009;206(5):263-4. pmid: 19287421. 34. pelka m, petschelt a. permanent mimic musculature and nerve damage caused by sodium hypochlorite: a case report. oral surgery, oral medicine, oral pathology, oral radiology, and endodontology. 2008;106(3):e80-e3. pmid: 18602848. 35. mehra p, clancy c, wu j. formation of a facial hematoma during endodontic therapy. j am dent assoc. 2000 jan;131(1):67-71. pmid: 10649874. 36. chaudhry h, wildan tm, popat s, anand r, dhariwal d. before you reach for the bleach. british dental journal. 2011;210(4):157-60. pmid: 21350524. 37. al-sebaei mo, halabi oa, el-hakim ie. sodium hypochlorite accident resulting in life-threatening airway obstruction during root canal treatment: a case report. clin cosmet investig dent. 2015 mar 4;7:41-4. pmid: 25767406. 38. elderton r. a modern approach to the use of rubber dam--1. the dental practitioner and dental record. 1971;21(6):187. pmid: 5278933. 39. palmer no, ahmed m, grieveson b. an investigation of current endodontic practice and training needs in primary care in the north west of england. br dent j. 2009 jun 13;206(11):e22; discussion 584-5. pmid: 19478812. 40. hill ee, rubel bs. do dental educators need to improve their approach to teaching rubber dam use? j dent educ. 2008 oct;72(10):1177-81. pmid: 18923098. 41. ahmad i. rubber dam usage for endodontic treatment: a review. international endodontic journal. 2009;42(11):963-72. pmid: 19825034. 42. kim sg, malek m, sigurdsson a, lin lm, kahler b. regenerative endodontics: a comprehensive review. int endod j. 2018 dec;51(12):1367-1388. pmid: 29777616. 43. european society of endodontology. quality guidelines for endodontic treatment: consensus report of the european society of endodontology. int endod j. 2006 dec;39(12):921-30. pmid: 17180780. 44. gilbert gh, litaker ms, pihlstrom dj, amundson cw, gordan vv; dpbrn collaborative group. rubber dam use during routine operative dentistry procedures: findings from the dental pbrn. oper dent. 2010 sep-oct;35(5):491-9. pmid: 20945739. 45. american academy on pediatric dentistry clinical affairs committee-pulp therapy subcommittee; american academy on pediatric dentistry council on clinical affairs. guideline on pulp therapy for primary and young permanent teeth. pediatr dent. 2008-... 46. ghasemi m, khoshakhlagh ah, mahmudi s, fesharaki mg. identification and assessment of medical errors in the triage area of an educational hospital using the sherpa technique in iran. int j occup saf ergon. 2015;21(3):382-90. pmid: 26327157. .47 jäggi m, magni e, eggmann f, elayouti a, connert t, weiger r. apical pressure generated using conventional syringe irrigation in immature teeth—an in vitro study. materials. 2021; 14(10):2580. pmid: 34063549 .48 silva wo, vianna silva macedo rp, nevares g, val rodrigues rc, grossi heleno jf, braga pintor av, almeida bm. recommendations for managing endodontic emergencies during coronavirus disease 2019 outbreak. j endod. 2021 jan;47(1):3-10. pmid: 33... microsoft word yang lee proof 4-6.docx   vol  3,  no  1  (2015)   issn  2167-­‐8677  (online)   doi  10.5195/d3000.2015.32           http://dentistry3000.pitt.edu     new  articles  in  this  journal  are  licensed  under  a  creative  commons  attribution  4.0  united  states  license.     this  journal  is  published  by  the  university  library  system,  university  of  pittsburgh  as  part  of  its  d-­‐scribe  digital  publishing  program  and  is  cosponored   by  the  university  of  pittsburgh  press.   familial  aggregation  of  mandibular  prognathism   april  lee1  and  chris  yang1     1  university  of  pittsburgh  school  of  dental  medicine,  pittsburgh  pa,  usa     abstract     mandibular  prognathism  is  a  hereditary  condi5on  where  there   is  an  excess  growth   of   the  mandible   in   rela/on  to  the  maxilla   that  can  be  associated  with  maxillary  retrusion,   mandibular  protrusion,  or  both.   skeletal  mandibular  prognathism  is  most  prevalent  in  eastern  asian  popula7ons.  this  paper   focuses   on   a   korean   family   with   skeletal   mandibular   prognathism   that   was   inherited   through   three   genera+ons.   apparently,   neither   mandible   nor   maxilla   is   retruded   in   the   affected   individuals,  but  there   is  a  concave  facial  profile.  the  den77on  has  a  class   i  occlu-­‐ sion  with  skeletal  mandibular  prognathism,  and  the  only  way  to  treat  this  case  would  be  or-­‐ thognathic  surgery  with  the  help  of  orthodon4c  appliances.       cita%on:   lee   a   and   yang   c.   (2015)   familial   ag-­‐ grega%on  of  mandibular  prognathism.  den$stry   3000.  1:a001  doi:10.5195/d3000.2015.32   received:  march  20,  2015   accepted:  march  26,  2015   published:  april    8,  2015   copyright:  ©2015   lee  a   and   yang  c.   this   is   an   open   access   ar!cle   licensed   under   a   crea!ve   commons   a!ribu%on   work   4.0   united   states   license.   email:  akl33@pi(.edu       introduction   mandibular  prognathism  is   a  complex,  hereditary  condition   where  the  skeletal  profile  is  con-­‐ cave:  the  mandible  is  large  or  there   is  underdeveloped  or  normal  maxil-­‐ la.  the  dentition  can  be  class  i  (not   necessarily  class  iii)  in  order  to   have  true  skeletal  mandibular   prognathism.     the  condition  can  be  pre-­‐ sented  with  distinct  cephalometric   features  such  as  a  short  anterior   cranial  base  length,  acute  cranial   base  angle,  short  retrusive  maxilla,   proclined  maxillary  incisors,  retro-­‐ clined  mandibular  incisors,  an  ex-­‐ cessive  lower  anterior  face  height   and  obtuse  gonial  angle.  it  can  be   associated  with  maxillary  retrusion   or  mandibular  protrusion  or  both   [1].     mandibular  prognathism   can  be  expressed  to  different  de-­‐ grees  based  on  sex  and  ethnicity.   even  though  esthetics  is  an   initial  concern  for  a  patient   with  malocclusion,  there  can   also  be  functional  concerns   (i.e.,  temporomandibular  joint   dysfunction),  and  psychologi-­‐ cal  problems  that  may  affect   the  patient  [2,3].  in  this  pa-­‐ per,  a  family  segregating   mandibular  prognathism  is   described  with  the  suggestion   there  is  a  strong  genetic  com-­‐ ponent  to  this  phenotype.        familial  aggrega%on  of  mandibular  prognathism   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.3 2    http://dentistry3000.pitt.edu   2   case  report   this  paper  describes  a  24-­‐ year-­‐old  female  who  has  been  con-­‐ cerned  about  her  esthetics  with   having  a  projected  mandible,  giving   her  a  concave  profile.  her  maxilla  is   positioned  slightly  backwards  in   regards  to  the  mandible.  this  gives   her  an  appearance  of  “flat  nose”  and   seemingly  smaller  cheekbones.  she   has  slight  frontal  bossing,  and  con-­‐ sequently  her  forehead  is  not  in  line   with  the  maxilla  or  mandible,  giving   her  the  concave  profile.     the  patient’s  mother  has   mandibular  prognathism.  the   mother’s  side  of  the  family  mem-­‐ bers  has  similar  profiles:  three  of   her  mother’s  sisters,  one  of  her   mother’s  brothers,  and  her  grand-­‐ mother.  the  mother’s  sisters  all   have  children  who  do  not  have   mandibular  prognathism,  but  her   brother  has  one  daughter  with  sub-­‐ tle  sign  of  class  iii  and  a  son  with   class  i.  however,  the  patient’s  father   and  her  younger  sister  both  have   straight  profiles  (figure  1,  2,  3).     the  patient  was  still  con-­‐ cerned  with  her  looks,  so  she  was   considering  getting  orthodontics  or   orthognathic  surgery.  however,  her   dentition  was  class  i  occlusion  with   perfectly  normal  function.  there-­‐ fore,  nothing  was  performed.         discussion   it  seems  that  mandibular   prognathism  is  most  prevalent  in   eastern  asian  population,  which   can  range  from  8-­‐40%.  there   seems  to  be  no  significant  sexual   preference  for  inheritability,  but   there  seems  to  be  significant  dif-­‐ ference  in  sexual  dimorphism.   there  can  be  five  distinct  sub-­‐ phenotypes  of  class  iii  malocclu-­‐ sion:  1)  prognathic  mandible   with  long  face,  2)  maxillary  defi-­‐ ciency  with  decreased  vertical   dimensions,  3)  maxillary  defi-­‐ ciency  with  increased  vertical   dimensions,  4)  mild  prognathic   mandible  with  nor-­‐ mal  vertical  dimen-­‐ sions,  and  5)  combi-­‐ nation  of  prognathic   mandible  and  maxil-­‐ lary  deficiency  with   normal  vertical  di-­‐ mension  [1].    of   those  subpheno-­‐ types,  this  family   seems  to  have  mild   prognathic  mandible   with  normal  vertical   dimensions.   according  to   the  literature,  man-­‐ dibular  prognathism  shows  auto-­‐ somal  dominance  with  incomplete   penetrance  with  variable  expressiv-­‐ ity,  or  it  could  be  multifactorial.  the   prevalence  varies  among  different   ethnicities,  and  it  can  show  variable   anatomic  characteristics  [1].  in  this   family,  no  one  on  her  father’s  side   was  affected  with  having  class  iii   malocclusion,  but  many  individuals   on  her  mother’s  side  of  the  family   were  affected.  there  seems  to  be  no   sexual  preference  in  inheritability.     as  a  dentist,  if  the  patient  is   concerned  with  esthetics,  the  den-­‐ tist  can  help  the  patient  to  feel  more   confident  by  considering  orthodon-­‐ tics  or  orthognathic  surgery,  but                      familial  aggrega%on  of  mandibular  prognathism   vol  3,  no  1  (2015)        doi  10.5195/d3000.2015.3 2    http://dentistry3000.pitt.edu   3   s/he  should  also  be  able  to  present   the  negative  aspects  of  the  treat-­‐ ment.    if  the  patient  was  not  con-­‐ cerned  with  esthetics  but  the  func-­‐ tion  was  an  issue,  then  the  dentist   should  foresee  the  problem  for  the   patient  in  the  long  term  and  give  all   the  options  available  for  the  patient   to  progress  with  treatment.     since  the  patient  had  no   functional  problems  with  occlusion,   the  dentist  should  discuss  with  pa-­‐ tient  the  pros  and  cons  of  orthodon-­‐ tics  or  orthognathic  surgery.  the   pros  would  be  improving  esthetics,   but  on  the  downside,  it  can  de-­‐ crease  function,  weaken  teeth,  long   recovery  period,  and  create  a  finan-­‐ cial  burden  for  the  patient.       references     1.    james  k.  hartsfield  jr.,  lorri  ann   morford  and  liliana  m.  otero  (2012).   genetic  factors  affecting  facial   growth,  orthodontics  -­‐  basic  aspects   and  clinical  considerations,  prof.  fa-­‐ rid  bourzgui  (ed.),  isbn:  978-­‐953-­‐51-­‐ 0143-­‐7,  intech,  available  from:   http://www.intechopen.com/books/ orthodontics-­‐basic-­‐aspects-­‐and-­‐ clinicalconsiderations/   genetic-­‐factors-­‐affecting-­‐facial-­‐ growth     2.  orthodontic-­‐surgical  treatment  of   class  iii  malocclusion  with  extraction   of  an  impacted  canine  and  multi-­‐ segmented  maxillary  surgery.   janson  m,  janson  g,  santana  e,  de   castro  rc,  de  freitas  mr.   am  j  orthod  dentofacial  orthop.   2010  jun;137(6):840-­‐9.  doi:   10.1016/j.ajodo.2007.12.036.   pmid:  20685541       3.  surgical-­‐orthodontic  treatment  of  a   skeletal  class  iii  malocclusion.  katiyar   r,  singh  g,  mehrotra  d,  singh  a.  natl   j  maxillofac  surg.  2010  jul-­‐dec;   1(2):143-­‐149.  pmcid:  pmc3304204       826 final novel surgical method for contour restora7on in pa7ents with micrognathia vol 13 no 1 (2025) doi 10.5195/d3000.2025.826 h#p://den*stry3000.pi#.edu novel surgical method for contour restoration in patients with micrognathia sabah hassan faculty of dentistry, al-mansour university college, baghdad, iraq abstract the bone gra+ procedure is the most popular approach that has been u5lized to repair and shape the micrognathia. several other treatments have also been employed. the new surgical technique is a comprehensive technique, it gives a ridge and permanent chin with a normal esthe5c appearance. keywords: facial asymmetry; mandibular bone; facial trauma. cita:on: hassan s. (2025) novel surgical method for contour restora:on in pa:ents with micrognathia. den:stry 3000. 1: a001 doi:10.5195/d3000.2025.826 received: january 14, 2025 accepted: february 12, 2025 published: february 14, 2025 copyright: ©2025 hassan s. this is an open access ar:cle licensed under a crea:ve commons awribu:on work 4.0 united states license. email: sabahbedi1@gmail.com introduction in this procedure, a novel surgical technique has been utilized, which involves the utilization of a newly modified problast, which is an artificial substance that is primarily utilized in plastic surgery [1]. in addition to having a low crashing strength and being resorbed by any direct external pressure, the newly redesigned problast is a porous substance that is between 70 and 90 percent porous by volume. subperiosteal implants have been utilized to sculpt the aesthetic of the micrognathia, and the new modified problast has been employed for this purpose. surgical procedures 12 patients have been hospitalized to our center. clinical finding, patients with micrognathia, no other complain, all vital signs are normal. patients have no drug allergy surgery was conducted on ten patients under general anesthesia with nasotracheal intubation. an incision made at the interior border of the mandible, to avoid the scar which may be a large and agley looking especially in a dark skin patient. the chine bone exposed by reflecting the periosteum and the mentalis muscle by a periosteal elevator [2]. the new modified prosthesis, fully immersed in a biocompatible mixture [3-4] was utilized to facilitate fibrous and bone formation through the porous prosthesis in the operating theatre, the modified implant was autoclaved and adapted as a subperiosteal implant to restore and reconstruct a normal aesthetic chin [5-6]. injecting a biocompatible mixture by dental syringe into the subperiosteal prosthesis may enhance and promote bone formation through the porous implant. the modified sub-periosteal implant does not require fixation to the bones; instead, fibrous ingrowth through the ultra-porous implant provides temporary fixation. bone ingrowth through the porous material leads to a robust permanent fixation of the implant, ensuring a lasting aesthetic appearance. before suturing a novel surgical method for contour restora7on in pa7ents with micrognathia vol 13 no 1 (2025) doi 10.5195/d3000.2025.826 h#p://den*stry3000.pi#.edu primary stabilization of the subperiosteal implant an achieved by resorbable suturing, wound closed in layers without tension results and discussion post-operative follow-up reveals that the clinical assessment of the implant demonstrates an aesthetically pleasing appearance of the restored chin. the prosthetic implants are radiolucent; in the initial phases of follow-up, no abnormalities were observed on the plain x-ray. bone spicules infiltrate the porous problast implant approximately three months post-surgery. three to four years' post-surgery, bone seen within the porous problast implant via a plane x-ray. after five years, the subperiosteal problast implant is fully resorbed and replaced by normal bone, followed by remodeling those results in a permanent, normal structure. this novel surgical approach may be executed intraorally under local anesthesia, by bilateral injections to the inferior dental nerve, mucosal infiltration sometimes needed to reduce any pain or hemorrhage. two cases were conducted intraorally, both resulting in failure owing to postoperative infection. table 1. surgical approach. this novel surgical treatment is straightforward, non-traumatic to the patient, and highly recommended for individuals who are contraindicated for bone graft surgery or those who have declined such procedures. the surgeon spends less time in the operating room, there is no fixation of the modified problast implant needed as in other techniques conclusion the novel surgical procedure provides a satisfactory and enduring cosmetic outcome, with no failures seen when executed via an extra oral route under general anesthesia. references 1. u.s. patent 3,992,725 and 4,129,470 corresponding foreign patents and pending applications. tx: proplast is registered trademark of vitek, inc houston; 1984. 2. puzovic d, konstan\novic vs, and dimitrijevic m. evalua\on of maxillofacial weapon injuries: 15 -year experience in belgrade. j craniofac surg. 2004; 15:543–546. haps://doi.org/10.1097/0000 1665-200407000-00003 3. podaropoulos l, veis aa, papadimitriou s, et al. bone regeneration using beta-tricalcium phosphate in a calcium sulfate matrix. j oral implantol. 2009; 35: 28 –36. https://doi.org/10.1563/15481336-35.1.28 4. shiria y, okuda k, kubota t, et al. the comparative effectiveness of granules or blocks of super-porous hydroxyapatite for treatment of intrabony periodontal defects. sci res. 2012; 2:81–87. no. of patient surgical approach result 2 adult patients intraoral approach failure 10 adult patients extra oral approach good result novel surgical method for contour restora7on in pa7ents with micrognathia vol 13 no 1 (2025) doi 10.5195/d3000.2025.826 h#p://den*stry3000.pi#.edu https://doi.org/10.4236/ojst.2012. 22015 5. earl ow, stephen mp, joe bd. mandibular replacements a review of embedded implants, the journal of prosthetic dentistry. 1976;35(2):207–210. https://doi.org/10.1016/00223913(76)90281-x 6. owens bd, kragh jf jr., wenke jc, et al. combat wounds in operation iraqi freedom and operation enduring freedom. j trauma. 2008; 64:295–299. https://doi.org/10.1097/ta.0b013 e318163b875 711+2024 management of gingival enlargement during orthodon5c treatment by diode lasers versus conven5onal method vol 12 no 2 (2024) doi 10.5195/d3000.2024.711 management of gingival enlargement during orthodontic treatment by diode lasers versus conventional method fatima h. almusawi¹*, shaimaa s. mahdi1, salah a. alkurtas2 ¹university of baghdad, baghdad, iraq 2al turath university, baghdad, iraq *corresponding author abstract objec&ve: the aim of this study was to assess the efficacy of diode lasers (810-980 nm) in trea=ng gingival enlargement caused by orthodon=c appliances. addi=onally, to compare the outcomes of diode laser treatment with the conven=onal method (scalpel) in managing gingival enlargement. materials and methods: for this study, a total of 20 pa=ents ranging in age from 14 to 28 years old were included. these individuals were divided into two groups: the laser group and the conven=onal group. in the laser group, surgery was performed on 10 pa=ents using an 810–980 nm diode laser in con=nuous wave mode with energy sejngs of 1.5–2 wals. on the other hand, the conven=onal group also consisted of 10 pa=ents who underwent gingival enlargement management due to an orthodon=c appliance using a scalpel. to assess pain and discomfort, pa=ents were provided with a ques=onnaire to record their experiences daily for the first seven days following the surgery. pain and discomfort were evaluated using a verbal ra=ng scale. during follow-up visits, plaque index, bleeding on probing, healing, and swelling were assessed. addi=onally, the dura=on of the surgery and bleeding scores were recorded during the surgical procedure. results: the ini=al postopera=ve bleeding score was significantly lower in the laser group compared to the conven=onal group. the dura=on of the procedure was shorter in the laser group than in the conven=onal group. addi=onally, the laser group exhibited lower levels of pi and bop percentages compared to the conven=onal group. notably, there was a significant different in the percentage of bop, with a p=0.03 in the first week, and a p=0.002 in the fourth week, as determined by t-tests. when comparing individuals in the laser group to those in the conven=onal group, it was observed that the pain and discomfort scores decreased. furthermore, the swelling score was significantly lower in the laser group compared to the conven=onal group, par=cularly in the third week (chi square sta=s=c = 4.9, p=0.05). lastly, there was a significant varia=on in healing scores in the laser group throughout all weeks of follow-up, as indicated by the t-test (p< 0.05). conclusion: based on the results obtained, it can be concluded that diode lasers opera=ng within the range of 810-980 nm, when set at the appropriate power level, prove to be an effec=ve method for managing gingival enlargement caused by orthodon=c appliances keywords: gingival enlargement, diode lasers, scalpel, gingivectomy. cita:on: almusawi fh, et al. (2024) management of gingival enlargement during orthodon:c treatment by diode lasers versus conven:onal method. den:stry 3000. doi:10.5195/d3000.2024.711 received: august 19, 2024 accepted: august 24, 2024 published: november 22, 2024 copyright: ©2024 abbas s, et al. this is an open access ar:cle licensed under a crea:ve commons auribu:on work 4.0 united states license. email: fa:ma.hasan2102m@ilps.uobaghdad.edu.iq introduction gingival enlargement is one of the most common soft tissue diseases related to fixed orthodontic equipment [1,2]. in addition to causing functional and aesthetic problems, gingival enlargement has been shown to interfere with orthodontic tooth movement and make it more difficult to maintain good dental hygiene, which in turn damages the periodontium more significantly [3]. with more individuals seeking orthodontic treatment, there is a greater risk of iatrogenic periodontal damage, which presents an ongoing challenge for orthodontists in maintaining a healthy periodontal condition for their patients [4]. the tested metal devices (brackets) showed a high affinity for bacteria and proteins. there were also differences in the adsorption profiles between the elastic devices. therefore, it is conceivable that different types of biofilms would form on these management of gingival enlargement during orthodon5c treatment by diode lasers versus conven5onal method vol 12 no 2 (2024) doi 10.5195/d3000.2024.711 orthodontic surfaces, as they are made of different materials and have different elasticity and morphology [5]. the management of gingival enlargement is contingent upon the cause and underlying pathology. the strategy employed is contingent upon the type of enlargement and is guided by the clinical and pathological manifestations. before any surgical measures are taken, phase i treatment should be implemented. addressing gingival enlargement necessitates an individualized approach that is tailored to the root cause. in cases where dental plaque is the culprit, nonsurgical treatment focuses on enhancing oral hygiene [6]. to achieve optimal oral health, it is crucial to undergo professional cleaning procedures such as scaling and root planning, which effectively eliminate plaque and calculus. equally important is patient education, which emphasizes the significance of proper brushing and flossing techniques, the utilization of antimicrobial mouthwashes, and the regularity of dental visits. additionally, it is essential to address local factors that contribute to plaque formation, such as making necessary adjustments or replacements to problematic restorations and modifying orthodontic appliances to minimize plaque buildup [6]. when nonsurgical methods prove to be ineffective, the appropriate course of action for treating gingival enlargement is surgical intervention. one such surgical procedure is gingivectomy, which entails the removal of excess free gingival tissue, allowing patients to maintain proper oral hygiene [7]. gingivectomy is defined as the surgical removal of the gingiva by removing the periodontal pocket walls, providing access and visibility for complete calculus removal, and smoothing the tooth roots, creating an environment conducive to the healing of the gingiva, and restoring the physiological the contour of the gingiva [8]. gingivectomy can be performed with a scalpel, electrosurgery, and laser [9]. traditional gingivectomy is still performed with a scalpel and is the most common method today. it has the advantages of being a being a low-cost and durable instrument, but hemostasis can be difficult to achieve [10,11]. in the past decade, lasers have received much attention as an adjunct to periodontal surgery because they offer a less invasive approach [12]. lasers are a relatively new modern technology, developed by maiman in 1960 [13]. researchers have studied the application of lasers in dental procedures encompasses both soft and hard tissues [14]. however, in the oral cavity, it was initially successfully applied in 1977 and has been improved and updated over time [15]. diode lasers generally do not interact with hard tooth tissue, making them excellent surgical lasers for soft tissues [16,17]. the advantages of diode lasers for soft tissues are their excellent cutting properties, cutting depths of 2–6 mm, obstruction of lymphatic and small blood vessels, which causes hemostasis, and minimalized edema following surgery [18]. it also disinfects the target tissue by local heating and the formation of a scab layer, and scarring is reduced due to reduced postoperative tissue contraction [19-21], accelerated wound healing, and pain relief [22]. the aim of this study was to assess the efficacy of diode lasers (810-980 nm) in treating gingival management of gingival enlargement during orthodon5c treatment by diode lasers versus conven5onal method vol 12 no 2 (2024) doi 10.5195/d3000.2024.711 enlargement caused by orthodontic appliances. materials and methods a total of 20 individuals, ranging in age from 14 to 28 years old, were selected to participate in the research. these patients had been diagnosed with gingival enlargement caused by orthodontic appliances and poor oral hygiene. the patients underwent an assessment of their plaque index (pi), bleeding on probing (bop) and probing pocket depth (ppd). following this evaluation, all patients received treatment in the form of scaling and root planning. they were also provided with instructions on proper tooth brushing techniques, the use of dental floss, and the utilization of a chlorhexidine (0.12%) mouthwash for one week to enhance their oral hygiene. subsequently, the patients were divided into two separate groups. group i: ten patients underwent gingivectomy by (810-980nm) diode lasers (quicklase uk), were used to removal gingival enlargement due to orthodontic appliance. group ii: ten patients underwent gingivectomy by conventional method used scalpel blade no. (12,15) to removal gingival enlargement due to orthodontic appliance. clinical intervention prior to the surgery during the evaluation of this visit, pi, bop percentage, and ppd were assessed. prior to the gingivectomy procedure as shown in figure 1, all patients in both groups underwent non-surgical treatment, which involved scaling with an ultrasonic scaler and root planning as shown figure 2, at least two weeks in advance. patients were provided with oral hygiene instructions, which included brushing their teeth a minimum of three times per day, using dental floss twice daily, and utilizing a 12% chlorhexidine mouthwash twice daily for one week. upon completion of a twoweek period of non-surgical treatment, it is expected that the patients' assessment of pi, ppd, and bop percentage will demonstrate a reduction to below 10%. figure 1. first visit (base line visit) evaluated gingival enlargement due to orthodontic appliance. figure 2. immediately after scaling and root planning. conventional surgical technique for the surgical procedure of the ten subjects, the site was numbed using local anesthesia, specifically 2.2 ml of a cartridge containing 2% lidocaine and 1:80,000 adrenaline (epinephrine). to investigate pseudo pockets, a periodontal probe was utilized to identify bleeding points, which were then marked using a pocket depth marker. the initial incision management of gingival enlargement during orthodon5c treatment by diode lasers versus conven5onal method vol 12 no 2 (2024) doi 10.5195/d3000.2024.711 was made 1 mm below the bleeding points, while the secondary incision was performed with blade no. 15. the excised tissue was carefully removed, and the surgical site was consistently cleansed with normal saline solution. following the removal of the tissue, the surgical site was delicately trimmed using a kirkland knife in a bevel direction, ensuring that only the necessary amount of tissue was removed to avoid exposing the bone. this approach allowed for the preservation of the natural shape of the gingiva once the cutting process was completed. any remaining fibrous tissue between the teeth was excised using blade no. 12. after the surgery, the surgical site was thoroughly cleaned with surgical gauze soaked in normal saline solution, and a periodontal pack, as shown in figure 3, was placed over the surgical area. this pack was left in place for a duration of one week. the duration of the surgery was determined prior to its commencement. once the operation was completed, we documented the surgery duration and bleeding score on the case sheet. the specialized dental center of al baladyat was where the surgical treatment took place. figure 3. conventional procedure (scalpel method) for gingival enlargement due to the orthodontic appliance. laser surgical technique for the surgical procedure of the ten subjects, the site was numbed with local anesthesia using a solution containing 2% lidocaine and 1:80,000 adrenaline, with a total of 2.2 ml administered per cartilage. the (cej) was determined using a periodontal probe, and bleeding points were marked using a pocket depth marker. the fiber tip was initiated by applying either articulating paper or dark chromophore, and the laser was activated to prime the fiber tip. the preparation of the fiber tip was considered complete when it ignited the articulation paper. to enhance thermal interaction with the tissue and expedite lesion removal, fiber priming was utilized to concentrate thermal energy at the fiber tip. the technique utilized quicklase uk's diode lasers, which emit light in the range of 810-980 nm. the diameter of the fiber tip used in the procedure was measured at 400 µm. the power output, ranging from 1.5 to 2w, seen in figure 4, was adjusted based on the thickness of the gingival tissue. to ensure safety and minimize unwanted reflections, both the surgical team and the patient were advised to wear protective eyewear, and the surgical area was cleared of any reflective objects such as jewelry, watches, and shiny medical equipment. tissue slices were created using a sweeping brush stroke motion with the fiber, resulting in beveled edges. throughout the surgical procedure, a member of the surgical team was assigned the task of positioning the suction tip near the surgical site in order to eliminate any smoke or odor that management of gingival enlargement during orthodon5c treatment by diode lasers versus conven5onal method vol 12 no 2 (2024) doi 10.5195/d3000.2024.711 may arise from the laser tissue ablation process. additionally, this individual was responsible for continuously irrigating the area with normal saline to maintain a cool environment. to prevent excessive heat generation and ensure optimal tissue condition, the laser was paused approximately every 20 seconds for thorough inspection of the gingival tissue. any tissue debris that accumulated on the operative site and the tip was carefully removed using wet gauze. notably, the surgical site was left uncovered without the use of a surgical pack, as depicted in figure 5. prior to the commencement of the surgery, the duration of the procedure was calculated. upon completion of the surgery, the duration and bleeding score were diligently recorded on the case sheet. figure 4. power se.ng in the diode laser (810-980). figure 5. laser procedure for management of gingival enlargement due to orthodontic appliance. the following parameters were assessed for both surgical techniques: patient satisfaction, pi, bop percentage, and ppd were assessed before the surgery, and in each visit, the patient was followed the surgery [23,24]. intraoperatively bleeding scores was assessed to determine the instrument performance for hemostasis, and it was graded as: (1) not bleeding, (2) self-limiting, (3) requiring light pressure, (4) requiring coagulation, and (5) requiring ligation or hemoclips [25]. healing scores: landry, tumbull and howely described an index to describe the extent of healing after periodontal surgery as (1) very poor, (2) poor, (3) good, (4) very good, and (5) excellent [26]. swelling score was assessed after the surgery in each visit of the patients according to following grades: (1) not swelling, (2) slight swelling, (3) moderate swelling, and (4) severe swelling [27]. statistical analysis spss v23 software was used for all statistical analysis. graphs and tables were utilized to illustrate results. the chi-square test was used to test if differences were significant between measurements of related numeric values. t-test and anova tests were utilized to determine the significance of differences between the tested mean of repetitive measurements. the significant differentiation criterion was a p value ≤0.05. results time duration laser surgery required a shorter duration per minute compared to conventional surgery (p=0.0001). this is supported by the data presented in table 1. table 1. comparative summary of the time duration in the study management of gingival enlargement during orthodon5c treatment by diode lasers versus conven5onal method vol 12 no 2 (2024) doi 10.5195/d3000.2024.711 groups. bleeding scores aker the complelon of the surgery, bleeding scores were measured. the laser group exhibited a significantly lower distribulon of bleeding scores compared to the convenlonal group (p=0.03), table 2. table 2. comparaive summary of bleeding scores in the study groups. pain and discomfort there was a decrease in pain and discomfort scores over lme in both methods. however, when comparing the two methods, the laser group tended to have a lower pain and discomfort scores than the convenlonal group. plaque index (pi) the average pi value following laser surgery was consistently lower than the pi value obtained through the conventional method in every week, as depicted in figure 6. the p-value obtained from the ttest for the conventional and laser methods were 0.87, 0.51, 0.43, and 0.9 for the first, second, third, fourth weeks, respectively. the results are shown in table 3. table 3. comparative statistical analysis of pi after the surgery in the study groups. figure 6. pi comparison. bleeding on probing the laser method consistently demonstrated a lower mean compared to the conventional method across all weeks, as illustrated in figure 7. the difference in means was found to be statistically significant, with pvalues of 0.03 and 0.002 in one week and four weeks, respectively, while p-values of 0.91 and 0.73 were observed at two and three weeks, respectively. furthermore, the anova-test revealed a statistically significant difference between the two methods, as indicated in table 4. management of gingival enlargement during orthodon5c treatment by diode lasers versus conven5onal method vol 12 no 2 (2024) doi 10.5195/d3000.2024.711 table 4. comparative statistical analysis of bop percentages in the study groups. figure 7. the distribution of mean bop percentages in the study groups. swelling scores the score for swelling exhibited a significant reduction throughout the duration of treatment in both the laser and conventional groups. the chi-square statistic yielded a value of 4.9, while the p-value obtained from the t-test was 0.005, indicating a significant difference between the laser group and the conventional group after three weeks. this disparity is clearly illustrated in table 5. however, the conventional group had three patients with mild swelling. after four weeks, only one patient in the laser group had mild swelling. these results are depicted in figure 8. table 5. comparative statistical analysis of swelling scores in the study groups. figure 8. the distribuion of mean values of the swelling scores in the study groups. healing scores differences in healing was statistically significant using t-test at one week between the two methods (p=0.005), two weeks (p=0.03), three weeks (p=0.03), and four weeks (p=0.02), respectively (table 6). as shown in figure 9, in one week after laser surgery, three out of 10 patients had a very good result in healing from the laser surgery, while no patient had a very good result in healing from the conventional surgery. in two weeks, four out of 10 patients after laser surgery had excellent results of healing, while one out of 10 patients had excellent results after conventional surgery. in three weeks, eight out of 10 patients had excellent results of healing after laser surgery, while two out of 10 had excellent results after the conventional method. in four weeks, eight patients out of 10 had excellent results of healing after laser surgery, while two patients out of 10 have excellent results after conventional surgery, so the healing result of the laser surgery was significantly different than that of the conventional method (figures 10 and 11). management of gingival enlargement during orthodon5c treatment by diode lasers versus conven5onal method vol 12 no 2 (2024) doi 10.5195/d3000.2024.711 table 6. comparative statistical analysis of healing scores in the study groups. figure 9. the distribuion of means of healing scores in the study groups. figure 10. follow up evaluation of healing in the laser surgery (a) after one week from the laser surgery, b) after two weeks from the laser surgery, (c) after three weeks of laser surgery, and (d) after four weeks of laser surgery. figure 11. follow up evaluaion of healing in the convenional surgery treatment: (a) axer one week of convenional surgery, (b) axer two weeks of convenional surgery, (c) axer three weeks of convenional surgery, and (d) axer four weeks of convenional surgery. discussion one of the most prevalent soft tissue issues associated with a fixed orthodontic appliance is gingival enlargement [28]. according to this study, the use of 810 nm wavelength energy was more effective in promoting coagulation in highly vascularized tissues following extensive procedures, as it significantly impacts blood factors. conversely, the use of 980 nm wavelength energy is more suitable for ablation, as it is highly absorbed by water and results in limited tissue involvement in areas with less vascularized tissue and narrower tissue involvement. the combination of these two wavelengths enhances both a b c d a b c d management of gingival enlargement during orthodon5c treatment by diode lasers versus conven5onal method vol 12 no 2 (2024) doi 10.5195/d3000.2024.711 coagulation and ablation while minimizing thermal damage [29]. in the process of photo-thermal laser-tissue interaction, the laser beam energy is absorbed by the tissue's main chromophores, which are the absorption centers. this absorption leads to an increase in tissue temperature within the area that is exposed to the laser, potentially resulting in tissue ablation and coagulation [30]. the diode laser with a wavelength of 980nm has an impact on temperature-gated calcium ion channels, while the 810nm wavelength primarily affects mitochondrial cytochrome c oxidase. when the near-surface water absorbs the 980nm wavelength, it causes changes in the temperature of protein domains. however, these temperature changes become insignificant when the overall bulk temperature is lowered. similarly, we hypothesized that raising the temperature to 42°c would render the microscopic temperature gradients caused by water absorption of the 980nm wavelength ineffective. the interesting observation from our experiments is that both heat and cold treatments nullified the effects of the 980nm laser, but not the effects of the 810nm laser. this suggests that the 980nm wavelength can activate heatgated ion channels, while the 810nm wavelength cannot. furthermore, the 810nm wavelength was found to be significantly effective in promoting healing, enhancing collagen accumulation, and facilitating complete re-epithelialization [31]. in this study, it was observed that there was no bleeding during laser surgery when compared to the conventional method. this lack of bleeding can be attributed to the increase in tissue temperature that occurs after the absorption of laser light by biological tissue. once the tissue temperature reaches 60°c, coagulation takes place through a process known as photo-coagulation. this process involves the instant denaturation of proteins, enzymes, and other bioactive molecules. as a result, there is a modification in the molecular structures of tissue collagen fibers, causing the irradiated tissue to constrict against the nearby blood vessels. additionally, the collagen in the blood vessel walls undergoes shrinkage, which aids in improving hemostasis. furthermore, the laser's impact on erythrocytes leads to enhanced platelet aggregation, further promoting hemostasis. consequently, the group that underwent intraluminal laser treatment experienced minimal blood loss compared to those who underwent scalpel surgery. diode lasers, due to their high absorption by hemoglobin and melanin, allow for precise cutting of soft tissue while ensuring excellent hemostasis. these lasers also can penetrate deeply, surpassing other types of lasers such as co2 lasers. as a result, the use of diode lasers reduces both the operation time and post-operative bleeding, eliminating the need for surgical packs or sutures [32]. however, there have been instances of bleeding during laser surgery in some cases specifically in the posterior teeth. on average, gingival enlargement is four times more likely to occur around posterior teeth compared to incisors and canines. this discrepancy is primarily attributed to the mechanical irritation caused by orthodontic bands, which have a higher chance of touching the gingiva around posterior teeth. additionally, factors such as increased food impaction in the posterior areas and a tendency for more thorough brushing of anterior teeth contribute to these differences. the presence of management of gingival enlargement during orthodon5c treatment by diode lasers versus conven5onal method vol 12 no 2 (2024) doi 10.5195/d3000.2024.711 exposed cement on the apical aspect of the bands may also lead to chemical irritation, while patients tend to have less effective oral hygiene practices for their posterior teeth [33]. as a result, patients may experience more inflammation and gingival enlargement in the posterior teeth due to orthodontic appliances compared to other areas. the findings of this study reveal that laser surgery has a shorter duration compared to conventional surgery. this is because the diode laser not only removes tissue but also cauterizes blood vessels, resulting in a bloodless surgical field. this reduces the time required for the procedure. in contrast, scalpel surgery often struggles to achieve proper hemostasis, leading to longer surgical times. additionally, gingivectomy using a surgical blade requires more instruments and steps to complete, whereas laser surgery utilizes a single surgical tip for both cutting and shaving the gingival tissue. to compare the levels of postoperative pain and discomfort among various patient groups, the present study utilized a visual analog scale (vas). the findings revealed that laser surgery resulted in lower levels of pain and discomfort compared to scalpel surgery at the first four days. this can be attributed to the heat produced by the laser, which inhibits the pain receptors, as well as the coagulation process that creates a dry and isolated environment, reducing the risk of infection in the wound [34,35]. in this study, pi and bop percentage after gingivectomy by diode lasers were lower than gingivectomy by scalpel. because the diode laser has an intense antibacterial effect by causing changes in the bacterial cell wall and destroying the cell membrane. diode laser exerts a photo-thermal effect on the reachable bacteria. it also exerts a photo-disruptive effect on the unreachable bacteria; where immediate cell death might not occur; but rather sublethal damage occurs inhibiting the cell growth through the destruction of cell wall integrity and accumulation of denatured proteins causing the cessation of bacterial growth and consecutive cell lysis. this effect on bacteria occurs with very small doses of heat [36]. the findings of this study demonstrated that swelling is reduced in laser surgery compared to conventional surgery. this is due to the laser's ability to effectively control bleeding during the procedure by blocking lymph vessels and sealing small blood vessels. as a result, postoperative complications like swelling and edema are significantly decreased [37,38]. the healing process following laser surgery in this study was found to be faster when compared to traditional surgical methods. the fundamental concept behind the modulation of cells through laser therapy is that targeted irradiation at a specific wavelength can modify cellular behavior by influencing either the mitochondrial respiratory chain or the calcium channels within the cell membrane. this, in turn, stimulates an enhancement in cell metabolism and promotes cell proliferation. as a result, these effects contribute to an exceptional coagulation ability and trigger unique healing mechanisms that differ significantly from those observed after the use of a scalpel [39,40]. conclusion 1.according to the obtained results, diode lasers 810-980nm can be used effectively at the management of gingival enlargement during orthodon5c treatment by diode lasers versus conven5onal method vol 12 no 2 (2024) doi 10.5195/d3000.2024.711 selected power setting in the management of gingival enlargement due to orthodontic appliance. 2.excellent hemostasis improve the vision without or with minimal bleeding in the posterior teeth in comparison to the conventional method, which does not have the hemostatic and bleeding effects during scalpel surgery. 3.less chair time and discomfort with the laser surgery make it more acceptable to the patients in comparison to the scalpel surgery. 4.less pain and swelling after the laser surgery. 5.faster healing is observed to the laser group. references [1] davis sm, plonka ab, fulks ba, taylor kl, bashutski j. consequences of orthodontic treatment on periodontal health: clinical and microbial effects. in seminars in orthodontics 2014 sep 1 (vol. 20, no. 3, pp. 139-149). wb saunders. [2] mazin h, salman sa, salah r. the effect of fixed orthodontic appliances on gingival health. iosr journal of dental and medical sciences 2016;15(11):82-88. [3] palomo l, palomo jm, bissada nf. salient periodontal issues for the modern biologic orthodontist. in seminars in orthodontics 2008 dec 1 (vol. 14, no. 4, pp. 229-245). wb saunders. [4] gomes sc, varela cc, da veiga sl, rösing ck, oppermann rv. periodontal conditions in subjects following orthodontic therapy. [5] a preliminary study. the european journal of orthodontics. 2007 oct 1;29(5):477-81. [5] fatani ej, almutairi hh, alharbi ao, alnakhli yo, divakar dd, alkheraif aa, khan aa. in vitro assessment of stainless steel orthodontic brackets coated with titanium oxide mixed ag for antiadherent and antibacterial properties against streptococcus mutans and porphyromonas gingivalis. microbial pathogenesis. 2017 nov 1;112:190-4. [6] steinberg d, eyal s. initial biofilm formation of streptococcus sobrinus on various orthodontics appliances. journal of oral rehabilitation. 2004 nov;31(11):1041-5. [7] sharma s, tomar n, rana n, kaushik m. comprehensive review on gingival enlargement and its management. educational administration: theory and practice. 2024 may 30;30(5):133731377.orthodontist. 2013 jan 1;83(1):43-7. [8] çayan t, hasanoğlu erbaşar gn, akca g, kahraman s. comparative evaluation of diode laser and scalpel surgery in the treatment of inflammatory fibrous hyperplasia: a split-mouth study. photobiomodulation, photomedicine, and laser surgery. 2019 feb 1;37(2):91-8. [9] newman mg, takei h, klokkevold pr, carranza fa. newman and carranza's clinical periodontology: newman and carranza's clinical periodontology e-book. elsevier health sciences; 2018 may 29. [10] fornaini c, rocca jp, bertrand mf, merigo e, nammour s, vescovi p. nd: yag and diode laser in the surgical management of soft tissues related to orthodontic treatment. photomedicine and laser surgery. 2007 oct 1;25(5):381-92. [11] musaa fe, awazli lg, alhamdani f. gingival enlargement management using diode laser 940 nm and management of gingival enlargement during orthodon5c treatment by diode lasers versus conven5onal method vol 12 no 2 (2024) doi 10.5195/d3000.2024.711 conventional scalpel technique (a comparative study). iraqi journal of laser 2017; 16(b), pp.1-9 [12] kumar p, rattan v, rai s. comparative evaluation of healing after gingivectomy with electrocautery and laser. journal of oral biology and craniofacial research. 2015 may 1;5(2):69-74. [13] kravitz nd, kusnoto b. softtissue lasers in orthodontics: an overview. american journal of orthodontics and dentofacial orthopedics. 2008 apr 1;133(4):s110-4. [14] maiman th. stimulated optical radiation in ruby. nature. 1960;187:493-494. [15] genovese md, olivi g. use of laser technology in orthodontics: hard and soft tissue laser treatments. european journal of paediatric dentistry. 2010 mar 1;11(1):44. [16] amaral mb, de ávila jm, abreu mh, mesquita ra. diode laser surgery versus scalpel surgery in the treatment of fibrous hyperplasia: a randomized clinical trial. international journal of oral and maxillofacial surgery. 2015 nov 1;44(11):1383-9. [17] marhab ei, al-bawi zf. use of laser in treatment of gingival fibromatosis: a case report. res militaris. 2022 nov 14;12(2):631520. [18] akram hm, ali oh, omran nk, ali ao. diode laser versus scalpel gingivectomy. biomedical and pharmacology journal. 2017 dec 21;10(4):1799-804. [19] govila v, gulati m, govila s. diode laser applications in periodontics. indian journal of dental sciences. 2011 dec 1;5(3):64-67. [20] al ghazali sr, ahmed ss, abdulhameed bs. the clinical evaluation of oral fibroma lesion removal utilizing two types of diode lasers 980 nm and 450 nm. ijl 2024 jun;23(1):80–89. [21] al-ani a, taher h, alalawi a. comparative evaluative study of erbium, chromium ysgg and wavelength-dual diode lasers in oral soft tissue incision morphology: a histological ex vivo study. ijl 2024 jun;23(1): 109– 117. [22] ize-iyamu in, saheeb bd, edetanlen be. comparing the 810nm diode laser with conventional surgery in orthodontic soft tissue procedures. ghana medical journal. 2013;47(3):107-11. [23] rebelo ma, queiroz ac. gingival indices: state of art. panagakos fs, davies rm. gingival diseases-their aetiology, prevention and treatment. london: intechopen limited. 2011 sep 22:41-54. [24] trombelli l, farina r, silva co, tatakis dn. plaque-induced gingivitis: case definition and diagnostic considerations. journal of clinical periodontology. 2018 jun;45:s44-67. [25] kumar p, rattan v, rai s. comparative evaluation of healing after gingivectomy with electrocautery and laser. journal of oral biology and craniofacial research. 2015 may 1;5(2):69-74. [26] mahtani aa, varghese s, jain r. periodontal dressing after flap surgery-a retrospective study. int j dent oral sci. 2021 mar 9;8(3):1932-8. [27] rosa ds, aranha ac, de paula eduardo c, aoki a. esthetic treatment of gingival melanin hyperpigmentation with er: yag laser: short-term clinical observations and patient followup. journal of periodontology. 2007 oct;78(10):2018-25. [28] to tn, rabie ab, wong rw, mcgrath cp. the adjunct management of gingival enlargement during orthodon5c treatment by diode lasers versus conven5onal method vol 12 no 2 (2024) doi 10.5195/d3000.2024.711 effectiveness of diode laser gingivectomy in maintaining periodontal health during orthodontic treatment: a randomized controlled clinical trial. the angle orthodontist. 2013 jan 1;83(1):43-7. [29] al-ani aj, al-alawi as, taher hj. analysis of the temperature elevation of the dual-wavelength diode laser and the er, cr: ysgg laser in oral soft tissue incisions. journal of lasers in medical sciences. 2023;14. [30] wang y, huang yy, wang y, lyu p, hamblin mr. photobiomodulation of human adipose-derived stem cells using 810 nm and 980 nm lasers operates via different mechanisms of action. biochimica et biophysica acta (bba)-general subjects. 2017 feb 1;1861(2):441-9. [31] fahim sz, ghali rm, hashem aa, farid mm. the efficacy of 2780 nm er, cr; ysgg and 940 nm diode laser in root canal disinfection: a randomized clinical trial. clinical oral investigations. 2024 feb 26;28(3):175. [32] cobb cm. lasers in periodontics: a review of the literature. journal of periodontology. 2006 apr;77(4):545-64. [33] giannopoulou c. inflammatory side effects associated with orthodontic tooth movement (doctoral dissertation, tesis: faculté de médecine université de genève). [34] hawkins d, houreld n, abrahamse h. low level laser therapy (lllt) as an effective therapeutic modality for delayed wound healing. annals of the new york academy of sciences. 2005 nov;1056(1):486-93. [35] majeed aa. expedited removal of pyogenic granuloma using 980nm diode laser (doctoral dissertation, institute of laser for postgraduate studies expedited removal of pyogenic granuloma using 980nm diode laser a thesis submitted to the institute of laser for postgraduate studies, university of baghdad). [36] bhagat m, tapashetti r, fatima g, bhutani n. lasers in periodontal therapy. surgery. 2020;6:7. [37] dompe c, moncrieff l, matys j, grzech-leśniak k, kocherova i, bryja a, bruska m, dominiak m, mozdziak p, skiba th, shibli ja. photobiomodulation—underlying mechanism and clinical applications. journal of clinical medicine. 2020 jun 3;9(6):1724. [38] mahdi ah. treatment of oral conditions by 810 nm diode laser. iraqi journal of laser. 2010 dec 15;9(b):21-5. [39] amaroli a, ferrando s, benedicenti s. photobiomodulation affects key cellular pathways of all life-forms: considerations on old and new laser light targets and the calcium issue. photochemistry and photobiology. 2019 jan;95(1):4559. [40] vitruk p. oral soft tissue laser ablative and coagulative efficiencies spectra. implant practice us. 2014;7(6):19-27 microsoft word 472 2023.docx effects of an,bio,cs on staphylococcus aureus biofilm forma,on associated with peri-implan,,s vol 11 no 1 (2023) doi 10.5195/d3000.2023.472 h#p://den*stry3000.pi#.edu effects of antibiotics on staphylococcus aureus biofilm formation associated with peri-implantitis aseel al-karawi1, abdulhameed hameed2, sally da’aj3, zeyad abed4 1periodontics, dentistry, bilad alrafidain university college, diyala, iraq 2oral and maxillofacial surgery, ministry of health, diyala, iraq 3pediatric dentistry, bilad alrafidain university college, diyala, iraq 4oral and maxillofacial surgery, bilad alrafidain university college, diyala, iraq abstract background: one hundred and twenty specimens were collected from pa,ents with peri-implan,,s in the period from august 2022 un,l september 2022 from baquba specialized dental center, in the province of diyala. the goal of this work was to es,mate the frequency of staphylococcus aureus and their ability to produce biofilms associated with peri-implan,,s. also, we aimed to assess the an,-biofilm property of ciprofloxacin and amoxicillin/clavulanic acid on staphylococcus aureus. the result showed growth of staphylococcus aureus in twenty isolates (16.6%), and 95% of the staphylococcus aureus isolates had the ability to produce biofilms. the results indicate that the minimum inhibitory concentra,on (mic) of ciprofloxacin for staphylococcus aureus was 32 to 512 μg/ml with the sub-mic 16 to 256 μg/ml. the mic for amoxicillin/clavulanic was 8 to 512 μg/ml, and the sub-mic 4 to 256) μg/ml. ciprofloxacin and amoxicillin/clavulanic acid had the ability to decrease biofilm forma,on and amoxicillin/clavulanic acid was more effec,ve than ciprofloxacin. keywords: staphylococcus aureus; biofilm; peri-implan,,s; minimum inhibitory concentra,on cita:on: al-karawi, a et al. (2023) effects of an:bio:cs on staphylococcus aureus biofilm forma:on associated with peri-implan::s den:stry 3000. 1:a001 doi:10.5195/d3000.2023.472 received: march 10, 2023 accepted: april 10, 2023 published: april 21, 2023 copyright: ©2023 al-karawi, a et al. this is an open access ar:cle licensed under a crea:ve commons axribu:on work 4.0 united states license. email: dr.aseel@bauc14.edu.iq introduction despite the fact that dental implants have a high success rate and are a safe and predictable therapeutic option, they are not exempt from biological and iatrogenic health problems caused by inadequate treatment planning, surgical and prosthetic execution, or element rejection, as well as maintenance issues [1]. peri-implant infections are classified as either peri-implant mucositis, which occurs when the initiated inflammation is limited to the peri-implant soft tissues, or periimplantitis, which occurs when the inflammation extends to the underlying bone and causes osteolysis [2]. there has been speculation that the biological side effects of peri-implant mucositis and peri-implantitis, which can cause soft and hard-tissue abnormalities, may be important for further marginal bone loss [3]. poly-microbial biofilms developing on the surfaces of the implanted area are thought the primary cause of the inflammatory illness conditions peri-implant mucositis and peri-implantitis [2]. peri-implant mucositis and periimplantitis impact more than 22 percent and more than 40 percent of implants, respectively [4]. periimplant mucositis and peri-implantitis effects of an,bio,cs on staphylococcus aureus biofilm forma,on associated with peri-implan,,s vol 11 no 1 (2023) doi 10.5195/d3000.2023.472 h#p://den*stry3000.pi#.edu are both "biofilm-associated pathological conditions" that affect the oral cavity. peri-implant mucositis is characterized by inflammation in the mucosa around dental implants, while peri-implantitis refers to its progression and subsequent progressive loss of supporting bone, and as a result, there is substantial evidence that the primary etiological cause for infections linked to dental implants is biofilm [2]. poly-microbial biofilm development on titanium surfaces is thought to be the primary contributor to the inflammatory reactions in the tissues around implant devices that frequently result in tooth loss [5]. because of its superior physical-chemical qualities and great bio-compatibility with host tissues, titanium has been chosen as the primary biomaterial for orthopedic and dental implant devices, promoting predictable longterm therapy [6]. but once exposed, implants are equally vulnerable to microbial adherence and biofilm development [7]. that leads to inflammation in the surrounding tissues [8]. although there is a human inflammatory response intended to stimulate immune responses and inhibit microbial development, this mechanism can also produce a biofilm that is highly infectious and drug-tolerant [9]. aim of the study the existing study's objective is to look at staphylococcus aureus biofilm formation around titanium dental implant in patients with periimplantitis, and to test approaches to prevent poly-microbial illness and lower microbial burdens on implantable devices. material and methods specimens’ collection this cross-sectional study included specimens from 120 patients. disposable sterile swabs were used for this purpose, and specimens were collected by taking a swab of the inflamed areas surrounding the implant. the specimens were collected from baquba specialized dental center in province of diyala during the time span between august 2022 until september 2022. gram stain, catalase checking, and coagulase verification and biochemical tests were used to diagnose the clinical specimens after all specimens were cultivated on blood agar and mannitol salt agar. biofilm formation we used the microtiter plate test to look for biofilm development [10]. the staphylococcus aureus isolates were cultured on a nutrient broth at 37°c for 24 h. then, two hundred microliter of staphylococcus aureus from the broth had been suspended in triplicates in the micro-titer plate and incubated for 24 hours at 37°c. after three times of using distilled water to clean each well, the plate was shaken violently until it was entirely dehydrated. two hundred μl of 100% methanol were added to fix the adhering staphylococcus aureus cells. subsequently, for 15 minutes, 200 μl of 0.5% crystal violet was used to stain each well. based on tang et al[11]. the biofilm density was calculated by measuring the od 630 nm using an elisa reader after removing the excessive amount of the stain by using 95% ethanol in each well. determination of minimum inhibitory concentration (mic) mic was estimated for staphylococcus aureus isolates exposed to ciprofloxacin (cip) and amoxicillin/clavulanic acid (aug) by the serial dilution method in muellerhinton broth. serial dilution of antibiotics between 2 to1024 μg/ml. bacterial suspensions with turbidity equivalent to 0.5 mcfarland was added to the tubes contained a different concentration of antibiotics. after incubation at 37°c for 18-24 hours, mic was determined as the lowest concentration of the antibiotic that inhibits bacterial growth. subminimum inhibitory concentrations (sub-mics) represent the lowest inhibitory concentration at which bacteria can grow [12]. antibiotics effect on the biofilm formation each antibiotic was tested at submics to study the change in the ability of staphylococcus aureus isolates in biofilm formation. microtiter plates were produced and then kept at 37°c for 24 hours. control plates were prepared in a free antibiotic-microtiter plate which effects of an,bio,cs on staphylococcus aureus biofilm forma,on associated with peri-implan,,s vol 11 no 1 (2023) doi 10.5195/d3000.2023.472 h#p://den*stry3000.pi#.edu was dispensed to the wells with 200 μl of nutrient broth without antibiotics [13]. the absorbency was measured in an elisa reader at 630 nm, according to a published protocol [11]. the absorbance of the wells was compared with the control wells, as follows: if o.d. ≤ o.d.c (regarded as a non-biofilm creator); if o.d.c ≤ o.d. ≤ 2* o.d.c (regarded as moderately biofilm creator); if 2* o.d.c ≤ o.d. (regarded as strongly biofilm creator). o.d. (display the investigated samples); o.d.c (display control wells). results and discussion the present results displayed that staphylococcus aureus’ growth was positive inn twenty isolates (16.6%) from the total of 120 specimens. this result is similar to [14], who found that the percentage of staphylococcus aureus that caused peri-implantitis was 18.75% based on culture dependent methods. for the phenotypic characterization of staphylococcus aureus on blood agar, gram stain, biochemical test, and mannitol salt agar were used, followed by confirmation on a vitek 2 system (figure 1). the occurrence of staphylococcus aureus an opportunistic pathogen in the initial phase of peri-implantitis in patients has additionally been validated by mombelli and décaillet [7]. moreover, canullo et al. [15] indicated that if the mounting edges are not cleansed prior screwing, staphylococcus aureus could be found on both the external and internal surfaces. peri-implantitis will affect around one-third of patients overall and one-fifth of all implants [16]. the mic and sub-mic were determined for two antibiotics, ciprofloxacin and amoxicillinclavulanic acid based on the clinical and laboratory standards institute (clsi-2020) guidelines breakpoint. mic values were estimated for each isolate by selecting the lowest concentrations in which no growth by the serial dilution method on mueller-hinton broth while the sub mic values were determined by selecting the lowest inhibitory concentration at which the bacteria could grow, the findings of the existing study indicated that the mic of ciprofloxacin for staphylococcus aureus was 32 to 512 μg/ml and the sub-mic 16 to 256 μg/ml while the mic for amoxicillin-clavulanic was 8 to 512) μg/ml and the sub-mic 4 to 256) μg/ml as mentioned in tables 1 and 2. the outcomes of the present study showed that 19 out of 20 isolates (95%) had the capability to create biofilm, 18 isolates were strong creators of biofilm and one isolate was a moderate biofilm producer as showed in table 3. the present results revealed that ciprofloxacin decreased the density of biofilm formation in 14 isolates after incubation for 24 hours, and only six isolates were not affected. amoxicillin/clavulanic acid decreased the density of biofilm formation in all the twenty isolates of staphylococcus aureus. the experiment on the antibiotic free samples showed that the staphylococcus aureus has the ability to form biofilm (p=0.001). generally, antibiotics reduce biofilm formation. however, several studies have shown that antibiotics can significantly induce biofilm formation depending on the antibiotic class and the bacterial strain [17]. peri-implant illnesses have appeared as a side effect of antibiotic therapy [2]. figure 1. staphylococcus aureus on blood agar and mannitol salt agar. effects of an,bio,cs on staphylococcus aureus biofilm forma,on associated with peri-implan,,s vol 11 no 1 (2023) doi 10.5195/d3000.2023.472 h#p://den*stry3000.pi#.edu no. mic sub-mic no. mic sub-mic 1 512 256 256 128 2 256 128 64 32 3 256 128 128 64 4 64 32 512 256 5 512 256 32 16 6 128 64 512 128 7 128 64 128 64 8 64 32 64 32 9 256 128 512 256 10 32 16 128 64 no. mic sub-mic no. mic sub-mic 1 256 512 11 256 128 2 512 128 12 32 16 3 64 32 13 128 64 4 512 128 14 512 128 5 64 32 15 64 32 6 32 16 16 128 64 7 8 4 17 32 16 8 128 64 18 128 64 9 256 128 19 512 128 10 16 8 20 64 32 no absorbance before treatment absorbance after treatment (cip) absorbance after treatment (aug) 1 0.173 (s) 0.140 (s) 0.034 (n) 2 0.142 (s) 0.102 (m) 0.042 (n) 3 0.157 (s) 0.144 (s) 0.051 (n) 4 0.121 (s) 0.103 (m) 0.044 (n) 5 0.159 (s) 0.117 (m) 0.040 (n) 6 0.052 (n) 0.051 (n) 0.046 (n) 7 0.219 (s) 0.162 (s) 0.051 (n) 8 0.112 (m) 0.109 (m) 0.044 (n) 9 0.186 (s) 0.145 (s) 0.041 (n) 10 0.190 (s) 0.154 (s) 0.045 (n) 11 0.184 (s) 0.161 (s) 0.048 (n) 12 0.197 (s) 0.170 (s) 0.044 (n) 13 0.152 (s) 0.112 (m) 0.041 (n) 14 0.169 (s) 0.120 (m) 0.047 (n) table 2. mic and sub-mic of amoxicillin/clavulanic for staphylococcus aureus. table 3. biofilm formation before and after treatment. table 1. mic and sub-mic of ciprofloxacin for staphylococcus aureus. effects of an,bio,cs on staphylococcus aureus biofilm forma,on associated with peri-implan,,s vol 11 no 1 (2023) doi 10.5195/d3000.2023.472 h#p://den*stry3000.pi#.edu 15 0.231 (s) 0.229 (s) 0.045 (n) 16 0.147 (s) 0.118 (m) 0.050 (n) 17 0.157 (s) 0.157 (s) 0.043 (n) 18 0.222 (s) 0.219 (s) 0.051 (n) 19 0.215 (s) 0.164 (s) 0.049 (n) 20 0.097 (m) 0.095 (m) 0.053 (n) conclusion staphylococcus aureus have a robust ability to produce biofilm on titanium which can affect the integrity of a dental implant. amoxicillin/clavulanic acid has decreased the density of biofilm formation by staphylococcus aureus and at a lower rate for ciprofloxacin. references 1. lang n, berglundh t, periimplant diseases: where are we now?– consensus of the seventh european workshop on periodontology. working group 4 of the seventh european workshop on periodontology. journal of clinical periodontology .2011;38:178181. 2. berglundh, t., armitage g, araujo m, avila-ortz g, blanco j, camargo p, zitzmann n. periimplant diseases and conditons: consensus report of workgroup 4 of the 2017 world workshop on the classificaton of periodontal and peri-implant diseases and conditons. journal of periodontology.2018;89:s313s318. 3. chan h, lin g, suarez f, maceachern m, wang h. surgical management of periimplantts: a systematc review and meta-analysis of treatment outcomes. journal of periodontology.2014; 85(8):1027-1041. 4. salvi g, cosgarea r, sculean a. prevalence and mechanisms of peri-implant diseases. journal of dental research.2017;96(1): 3137. 5. souza j, bertolini m, costa r , nagay b, dongari-bagtzoglou a, barão v. targetng implantassociated infectons: ttanium surface loaded with antmicrobial. iscience.2021;24(1): 102008. 6. spriano s, yamaguchi s, baino f, ferraris s. a critcal review of multfunctonal ttanium surfaces: new fronters for improving osseointegraton and host response, avoiding bacteria contaminaton. acta biomaterialia.2018;79: 1-22. 7. mombelli a, & décaillet f.the characteristcs of biofilms in peri-implant disease. journal of clinical periodontology.2011;38: 203-213. 8. arciola c, campoccia d, montanaro l. implant infectons: adhesion, biofilm formaton and immune evasion. nature reviews microbiology.2018;16(7):397409. 9. marsh p, devine d. how is the development of dental biofilms influenced by the host. journal of clinical periodontology.2011; 38:28-35. 10. almeida c, azevedo n, santos s, keevil c, vieira m. correcton: discriminatng mult-species populatons in biofilms with peptde nucleic acid fluorescence in situ hybridizaton (pna fish). plos one.2013;8(6): 11. tang, j., kang m, chen h, shi x, zhou r, chen j, du y. the staphylococcal nuclease prevents biofilm formaton in staphylococcus aureus and other biofilm-forming bacteria. science china life sciences.2011;54(9): 863-869. 12. andersson d, hughes d. microbiological effects of sublethal levels of antbiotcs. nature reviews microbiology.2014;12(7): 465478. 13. hemat s , sadeghifard n, ghafurian s, maleki f , mahdavi z, hassanvand a, hatami v. the associaton of biofilm formaton and sub-minimal inhibitory concentratons of antmicrobial agents. j bas res med sci 2016; 3(1):26-30. 14. passariello c, lucchese a, pera f, gigola p. clinical, microbiological and inflammatory evidence of the efficacy of combinaton therapy including serratopeptdase in the treatment of periimplantts. european journal of inflammaton.2012;10(3): 463472 15. canullo l, penarrocha-oltra d, soldini c, mazzocco f, penarrocha m, covani u. microbiological assessment of the implant-abutment interface in different connectons: crosssectonal study aner 5 years of functonal loading. clinical oral od of the control 0.060, s (strong biofilm producer), m (moderate biofilm producer), n (non-biofilm producer). effects of an,bio,cs on staphylococcus aureus biofilm forma,on associated with peri-implan,,s vol 11 no 1 (2023) doi 10.5195/d3000.2023.472 h#p://den*stry3000.pi#.edu implants research.2015;26(4): 426-434. 16. kordbacheh c, finkelstein j, papapanou p. peri-implantts prevalence, incidence rate, and risk factors: a study of electronic health records at a us dental school. clinical oral implants research.2019; 30(4): 306-314. 17. zhou, l, li t, an j, liao c, li n, wang x. subminimal inhibitory concentraton (sub-mic) of antbiotc induces electroactve biofilm formaton in bioelectrochemical systems. water research.2017;125: 280287. 825 final effec&veness of behavioral and lifestyle changes in reducing tmd symptoms: evidence from a structured interven&on vol 13 no 1 (2025) doi 10.5195/d3000.2025.825 h#p://den*stry3000.pi#.edu effectiveness of behavioral and lifestyle changes in reducing tmd symptoms: evidence from a structured intervention dunya malhan hanweet, karar abdulzahra mahdi, ansam mahdi khalel, salah m. ibrahim faculty of dentistry, kufa university, najaf, iraq abstract objec>ve: to assess the effec+veness of behavioral measures in managing tmds with an emphasis on pain, joint mobility and psychosocial status. methods: forty-eight subjects with tmds were enrolled based on the rdc/tmd criteria. stress management, ergonomics training, dietary changes and stretching exercises were recommended as individualized lifestyle plans. the clinical, psychosocial and func+onal evalua+ons were done before and ager three months of interven+on. results: the frequency of myofascial pain without limited mouth opening was 45. 8% which was reduced to 20. the percentage of subjects with right and leg tmj arthralgia was also seen to have reduced significantly (70. 8% to 43. 7%, p = 0. 0133; 75% to 41. 6%, p = 0. 001). right tmj osteoarthrosis and osteoarthri+s showed a considerable improvement (p < 0. 05) while the changes in the leg tmj were not significant. disc displacement with reduc+on on the right and without reduc+on on the leg side was also found to have improved markedly (p = 0. 036; p = 0. 041). depression severity was also found to have reduced significantly with the normal levels increasing from 45. 8% to 83. 3% (p < 0. 05). pain intensity as measured by vas also reduced from 4. 72 ± 1. 44 to 3. 43 ± 1. 38 (p = 0. 001). there was also an enhancement in the non-specific physical symptoms but the differences were not sta+s+cally significant. conclusions: the present study demonstrated that the implementa+on of lifestyle modifica+ons helps in decreasing the symptoms of tmd, increases the joint func+on and has a posi+ve effect on the psychosocial status of the pa+ents and therefore should be considered as the firstline treatment in the management of tmd. keywords: stress management; ergonomics; dietary changes; stretching exercises. cita9on: hanweet dm, et al. (2025) effec9veness of behavioral and lifestyle changes in reducing tmd symptoms: evidence from a structured interven9on. den9stry 3000. 1:a001 doi:10.5195/d3000.2025.825 received: january 12, 2025 accepted: february 11, 2025 published: february 14, 2025 copyright: ©2025 hanweet dm, et al. this is an open access ar9cle licensed under a crea9ve commons azribu9on work 4.0 united states license. email: salahm.abrahem@uokufa.edu.iq introduction temporomandibular disorders, or tmds, are a group of diseases that affect the temporomandibular joint, masticatory muscles, and the other structures around it. tmds are defined by pain, limited functionality, and auditory manifestations, including clicking or crepitus [1]. they are common in the international community with important consequences for oral health, quality of life, and health care expenses. causes of tmd include occlusal irregularities, parafunctional habits, stress, and some systemic diseases [2]. however, noninvasive treatments are still the mainstay of managing these disorders even with the new pharmacological and surgical management [3]. lifestyle changes have been identified as a way of managing tmds, as they help manage the variables that can be altered [4]. changes in behavior that include stress management, proper posture, changes in diet, and regular exercise have been seen to decrease muscle tension and effec&veness of behavioral and lifestyle changes in reducing tmd symptoms: evidence from a structured interven&on vol 13 no 1 (2025) doi 10.5195/d3000.2025.825 h#p://den*stry3000.pi#.edu improve the joint’s flexibility, thus improving the overall health of patients [5]. these include relaxation techniques, ergonomic measures, and certain stretching and strengthening exercises for the tmj and surrounding muscles that may be helpful in the management of symptoms in the long term [6]. this paper highlights the need to adopt a holistic view when managing tmds, as evidenced by the relationship between lifestyle and tmj health. knowing which lifestyle factors may aggravate or reduce tmd symptoms will help patients and healthcare providers find the right direction for a noninvasive treatment. this research therefore seeks to assess the effectiveness of lifestyle alterations on tmj health and their efficacy in decreasing tmd symptoms to suggest best practice for their application in the clinical setting. to enable individuals with tmds to be more involved in the management of their condition and enhance their quality of life, this study seeks to encourage and support the implementation of cost-efficient measures. material and methods study design a prospective interventional study was used to determine the efficacy of lifestyle changes in managing tmd. the study design involved 62 participants who were followed up for three months during which the research diagnostic criteria for temporomandibular disorders (rdc/tmd) was used to determine the diagnosis and intensity of the symptoms of the participants before and after the lifestyle changes. at the end of the assessment period, only 48 patients committed to the instructions, and all other uncommitted participants were excluded. the inclusion criteria were as follows: adults between the ages of 18-60 years with primary tmd as per rdc/tmd, reporting at least one of the symptoms which include pain, joint sounds or limited jaw function. the history of tmj surgery, systemic diseases that affect the tmj including rheumatoid arthritis, or concurrent medication or physical therapy for tmds was excluded from the study. baseline assessment: 1. clinical examination: the rdc/tmd axis 1 protocol [7] involved a trained oral medicine specialist who conducted a structured procedure for assessing pain (through palpation), joint sounds like clicking or crepitus, and the range of motion of the mandible. 2. symptom severity: pain intensity was measured using the visual analog scale (vas). 3. psychosocial assessment: rdc/tmd axis ii criteria were used to assess psychological distress and parafunctional habits. intervention: all participants were given individualized lifestyle change guidelines that included the following: all participants received a written personalized lifestyle modification plan, including: 1. stress management measures such as mindfulness, deep breathing, and meditation. 2. ergonomic training with video record reference to teach participants correct posture. 3. dietary changes that entail avoiding hard or chewy foods and using small bites during food chewing. 4. exercises for the neck and tmj region to be done regularly. 5. sleep quality improvement. the participants were then reassessed for myofascial pain using the same rdc/tmd criteria and scales three months after the intervention. effec&veness of behavioral and lifestyle changes in reducing tmd symptoms: evidence from a structured interven&on vol 13 no 1 (2025) doi 10.5195/d3000.2025.825 h#p://den*stry3000.pi#.edu chi-square test and fisher’s exact test were used to test the differences between the records before and after intervention, which was significant at a level of a p-value < 0.05. results as shown in table 1, iniaally, a large proporaon of paaents had myofascial pain without limited mouth opening (45.8%), which significantly decreased to 20.8% (p=0.0172). a reducaon in number of right tmj arthralgia cases also was recorded from 70.8% to 43.7% (p=0.0133), while lee tmj arthralgia decreased from 75% to 41.6% (p=0.001). similar records were observed for right tmj osteoarthrosis (68.7% to 45.8%) with significant difference (p<0.05) and right tmj osteoarthrias, which also was significantly reduced in the second records (p-value <0.05). while the reducaons in lee tmj osteoarthrosis and osteoarthrias were not staasacally significant (p-value >0.05). table 1. tmd symptoms (rdc/tmd axis i for paaents before and aeer 3 months from intervenaon (* chi-square test. p-value a:er 3 months (n=48) before intervendon (n=48) 0.0172* 10(20.8%) 22(45.8%) myofascial pain without limited mouth opening 0.0133* 21(43.7%) 34(70.8%) right tmj arthralgia 0.001* 20(41.6%) 36(75%) lee tmj arthralgia 0.0107* 22(45.8) 33(68.7) right tmj osteoarthrosis 0.25* 7(14.5%) 12(25%) lee tmj osteoarthrosis 0.022* 6(12.5%) 17(35.4%) right tmj osteoarthrias effec&veness of behavioral and lifestyle changes in reducing tmd symptoms: evidence from a structured interven&on vol 13 no 1 (2025) doi 10.5195/d3000.2025.825 h#p://den*stry3000.pi#.edu 0.317* 6(12.5%) 18(37.5%) lee tmj osteoarthrias 0.036* 18(37.5%) 33(68.7) right disc displacement with reducaon 0.157* 6(12.5%) 12(25%) right disc displacement without reducaon 0.128* 13(27%) 22(45.8) lee disc displacement with reducaon 0.041* 9(18.7%) 20(41.6%) lee disc displacement without reducaon effec&veness of behavioral and lifestyle changes in reducing tmd symptoms: evidence from a structured interven&on vol 13 no 1 (2025) doi 10.5195/d3000.2025.825 h#p://den*stry3000.pi#.edu table 2. rdc/tmd axis ii levels of depression and non-specific physical symptoms for paaents before and aeer 3 months of intervenaon (*chi-square test, **fisher’s exact test). table 3. visual analogue scale before and aeer intervenaon (paired t-test). . depression p-value non-specific physical symptoms p-value iniaal records n=48 aeer 3 months n=48 iniaal records n=48 aeer 3 months n=48 normal 22(45.8) 40(83.3%) 0.022* 26 30 0.596* moderate 16(33.3%) 6(12.5%) 0.033* 13 12 0.841* severe 10(20.8%) 2(4.2%) 0.016** 9 6 0.439* n vas mean (sd) p-value before treatment 48 4.72 (1.44) 0.001* aeer treatment 48 3.43 (1.38) effec&veness of behavioral and lifestyle changes in reducing tmd symptoms: evidence from a structured interven&on vol 13 no 1 (2025) doi 10.5195/d3000.2025.825 h#p://den*stry3000.pi#.edu for disc displacement condiaons there was a notable change. the number of paaents with right disc displacement with reducaon declined from 68.7% to 37.5% (p=0.036), while the number with lee disc displacement without reducaon decreased significantly too (41.6% to 18.7%, p=0.041). depression levels also showed improvement, the percentage of paracipants with normal levels increased significantly from 45.8% to 83.3% (p-value < 0.05). moderate and severe depression raaos showed significant reducaons too (pvalue < 0.05). although there were slight improvements in non-specific physical symptoms, changes were not staasacally significant across severity levels (e.g., severe symptoms reduced slightly from 9 to 6 paracipants, p=0.439) (table2). as in table 3, pain intensity measured by vas scores significantly decreased from a mean of 4.72 (±1.44) before treatment to 3.43 (±1.38) in the final record (p=0.001). discussion the results of this research also show that there are huge advantages of lifestyle changes in managing symptoms and clinical diagnoses of temporomandibular joint disorder. tmj disorders are acute and chronic pain, dysfunction and structural changes of the joint which are often complicated for the patient and the healthcare giver. it shows that focusing on the factors that can be changed can help to decrease the symptoms and increase the quality of life of the patient. the best result was the enhancement of tmj-related diagnosis after the intervention. some of the diseases that were observed include myofascial pain, arthralgia and osteoarthrosis which revealed a decrease especially on the right side. these changes depict that non-invasive measures can also be used in the management of not only muscular aches but also joint dysfunctions [8]. the reduction in osteoarthritis severity also supports the notion that lifestyle changes may help in lessening degenerative processes which may include reduction of joint stress and inflammation [9]. however, there were no significant changes in some of the conditions on the left side suggesting possibly different responses which could be attributed by factors such as the asymmetry of tmj loading or differing ability of individuals to adapt to the intervention. about disc displacement conditions, the improvements seen point to the potential of behavioral changes to enhance joint dynamics. changes in parafunctional habits and mandibular positioning using stress management, posture alignment, and dietary changes may be useful in the restoration of disc positioning and effec&veness of behavioral and lifestyle changes in reducing tmd symptoms: evidence from a structured interven&on vol 13 no 1 (2025) doi 10.5195/d3000.2025.825 h#p://den*stry3000.pi#.edu enhancement of joint stability [10]. these results also support the notion that the management of tmj disorders should encompass the treatment of the biomechanical and behavioral factors that lead to the condition, in an addition to other noninvasive types of management like sleep quality improvement and diet habit correction [11,12]. pain and psychological wellbeing were also found to have improved markedly after the intervention. the reduction in pain intensity is very promising especially because pain is a major factor that affects the quality of life of people with tmj disorders [13]. the decrease in depressive symptoms also supports the link between psychological health and pain. some of the lifestyle changes that are recommended also help in reducing stress and thus have the potential of improving the physical wellbeing of an individual and the mental health as well. this is an important finding because mood disorders are known to worsen the tmd and may interfere with the treatment process [14]. however, the changes in the non-specific physical symptoms were not statistically significant therefore suggesting that these changes might not have been significant or consistent within the study sample. this shows that there is still a need to explore more about these symptoms and how they are related to tmd. further research could also examine whether longer durations of intervention or more individualized management plans may produce better results in this regard. the findings of this study support the need to take a systemic approach when managing tmj disorders. many surgical and pharmacological strategies were tested in previous research for the management of tmd [15-17]. this paper shows that behavioral, lifestyle and dietary changes can be as effective as pharmacological and surgical interventions but at a cheaper and with no side effects [18-20]. modifying these behavioral, postural and dietary factors can help manage the various symptoms of tmds therefore offering a complete management plan [21-23]. this implies that there could be differences in the response rates based on the diagnosis and the symptom profiles hence possibly needing tailored treatment plans to enhance the best results. conclusion this research shows that implemenang lifestyle changes is an effecave way of enhancing the tmj and thus reducing the severity of tmds. the reducaon of pain, psychological distress and the pathologies of the tmj demonstrates the effecaveness of these intervenaons to effec&veness of behavioral and lifestyle changes in reducing tmd symptoms: evidence from a structured interven&on vol 13 no 1 (2025) doi 10.5195/d3000.2025.825 h#p://den*stry3000.pi#.edu improve the quality of life of the sufferers. further research should also entail the enhancement of these measures, the evaluaaon of their maintenance over ame and the assessment of the results in different people and environments. a ailability o data data are v e o re e t to t e corres o g t or financial support and sponsorship no e con icts o interest ere re o conflict of tere t. references 1. kapos fp, exposto fg, oyarzo jf, durham j. temporomandibular disorders: a review of current concepts in eaology, diagnosis and management. oral surg. 2020 nov;13(4):321-34. 2. matheson em, fermo jd, blackwelder rs. temporomandibular disorders: rapid evidence review. am fam physician. 2023 jan;107(1):528. pmid: 36689971. 3. vaira la, de riu g. temporomandibular joint disorders: funcaonal and conservaave treatment. j clin med. 2023 jul 19;12(14):4772. 4. tabrizi r, shourmaej y, pourdanesh f, shafiei s, moslemi h. does lifestyle modificaaon (physical exercise and listening to music) improve symptoms in paaents with temporomandibular disorder? a randomized clinical trial. natl j maxillofac surg. 2024 janapr;15(1):55-8. doi: 10.4103/njms.njms_23_23. 5. bianiene d, zamaliauskiene r, kubilius r, leketas m, gailius t, smirnovaite k. quality of life in paaents with temporomandibular disorders: a systemaac review. stomatologija. 2018;20:3-9. 6. keefe fj, somers tj, marare lm. psychologic intervenaons and lifestyle modificaaons for arthrias pain management. rheum dis clin north am. 2008;34:351-68. 7. dworkin sf, leresche l. research diagnosac criteria for temporomandibular disorders: review, criteria, examinaaons and specificaaons, criaque. j craniomandib disord facial oral pain. 1992;6(4):301-55. pmid: 1298767. 8. meyer jd, torres er, grabow ml, zgierska ae, teng hy, coe cl, et al. benefits of 8week mindfulness-based stress reducaon or aerobic training on seasonal declines in physical acavity. med sci sports exerc. 2018;50:1850-8. 9. dahlström l, carlsson ge. temporomandibular disorders and oral healthrelated quality of life: a systemaac review. acta odontol scand. 2010;68:80-5. 10. mander dk, beniwal sk, kam n, singh h, sharma a. conservaave management of temporomandibular joint disorders: a review. j adv med dent sci res. 2023 sep;11(9):50-5. 11. rener-sitar k, john mt, pusalavidyasagar ss, bandyopadhyay d, schiffman el. sleep quality in temporomandibular disorder cases. sleep med. 2016;25:10512. 12. safour w. understanding the influence of changes to diet on individuals living with temporomandibular disorder (tmd): an interpreave phenomenological study [dissertaaon]. canada: mcgill university; 2018. 13. de freitas rf, ferreira ma, barbosa ga, calderon ps. counselling and selfmanagement therapies for temporomandibular disorders: a systemaac review. j oral rehabil. 2013 nov;40(11):86474. 14. qamar z, alghamdi am, haydarah nk, balateef aa, alamoudi aa, abumismar ma, et al. impact of temporomandibular disorders on oral health-related quality of life: a systemaac review and meta-analysis. j oral rehabil. 2023 aug;50(8):706-14. effec&veness of behavioral and lifestyle changes in reducing tmd symptoms: evidence from a structured interven&on vol 13 no 1 (2025) doi 10.5195/d3000.2025.825 h#p://den*stry3000.pi#.edu 15. mahdi ka, al-mudhafar mh, hanweet dm. platelet-rich plasma injecaon modulates inflammatory cytokines in temporomandibular joint disorders: a cross-secaonal study. j indian acad oral med radiol. 2024 oct-dec;36(4):4225. doi: 10.4103/jiaomr.jiaomr_90_24. 16. chrisadis n, al-moraissi ea, barjandi g, svedenlöf j, jasim h, chrisadis m, et al. pharmacological treatments of temporomandibular disorders: a systemaac review including a network meta-analysis. drugs. 2024 jan;84(1):59-81. 17. andre a, kang j, dym h. pharmacologic treatment for temporomandibular and temporomandibular joint disorders. oral maxillofac surg clin north am. 2022 feb;34(1):49-59. 18. ibrahim sm, almizraqchi as. comparison of the anabacterial acavity of panax ginseng and symphytum officinale with metronidazole against p. gingivalis: an mic and mbc analysis. the open denastry journal. 2024 may 7;18(1). 19. ibrahim sm, almizraqchi as, haider j. metronidazole potenaaaon by panax ginseng and symphytum officinale: a new strategy for p. gingivalis infecaon control. anabioacs. 2023 aug 4;12(8):1288. 20. ibrahim sm, al-hmedat sj, alsunboli mh. histological study to evaluate the effect of local applicaaon of myrtus communis oil on alveolar bone healing in rats. the open denastry journal. 2024 may 7;18(1). 21. khalel am, ali mb, sadiq ma, ibrahim sm, ali sh. dmft and pufa indices in first permanent molars of iraqi children in najaf city. denastry 3000. 2024 nov 22;12(2). 22. hassan rs, hanoon za, ibrahim sm, alhusseini nb. assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs: a cross-secaonal study. denastry 3000. 2024 nov 22;12(2). 23. ibrahim sm, abdulzahra al-hmedat sj. role of manual and powered tooth brushes in plaque removal and oral health status (a comparaave study). indian journal of public health research & development. 2019 aug 1;10(8). 176 2022 tridimensional roughness and morphology of sound den3n surfaces a5er papain-gel treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.176 h#p://den*stry3000.pi#.edu tridimensional roughness and morphology of sound dentin surfaces after papain-gel treatment paula maciel pires, paula martins bravo miranda, paula helena de accioly costa, amanda souza nunes monteiro, adílis kalina alexandria, lucianne cople maia, aline de almeida neves universidade federal do rio de janeiro, universidade federal fluminense, universidade esadual do rio de janeiro, brazil abstract purpose: to inves3gate the effect of chemomechanical caries removing agents (ccras) based on papain (papacárie duo gel® and brix3000®) over den3n surfaces compared with 37% phosphoric acid and 11.5% polyacrylic acid. materials and methods: sound human molars were sec3oned at the crown level, resul3ng in 48 den3n blocks, which were randomly divided into 4 groups (n=12): 1) papacárie duo gel®; 2) brix3000®; 3) 11.5% polyacrylic acid solu3on and 4) 37% phosphoric acid gel. all products were applied for 30s. ten blocks per group were analyzed by a noncontact 3d profilometer before and a5er treatments for linear (ra) and volumetric roughness (sa). the superficial morphology of the remaining blocks in each group (n=2) was evaluated by scanning electron microscopy (sem). normality was rejected for the data (shapiro-wilk test) and therefore, kruskal-wallis test, followed by dunn’s proof or wilcoxon signed rank test with its respec3ve effect size calcula3on were used to compare the results with α=5%. results: ra and sa values for specimens submiaed to papacárie duo gel® and brix3000® were sta3s3cally similar to baseline values. applica3on of phosphoric and polyacrylic acid resulted in a sta3s3cally increase in roughness compared to the ccras. sem evalua3on showed that papacárie duo gel® resulted in surface debris. polyacrylic acid and brix3000® resulted in par3al opening of the tubules but den3n exposed to polyacrylic was able to remove more smear layer than brix3000®, while phosphoric acid resulted in total opening of the den3nal tubules. conclusion: both papacárie duo gel® and brix3000® did not result in roughness changes when applied in sound den3n. keywords: dental caries; chemomechanical caries removal; dental materials; den3n; papain; pediatric den3stry cita:on: pires, p et al. (2022) tridimensional roughness and morphology of sound den:n surfaces aker papaingel treatment den:stry 3000. 1:a001 doi:10.5195/d3000.2022.176 received: april 28, 2021 accepted: august 4, 2021 published: march 25, 2022 copyright: ©2022 pires, p et al. this is an open access ar:cle licensed under a crea:ve commons auribu:on work 4.0 united states license. email: aline.dealmeidaneves@gmail.com introduction non-treated caries lesions are still a burden to dental services around the world [1]. chemomechanical caries removal agents (ccras) gained interest over the last twenty years due to many factors, including: 1) increased dental tissue preservation due to selective tissue removal; 2) ability to provide a more objective caries removal threshold; 3) reduced need for anesthesia during operative procedures and 4) possibility of undertaking operative procedures in non-dental settings [2-4]. initially, hypochlorite-based ccras such as carisolv® (rls global, mölndal, sweden) where developed, in which good effectiveness and a selective action in removal of carious dentin has been demonstrated [4]. in brazil however, papain-based ccras became more commercially available and therefore, much commonly used tridimensional roughness and morphology of sound den3n surfaces a5er papain-gel treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.176 h#p://den*stry3000.pi#.edu (papacárie duo gel®, fórmula & ação, são paulo, sp, brazil). this product is a gel containing 3% papain, 0.5% chloramine and toluidine blue and has been also found to be effective in removing infected carious dentin [5] with a user-friendly technique [6] and being more comfortable to the child dental patient than the drilling method [7]. more recently, another papain-based ccra has been developed (brix3000®, brix medical science, carcarañá, santa fe, argentina) which allegedly contain an increased papain concentration (3.000 u/mg or 10%). according to the manufacturer, in this gel, the enzyme is protected by a buffer emulsion technology, which immobilizes and stabilizes it at an ideal ph, releasing papain only at the time of collagen proteolysis, resulting in an increase of the enzymatic activity by up to 60%. differently from papacárie duo gel®, brix3000® does not contain chloramine in its composition [8]. from the currently available minimally invasive caries removal methods, ccras agents result in the best threshold for conservative caries removal [9,10]. papain is an enzyme extracted from papaya fruit with broad proteolytic, bactericidal, bacteriostatic and anti-inflammatory activity [11]. papain is able to breakdown infected or necrotic tissues because degraded collagen lacks α1-anti-trypsin, which normally inhibits protein digestion in healthy tissues [12,13], guaranteeing thus, the selectivity of the ccra. the lack of anti-trypsin in infected/necrotic tissues will allow papain to further breakdown this substrate, enabling easy removal with hand instruments [14]. the concept of minimal invasive dentistry (mid) means removing only infected and irreversibly destroyed dentin and leaving, as residual substrate for further adhesive procedures, a slightly demineralized caries-affected dentin [15]. like other active substances in ccras, papain is supposed to acts exclusively on the breakdown of the partially degraded collagen in carious dentin, without damaging intact collagen fibrils [16], leaving thus, sound dentin intact. keeping with the mid approach, ccras are constantly used over sound dentin surfaces, especially if the enamel-dentin junction is prepared to “scratchy” dentin to ensure appropriate sealing of the restoration [17]. while hypochloritebased ccras have been investigated on its topographic effects over sound dentin [18,19], the possible effects of papain-based ccras sound dentin surfaces have not yet been investigated. to evaluate the effect of papacárie duo gel® and brix3000® on the superficial morphology and roughness of sound dentin compared to commonly used dentin conditioning agents (37% phosphoric acid and 11.5% polyacrylic acid), which purposely cause exposure of dentin collagen. surface roughness and morphological features have been investigated by non-contact profilometry measurements and scanning electron microscope, respectively. material and methods sound human third molars, extracted for clinical reasons were used after the patient has given written consent. this protocol has been approved by the institution’s ethical committee and it is registered at “plataforma brazil” (17389213.5.0000.5257). the teeth were selected according to the following inclusion criteria: absence of caries lesions or restorations/sealing material. after sectioning, the specimens were examined under a stereomicroscope for presence of any defects or areas of sclerotic dentin, and if present, they were excluded from the sample. excluded teeth were discarded via standardized protocol for biological material. sample size was estimated for comparison of differences in more than two experimental groups considering a 0.05 alpha value and a 0.9 power of the test, based on results of a previous study on the effect of a hypochlorite-based chemo-mechanical caries removing method on the roughness of sound dentin [18]. the following information was input to the software (bioestat v.5.3, instituto mamirauá, manaus, am, brazil): minimum difference between mean of groups = 0.002; standard deviation tridimensional roughness and morphology of sound den3n surfaces a5er papain-gel treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.176 h#p://den*stry3000.pi#.edu of the experimental error = 0.001, which resulted in a minimum of 8 specimens per group. the tooth crowns were cut, with the aid of a diamond disk mounted on a low speed cutting machine (isomet, buehler, lake bluff, il, usa) into dentin blocks (approximately 4 x 4 x 2 mm) in which the occlusal dentin was selected as the test surface. forty-eight dentin blocks were selected among those obtained and attached with sticky wax to polyethylene devices with the test surface exposed at the top. the specimens were further polished with 400, 800 and 1200 grit sandpaper, in this order, under water-cooling with the aid of a semi-automatic polishing machine (plf, fortel, são paulo, sp, brazil). after that, twelve specimens were randomly allocated to each four experimental groups: 1) application of papacárie duo gel® (fórmula & ação, são paulo, sp, brazil); 2) application of brix3000® (brix medical science, carcarañá, santa fe, argentina); 3) application of a 11.5% polyacrylic acid solution (vitro condicionador, dfl, rio de janeiro, rj, brazil) 4) application of a 37% phosphoric acid gel (condac, fgm, joinville, santa catarina, brazil). application time for all products was standardized in 30s. after each application time, and for all groups, the specimens were washed in distilled water for 60s and stored under 100% humidity until further analysis. before and after the treatment regimens, 10 specimens in each group were analyzed in a 3d noncontact chromatic confocal optical profilometry (nanovea ps50 optical, nanovea inc., irvine, california, united states). linear roughness (ra) was obtained by averaging three linear readings from each sample while volumetric roughness (sa) was obtained from one volumetric reading over each specimen. the remaining two blocks per group were used for surface topographic analysis. the blocks were fixed on stubs with double-faced carbon tape, gold-sputtered (30µm) and analyzed in a scanning electronic microscope (6460lv, jeol, tokyo, japan) in secondary electrons mode after the treatment regimens. shapiro-wilk’s test was used to evaluate normality of the data. as normality was rejected, kruskallwallis test, followed by dunn’s proof was used to disclose statistical significance among the roughness values within the experimental time (baseline or treated) and an epsilon squared effect size was calculated [19]. wilcoxon signed rank test was used to test differences in each experimental group between time (baseline or treated) and the effect size was calculated as the r estimate [19, 20]. the α-level chosen was 5% and all analysis were undertaken using bioestat v.5.3 (instituto mamirauá, manaus, am, brazil). results both surface roughness parameters evaluated (ra and sa) were similar among the groups at baseline. for the treated specimens, polyacrylic acid and phosphoric acid treated specimens showed statistically significant higher ra and sa values compared to both papain-treated groups, with large effect sizes (tables 1 and 2). intra-group comparison of tridimensional surface roughness analysis disclosed that specimens submitted to the papain-based caries removing gels did not show significant changes in both roughness parameters tested (ra and sa) after the treatments (tables 1 and 2). on the other hand, specimens treated with phosphoric acid or polyacrylic acid resulted in a statistically significant higher linear (ra) and volumetric (sa), with very strong or strong effect sizes. tridimensional roughness and morphology of sound den3n surfaces a5er papain-gel treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.176 h#p://den*stry3000.pi#.edu sem analysis of the superficial morphology of the dentinal tubules revealed that phosphoric acid resulted in full opening of the dentin tubules (figure 1a). polyacrylic acid and brix3000® resulted in partial opening of the dentin tubules (figure 1b and 1d) while papacárie duo gel® resulted in the presence of debris, probably resulting from the polishing procedures undertaken during specimen preparation which remained over the tubules (figure 1c). discussion the results of the present study revealed no statistically significant changes on linear (ra) and volumetric (sa) roughness in specimens submitted to the papain-based ccras before and after the treatments. specimens treated with phosphoric acid or polyacrylic acid resulted in a statistically significant increase in both ra and sa. these results are in agreement with previous investigations of the effect of carisolv® in sound dentin surfaces. roughness parameters has also not experimental group ra baseline treated difference phosphoric acid 0.45 ± 0.11 a, a 1.34 ± 0.48 a, b 0.89 ± 0.53 a polyacrylic acid 0.48 ± 0.04 a, a 1.84 ± 0.79 a, b 1.37 ± 0.79 a papacárie duo gel® 0.41 ± 0.09 a, a 0.47 ± 0.09 b, a 0.07 ± 0.05 b brix3000® 0.40 ± 0.04 a, a 0.39 ± 0.04 b, a 0.03 ± 0.03 b experimental group sa baseline treated difference phosphoric acid 0.61 ± 0.51 a, a 2.12 ± 1.38 a, b 1.51 ± 1.42 a polyacrylic acid 0.88 ± 0.25 a, a 4.70 ± 3.75 a, b 3.82 ± 3.65 a papacárie duo gel® 0.59 ± 1.79 a, a 0.65 ± 0.16 b, a 0.06 ± 0.17 b brix3000® 0.96 ± 0.62 a, a 1.21 ± 1.69 b, a 0.25 ± 1.30 b table 1: mean (±sd) of linear (ra) roughness measurements (µm) before and after treatments in each experimental group. * different lowercase superscript letters indicate statistical significance in the same column (kruskal-wallis followed by dunn’s test, p<0.05). effect size was 0.76 (very strong) and 0.69 (very strong) for the column “treated” and “difference”, respectively [19]. different uppercase superscript letters indicate statistical significance between baseline and treated specimens in the same group (wilcoxon signed rank test). effect size was 0.83 (large) and 0.84 (large) for phosphoric acid and polyacrylic acid respectively [20]. table 2: mean (±sd) of volumetric (sa) roughness measurements (µm) before and after treatments in each experimental group. * different lowercase superscript letters indicate statistical significance in the same column (kruskal-wallis followed by dunn’s test, p<0.05). effect size was 0.63 (strong) and 0.51 (strong) for the column “treated” and “difference”, respectively [19]. different uppercase superscript letters indicate statistical significance between baseline and treated specimens in the same group (wilcoxon signed rank test). effect size was 0.84 (large) and 0.81 (large) for phosphoric acid and polyacrylic acid respectively [20]. tridimensional roughness and morphology of sound den3n surfaces a5er papain-gel treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.176 h#p://den*stry3000.pi#.edu been changed after carisolv® application but increased substantially when phosphoric acid was applied in this substrate [18]. carisolv® is a ccra based on chloramine, while the ccras used in the present study are based on papain. therefore, an investigation on the effect of these on the sound topography of dentin was warranted. the morphology of the dentin surface as revealed by sem showed that phosphoric acid resulted in full opening of the dentin tubules (figure 1a). polyacrylic acid and brix3000® resulted in partial opening of the dentin tubules (figures 1b and 1d) but dentin exposed to polyacrylic acid resulted in less smear layer than brix3000®. papacárie duo gel® resulted in a relatively smeared dentin surface the presence of debris (figure 1c). a previous sem study comparing carisolv® and other dentin conditioners over sound dentin also showed that this ccra was unable to remove smear layer and completely open dentin tubules [21]. however, when carisolv® has been used over carious dentin, the surface generally appeared clean, with open tubules [22]. the same is likely to be true for papacárie, since many studies have also reported similar residual carious dentin surface morphology after it use, with open tubules and reduced smear layer [23-25]. nonetheless, one study showed presence of smear layer and debris partially occluding the dentin tubules in residual carious primary teeth dentin after caries removal with papacárie duo gel® [26]. it has been claimed that chloramine present in papacárie duo gel® would be responsible for opening the dentinal tubules of the outer surface of the carious dentin, softening it chemically and facilitating its removal [27]. in the present study however, sound dentin surfaces treated with this product still showed a smear layer covered surface. the main function of phosphoric acid 37% and polyacrylic acid 11.5%, used during conditioning of dentin surfaces prior to composite and glass ionomer restorations, is to guarantee removal of the smear layer, which is accompanied by the superficial mineral dissolution of the dentin, exposure of collagen fibers and opening of the superficial dentin tubules to allow composite tag formation [28]. the sem observations of the present study corroborate the effect of both phosphoric and polyacrylic acid on sound dentin. although complete removal of the smear layer is frequently seen with phosphoric acid [29], this is invariably followed by great peritubular dentin dissolution and exposure of collagen fibers [30]. polyacrylic etched sound dentin surfaces normally retain part of the smear layer [31] and also result in calcium-enriched surfaces [29]. there have been claims that nonmineralized type i collagen fibrils would be partially degraded (without fiber rupture) by a papain-gel, as shown by atomic force evaluation of the surface of collagen fibers in sound dentin [32], what could be evidence of a detrimental effect of the gel in sound dentin. others, however, using fourier-transformed infrared spectroscopy claimed no collagen degradation to occur [33] and no removal of calcium of sound dentin after papacárie application [34]. regarding surface roughness and morphological characteristics, the results of the present study support the selective action of papain gels in carious dentin. the restoration of cavities using a mid approach requires adhesive materials such as composite resins or glass ionomer cements, which directly bond to the dentin surfaces. several studies have reported that papain-based gel treatment does not interfere with the bond strength of the adhesive restorative materials to figure 1. sem images of dentin surfaces after experimental treatments. a) 37% phosphoric acid treatment. b) 11.5% polyacrylic acid treatment. c) papacárie duo gel® treatment. d) brix3000® treatment. tridimensional roughness and morphology of sound den3n surfaces a5er papain-gel treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.176 h#p://den*stry3000.pi#.edu sound and demineralized dentin [3537]. however, these ccras do not promote enough surface roughness in sound dentin; thus, it’s use probably does not replace the use of conditioning agents before restoration. more studies are indeed, necessary to investigate possible ultrastructural changes in sound and carious dentin after use of papainbased ccras in order to optimize its use in light of the mid approach. limitations of this study include the fact that the dentin quality among the experimental groups were not fully standardized. factors such as age of the patient, anatomical location of the tooth or distance of the cut from the dentin-pulp junction, might result in morphological differences at the dentin level that could have influenced the results (i.e., the high variability of data obtained). however, as distribution of the dentin specimens was randomized among the groups in the present study, this effect was probably evenly distributed among the groups. papain-based caries removing gels did not induce significant roughness changes in sound dentin. regarding the morphologic characteristics of the surfaces, application of brix3000® resulted in partially opening of dentin tubules, similar to that caused by polyacrylic acid treatment but this removed more smear layer. papacárie duo gel® did not remove smear layer and left dentin tubules obliterated. this study has shown that papainbased gels are harmless when applied in sound dentin surfaces. acknowledgement paula maciel pires is supported by a phd grant from capes, finantial code 001. paula martins bravo miranda and paula helena de accioly costa were supported by ic grants from faperj which has also partially supported this project with jcne grant number e-26/203.185/2016. references 1. the global prevalence of dental healthcare needs and unmet dental needs among adolescents: a systematic review and meta-analysis. ghafari m, bahadivand-chegini s, nadi t, doosti-irani a. epidemiol health. 2019;41:e2019046. http://doi.org/10.4178/epih.e20 19046. pmid: 31778605. 2. current update of chemomechanical caries removal methods. hamama h, yiu c, burrow m. aust dent j. 2014;59(4):446-56. http://doi.org/10.1111/adj.1221 4. pmid: 25131424. 3. current concepts and techniques for caries excavation and adhesion to residual dentin. neves aa, coutinho e, cardoso mv, lambrechts p, van meerbeek b. j adhes dent. 2011;13(1):7-22. http://doi.rg/10.3290/j.jad.a184 43. pmid: 21403932. 4. caries removal by chemomechanical (carisolv) vs rotary drill: a systematic review. maru vp, shakuntala bs, nagarathna c. open dent j. 2015;9:462-72. http://doi.org/10.2174/1874210 601509010462. pmid: 26962375. 5. caries-removal effectiveness of a papain-based chemomechanical agent: a quantitative micro-ct study. neves aa, lourenco ra, alves hd, lopes rt, primo lg. scanning. 2015;37(4):258-264. http://doi.org/10.1002/sca.2120 6. pmid: 25809787. 6. chemo-mechanical caries removal with papacarie: case series with 84 reports and 12 months of follow-up. bussadori sk, godoy ch, alfaya ta, fernandes kp, mesquita-ferrari ra, motta lj. j contemp dent pract. 2014;15(2):250-3. http://doi.org/ 10.5005/jpjournals-10024-1523. pmid: 25095852. 7. pain during removal of carious lesions in children: a randomized controlled clinical trial. motta lj, bussadori sk, campanelli ap, da silva al, alfaya ta, de godoy ch, et al. int j dent. 2013;2013:896381. http://doi.org/10.1155/2013/89 6381. 8. comparing the efficacies of two chemo-mechanical caries removal agents (2.25% sodium hypochlorite gel and brix 3000), in caries removal and patient cooperation: a randomized controlled clinical trial. alkhouli mm, al nesser sf, bshara ng, almidani an, comisi jc. j dent. 2020;93:103280. http://doi.org/10.1016/j.jdent.2 020.103280. pmid: 31981604. 9. caries-removal effectiveness and minimal-invasiveness potential of caries-excavation techniques: a micro-ct investigation. neves aa, coutinho e, de munck j, van meerbeek b. j dent. tridimensional roughness and morphology of sound den3n surfaces a5er papain-gel treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.176 h#p://den*stry3000.pi#.edu 2011;39(2):154-62. http://doi.org/10.1016/j.jdent.2 010.11.006. pmid: 21111770. 10. evaluation of the efficiency and effectiveness of three minimally invasive methods of caries removal: an in vitro study. boob ar, manjula m, reddy r, srilaxmi r, rani t. int j clin ped dent. 2014;7(1):11-8. http://doi.org/ 10.5005/jpjournals-10005-1226. pmid: 25206231. 11. papain-like cysteine proteases. bromme d. curr prot protein sci. 2001;21(1):1-14. http://doi.org/10.1002/0471140 864.ps2102s21 12. chemo-mechanical removal of caries in an adolescent patient using a papain gel: case report. bussadori sk, guedes cc, hermida bruno ml, ram d. j clin pediatr dent. 2008;32(3):177-80. http://doi.org/10.17796/jcpd.32 .3.1168770338617085. pmid: 18524264. 13. biocompatibility analysis of chemomechanical caries removal material papacarie on cultured fibroblasts and subcutaneous tissue. martins md, fernandes kp, motta lj, santos em, pavesi vc, bussadori sk. j dent child. 2009;76(2):1239. pmid: 19619425. 14. clinical and radiographic study of chemical-mechanical removal of caries using papacarie: 24month follow up. bussadori sk, guedes cc, bachiega jc, santis to, motta lj. j clin pediatr dent. 2011;35(3):251-4. http://doi.org/ 10.17796/jcpd.35.3.75803m025 24625h5. pmid: 21678665. 15. clinical threshold for carious tissue removal. kidd ea. dent clin north am. 2010;54(3):5419. http://doi.org/10.1016/j.cden.20 10.03.001. pmid: 20630195. 16. papain gel: a new chemomechanical caries removal agent. bussadori sk, castro lc, galvao ac. j clin pediatr dent. 2005;30(2):115-9. http://doi.org/ 10.17796/jcpd.30.2.xq641w720u 101048. pmid: 16491964. 17. managing carious lesions: consensus recommendations on carious tissue removal. schwendicke f, frencken je, bjorndal l, maltz m, manton dj, ricketts d, et al. adv dent res. 2016;28(2):58-67. http://doi.org/10.1177/0022034 516639271. pmid: 27099358. 18. the influence of carisolv on enamel and dentine surface topography. wennerberg a, sawase t, kultje c. eur j oral sci. 1999;107(4):297-306. http://doi.org/ 10.1046/j.09098836.1999.eos107410.x. pmid: 10467946. 19. tomczak m, tomczak e. the need to report effect size estimates revisited: an overview of some recommended measures of effect size. trend sport sci. 2014;1(21):19-25. 20. cohen j. a power prime. psychol bull. 1992;112(1):155159. 21. effect of a chemo-mechanical caries removal system (carisolvtm) on dentin topography of non-carious dentin. cederlund a, lindskog s, blomlof j. acta odontol scand. 1999;57(4):185-9. https://doi.org/10.1080/000163 599428751. pmid: 10540927. 22. scanning electron microscopic observations of human dentine after mechanical caries excavation. banerjee a, kidd eam, watson tf. j dent. 2000;28(3):179-86. http://doi.org/10.1016/s03005712(99)00064-0. pmid: 10709339. 23. comparative evaluation of effects of chemo-mechanical and conventional caries removal on dentinal morphology and its bonding characteristics: an sem study. arora r, goswami m, chaudhary s, chaitra tr, kishor a, rallan m. eur arch paediatr dent. 2012;13(4):179-84. http://doi.org/10.1007/bf03262 867. pmid: 22883356. 24. sem analysis of residual dentin surface in primary teeth using different chemomechanical caries removal agents. thakur r, patil sds, kush a, madhu k.. j clin pediatr dent. 2017;41(4):289-93. http://doi.org/10.17796/10534628-41.4.289. pmid: 28650784. 25. dentin topographic features following chemomechanical caries removal in primary teeth. kotb rm, elkateb ma, ahmed am, kawana ky, el meligy oa. j clin pediatr dent. 2016;40(6):472-9. http://doi.org/10.17796/10534628-40.6.472. pmid: 27805895. 26. evaluation of residual dentin after conventional and chemomechanical caries removal using sem. correa fn, rodrigues filho le, rodrigues cr. j clin pediatr dent. 2008;32(2):115-20. http://doi.org/ 10.17796/jcpd.32.2.44n2787118 133880. pmid: 18389676. 27. chemomechanical caries removal: a comprehensive review of the literature. maragakis gm, hahn p, hellwig e. int dent j. 2001;51(4):291-9. http://doi.org/10.1002/j.1875tridimensional roughness and morphology of sound den3n surfaces a5er papain-gel treatment vol 10 no 1 (2022) doi 10.5195/d3000.2022.176 h#p://den*stry3000.pi#.edu 595x.2001.tb00841.x. pmid: 11570545. 28. buonocore memorial lecture adhesion to enamel and dentin: current status and future challenges. van meerbeek b, de munck j, yoshida y, inoue s, vargas m, vijay p, et al. oper dent. 2003;28(3):215-35. pmid: 12760693. 29. effectiveness of etching by three acids on the morphological and chemical features of dentin tissue. kharouf n, mancino d, naji-amrani a, eid a, haikel y, hemmerle j. j contemp dent pract. 2019;20(8):915-9. pmid: 31797847. 30. correlative transmission electron microscopy examination of nondemineralized and demineralized resin-dentin interfaces formed by two dentin adhesive systems. van meerbeek b, conn lj, duke es, eick jd, robinson sj, guerrero d. j dent res. 1996;75(3):879-88. http://doi.org/10.1177/0022034 5960750030401. pmid: 8675798. 31. the effect of dentine pretreatment using bioglass and/or polyacrylic acid on the interfacial characteristics of resin-modified glass ionomer cements. sauro s, watson t, moscardo ap, luzi a, feitosa vp, banerjee a. j dent. 2018;73(1):32-9. http://doi.org/10.1016/j.jdent.2 018.03.014. pmid: 29609016. 32. papain-gel degrades intact nonmineralized type i collagen fibrils. bertassoni le, marshall gw. scanning. 2009;31(6):253-8. http://doi.org/10.1002/sca.2017 1. pmid: 20205185. 33. effect of papain-based gel on type i collagen spectroscopy applied for microstructural analysis. junior zs, botta sb, ana pa, franca cm, fernandes kp, mesquita-ferrari ra, et al. scientific reports. 2015;5:11448. https://doi.org/10.1038/srep114 48. 34. mineral content removal after papacarie application in primary teeth: a quantitative analysis. bittencourt st, pereira jr, rosa aw, oliveira ks, ghizoni js, oliveira mt. j clin pediatr dent. 2010;34(3):229-31. doi: 10.17796/jcpd.34.3.k15t8q1805 538524. pmid: 20578660. 35. microtensile bond strength of etch-and-rinse and self-etch adhesive systems to demineralized dentin after the use of a papain-based chemomechanical method. gianini rj, do amaral fl, florio fm, basting rt. am j dent. 2010;23(1):23-8. pmid: 20437723. 36. morphology and microtensile bond strength of adhesive systems to in situ-formed cariesaffected dentin after the use of a papain-based chemomechanical gel method. botelho amaral fl, martao florio f, bovi ambrosano gm, basting rt. am j dent. 2011;24(1):13-9. pmid: 21469401. 37. effect of a papain-based gel for chemomechanical caries removal on dentin shear bond strength. lopes mc, mascarini rc, da silva bm, florio fm, basting rt.. j dent child. 2007;74(2):93-7. pmid: 18477426. chemopreventive effects of curcumin against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. vol 9 no 1 (2024) doi 10.5195/d3000.2024.559 http://dentistry3000.pitt.edu chemopreventive effects of curcumin against 7,12dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. maher al-assaf 1, nabil kochaji 1, shaza al laham 2, issa al-assaf 3, caroline mousallam 4, charif barakat 1 1 department of oral histology and pathology, faculty of dentistry, damascus university, damascus, syria. 2 department of pharmacology & toxicology, faculty of pharmacy, damascus university, damascus, syria. 3 department of pharmacognosy, faculty of pharmacy, damascus university, damascus, syria. 4 department of pediatric dentistry, faculty of dentistry, damascus university, damascus, syria. abstract background: chemoprevention is an important means that has a high potential to reverse, prevent or suppress the development of cancer in initial stages or the development of pre-cancerous cells to the cancer stage. objective: this study aimed to investigate the potential protective effect of curcumin as a chemopreventive agent against induced oral squamous cell carcinoma in syrian hamster. materials and methods: in this experimental study, forty male syrian hamsters were divided equally into two main groups. the carcinogenic dmba was only applied to the control group (g1 = 20 hamsters) by topical painting in the buccal pouch, as for the experimental group (g2 = 20 hamsters) curcumin was administered orally (80 mg\kg) concurrently with the dmba. the animals were sacrificed at consecutive periods (after 2, 6, 10 and 14 weeks), then the histopathological changes in the buccal pouches were studied by h&e staining, and the vegf expression during the previous sacrifice periods was studied. the kruskal–wallis test was used to study the significance of differences in vegf expression between the two groups at the level of confidence 95%. results: p-value = 0.116>0.05 0.005<0.05 0.007<0.05 0.006<0.05, in 2, 6, 10 and 14 weeks respectively. thus, the statistically significant differences in vegf expression were found in the second, third and fourth sacrifice period between group 1 and group 2. conclusion: curcumin had chemopreventive effects against dmbainduced buccal pouch oscc through the modulating effect on the abnormal expression of vegf. keywords: oral squamous cell carcinoma; curcumin; chemoprevention. citation: al-assaf m, et al. (2024) chemopreventive effects of curcumin against 7,12dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis dentistry 3000. 1:a001 doi:10.5195/d3000.2024.559 received: august 10, 2023 accepted: january 14, 2021 published: may 9, 2024 copyright: © al-assaf m, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: maher2.maher@damascusuniversity.edu.sy introduction oral squamous cell carcinoma (oscc) is defined according to the world health organization (who) as an invasive carcinoma with varying degrees of squamous differentiation and with a tendency to metastasize to lymph nodes in early stages. it arises from the oral mucosal epithelium, and is the most common oral cancers. it constitutes more than ninety percent of the malignancies that affect it [1]. oscc arises from the accumulation of many mutations that affect the cell life cycle, cellular proliferation, and angiogenesis [2, 3]. angiogenesis is defined as the process of creating new blood vessels from pre-existing vasculature [4, 5]. angiogenesis occurs physiologically in many conditions such as pregnancy, development, and wound healing. as for angiogenesis disorders, they are involved in many diseases, such as chronic inflammation, cardiovascular diseases, and carcinogenesis [2, 3]. angiogenesis is an essential part of solid tumors evolution larger than 2mm in diameter, and the vascular http://dentistry3000.pitt.edu/ chemopreventive effects of curcumin against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. vol 9 no 1 (2024) doi 10.5195/d3000.2024.559 http://dentistry3000.pitt.edu endothelial growth factor (vegf) is the main factor in this process which make the detection of the role of this factor becomes a topic of high importance [6]. currently, most cancers are being treated and may be cured, but it is common to encounter poor therapeutic response and recurrence despite great progress in surgery, radiotherapy and chemotherapy [7, 8]. chemotherapy has suffered from many problems, the most important of which is the non-selectivity, as it attacks both healthy and cancerous cells, so it has a highly toxic effect. in addition to noting the phenomenon of multi-drug resistance (mdr) in cancer cells at times, which makes it ineffective. in addition to the possibility of creating new mutations in the dna, may lead to the formation of new cases of cancer [9]. therefore, the low efficacy and high toxicity of chemotherapy led to resort to another type of treatment that includes what is called nutraceutical, which combines nutrition and pharmaceutical and is defined as a substance isolated or purified from plants, that has proven effective in the treatment or chemoprevention of cancer [10, 11]. chemoprevention has been described as the use of agents that allow suppression, regression, or delay the progression of carcinogenesis [12, 13]. the current chemopreventive agents have low side effects and low toxicity, and most of these agents are plant extracts that are divided into two categories: (i) blocking agents that prevent the initiation step by inhibiting the activation of carcinogens, (ii) suppressing agents that inhibit the cancerous cell proliferation during the evolution of carcinogenesis. however, there is a distinct class of chemopreventive agents including curcumin (from the turmeric plant) belong to both classes because they represent multiple mechanisms of action [14]. turmeric is an herbaceous plant with a yellow rhizome that belongs to the ginger family, native to southern india. it is known to contain compounds that have an important effect on public health, the most important and effective of which is curcumin. curcumin, is a polyphenolic compound found in the turmeric plant (curcuma longa) with the chemical formula c21h20o6 [15-17]. the decrease in cancer incidence, especially colon cancer, was noted in india, where turmeric is widely used as a spice. researchers have linked turmeric intake to this decrease in cancerous cases number. this prompted them to investigate the effectiveness of its active component "curcumin" through in-vitro, in-vivo, and clinical studies. indeed, this compound has proven highly effective in curbing carcinogenesis in addition to its safety, as high doses of it can reach 12 g per day without leaving any toxic side effects [18-20]. curcumin has been studied extensively over the past years, with most studies focusing on its antiinflammatory and anti-cancer effects. shanmugam et al. reviewed most of the experimental studies conducted on curcumin to confirm its efficacy as a safe treatment for cancer patients. it was concluded that this substance has supportive efficacy for most types of cancer, including multiple myeloma, pancreatic, colon, and lung cancer [21]. curcumin plays a crucial role in cancer prevention by inhibiting many mechanisms that can cause the initiation of cancer cell formation. the most important of these mechanisms is the inhibition of the nuclear factor nf-κb pathway, which is responsible for inflammation. in addition, curcumin is curbing reactive oxygen species (ros), which is one of the most important factors causing mutations and forming abnormal cells with a high ability to divide abnormally [19, 22]. furthermore, curcumin has been described as an important antiangiogenic agent, this phenomenon could be attributed to vegf and angiopoietin 1 and 2 inhibition, and also by inhibition of kdr/ flk-1 (vegf receptor-2) [23, 24]. moreover, curcumin inhibits angiogenesis through downregulation of the expression of cox-2 and hypoxiainducible factor 1 (hif-1), which in turn leads to inhibition of the vegf expression, suggesting that the curcumin possesses antiangiogenic effects, which warrants further http://dentistry3000.pitt.edu/ chemopreventive effects of curcumin against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. vol 9 no 1 (2024) doi 10.5195/d3000.2024.559 http://dentistry3000.pitt.edu investigation as a chemopreventive agent [25-27]. some studies have demonstrated the ability of curcumin to inhibit abnormal cell division and induce apoptosis in different types of cancer cells [28]. among the anti-cancer effects is its ability to inhibit the pathway of cyclooxygenase (cox-2) and lipoxygenase (lox), which in turn also helps to inhibit cell proliferation [29]. curcumin has been confirmed to be active in several head and neck squamous cell carcinoma cell lines (hnscc), including cal27 and ccl23 (laryngeal) and umscc14a (oral) by affecting mainly the nf-κb signaling pathway through decreasing its expression, in addition, inhibiting its nuclear localization resulting in sequestering the nf-κb in the cytoplasm [30, 22] and through suppressing the expression of several proteins regulated by nf-κb including interleukin-6 and 8, matrix metalloproteinase-9 (mmp-9), cyclin d1, c-myc, cyclooxygenase-2 (cox-2) and bcl-xl [31-33]. the exact mechanism of action of curcumin is still under research and study, for example, in 2009, manoharan et al. [34] induced oral squamous cell carcinoma in the buccal pouches of hamsters that were administered orally (80 mg\kg) concurrently with the dmba carcinogen, and the results of this experimental study indicated an effective preventive role of curcumin in delaying the occurrence of epithelial dysplasia in the buccal pouch mucosa, due to its antioxidant potential in addition to its effect on the detoxification process of carcinogens. saleh et al. [35] concluded that curcumin alone has an important chemopreventive role, and it was also confirmed that this protective effect of curcumin increases when combined with green tea extract. this study aimed to investigate the potential protective effect of curcumin against oscc induced in the buccal pouch of syrian golden hamsters by evaluating vegf expression levels. materials and methods ❖ animals: an approval was gained from the ethics committee at damascus university to conduct this study (approval ref: 3574/2019). figure 1: histopathological photomicrograph shows the mild dysplasia after 6 weeks of dmba in group 1 (a), and after 6 weeks of dmba + curcumin in group 2 (b). h&e staining, magnification 400. figure 2: histopathological photomicrograph shows the moderate dysplasia after 10 weeks of dmba in group 1 (a), and after 10 weeks of dmba + curcumin in group 2 (b). h&e staining, magnification 400. http://dentistry3000.pitt.edu/ chemopreventive effects of curcumin against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. vol 9 no 1 (2024) doi 10.5195/d3000.2024.559 http://dentistry3000.pitt.edu this study was conducted on syrian golden hamsters (mesocricetus auratus) at department of oral histology and pathology, damascus university. inclusion criteria: healthy male hamsters, aged about 7-10 weeks old, weight about 90-110 g, no anatomical deformities and were quarantined previously for adaptation purposes (for one week prior to the start of this study). afterwards, all hamsters that fulfilled the inclusion criteria (40 hamsters) were divided equally into two main groups: the first main group, the control group, included 20 hamsters to which only the carcinogenic dmba was topically applied (g1 dmba alone), while in the second main group, the experimental group, the curcumin was administered orally (80 mg\kg) concurrently with the dmba (g2 curcumin + dmba). ❖ 7,12dimethylbenz[a]anthracene (dmba): dmba was obtained from sigmaaldrich chemical company. (st. louis, mo, usa). this chemical substance can bind to dna and cause mutations that play a role in malignant transformation. oscc was induced in a buccal pouch through topical application of 0.5% dmba dissolved in mineral oil three times a week for fourteen weeks by a paintbrush (size 4). dmba was applied only on the left buccal pouch to ensure the ability of the hamster to eat and store its food using the right one, which helps to preserve the life of the hamster and prevent it from dying because of nutritional deficiency [36, 37]. ❖ curcumin: the curcumin powder (sigma-aldrich co. st. louis, mo, usa) was administered orally (80 mg of body weight) concurrently with the dmba (3 times per week) by a 1 cm3 syringe after dissolving it in 1 ml of almond oil according to the recommended dose to hamsters to avoid toxic effects [38]. ❖ experimental design: each main group was divided according to the sacrifice time into four sub-groups, each of which included five hamsters. the first main group (g1 dmba alone) was subdivided into four groups: five hamsters were sacrificed after two weeks, five hamsters after 6 weeks, five hamsters after 10 weeks, and five hamsters after 14 weeks. and the same was applied on the second group (g2 curcumin + dmba). the potential protective effect of curcumin against the development of oscc induced during these consecutive periods was investigated by evaluation of vegf expression. the hamsters were sacrificed according to ethical procedures by injecting sodium pentobarbital (180 mg/kg body weight) by using a 26-g syringe and then the buccal pouches were removed unilaterally for each hamster's side which was exposed to the dmba or dmba + curcumin. ❖ immunohistochemical evaluation (ihc): immunohistochemical detection of vegf was carried out by using bio sb, inc. co. detection system. the vegf antibody (rabbit monoclonal antibody) was used, with figure 3: histopathological photomicrograph shows the severe dysplasia after 10 and 14 weeks of dmba in group 1 (a), and after 14 weeks of dmba + curcumin in group 2 (b). h&e staining, magnification 400. http://dentistry3000.pitt.edu/ chemopreventive effects of curcumin against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. vol 9 no 1 (2024) doi 10.5195/d3000.2024.559 http://dentistry3000.pitt.edu brown cytoplasmic\membranous expression. all steps of ihc staining were followed according to the manufacturer’s instructions. the staining evaluation was done in epithelial cells of the buccal pouch mucosa by using a light microscope (olympus –nikon cx21). any epithelial cell that exhibited brown cytoplasmic\membranous immunopositivity to vegf was considered positive. the staining score of vegf was evaluated by allred’s modified semi‑quantitative immunoreactive score as following [39]: quantitative assessment (percentage %): was graded as following: (1) <25% of positive cells, (2) 25%–50% of positive cells, (3) 50%–75% of positive cells, and (4) >75% of positive cells. no counts were done in areas of necrosis. qualitative assessment (staining intensity): was rated as following: (0) no staining, (1) weak staining, (2) moderate staining, and (3) strong staining. the final score was obtained by adding qualitative and quantitative indices as following: (0) negative, (1– 2–3) weak, (4–5) moderate, and (6–7) strong. ❖ statistical analysis: spss software (v.23; ibm, armonk, new york) was used to analyze the data obtained statistically and g*power software was used to calculate the sample size. the normality distribution was checked by using the kolmogorov-smirnov test. the kruskal–wallis test was used to study the significance of differences in vegf expression between the two groups. results: all developmental sequential stages of well-differentiated oscc were observed in the control group (g1 dmba alone), starting from hyperplasia of the buccal pouch epithelium, passing through mild and moderate dysplasia, to severe dysplasia and up to the welldifferentiated invasive carcinoma, while in the experimental group (g2 curcumin + dmba), the progression of oscc had stopped at the severe dysplasia after the fourteenth week of the experiment where no cases of carcinoma were observed in the last week of the experiment (14th week) (figures 1,2,3,4). the kruskal–wallis test was used to analyze and compare the results of both groups in order to evaluate the effectiveness of curcumin as a chemopreventive agent. the kruskal– wallis test indicated that the p-value in the second, third, and fourth sacrifice period (6-10-14 weeks) was smaller than 0.05 (p-value = 0.005 0.007 – 0.006, respectively), and therefore there were statistically significant differences between both groups at the confidence level of 95%. while there were no statistically figure 5: histopathological photomicrograph shows the weak expression of vegf: (a) group 1, (b) group 2. magnification 400. figure 4: histopathological photomicrograph shows the invasive oscc after 14 weeks of dmba in group 1. h&e staining, magnification 400. http://dentistry3000.pitt.edu/ chemopreventive effects of curcumin against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. vol 9 no 1 (2024) doi 10.5195/d3000.2024.559 http://dentistry3000.pitt.edu significant differences in the first sacrifice periods (two weeks) because the p-value = 0.116 > 0.05 (table 1), (figures 5,6,7). discussion the better practical approach to decrease the morbidity of cancer is delaying the process of carcinogenesis by using chemopreventive agents. this necessitates that compounds extracted from natural sources must be critically evaluated for chemoprevention [20]. this study focused on the potential protective potential of curcumin (diferuloylmethane) in reducing the development of oscc induced in the buccal pouch mucosa of syrian hamsters by dmba, as this substance produces "dihydrodiol epoxide" a compound that mediates the carcinogenesis through chronic inflammation, overproduction of ros, activation of protooncogenes, inactivation of tumor suppressor genes, induction of extensive dna damage, reduction of dna damage repair, and reduced ability to induce apoptosis [37]. the process of oral carcinogenesis induced by the application of dmba is very similar histologically and morphologically to human oscc [36, 40]. in this study, it was noted the similarity between signs of dysplasia in the buccal pouch of hamsters and the pathological changes that occur in the human oral mucosa, and this was consistent with several previous studies [41-43]. dmba was also chosen as a carcinogen in the animal models because it has similar etiological effects as alcohol and tobacco do in human oral cancer therefore, the carcinogenesis in the animal models induced by dmba can be used as an ideal model for studying the chemoprevention [37,40]. the poor solubility and low oral bioavailability of curcumin are common impediments to its clinical benefit despite its multidirectional pharmacological properties. however, studies have shown that stability increases in acidic environments such as the stomach [44]. curcumin at a dose of 80 mg/kg was used in this study in line with most papers in the context [38, 45, 46, 34], but on the other hand, some papers showed that although low concentrations of curcumin stimulate figure 6: histopathological photomicrograph shows the moderate expression of vegf: (a) group 1, (b) group 2. magnification 400. figure 7: histopathological photomicrograph shows the strong expression of vegf: (a) group 1, (b) group 2. magnification 400. http://dentistry3000.pitt.edu/ chemopreventive effects of curcumin against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. vol 9 no 1 (2024) doi 10.5195/d3000.2024.559 http://dentistry3000.pitt.edu its antioxidant effects, the higher concentrations can increase cellular levels of ros [47]. referring to the results of the kruskal–wallis test, it was found that the differences between both groups in each of the second, third, and fourth sacrifice periods were statistically significant, while they were not in the first sacrifice period, and this indicates that the chemopreventive effect of curcumin was greatest in most of the experiment periods, especially at the end of the fourteenth week when no incidence of oscc in the g2 curcumin + dmba was observed, while it did not have any positive role within two weeks of the application of the dmba. these findings were consistent with manoharan et al. [34]. manoharan had studied the protective role of curcumin and piperine against oscc induced by dmba in the buccal pouch group n mean std. deviation mean rank chisquare p-value 2 weeks g1 dmba alone 5 2.4 .548 10 4.31 0.116>0.05 ns g2 curcumin + dmba 5 2 .0 7 6 weeks g1 dmba alone 5 4.6 .548 13 10.75 0.005<0.05 * g2 curcumin + dmba 5 2.2 .447 5 10 weeks g1 dmba alone 5 6.6 .447 13 9.99 0.007<0.05 * g2 curcumin + dmba 5 3.6 .894 5.1 14 weeks g1 dmba alone 5 7 .0 13 10.18 0.006<0.05 * g2 curcumin + dmba 5 4.4 1.14 5.2 table 1: results of the kruskal–wallis test to study the significance of differences in vegf expression between the two groups. ns = not significant, * = significant, std. deviation = standard deviation, g1 = group 1, g2 = group 2, http://dentistry3000.pitt.edu/ chemopreventive effects of curcumin against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. vol 9 no 1 (2024) doi 10.5195/d3000.2024.559 http://dentistry3000.pitt.edu in hamsters and it was concluded that feeding on curcumin at a dose of 80 mg/kg had played an important chemopreventive efficacy in delaying the occurrence of signs of dysplasia in the buccal pouch due to its antilipidperoxidative potency as well as its modulating effect on the detoxification process of carcinogens. this study was also agreed with balakrishnan et al. [45]. it was demonstrated by balakrishnan that the curcumin at a dose of 80 mg/kg and ferulic acid had a modulating effect on the abnormal expression of p53 and bcl-2 proteins induced by dmba in the buccal pouch mucosa of syrian hamsters. these results were in line with saleh et al. [35], as this study concluded that curcumin alone has an important chemopreventive role, and it was also confirmed that this protective effect of curcumin increases when it is shared with green tea. whereas the difference was made with maulina et al. [38], which did not prove the protective efficacy of curcumin despite the use of a similar dose (80 mg/kg). this difference can be explained through several points, the first of which was the difference in the animal model, where male white sprague dawley rats were used, and the second was the difference in the protocol of applying the dmba, maulina scratched the buccal mucosa of the rat by using a 27-g syringe that contained dmba, while in this study, the application was done through topical painting in the buccal pouch, and the third and most important point was the short period of administration of curcumin, which amounted to only about four weeks, while the period of administration in the current study lasted fourteen weeks concurrently with the dmba. last but not least, in regards to the results of the current study, further investigations are required to better understand the mechanisms of the chemopreventive effects of curcumin. conclusion: based on the results of this study, it can be concluded that the oral administration of curcumin at a dose of 80 mg/kg had chemopreventive effects against dmba-induced buccal pouch oscc through the modulating effect on the abnormal expression of vegf. acknowledgments: none. references 1. worst pattern of invasion and other histopathological features in oral cancer as determinants of prognosis and survival rate: a retrospective cohort analysis. marzouki hz, bukhari af, al-ghamdi da, abdullah rm, al-hajeili m, khayyat s, alzahrani rm, alotaibi yr, al-wassia r, al-marzouki h, merdad m. oncol lett. 2023 jan;25(2):75-85. doi: 10.3892/ol.2023.13661. pmid: 36688107. 2. impact of vascular endothelial growth factor gene-gene and gene-smoking interaction and haplotype combination on bladder cancer risk in chinese population. fu d, li p, cheng w, tian f, xu x, yi x, tang c, wang y, hu q, zhang z. oncotarget. 2017 apr;8(14):2292722935. doi: 10.18632/oncotarget.15287. pmid: 28206971. 3. expression of vascular endothelial growth factor in oral squamous cell carcinoma. kim sk, park sg, kim kw. j korean assoc oral maxillofac surg. 2015;41(1):11-18. 4. molecular basis of angiogenesis and cancer. tonini t, rossi f, claudio pp. oncogene. 2003 sep;22(42):6549-2556. doi: 10.1038/sj.onc.1206816. pmid: 14528279. 5. immunohistochemical evaluation of tumor angiogenesis and the role of mast cells in oral squamous cell carcinoma. kabiraj a, jaiswal r, singh a, gupta j, singh a, samadi fm. j cancer res ther. 2018 apr-jun;14(3):495-502. doi: 10.4103/0973-1482.163693. pmid: 29893305. 6. the hypoxia-inducible factorresponsive proteins semaphorin 4d and vascular endothelial growth factor promote tumor growth and angiogenesis in oral squamous cell carcinoma. zhou h, yang yh, binmadi no, proia p, basile jr. exp cell res. 2012 aug;318(14):1685-1698. doi: 10.1016/j.yexcr.2012.04.019. pmid: 22652457. 7. the molecular pathology of cancer. harris tj, mccormick f. nat rev clin oncol. 2010 may;7(5):251http://dentistry3000.pitt.edu/ chemopreventive effects of curcumin against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. vol 9 no 1 (2024) doi 10.5195/d3000.2024.559 http://dentistry3000.pitt.edu 265. doi: 10.1038/nrclinonc.2010.41. pmid: 20351699. 8. biomarkers in diagnosis and therapy of oral squamous cell carcinoma: a review of the literature. blatt s, krüger m, ziebart t, sagheb k, schiegnitz e, goetze e, al-nawas b, pabst am. j craniomaxillofac surg. 2017 may;45(5):722-730. doi: 10.1016/j.jcms.2017.01.033. pmid: 28318929. 9. autophagy and multidrug resistance in cancer. li yj, lei yh, yao n, wang cr, hu n, ye wc, zhang dm, chen zs. chin j cancer. 2017 jun;36(1):52-61. doi: 10.1186/s40880-017-0219-2. pmid: 28646911. 10. new concepts in nutraceuticals as alternative for pharmaceuticals. nasri h, baradaran a, shirzad h, rafieian-kopaei m. int j prev med. 2014 dec;5(12):1487-1499. pmid: 25709784. 11. the role of nutraceuticals in chemoprevention and chemotherapy and their clinical outcomes. saldanha sn, tollefsbol to. j oncol. 2012;2012:192464. doi: 10.1155/2012/192464. pmid: 22187555. 12. mechanistic considerations in chemopreventive drug development. kelloff gj, boone cw, steele ve, fay jr, lubet ra, crowell ja, sigman cc. j cell biochem suppl. 1994;56(20):124. doi: 10.1002/jcb.240560903. pmid: 7616736. 13. prevention of chemical carcinogenesis by vitamin a and its synthetic analogs (retinoids). sporn mb, dunlop nm, newton dl, smith jm. fed proc. 1976 may;35(6):13321338. pmid: 770206. 14. chemopreventive and therapeutic effects of curcumin. duvoix a, blasius r, delhalle s, schnekenburger m, morceau f, henry e, dicato m, diederich m. cancer lett. 2005 jun;223(2):181-190. doi: 10.1016/j.canlet.2004.09.041. pmid: 15896452. 15. anticancer potential of curcumin: preclinical and clinical studies. aggarwal bb, kumar a, bharti ac. anticancer res. 2003 janfeb;23(1a):363-398. pmid: 12680238. 16. potential anticancer activity of turmeric (curcuma longa). kuttan r, bhanumathy p, nirmala k, george mc. cancer lett. 1985 nov;29(2):197202. doi: 10.1016/03043835(85)90159-4. pmid: 4075289. 17. turmeric and curcumin: biological actions and medicinal applications. chattopadhyay i, biswas k, bandyopadhyay u, banerjee rk. current science. 2004;87:44-53. 18. phase i clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or premalignant lesions. cheng al, hsu ch, lin jk, hsu mm, ho yf, shen ts, ko jy, lin jt, lin br, ming-shiang w, yu hs, jee sh, chen gs, chen tm, chen ca, lai mk, pu ys, pan mh, wang yj, tsai cc, hsieh cy. anticancer res. 2001 jul-aug;21(4b):2895-2900. pmid: 11712783. 19. curcumin, a multifaceted hormetic agent, mediates an intricate crosstalk between mitochondrial turnover, autophagy, and apoptosis. rainey ne, moustapha a, petit px. oxid med cell longev. 2020 jul;2020:3656419. doi: 10.1155/2020/3656419. pmid: 32765806. 20. curcumin for chemoprevention of colon cancer. johnson jj, mukhtar h. cancer lett. 2007 oct;255(2):170-181. doi: 10.1016/j.canlet.2007.03.005. pmid: 17448598. 21. the multifaceted role of curcumin in cancer prevention and treatment. shanmugam mk, rane g, kanchi mm, arfuso f, chinnathambi a, zayed me, alharbi sa, tan bk, kumar ap, sethi g. molecules. 2015 feb;20(2):2728-2769. doi: 10.3390/molecules20022728. pmid: 25665066. 22. biological and therapeutic activities, and anticancer properties of curcumin. perrone d, ardito f, giannatempo g, dioguardi m, troiano g, lo russo l, de lillo a, laino l, lo muzio l. exp ther med. 2015 nov;10(5):1615-1623. doi: 10.3892/etm.2015.2749. pmid: 26640527. 23. curcumin is an in vivo inhibitor of angiogenesis. arbiser jl, klauber n, rohan r, van leeuwen r, huang mt, fisher c, flynn e, byers hr. mol med. 1998 jun;4(6):376-383. pmid: 10780880. 24. molecular mechanisms of anti-angiogenic effect of curcumin. gururaj ae, belakavadi m, venkatesh da, marmé d, salimath bp. biochem biophys res commun. 2002 oct;297(4):934-942. doi: 10.1016/s0006-291x(02)02306-9. pmid: 12359244. 25. curcumin inhibits vegfmediated angiogenesis in human intestinal microvascular endothelial cells through cox-2 and mapk inhibition. binion dg, otterson mf, rafiee p. gut. 2008 nov;57(11):1509http://dentistry3000.pitt.edu/ chemopreventive effects of curcumin against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. vol 9 no 1 (2024) doi 10.5195/d3000.2024.559 http://dentistry3000.pitt.edu 1517. doi: 10.1136/gut.2008.152496. pmid: 18596194. 26. curcumin: a potential candidate in prevention of cancer via modulation of molecular pathways. rahmani ah, al zohairy ma, aly sm, khan ma. biomed res int. 2014;2014:761608. doi: 10.1155/2014/761608. pmid: 25295272. 27. curcumin inhibits hypoxiainduced angiogenesis via downregulation of hif-1. bae mk, kim sh, jeong jw, lee ym, kim hs, kim sr, yun i, bae sk, kim kw. oncol rep. 2006 jun;15(6):1557-1562. pmid: 16685395. 28. anti cancer effects of curcumin: cycle of life and death. sa g, das t. cell div. 2008 oct;3:1-14. doi: 10.1186/1747-1028-3-14. pmid: 18834508. 29. inhibitory effects of curcumin on in vitro lipoxygenase and cyclooxygenase activities in mouse epidermis. huang mt, lysz t, ferraro t, abidi tf, laskin jd, conney ah. cancer res. 1991 feb;51(3):813-819. pmid: 1899046. 30. curcumin suppresses growth of head and neck squamous cell carcinoma. lotempio mm, veena ms, steele hl, ramamurthy b, ramalingam ts, cohen an, chakrabarti r, srivatsan es, wang mb. clin cancer res. 2005 oct;11(19 pt 1):6994-7002. doi: 10.1158/10780432.ccr-05-0301. pmid: 16203793. 31. curcumin inhibits nfkappab mediated radioprotection and modulate apoptosis related genes in human neuroblastoma cells. aravindan n, madhusoodhanan r, ahmad s, johnson d, herman ts. cancer biol ther. 2008 apr;7(4):569576. doi: 10.4161/cbt.7.4.5534. pmid: 18305409. 32. curcumin downregulates the constitutive activity of nf-kappab and induces apoptosis in novel mouse melanoma cells. marín ye, wall ba, wang s, namkoong j, martino jj, suh j, lee hj, rabson ab, yang cs, chen s, ryu jh. melanoma res. 2007 oct;17(5):274-283. doi: 10.1097/cmr.0b013e3282ed3d0e. pmid: 17885582. 33. liposome-encapsulated curcumin suppresses growth of head and neck squamous cell carcinoma in vitro and in xenografts through the inhibition of nuclear factor kappab by an akt-independent pathway. wang d, veena ms, stevenson k, tang c, ho b, suh jd, duarte vm, faull kf, mehta k, srivatsan es, wang mb. clin cancer res. 2008 oct;14(19):6228-6236. doi: 10.1158/1078-0432.ccr-07-5177. pmid: 18829502. 34. chemopreventive efficacy of curcumin and piperine during 7,12dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. manoharan s, balakrishnan s, menon vp, alias lm, reena ar. singapore med j. 2009 feb;50(2):139-146. pmid: 19296028. 35. chemopreventive effect of green tea and curcumin in induced oral squamous cell carcinoma: an experimental study. saleh mm, darwish ze, el nouaem mi, mourad gm, ramadan or. alexandria dental journal. 2020;45(3):74-80. 36. inhibition of dmba-induced oral squamous cells carcinoma growth by brazilian red propolis in rodent model. ribeiro dr, alves âv, dos santos ep, padilha ff, gomes mz, rabelo as, cardoso jc, massarioli ap, de alencar sm, de albuquerquejúnior rl. basic clin pharmacol toxicol. 2015 aug;117(2):85-95. doi: 10.1111/bcpt.12374. pmid: 25556639. 37. effect of docosahexaenoic acid as a chemopreventive agent on experimentally induced hamster buccal pouch carcinogenesis. alqalshy em, ibrahim am, abdel-hafiz aa, kamal kae, alazzazi ma, omar mr, abdel-wahab as, mohammed ss. cancer treat res commun. 2022;31:100558. doi: 10.1016/j.ctarc.2022.100558. pmid: 35443225. 38. the usage of curcumin as chemopreventive agent for oral squamous cell carcinoma: an experimental study on spraguedawley rat. maulina t, widayanti r, hardianto a, sjamsudin e, pontjo b, yusuf hy. integr cancer ther. 2019 jan-dec;18:1534735418822094. doi: 10.1177/1534735418822094. pmid: 30616418. 39. bcl-2 and c-myc expression in oral dysplasia and oral squamous cell carcinoma: an immunohistochemical study to assess tumor progression. pallavi n, nalabolu grk, hiremath sks. j oral maxillofac pathol. 2018 sep-dec;22(3):325-331. doi: 10.4103/jomfp.jomfp_197_18. pmid: 30651675. 40. evaluation of anticarcinogenic effects of clerodendron inerme on 7,12-dimethylbenz(a) anthraceneinduced hamster buccal pouch carcinogenesis. manoharan s, kavitha k, senthil n, renju gl. singapore med j. 2006 dec;47(12):1038-1043. pmid: 17139399. http://dentistry3000.pitt.edu/ chemopreventive effects of curcumin against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. vol 9 no 1 (2024) doi 10.5195/d3000.2024.559 http://dentistry3000.pitt.edu 41. anti-tumor initiating potential of andrographolide in 7,12dimethylbenz[a]anthracene induced hamster buccal pouch carcinogenesis. manoharan s, singh ak, suresh k, vasudevan k, subhasini r, baskaran n. asian pac j cancer prev. 2012;13(11):5701-5708. doi: 10.7314/apjcp.2012.13.11.5701. pmid: 23317242. 42. carnosic acid: a potent chemopreventive agent against oral carcinogenesis. manoharan s, vasanthaselvan m, silvan s, baskaran n, kumar singh a, vinoth kumar v. chem biol interact. 2010 dec;188(3):616-622. doi: 10.1016/j.cbi.2010.08.009. pmid: 20816777. 43. chemopreventive potential of apigenin in 7,12dimethylbenz(a)anthracene induced experimental oral carcinogenesis. silvan s, manoharan s, baskaran n, anusuya c, karthikeyan s, prabhakar mm. eur j pharmacol. 2011 nov;670(2-3):571-577. doi: 10.1016/j.ejphar.2011.09.179. pmid: 21970806. 44. regulation of polyamine metabolism by curcumin for cancer prevention and therapy. murraystewart t, casero ra. med sci (basel). 2017 dec;5(4):38-51. doi: 10.3390/medsci5040038. pmid: 29258259. 45. effect of curcumin and ferulic acid on modulation of expression pattern of p53 and bcl-2 proteins in 7,12-dimethylbenz[a]anthraceneinduced hamster buccal pouch carcinogenesis. balakrishnan s, manoharan s, alias lm, nirmal mr. indian j biochem biophys. 2010 feb;47(1):7-12. pmid: 21086748. 46. antigenotoxic effects of curcumin and piperine alone or in combination against 7,12dimethylbenz(a)anthracene induced genotoxicity in bone marrow of golden syrian hamsters. balakrishnan s, vellaichamy l, menon vp, manoharan s. toxicol mech methods. 2008 jan;18(9):691-696. doi: 10.1080/15376510701781520. pmid: 20020926. 47. the dark side of curcumin. burgos-morón e, calderón-montaño jm, salvador j, robles a, lópez-lázaro m. int j cancer. 2010 apr;126(7):1771-1775. doi: 10.1002/ijc.24967. pmid: 19830693. http://dentistry3000.pitt.edu/ 830 final parents' perspectives and awareness concerning oral health care of pediatric patients receiving chemotherapy vol 13 no 1 (2025) doi 10.5195/d3000.2025.830 h#p://den*stry3000.pi#.edu parents' perspectives and awareness concerning oral health care of pediatric patients receiving chemotherapy ali najed fandy, kahtan h. hussein al jebori babylon university, al hela, iraq abstract objec;ve: assess and determine the rela.onship between the parent’s awareness and perspec.ves on the oral health of their children receiving chemotherapy and demographics data. methods: a descrip.ve (cross-sec.on) design study conducted in holly karbala teaching hospitals (alturkish and al-wareth oncology) from 24th of november 2023 to 30th june 2024. results: this study showed that 57% of par.cipa.ng parents had a low level of awareness regarding oral care, 49% regarding gum and tooth care, and 46% regarding nutri.on. the study also found that 49% of parents had a low level of awareness overall regarding pediatric pa.ents receiving chemotherapy need oral health care and the correla.on show that it was significant correla.on among parents' awareness (r= 0.9, p = 0.000). conclusion: there is a low level of awareness of parents of children undergoing chemotherapy regarding their oral health. keywords: cancer; oral hygiene; oral health literacy; pediatrics. cita9on: fandy an, et al. (2025) parents’ perspec9ves and awareness concerning oral health care of pediatric pa9ents receiving chemotherapy. den9stry 3000. 1:a001 doi:10.5195/d3000.2025.830 received: january 18, 2025 accepted: march 25, 2025 published: april 23, 2025 copyright: ©2025 al-aaloosi sra, et al. this is an open access ar9cle licensed under a crea9ve commons awribu9on work 4.0 united states license. email: aljborykahtan@gmail.com introduction according to the world health organization (who), around 4% of children will get cancer [1]. chemotherapy, surgery, and radiation treatment are the main therapeutic modalities used to treat cancer in children [2]. oral mucositis, xerostomia, neuropathy, oral infection, tooth decay, oral bleeding, lip care are side effects of the mouth and teeth during cancer treatment [3]. improvement in childhood cancer services was achieved over the past two decades through better availability of who essential chemotherapy drugs, introduction of advanced diagnostic/screening tools and bone marrow transplant services, provision of satellite telemedicine e-learning training program [4]. the burden of cancer will increase substantially to a prediction of 961,000 new annual cancer cases by 2030 [5]. mothers play a crucial role in the management of the children’s needs during the treatment period [6]. cultural and regional distinctions in childhood cancer experiences are critical, including communication of diagnosis, hierarchies of family decisionmaking [7]. children with cancer sometimes require extensive hospital stays, the long-term adverse effects of at-home therapy, and these factors make ongoing intervention necessary. enabling parents to take care of their children is one of the primary objectives of pediatric oncology nursing [8]. who recommended that pediatric palliative care (ppc) should start as soon as a child is diagnosed with a severe or lifethreatening illness and continue whether the child receives treatment intended to improve their comfort level [9]. family education is an essential and critical part of pediatric oncology nursing care, experts have been created to guide the education of relatives of newly diagnosed patients. patient and family parents' perspectives and awareness concerning oral health care of pediatric patients receiving chemotherapy vol 13 no 1 (2025) doi 10.5195/d3000.2025.830 h#p://den*stry3000.pi#.edu education in pediatric oncology should begin as soon as a child receives a cancer diagnosis [10]. understanding the parents' views on the importance of oral care is necessary to propose solutions and develop programs to ensure the best level of care for cancer children and protect them from such disorders. material and methods design of study a descriptive (cross-sectional) study. ethical considerations and administrative arrangement the investigator obtained an approval from the university of babylon's college of nursing and its ethical committee. official permissions were secured from the ministry of health's holly karbala health office and development and training center. agreements were obtained from three medical facilities in the region, including imam hussein medical city, imam hassan al mujtaba hospital, and others, to facilitate data collection. study setting the study was carried out at the imam hassan al-mujtaba hospital (al-turkish) / al-wareth oncology hospital. study sample a sample of 100 parents who were present to the treatment of their injured children was selected for the purpose of assessing their awareness and views regarding oral care for their children. study instrument the measurement instrument was organized into four parts: part i: social, demographic, and personal characteristics of the parents. this part includes sociodemographic data such as age, gender, marital status, number of family members, residence, educational level, and economic status. part ii: demographics of the child receiving chemotherapy. part iii: clinical data of the child. part iv: contains domains for assessment of awareness and perspectives of parents regarding oral health status (oral care, gum and dental statuses, diet) of their children under chemotherapy. the parents’ awareness levels were classified based on the mean score of their answers to the items of the awareness form, where their answers were given a score of 2 (not sure), 3 (i don't know), and 1 (i know). parents' perspectives concerning oral health care of pediatric patients receiving chemotherapy were measured using a likert scale with three points: disagree = 1, unclear = 2, and agree = 3. rating and scoring for parents' awareness the section regarding a scale for parents' awareness concerning oral health care of pediatric patients receiving chemotherapy contains 26 items each item is measured on a 3-point likert scale as (not sure = 2, i don't know = 3, and i know = 3). cutoff points used are l (low level of awareness mean = 2.34-3), m (median level of awareness mean = 1.67-2.33) and h (high level of awareness mean = 1-1.66). rating and scoring for parents' awareness the section regarding a scale for parents' awareness concerning oral health care of pediatric patients receiving chemotherapy contains 26 items each item is measured on a 3-point likert scale as (not sure = 2, i don't know = 3, and i know = 3). and cutoff point l: low level of awareness mean =2.34 -3, m: median level of awareness mean= 1.672.33, h: high level of awareness mean = 11.66. parents' perspectives and awareness concerning oral health care of pediatric patients receiving chemotherapy vol 13 no 1 (2025) doi 10.5195/d3000.2025.830 h#p://den*stry3000.pi#.edu rating and scoring for negative elements: this section regarding a scale for parents' perspectives concerning oral health care of pediatric patients receiving chemotherapy contains 21 items. each item is scored using a likert scale with three points: disagree = 1, unclear = 2, and agree = 3. pilot study a pilot study was conducted from may 9th -15th, 2024. it took subjects 10 to 15 minutes to complete each self-report. ten parents participated in the pilot study. the purpose was to ascertain the validity and reliability of the questionnaire. validation validation was obtained through exposure of the tool to 10 panel of experts, from different fields, with no less than 10 years of experience in investigating specificity of research tools. the reliability coefficient was used to calculate the agreement between the items of the questionnaire using the reliability test as a statistical analysis tool. cronbach's alpha and the result of an internal consistency method. the reliability of awareness and perspectives questionnaire was 0.98 and 0.88 (cronbach's alpha values). this indicates that the questionnaire was reliable in measuring this variable. the results of the pilot study revealed that the questionnaire was valid and reliable for studying the phenomenon on the same population at any time in the future. data collection methodology a self-administered, arabiclanguage questionnaire was used to collect data from parents across two hospitals (may 16th to 30th, 2024). the researcher obtained verbal consent after explaining the study's purpose. each one independently completed the questionnaire (10-15 minutes), and hospital health directors provided prior authorization. statistical analysis data analysis used spss v. 27. descriptive statistics (frequency, percentage, mean, standard deviation) and inferential statistics (cronbach's alpha, independent samples t-test, pearson correlation, one-way anova) were employed results table 1 shows that ninety percent of kids live with both parents and 45% of the fathers were between 40-49 years old. the study also showed that 50% of the fathers had intermediate education, while 48% of the mothers had primary education. study showed that 74% of the participants had family members between 6-10 people and did not have any previous infection with any child. table 2 shows that 42% of the children were between 6-10 years old, 62% of them were male, and 89% of them were fully certified on their parents.the study showed that 95% of the children participating in this study fed orally. table 3 shows that 94% of them were due to receiving regular, periodic doses for several hours without admission. the study also found that 44% of the children participating in this study were suffering from leukemia, with 77% of them being treated with chemotherapy. table 4 show that the study indicated that 29.7% of parents knew about 26.8% of pediatric chemotherapy patients received oral health care. were unsure, and 43.5% of them did not know. this means that nearly half of them do not know, and thus their awareness is low. table 5 shows that this study found a statistically significant correlation between parents' perspectives and the number of family members, economic status, parents' perspectives and awareness concerning oral health care of pediatric patients receiving chemotherapy vol 13 no 1 (2025) doi 10.5195/d3000.2025.830 h#p://den*stry3000.pi#.edu father's and mother's educational levels, etc. table 6 shows that this study indicated that a statistically meaningful correlation exists between parents’ perspectives, sex, and child dependence on parents, school-age child, and arrangement of the child in the family, feeding method, therapeutic modalities and diet. table 1. distribution of the parent’s demographic characteristics. demographic characteristics f % cumulative percent who is with the child father 4 4 4 mother 6 6 10 both 90 90 100 total 100 100 age father group mean ± sd 46.4 ± 8.3 30-39 18 18 18.0 40-49 45 45 63.0 50-60 37 37 100.0 total 100 100 age mother group mean ± sd 41.1 ± 7.7 30-39 39 39.0 39.0 40-49 44 44.0 83.0 50-60 17 17.0 100.0 total 100 100.0 educational level of father uneducated 2 2.0 2.0 primary education 30 30.0 32.0 secondary education 50 50.0 82.0 diploma and above 18 18.0 100.0 total 100 100.0 educational level of mother uneducated 10 10.0 10.0 primary education 48 48.0 58.0 secondary education 33 33.0 91.0 diploma and above 9 9.0 100.0 parents' perspectives and awareness concerning oral health care of pediatric patients receiving chemotherapy vol 13 no 1 (2025) doi 10.5195/d3000.2025.830 h#p://den*stry3000.pi#.edu total 100 100.0 economic status enough 30 30.0 30.0 somewhat adequate 38 38.0 68.0 not enough 32 32.0 100.0 total 100 100.0 family status of parents separated couple 9 9.0 9 together 90 90.0 99 death of one of them 1 1.0 100 total 100 100.0 number of family members 3-5 16 16.0 16 6-10 74 74.0 90 more than 10 10 10.0 100 total 100 100.0 the presence of a previous injury to the child or a family member yes 14 14.0 14 no 86 86.0 100 total 100 100.0 %: percentage, f: frequency table 2. distribution of the child’s demographic characteristics. demographic characteristics f % cumulative percent age group child mean ± sd 7.7 ± 4.1 1-5 years 32 32.0 32 6-10 years 42 42.0 74 11-18 years 26 26.0 100 total 100 100.0 sex home 62 62.0 62 parents' perspectives and awareness concerning oral health care of pediatric patients receiving chemotherapy vol 13 no 1 (2025) doi 10.5195/d3000.2025.830 h#p://den*stry3000.pi#.edu woman 38 38.0 100 total 100 100.0 child dependence on parents fully certified 89 89.0 89 partially approved 8 8.0 97 unapproved 3 3.0 100 total 100 100.0 a child of school age study follower 57 57.0 57 study intermittently 26 26.0 83 leaving for study 17 17.0 100 total 100 100.0 sequence of the child in the family 3rd 15 15.0 15.0 4th 9 9.0 24.0 6th 27 27.0 51.0 7th 24 24.0 75.0 8th 25 25.0 100.0 total 100 100.0 the child's injury first 97 97.0 97 second 3 3.0 100 total 100 100.0 feeding method orally 95 95.0 95.0 parents' perspectives and awareness concerning oral health care of pediatric patients receiving chemotherapy vol 13 no 1 (2025) doi 10.5195/d3000.2025.830 h#p://den*stry3000.pi#.edu ng tube 4 4.0 99.0 iv 1 1.0 100.0 total 100 100.0 table 3. distribution of children according to their clinical data. clinical data f % cumulative percent duration of treatment less than 5 years 77 77.0 77 5 years or more 23 23.0 100 total 100 100.0 the baby presence for the hospital was due to the baby condition is critical and requires hospitalization 6 6.0 6 receiving regular, periodic doses for several hours without admission 94 94.0 100 total 100 100.0 child's diagnosis leukemia 44 44.0 44.0 solid tumor 11 11.0 55.0 tumors of the central nervous system 17 17.0 72.0 lymphoma 13 13.0 85.0 other 15 15.0 100.0 total 100 100.0 therapeutic modalities radiation 7 7.0 7 chemotherapy 77 77.0 84 surgical and chemotherapy 16 16.0 100 parents' perspectives and awareness concerning oral health care of pediatric patients receiving chemotherapy vol 13 no 1 (2025) doi 10.5195/d3000.2025.830 h#p://den*stry3000.pi#.edu total 100 100.0 diet follow a normal diet 88 88.0 88 follows a special diet specified by the health care provider 12 12.0 100 total 100 100.0 table 4. identification of the relationship between perspectives and awareness of parents the importance of oral health care for children undergoing chemotherapy. 2-tailed correspondence average variance person correlation r p-value parents' awareness 2.13 0.68 0.915** 0.000 parents' perspectives 2.18 0.58 at the 2-tailed 0.01 significance level, the correlation is significant. table 5. identification of the relationship between the knowledge of parents of the oral health care and demographics of pediatric chemotherapy patients. demographic characteristics chi-square test of perspectives p-value p-value who is with the child 0.003 0.003 age father group 0.006 0.062 age mother group 0.008 0.074 educational level of father 0.006 0.000 educational level of mother 0.028 0.000 economic status 0.119 0.000 family status of parents 0.61 0.19 parents' perspectives and awareness concerning oral health care of pediatric patients receiving chemotherapy vol 13 no 1 (2025) doi 10.5195/d3000.2025.830 h#p://den*stry3000.pi#.edu number of family members 0.431 0.000 the presence of a previous injury to the child or a family member 0.062 0.000 table 6. identification of the relationship between parents' knowledge of the oral health needs of young chemotherapy patients, as well as the child's clinical information and demographics. child's clinical information and demographics chi-square test of perspectives p-value p-value age group of children 0.453 0.478 sex 0.000 0.000 child dependence on parents 0.134 0.000 a child of school age 0.000 0.000 sequence of the child in the family 0.000 0.000 the child's injury 0.953 0.296 feeding method 0.061 0.000 duration of treatment 0.000 0.000 the child's presence in the hospital was due to 0.145 0.000 child's diagnosis 0.000 0.000 therapeutic modalities 0.000 0.000 diet 0.001 0.002 discussion distribution of the parent’s demographic characteristics the study indicates that 90% of children live with both parents and 45% of the fathers were between 40-49 years old, and 44% of the mothers were as well. the study also showed that 50% of the parents' perspectives and awareness concerning oral health care of pediatric patients receiving chemotherapy vol 13 no 1 (2025) doi 10.5195/d3000.2025.830 h#p://den*stry3000.pi#.edu fathers had intermediate education, while 48% of the mothers had primary education. the study indicated that 38% of the participants had an economic status of income and did not have any previous infection with any child or other family member, with a percentage of 86% and the findings of this study are consistent with a previous study [11], which showed that the mother of the children under observation was on average 33.73±4.06 year, while the mean age of the studied children’s father was 40.37±2.97 year. regarding parents’ education, 66.7% of the studied children’ mother had secondary education, while 40.0% of the studied children’ father had secondary education. related to children’s parent income level, 85.0% of them were having insufficient income. the findings of this study conflict with a study conducted earlier [12]. this indicates that most mothers in this study are between the ages of 20 and 40, the majority have completed their university degree (87 % of the population). distribution of the child’s demographic characteristics the study found that 42% of the children were between 6-10 years old, 62% of them were male, and 89% of them were fully certified on their parents. the study showed that 95% of the children participating in this study fed orally and the findings agree with previous work [12]. the results of this study are consistent with studies from the united states, which showed that although the survival rate of child cancer was the same for both sexes and disagree with research performed by others [13]. the dietary counseling of a patient undergoing chemotherapy is usually given by the dietitian. ensuring a proper nutritional status through good hydration and balanced healthy diet is important for those patients. distribution of the child’s clinical data the study showed that 77% of the children's treatment duration was less than 5 years, and 94% of them were due to receiving regular, periodic doses for several hours without admission. the study also found that 44% of the children participating in this study were suffering from leukemia, with 77% of them being treated with chemotherapy and 88% of them were following a normal die. these results agreed with a study previously done [14], which revealed leukemia was the most common malignancy among children. childhood brain and central nervous system cancer were the second most prevalent kind of cancer, which demonstrates the frequent childhood cancers in boys in the order they occur. the study results showed disagreement with study done by others [13]. about 88% of the parents were given dietary counseling by pediatrician or dietitian. however, 85.7% of the children consumed sweetened beverages and/or snacks between the meals. relationship between perspectives and awareness of parents on the topic of children's oral health care undergoing chemotherapy pearson’s correlation tests showed that there was a significant correlation between parents' awareness and parents' perspectives (r= 0.9, p = 0.000). these results agreed with previous work done [18]. one study showed that parents of pediatric cancer patients receiving chemotherapy were unaware of dental health and most of them never took their child to the dentist. previous findings of the generalized linear model's analysis of the correlation indicated that the mean knowledge and attitude scores increased with parent age [17]. only knowledge was associated with statistically significant parents' perspectives and awareness concerning oral health care of pediatric patients receiving chemotherapy vol 13 no 1 (2025) doi 10.5195/d3000.2025.830 h#p://den*stry3000.pi#.edu changes in income, compared to high-income groups. nevertheless, there was no statistically significant correlation found between scores and educational attainment, or household size [13]. concern exists in parents understanding the essential care for oral hygiene, particularly among children [19]. the risks of cancer are considerably higher for children whose mothers have less education than those children whose mothers that have completed high school. regarding the father’s educational, four individuals declined to respond. there is concern regarding children not knowing how important it is to take care of their dental hygiene [13,19]. our study findings disagreed with research [15], that showed that while most parents of children with cancer are aware of the significance of dental health. furthermore, every parent mentioned that the oncology team needed to include a dentist. parents' understanding and attitudes during chemotherapy and immunosuppression will have a significant influence on their children's oral health as well as fundamental oral hygiene routines. conclusion the purpose of this study was to evaluate oral hygiene behaviors, such as how often people clean their teeth and whether they take any further precautions. the questionnaire also evaluated dietary practices, including the kinds of food, sweetened snacks and beverages, and how often they were consumed. furthermore, the goal was also to assess the parents' desire to keep their immune compromised child's teeth clean. conflicts of interest the authors declare no competing interest. references 1. acs (american cancer society). key statistics for childhood cancers. 2020a. [august 17, 2020]. 2. lewandowska, anna. “the needs of parents of children suffering from cancercontinuation of research.” children (basel, switzerland) vol. 9,2 144. 23 jan. 2022, doi: 10.3390/children9020144. 3. damen, h., scholte, r. h., vermulst, a. a., van steensel, p., & veerman, j. w. parental empowerment as a buffer between parental stress and child behavioral problems after family treatment. children and youth services review, 124, (2021). 4. moleti, maria luisa et al. “longterm results with the adapted lmb 96 protocol in children with b-cell non hodgkin lymphoma treated in iraq: comparison in two subsequent cohorts of patients.” leukemia & lymphoma vol. 60,5 (2019): 12241233. doi:10.1080/10428194.2018.1519 810 5. lyons, gemma et al. “scaling up cancer care in the who eastern mediterranean region.” eastern mediterranean health journal = la revue de sante de la mediterranee orientale = al-majallah al-sihhiyah li-sharq al-mutawassit vol. 24, 1 104-110. 5 apr. 2018 6. alaa ahmed, h., ahmed mersal, f., khalil ibrahim, w. mothers' role towards management of their children with burkitt's lymphoma. egyptian journal of health care, 2024; 15(1): 614-624. doi: 10.21608/ejhc.2024.343028 7. mezgebu, esubalew et al. “pediatric oncology nursing research in lowand middleincome countries: exemplars from three regions.” seminars in oncology nursing vol. 37,3 (2021): 151168. doi:10.1016/j.soncn.2021.151168 8. ashcraft, laura ellen et al. “parent empowerment in pediatric healthcare settings: a systematic review of observational studies.” the patient vol. 12,2 (2019): 199-212. doi:10.1007/s40271-018-0336-2. 9. newman, a., crane, s., spruit, j., alharrasi, s., & bell, c. palliative care in pediatric oncology, pediatric oncology nursing, and parents' perspectives and awareness concerning oral health care of pediatric patients receiving chemotherapy vol 13 no 1 (2025) doi 10.5195/d3000.2025.830 h#p://den*stry3000.pi#.edu defining care through science pp: 191-196, (2020). 10. haugen, m., zupanec, s., & landier, w. (2020) improving care through patient and family education in pediatric oncology. in: hinds p., linder l. (eds) pediatric oncology nursing. pediatric oncology. springer, cham. https://doi.org/10.1007/978-3030-25804 11. mohamed, maha ahmed et al. “nurses knowledge and practice regarding care of comatose children at pediatric intensive care units.” (2019). 12. hasnani et al asian journal of pharmaceutical research and development. 2022; 10(5): 06-09. 13. gupta, d. k., palma, j. m., & corpas, f. j. (eds.). (2016). redox state as a central regulator of plant-cell stress responses (pp. 1386). cham: springer. 14. aamir jalal al-mosawi. advisor, pediatrics/psychiatry, children teaching ... science world journal of pharmaceutical sciences 1 (1), 14, 2020. 25, 2020. 15. pedrosa, et al. parents’ knowledge about the oral health care of oncological children. rgorevista gaúcha de odontologia, 67, (2019). 16. leventakkaş, (2019) https://katalog.marmara.edu.tr/ve riler/yordambt/cokluortam/c/d/b /c/a/5d19e3aeb1db4.pdf http://hdl.handle.net/11424/2044 16. 17. benghasheer, h. f., & saub, r. (2022). oral health knowledge, attitude, practice, perceptions and barriers to dental care among libyan parents. journal of oral research, 11(1), 1-14 18. alkhuwaiter ss. parents' awareness and oral health care measures of pediatric patients receiving chemotherapy. j pediatr dent 2021. 19. hassan, g.a., & ibrahim, h.s, the effect of supportive nursing intervention on the burden and coping strategies of caregivers of children with cancer. journal of nursing education and practice. 8(7):125. v8n7p125. (2018). a randomized clinical trial on the clinical and immunological efficacy of a mouth rinse containing hydrogen peroxide and hyaluronic acid in pa?ents with gingivi?s. vol 11 no 1 (2023) doi 10.5195/d3000.2023.556 h#p://den*stry3000.pi#.edu a randomized clinical trial on the clinical and immunological efficacy of a mouth rinse containing hydrogen peroxide and hyaluronic acid in patients with gingivitis. ahmed hashim khudhair1, maha abdul aziz ahmed1 1department of periodontics, college of dentistry, university of baghdad abstract objective: the present study aimed to inves?gate the impact of ha/h2o2 (hyaluronic acid/hydrogen peroxide) mouthwash on pro-inflammatory cytokines, specifically il-1b and il-6, in individuals diagnosed with gingivi?s. furthermore, a comparison was made between the effects of ha/h2o2 and those of chx (chlorhexidine) and placebo. materials and methods: this clinical trial was randomized, double-blinded, and parallel. fiuy-four students with biofilm-induced gingivi?s (18 to 23 years old) were randomly assigned to three mouthwash groups: ha/h2o2, chx, and placebo. uniden?fied bo[les were delivered to par?cipants. for 14 days, 10 millilitres of ha/h2o2 mouthwash formula?on, chx (0.12%), and placebo were used twice a day. all subjects were assessed twice, once at baseline and once auer 14 days. before and auer each par?cipant’s mouthwash usage, bleeding on probing was measured as a clinical parameter for inflamma?on, salivary il-1b and il-6 were also assessed by elisa. changes in salivary interleukins and bleeding between baseline and auer treatment were determined and compared by the t test with an alpha of 0.05. results: there was a sta?s?cally significant decrease in salivary interleukin-1β and il-6 in all three groups. however, intergroup comparisons at second visits showed no significant difference between ha/h2o2 and chx groups (p>0.05) for the interleukins tested. furthermore, all treatments considerably reduced bleeding on probing, although mouthwash with hyaluronic acid (ha) and hydrogen peroxide (h2o2) had a greater impact than the other two treatments. conclusion: mouthwash containing ha/h2o2 demonstrated an an?inflammatory effect, sugges?ng they are immunomodulatory agents. these findings may be beneficial and encouraging for the use of ha/h2o2 in the treatment of gingivi?s; thus, ha/h2o2 rinse has the poten?al to serve as an appropriate replacement for chlorhexidine (chx). keywords: hyaluronic acid; interleukin-1b; hydrogen peroxide; gingivi?s; saliva cita9on: khudhair, ah et al. (2023) a randomized clinical trial on the clinical and immunological efficacy of a mouth rinse containing hydrogen peroxide and hyaluronic acid in pa9ents with gingivi9s. den9stry 3000. 1:a001 doi:10.5195/d3000.2023.556 received: july 7, 2023 accepted: august 7, 2023 published: november 21, 2023 copyright: ©2023 khudhair, ah et al. this is an open access ar9cle licensed under a crea9ve commons azribu9on work 4.0 united states license. email: ahmedalyasari92@gmail.com introduction gingivitis is an inflammatory reaction caused by the accumulation of bacteria-in the plaque around the margin of the gingival tissue [1,2].the prevalence of biofilm-induced gingivitis is widespread among human populations and represents a primary periodontal ailment. if left untreated during its initial stages, this condition has the capacity to escalate into more severe forms of periodontal disease [3]. cytokines are a category of soluble proteins with small molecular weight that are synthesized in response to an antigenic stimulus. they serve as chemical mediators that regulate the immune system that is innate as well as adaptive [4]. interleukin-1 beta (il-1) is a potent http://dentistry3000.pitt.edu/ a randomized clinical trial on the clinical and immunological efficacy of a mouth rinse containing hydrogen peroxide and hyaluronic acid in pa?ents with gingivi?s. vol 11 no 1 (2023) doi 10.5195/d3000.2023.556 h#p://den*stry3000.pi#.edu pro-inflammatory mediator that binds to il-1 receptor and is essential for host defense responses to infection and injury [5]. it has been reported that il-1 in the inflammatory site is responsible for increased local blood flow, leucocyte recruitment, and neutrophil infiltration [6]. interleukin-6 (il-6) is a cytokine that exhibits diverse physiological effects, such as the differentiation and activation of macrophages and t cells, the growth and differentiation of b cells, the stimulation of haematopoiesis, the differentiation of osteoclasts, and the resorption of bone [7]. the mouthwash containing chlorhexidine (chx) is considered the preferred option for complementing oral hygiene due to its exceptional effectiveness in preventing the development of dental biofilm [8,9]. nevertheless, the utilisation of this substance may result in various adverse effects, such as discoloration, modified gustatory perception, and infrequently enlargement of the parotid gland [10].the development of alternative mouthwashes with comparable efficacy, but without these adverse effects is desirable. the present study aimed to examine the efficacy of a mouth rinse solution comprising of hydrogen peroxide (h2o2) and hyaluronic acid (ha). h2o2 possesses oxygenating properties and exhibits antimicrobial activity by releasing oxygen. it has been demonstrated to be efficacious against both gram-positive and gram-negative bacterial strains [11,12]. ha is a linear polysaccharide that occurs naturally and serves as a crucial constituent of the extracellular matrix in various human tissues [13]. additionally, ha is present in the periodontal ligament, as well as gingivae, while the cementum and alveolar bone contain minimal amounts of this substance [14]. ha is involved in phagocytosis, cell migration, adhesion, and wound healing due to its anti-inflammatory and anti-bacterial characteristics [15]. the objective of this investigation was to assess the impact of a rinse containing (ha+ h2o2) on proinflammatory cytokines, specifically il-1b and il-6, among individuals diagnosed with gingivitis, and to juxtapose these results with those obtained from chx and a placebo solution. material and methods experimental design this parallel three-arm doubleblinded clinical trial was conducted between march and april 2023 at university of baghdad's faculty of dentistry. the helsinki declaration and consort 2010 statement for multi-arm studies were followed. volunteers visited the hospital's dentistry clinics at the start and end of the 14-day study. the research ethics committee of the university of baghdad, faculty of dentistry approved the study (project no. 748622, december 28-2022). in 2023, “clinicaltrials.gov” registered this clinical trial under protocol number “nct05787600”. study subjects the trial's sample comprised of undergraduate students who were receiving treatment at the dental clinics of university of baghdad's faculty of dentistry. a notice was displayed in the reception area of the clinics, soliciting the participation of students as volunteers in the research investigation. individuals who satisfied the predetermined criteria for inclusion were extended an invitation to partake in the present investigation. the study's inclusion criteria encompassed individuals who were in good systemic health, did not smoke, possessed over 20 natural teeth, and had been diagnosed with biofilminduced generalised gingivitis. this diagnosis was characterised by the presence of over 30% bleeding sites with no ppd greater than 3 mm, an intact periodontium, and no loss of periodontal attachment [16]. furthermore, participants were required to have no visible supraor sub-gingival calculus and to regularly perform brushing their teeth. the study's exclusion criteria comprised individuals with periodontitis, current use of oral rinse, habitual smoking behaviour, extensive neglected dental caries, dental appliance wearers, individuals who underwent http://dentistry3000.pitt.edu/ a randomized clinical trial on the clinical and immunological efficacy of a mouth rinse containing hydrogen peroxide and hyaluronic acid in pa?ents with gingivi?s. vol 11 no 1 (2023) doi 10.5195/d3000.2023.556 h#p://den*stry3000.pi#.edu periodontal therapy within the past six months, those who received antibiotic therapy three months prior to the study, pregnant or breastfeeding women, and individuals with a previous record of allergic reactions to any of the items used in the investigation. following the process of participant selection, the objective of the inquiry was communicated to every potential participant, and written consent was procured. the decision was made to refrain from providing additional guidance on oral hygiene in order to preserve the participants' current dental hygiene routines. calibration the reproducibility of the examiner's recording of clinical periodontal parameters (bleeding on probing) as determined by intra-examiner calibration. the examiner recorded periodontal parameters twice with a 2-hour interval for five participants. the acceptable limit for agreement for all clinical parameters was > 0.75. in this research, the kappa value for parameter was 0.91. sample size to calculate sample size, il-6 was used as a primary outcome of the study. the concentration of the biomarker in health is estimated to be equal to 6.69 ±0.89 and during gingivitis is equal to 7.85±0.82 (20). the software g*power version 3.1.9.2 was utilised to determine the appropriate sample size with a 95 % power of study, an alpha error of probability of 0.05. the results indicated that each study arm should consist of 18 participants, which includes 16 calculated individuals and an additional 2 to account for any potential dropouts. therefore, the total sample size for the study was 54 participants. study interventions the clinical trial employed a threearm design, utilising three different mouth rinses. the first is a positivecontrol rinse, consisting of 0.12% chlorhexidine and marketed under the brand name kin gingival by kin, spain. the second rinse was a placebo, consisting of distilled water with added food colouring to ensure double-blind conditions. the third rinse, known as perhyal, was used as the test intervention. perhyal is manufactured by bmg pharma in milan, italy and contains h2o2 (1.80%) and ha (0.10%) as active ingredients, with the remaining portion consisting of water (97.3%) and inert additives. the mouthwashes were stored in impermeable bottles that were uniform in appearance and were randomly labelled with letters (a, b, and c) by an individual who was not involved in the clinical trial. therefore, maintaining volunteer and investigator blindness throughout the clinical investigation was crucial. the process of decoding was conducted subsequent to the conclusion of the inquiry. the randomization, blinding, and intervention allocation procedures were followed as reported in previous investigations [17]. clinical research design one experienced, calibrated examiner selected, enrolled, and evaluated the individuals. 54 students volunteered and agreed to the research criteria. a comprehensive intraoral examination followed. bleeding on probing, gingival index and plaque index were assessed with sterile williams' periodontal probes (medesy 546-1, italy). clinical examiners would clarify the study's aims and protocols if individuals met inclusion criteria. participants who accepted the conditions and guidelines signed the informed consent form and started the investigation. in the baseline visit at the beginning of the study period, saliva samples were taken from all 54 participants before starting with measures of the clinical parameters. the collection of unstimulated whole saliva was conducted utilizing a previously described technique [18]. the study's participants were directed to abstain from engaging in oral hygiene practices, such as brushing, flossing, and using mouth rinses, as well as consuming food, beverages, or chewing gum for a period of one hour prior to the collection of saliva samples. subsequently, all the subjects underwent a 30-second rinse http://dentistry3000.pitt.edu/ a randomized clinical trial on the clinical and immunological efficacy of a mouth rinse containing hydrogen peroxide and hyaluronic acid in pa?ents with gingivi?s. vol 11 no 1 (2023) doi 10.5195/d3000.2023.556 h#p://den*stry3000.pi#.edu with 10 ml of tap water, followed by expectoration. the participants expectorated at least 5 ml of unstimulated whole saliva in sterile containers containing lyophilized protease inhibitor solution. on the ice, saliva samples were collected. before analysis, aliquots were prepared, and samples were frozen at -80°c . whole saliva samples were tested for the presence of interleukin (il)-6 and interleukin (il)-1β by using an enzyme-linked immunoassay (elisa) kit (uscn business co., ltd., wuhan, china), used for determining protein levels in salivary samples. the analysis was done following the manufacturer’s instructions for each kit. the elisa procedure for this study was the sandwich elisa technique. then, measurement of bleeding of probing (bop) for the six surfaces by gently introducing the periodontal probe to the level of the gingival sulcus removed coronally and waiting thirty seconds to see whether there is any bleeding (1: bleeding, 0: no bleeding) [19]. 14-days after baseline (the first visit) saliva collection and clinical periodontal parameter scoring (bop) were repeated. examiners regulated mouthwash usage by giving one patient a 140ml bottle at the start of each week and asking for the returning the previous week's bottle. the mouth rinse bottles were recovered, and the remaining liquid was computed to determine usage compliance (280 ml/bottle for two weeks). randomization the simple randomization method was used to allocate patients into three distinct groups for enrollment. each cohort of subjects was allocated a letter (a, b, or c) to match up with the intervention. a computer-based random number generator, implemented in microsoft excel 2016, was employed to generate a set of random numbers. these numbers were subsequently utilised to rearrange the order of participants (n=54) and groups, which were predetermined. each group was assigned an equal number of participants (n=18) in a 1:1:1 allocation. subsequently, bottles that were appropriately labelled with the corresponding interventions were dispensed to the patients, accompanied by detailed guidelines for their administration. the participants were directed to perform a mouth rinse for a duration of one minute, using 10 ml of undiluted mouthwash, after brushing their teeth. they were also advised to refrain from consuming any food or beverage for a period of 30 minutes following the mouth rinse. in order to mitigate potential interference with the effectiveness of the interventions, participants were instructed to perform oral hygiene procedures twice a day, with a 12-hour time interval between each session. every individual employed a toothbrush featuring filaments of a soft hardness and toothpaste from the brand colgate® by colgate-palmolive in india. the tooth-brushing method of the subjects was not allowed to be modified. statistical analysis the statistical package for the social science (spss) version 21, developed in chicago, illinois, united states of america, was utilised for the purposes of data description, analysis, and presentation. the agreement between investigators was assessed by means of cohen’s kappa coefficient. the shapiro-wilk test was employed to assess the normality of the distributions. to compare interventions, statistical measures such as mean and standard deviation are utilised to describe the scores of bleeding on probing %. the study utilised a parametric analysis, specifically a paired t-test, to compare the aforementioned variables within the same group. the researchers employed a one-way analysis of variance (anova) to detect potential variations among the interventions. subsequently, the tukey hsd posthoc test was employed to ascertain the presence of a statistically significant distinction between every intervention pair. the tukey hsd posthoc test was employed to determine the proportion of participants who achieved a state of good health. the effect size of the interventions in comparison to the placebo was calculated. p < 0.05 was http://dentistry3000.pitt.edu/ a randomized clinical trial on the clinical and immunological efficacy of a mouth rinse containing hydrogen peroxide and hyaluronic acid in pa?ents with gingivi?s. vol 11 no 1 (2023) doi 10.5195/d3000.2023.556 h#p://den*stry3000.pi#.edu considered to indicate statistical significance. results the eligibility of 75 individuals was assessed, and 54 of them were selected following the application of inclusion and exclusion criteria. thus, the final analysis included a total of 54 individuals. the study reports that the ha+h2o2 mouthwash group had a mean age of 21.22 ±0.87 years, and the placebo group had a mean age of 21.16 ±0.92 years. the distribution of participants based on sex and treatment are shown in table 1. study groups demographic characteristics ha/h2o2 n=18 chx n=18 placebo n=18 p-value age (years) range (20-23) (20-23) (22-23) 0.93ns* mean ± sd 21.11±0.83 21.2±0.87 21.16±0.92 sex female (n) 10 10 11 0.92ns** male (n) 8 8 6 bleeding on probing non-significant differences (p< .05) were detected among the groups at baseline. on day 14, the mean bleeding on probing values were significantly decreased; they were 10.222 ± 0.943, 9.500 ±1.043, and 32.944 ±1.392 for ha+ h2o2, chx, and placebo, respectively, with statistically significant differences among groups. still, the effect sizes were higher (20.478) in the ha+ h2o2 group than in the chx group (15.761), with a non-significant difference between them and significant differences when each were compared to the placebo group, as shown in table 2. ha/ h2o2 0.12%chx placebo mean ±sd mean ±sd mean ±sd p value* baseline 34.778 ±1.309 35.000±1.188 34.556±1.149 0.553 14 days 10.222±.943 9.500 ±1.043 32.944 ±1.392 0.000 paired t test 86.880 66.868 7.459 p value** 0.000 0.000 0.000 effect size 20.478 15.761 1.758 multiple comparisons (i) groups (j) groups mean difference (i-j) p value*** ha/h2o2 0.2%chx 0.722 0.150 placebo -22.722 0.000 table i: demographic variables of the study groups. chx: chlorhexidine; * one-way anova test, **chi-square test; ns: not significant difference p > 0.05, sd: standard deviation table 2: bleeding on probing by study groups and intervals. http://dentistry3000.pitt.edu/ a randomized clinical trial on the clinical and immunological efficacy of a mouth rinse containing hydrogen peroxide and hyaluronic acid in pa?ents with gingivi?s. vol 11 no 1 (2023) doi 10.5195/d3000.2023.556 h#p://den*stry3000.pi#.edu interleukins -6 il-6 levels were measured in the study groups at baseline and after 41 days, as shown in table 3. in the ha/ h2o2 group, there was significant difference between mean salivary level of il-6 before (70.956 ±3.177) pg/ml and after (29.186 ±5.716) pg/ml, with a 7.03 of effect size. in the chx group, there was significant difference between mean salivary level of il-6 before (71.529 ±1.485) pg/ml and after (28.118 ±2.892) pg/ml, with a 14.14 effect size. while in the placebo group, there was significant difference between mean salivary level of il-6 before (72.09±1.29) pg/ml and after (69.50±1.05) pg/ml, with a 1.63 effect size. no significant difference between mean il-6 changes after rinsing with ha/ h2o2 and chx (p > 0.05) were seen. groups ha/ h2o2 0.12%chx placebo mean ±sd mean ±sd mean ±sd p value* baseline 70.957±3.177 71.529±1.485 72.096±1.291 0.294 14 days 29.186±5.716 28.118±2.892 69.502±1.055 0.000 paired t test 29.834 59.993 6.928 p value** .000 .000 .000 effect size 7.032 14.140 1.633 multiple comparisons (i) groups (j) groups mean difference (i-j) p value*** ha+ h2o2 0.12%chx 1.067 0.671 placebo -40.317 0.000 0.2%chx placebo -41.384 0.000 interleukins -1β il-1β levels were measured in the study groups at baseline and after 14 days, as shown in table 4. in the ha/ h2o2 group, there was significant difference between mean salivary level of il1β before (181.704 ±8.018) pg/ml and after (70.013 ±1.307) pg/ml, with a 13.133 effect size. in the chx group, there was significant difference between mean salivary level of il-1β before (184.606 ±7.112) pg/ml and after (69.839 ±3.469) pg/ml, with a 13.549 effect size. while in the placebo group, there was significant difference between mean salivary level of il1β before (182.835±5.314) pg/ml and after (171.528±5.353) pg/ml, with a 1.833 effect size. no significant differences between mean il1β changes after rinsing with ha/ h2o2 and chx (p > 0.05) were seen. groups ha/ h2o2 0.12%chx placebo table 3: il-6 levels by study groups and intervals. *one-way anova test; ** paired t-test; *** tukey hsd test table 4: il-1β levels by study groups and intervals. http://dentistry3000.pitt.edu/ a randomized clinical trial on the clinical and immunological efficacy of a mouth rinse containing hydrogen peroxide and hyaluronic acid in pa?ents with gingivi?s. vol 11 no 1 (2023) doi 10.5195/d3000.2023.556 h#p://den*stry3000.pi#.edu mean ±sd mean ±sd mean ±sd p value* baseline 181.704±8.018 184.606±7.112 182.835±5.314 0.452 14 days 70.013±1.307 69.839±3.469 171.528±5.359 0.000 paired t test 55.719 57.485 7.776 p value** 0.000 0.000 0.000 effect size 13.133 13.549 1.833 multiple comparisons (i) groups (j) groups mean difference (i-j) p value*** ha/ h2o2 0.12%chx 0.174 0.989 placebo -101.516 0.000 0.2%chx placebo -101.689 0.000 discussion the objective of our investigation was to assess a new composition including ha and h2o2 in mitigating the salivary concentrations of il-1b and il-6 among individuals afflicted with gingivitis caused by plaque accumulation. both of these actives have been researched separately in vitro in addition to in vivo models, and their features are well recognized. as an instance, rodrigues et al. [13] found that ha exhibited antibacterial properties by inhibiting the growth of two prevalent periodontopathogens, namely aggregatibacter actinomycetemcomitans as well as prevotella intermedia, to a comparable extent as chlorhexidine. on the other hand, a recently published randomized clinical trial conducted by yaneva et al. [12] has provided confirmation of the debated antibacterial and anti-inflammatory effects of h2o2. the study's results indicated that a statistically significant decrease in salivary il-1β and il-6 levels in the group that used ha/ h2o2 mouthwash before and after use. the observed decrease in il-1β and il-6 concentrations may be attributed to the synergistic action of ha and h2o2, which are potent antiinflammatory agents known to scavenge reactive free radicals and exhibit efficacy against both grampositive and gram-negative bacterial strains. these factors are directly associated with the upregulation of cytokine levels [15,21]. as this is a new substance, being the first to investigate the effectiveness of the combination of ha and h2o2 on salivary cytokine levels, it is not feasible to make a direct comparison with previous studies. nonetheless, there are further studies that have examined the impact of ha on il-1β levels in a different kind of inflammation. mohammad et al. [22] investigated the possibility of using ha gel application in treatment patient with periodontitis. their results indicated that the greatest mean difference in il-1β level was observed subsequent to the administration of hyaluronic acid gel locally in the ha group, with chx gel application in the chx group following closely behind. regarding h2o2 is the other ingredient in the *one-way anova test; ** paired t-test; *** tukey hsd test http://dentistry3000.pitt.edu/ a randomized clinical trial on the clinical and immunological efficacy of a mouth rinse containing hydrogen peroxide and hyaluronic acid in pa?ents with gingivi?s. vol 11 no 1 (2023) doi 10.5195/d3000.2023.556 h#p://den*stry3000.pi#.edu solution, the efficacy of h2o2 on cytokines in the human body has not been previously studied. on the other hand, the present study's results indicated a statistically significant decrease in salivary il-1β and il-6 levels among participants who used chx mouthwash before and after administration. this finding is consistent with prior study on the impact of chx on gingivitis patients' il-1β levels and il-6 [23]. yoshida et al. discovered that periodontal therapy, which included dental hygiene instructions, conventional mechanical treatment, and 0.12% chlorhexidine used as an adjunct for 15 days, reduced salivary il-1b and 1l6 in gingivitis patients [23]. il-1β secretion requires cellular alterations or stress signals to activate inflammatory cells, which activate caspase-1 and activate il-1β [24]. high levels of this cytokine are linked to periodontal disease's pathogenesis and progression. overall, il-1β is a significant contributor to the neutrophil migration capability as it prompts the dissemination of these cells via the bloodstream. diminishing the concentration of the aforementioned cytokine could potentially lead to a decrease in migratory requirements, as the inflammatory process would be mitigated. through the mechanical elimination of the biofilm as a component of periodontal therapy, the difficulty was diminished, resulting in a decrease in the production of these cytokines. a recent study found the resolution of gingivitis is associated with significant reductions in both gingival inflammation and plaque levels, as well as systemic inflammatory markers [25]. in a comparison between ha/ h2o2 with chx mouthwashes, the present findings revealed that chx mouthwash had the highest effect in lowering il-1b and il-6 levels when compared to ha/ h2o2 mouthwash. moreover, these results revealed that there was no statistically significant difference between ha/ h2o2 mouthwash and chx in reducing of il-1b levels at day 14. the findings of our study indicated a statistically significant variance in the average bleeding on probing index percentage among all mouthwash treatments (ha/ h2o2, chx, and placebo) prior to and following a 14day period of use. nevertheless, the intergroup comparisons regarding bleeding on probing (bop) during second visits did not exhibit any significant disparity between the ha/ h2o2 and chx groups (p>0.05). the results of our study indicated a significant reduction in bop scores when using the ha+ h2o2 mouthwash in comparison to both the baseline data and the negative control. this finding can be explained by antiinflammatory properties of both constituent (ha, h2o2) and efficacy against both gram-positive and gram-negative bacterial strains of both ingredients [13,15] .the present data are compatible with abdulkareem et al. [26], who found that a new formulation containing 0.025% ha with a 0.12% chlorhexidine mouth rinse compared to placebo resulted in a statistically significant reduction in gingival inflammation when compared to both the baseline and the placebo. furthermore, the findings indicated that there was no significant statistical disparity between the effectiveness of ha/ h2o2 mouthwash and chx in diminishing the bop score on the 14th day. on the other hand, the third (placebo) group in both biomarkers showed positive results. it was expected that the placebo group would also experience a decrease in gingival inflammation scores, as the patients were motivated to uphold optimal oral hygiene. the hawthorne effect, a well-known phenomenon, could have played a role in the enhanced results observed in both cohorts. this may be attributed to an increased consciousness of oral health care [27] and the influence of the regular monitoring visits. the trial's limitations included a short 14-day evaluation interval and the absence of instances of supra-sub calculus in the intervention group, as well as healthy controls. in addition, the trial was limited to students between the ages of 18 and 23. in addition, no effort was made to modify the subjects' diets, which http://dentistry3000.pitt.edu/ a randomized clinical trial on the clinical and immunological efficacy of a mouth rinse containing hydrogen peroxide and hyaluronic acid in pa?ents with gingivi?s. vol 11 no 1 (2023) doi 10.5195/d3000.2023.556 h#p://den*stry3000.pi#.edu could have had a long-term impact on the results, particularly salivary cytokines; additional research is required to determine the extent of this influence. financing. conclusion mouthwash containing ha/h2o2 demonstrated an anti-inflammatory effect, suggesting an immunomodulatory effect. these findings may be beneficial and encouraging for the use of ha/ h2o2 in the treatment of gingivitis; thus, ha/h2o2 rinse has the potential to serve as an appropriate replacement for chlorhexidine (chx). data availability the data used to support the findings of this trail are available from the corresponding author upon request. conflict of interest the authors declare that they have no conflicts of interest. financial support the study was completely selffinancing. acknowledgements the authors would like to express their gratitude to the staff of the hospital’s dental clinics of the faculty of dentistry, university of baghdad, for their collaboration. references 1. dental plaque–induced gingival condi4ons. journal of clinical periodontology. murakami s, mealey bl, marioa a, chapple il. 2018 jun;45: s17-27. 2. evalua4on of entamoeba gingivalis and trichomonas tenax in pa4ents with periodon44s and gingivi4s and its correla4on with some risk factors. ibrahim s, abbas r. j bagh coll den4stry. 2012;24(3):158–62. 3. prevalence and severity of gingivi4s in american adults. li y, lee s, hujoel p, su m, zhang w, kim j, zhang yp, de vizio w. american journal of den4stry. 2010 feb 1;23(1): 9. 4. serum levels of interlukine-1beta and interlukine-2 in chronic periodon44s. al-ghurabei bh, shaker zf, fadhel r, al-khayli ng, mustafa lk. al-mustansiriya j. sci. 2012;23(3):55-62. 5. biologic basis for interleukin-l in disease.dinarello ca. the journal of the american society of hematology. 1996. vol. 87. 6. interleukin-1 func4on and role in rheuma4c disease. schef g, dayer jm, manger b. nature reviews rheumatology. 2016 jan;12(1):14-24. 7. biological effects of interleukin-6 on gingival fibroblasts: cytokine regula4on in periodon44s .naruishi k, nagata t. journal of cellular physiology. 2018 sep;233(9):6393400. 8. effect of a chlorhexidine mouthrinse on plaque, gingival inflamma4on and staining in gingivi4s pa4ents: a systema4c review. an strydonck dac, slot de, van der velden u, van der weijden f. j clin periodontol. 2012 nov;39(11):1042–55. 9. an4bacterial efficiency of chlorhexidine digluconate 0.2% against oral β-hemoly4c streptococci and oral staphylococcus aureus in immunocompromisedpa4ents .qanbar fh. j bagh college den4stry. 2012;24(2):166-9. 10. evalua4on of salvadora persica l. and green tea an4-plaque effect: a randomized controlled crossover clinical trial. abdulbaqi hr, himratulaznita wh, baharuddin na. bmc complementary and alterna4ve medicine. 2016 dec;16(1):1-7. 11. a systema4c review of the effect of oral rinsing with h 2 o 2 on clinical and microbiological parameters related to plaque, gingivi4s, and microbes.muniz fw, cavagni j, langa gp, stewart b, malheiros z, rösing ck. interna4onal journal of den4stry. 2020 oct 31;2020. 12. randomised controlled trial comparing the clinical effec4veness of mouthwashes based on essen4al oils, chlorhexidine, hydrogen peroxide and prebio4c in gingivi4s treatment.yaneva bk, dermendzhieva yb, mutafchieva mz, stamenov nv, kavlakova lb, tanev mz, karaslavova e, tomov gt. folia medica. 2022 aug 31;64(4):588-95. 13. hyaluronan-containing mouthwash as an adjunc4ve plaque-control agent. rodrigues sv, acharya ab, bhadbhade s, thakur sl. oral health & preven4ve den4stry. 2010 jan 1;8(4):389-94. 14. hyaluronan: its nature, distribu4on, func4ons and turnover.fraser jr, laurent tc, laurent ub. journal of internal medicine. 1997 jul;242(1):27-33. 15. hyaluronic acid: a boon in periodontal therapy. dahiya p, kamal r.north american journal of medical sciences. 2013 may;5(5):309. 16. plaque-induced gingivi4s: case defini4on and diagnos4c considera4ons. trombelli l, farina r, silva co, tatakis dn. journal of clinical periodontology. 2018 jun;45:s44-67. http://dentistry3000.pitt.edu/ a randomized clinical trial on the clinical and immunological efficacy of a mouth rinse containing hydrogen peroxide and hyaluronic acid in pa?ents with gingivi?s. vol 11 no 1 (2023) doi 10.5195/d3000.2023.556 h#p://den*stry3000.pi#.edu 17. an4-inflammatory effects of manuka honey on salivary cytokines (clinical study). al-kubaisi mw, al-ghurabi bh, alkubaisy w, abdullah nn. journal of baghdad college of den4stry. 2023 mar 15;35(1):10-9. 18. targeted salivary biomarkers for discrimina4on of periodontal health and disease (s). ebersole jl, nagarajan r, akers d, miller cs. fron4ers in cellular and infec4on microbiology. 2015 aug 19;5:62. 19. problems and proposals for recording gingivi4s and plaque. ainamo j, bay i. interna4onal dental journal. 1975 dec 1;25(4):22935. 20. changes of four pro-inflammatory proteins in whole saliva during experimental gingivi4s. zhou m, meng hx, zhao yb, chen zb. chinese journal of dental research. 2012 jan 1;15(2):121. 21. the effects of hydrogen peroxide mouthwashes on the preven4on of plaque and gingival inflamma4on: a systema4c review. hossainian n, slot de, afennich f, van der weijden ga. interna4onal journal of dental hygiene. 2011 aug;9(3):171-81. 22. the effect of hyaluronic acid gel on periodontal parameters, proinflammatory cytokines and biochemical markers in periodon44s pa4ents. mohammad ca, mirza ba, mahmood zs, zardawi fm. gels. 2023 apr 12;9(4):325. 23. inflammatory markers in the saliva of cerebral palsy individuals with gingivi4s aser periodontal treatment. yoshida ra, gorjão r, mayer mp, corazza pf, guare ro, ferreira ac, santos mtbrazilian oral research. 2019 jul 1;33. 24. associa4on between interleukin-8 levels and chronic periodontal disease: a prisma-compliant systema4c review and meta-analysis. fino4 ls, nepomuceno r, pigossi sc, corbi sc, secolin r, scarel-caminaga rmmedicine. 2017 jun;96(22). 25. treatment of gingivi4s is associated with reduc4on of systemic inflamma4on and improvement of oral health-related quality of life: a randomized clinical trial. perić m, marhl u, gennai s, marrugan4 c, graziani f. journal of clinical periodontology. 2022 sep;49(9):899910. 26. a randomized double-blind clinical trial to evaluate the efficacy of chlorhexidine, an4oxidant, and hyaluronic acid mouthwashes in the management of biofilm-induced gingivi4s. abdulkareem aa, al marah za, abdulbaqi hr, alshaeli aj, milward mr interna4onal journal of dental hygiene. 2020 aug;18(3):26877. 27. the" hawthorne effect"—what did the original hawthorne studies actually show? wickström g, bendix t. scandinavian journal of work, environment & health. 2000 aug 1:363-7. http://dentistry3000.pitt.edu/ comparison of the effect of ketorolac and betamethasone injec8on on post complica8ons of third molar surgery (a clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.513 h#p://den*stry3000.pi#.edu comparison of the effect of ketorolac and betamethasone injection on post complications of third molar surgery (a clinical trial) parnian tarabosi1, hamed nazari2, neda omidpanah3 1general dentist, school of dentistry, kermanshah university of medical sciences, kermanshah, iran 2department of oral and maxillofacial surgery, school of dentistry, kermanshah university of medical sciences, kermanshah, iran 3department of oral medicine, school of dentistry, kermanshah university of medical sciences, kermanshah, iran abstract background: the aim of this study was to evaluate the effect of preopera8ve ketorolac and betamethasone injec8ons on reducing postopera8ve complica8ons mandibular third molar surgery. materials and methods: in this randomized clinical trial, 134 pa8ents at the age group of 18-40 years who required mandibular molars surgery were studied. pa8ents were randomly divided into two groups. in a group, 1 ml of betamethasone la was injected into the master muscle before surgery, and in the a group, 30 mg of ketorolac per 1 ml was injected before surgery. on the second and seventh days aser surgery, pa8ents were evaluated for the variables of maximum mouth opening, pain, swelling, and general sa8sfac8on with the surgery through telephone calls on the second and seventh days. independent t-test and mann-whitney test were used to evaluate the variables. results: the mean of pain intensity and the swelling variables in the betamethasone group were lower than the ketorolac group in second and seventh days (p<0.05). the mean of maximum mouth opening and the pa8ent sa8sfac8on variables in the betamethasone group were higher than the ketorolac group in second day (p<0.05). there was no sta8s8cally significant difference in the pa8ent sa8sfac8on and maximum mouth opening variables on the seventh day (p>0.05). conclusion: this study concluded that betamethasone performed significantly bezer in reducing swelling, pain, maximum mouth opening limita8on, and general sa8sfac8on. keywords: ketorolac; injec8on; masseter muscle; betamethasone la; third molar surgery cita9on: tarabosi, p. et al. (2024) comparison of the effect of ketorolac and betamethasone injec9on on post complica9ons of third molar surgery (a clinical trial) den9stry 3000. 1:a001 doi:10.5195/d3000.2024.513 received: may 27, 2023 accepted: july 14, 2023 published: april 24, 2024 copyright: ©2024 tarabosi, p. et al. this is an open access ar9cle licensed under a crea9ve commons axribu9on work 4.0 united states license. email: n.omidpanah20000@gmail.com introduction impacted mandibular third molar surgery is one of the most common interventions and that is a traumatic process in the field of oral and maxillofacial surgery, which has many complications such as pain, swelling, and trismus .these phenomena are caused by excessive inflammatory processes [1,2]. pain is an unpleasant sensory experience that is connected to real or potential tissue damage and is originated from inflammatory mediators such as bradykinin, histamine, and prostaglandin. prostaglandins and leukotrienes play a role in the inflammatory process and pain production. these substances are produced from arachidonic acid, which is caused by http://dentistry3000.pitt.edu/ comparison of the effect of ketorolac and betamethasone injec8on on post complica8ons of third molar surgery (a clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.513 h#p://den*stry3000.pi#.edu the cell membrane destruction and damage due to surgery [3,4]. edema or swelling is the body's expected physiological response to injury, which is due to the accumulation of fluids at the injury site. accumulation of fluids and extensive inflammation in the masticatory muscles after surgery can lead to mouth opening restriction or trismus [5,6]. third molar extraction can reduce the life quality in patients who experience pain and swelling up to 3 times [7]. it seems that factors such as age, sex, medical history, oral contraceptives, pericoronitis, inappropriate oral hygiene, smoking, type of impaction, the third molar relationship with inferior alveolar nerve, surgical time, surgical technique, surgeon's experience, using postoperative antibiotics, local antiseptics, intraoral drugs, anesthetic techniques [8], individual physiological inflammatory response, tissue trauma degree and bone manipulation [9] effect on the third molar extraction post complications. different medical treatment methods have been proposed to control the impacted third molar surgery post complicationswhich are caused by inflammationto reduce the complications by inhibiting the inflammatory pathway, including non-steroidal anti-inflammatory drugs and corticosteroids [4,10]. corticosteroids inhibit the phospholipase a2 enzyme, which plays a role in the inflammatory pathway and the conversion of cell membrane phospholipids into arachidonic acid. this inhibitory effect reduces the release of arachidonic acid and ultimately leads to a reduction in inflammatory mediators (bradykinin, prostaglandin, etc.) production [4]. long-acting betamethasone is a combination of short-acting (betamethasone diphosphate) and long-acting (betamethasone acetate), two forms of corticosteroids, and its acting duration is more than 36 hours, and its anti-inflammatory power is 25 times of body's natural cortisol , and it is similar to the dexamethasone power [11,12]. using corticosteroids in dentistry in low doses and for a short period does not cause significant side effects [4,13]. nsaids inhibit the inflammatory mediators’ productions by inhibiting cyclooxygenases 1 and 2. ketorolac from the nsaid category has a moderate to strong analgesic effect and has been useful for acute and chronic pain and it has antiinflammatory effects and has shown a good analgesic effect on tooth extraction [14]. ketorolac is more potent among different anti-inflammatory drugs under similar experimental conditions [15]. intramuscular corticosteroid injection is very effective for reducing pain, and while it is injected into the masseter muscle, there is no need for additional anesthesia injection to the patient and it is easily available for the dentist [16-18]. grossi et al. showed that injection in the anesthetized area of the mouth is comfortable for the patient and the surgeon, while the injection in the gluteal muscle requires special facilities and equipment [19]. in addition, a study by selvaraj et al. showed that there is no statistically significant difference between methylprednisolone injection into the masseter muscle and gluteal muscle injection in terms of pain after impacted third molar surgery [20]. skjelbred et al. showed in their study that betamethasone can be effective in reducing pain after mandibular third molar surgery [21]. rao et al. have shown in their study that ketorolac provides better analgesia than acetaminophen in tooth extraction [14]. ketorolac has a similar effect to morphine [22] and is even better than opioids in pain relief [23]. in a study, majid et al. showed that the use of corticosteroids can be useful in improving the complications and quality of life after mandibular third molar surgery [6]. more studies are necessary to compare the effect of corticosteroids with nsaids on the wisdom teeth surgery post complications . the purpose of this study is to compare the effect of betamethasone long acting (la ) or ketorolac injection on the masseter muscle immediately after mandibular block anesthesia injection, on pain, http://dentistry3000.pitt.edu/ comparison of the effect of ketorolac and betamethasone injec8on on post complica8ons of third molar surgery (a clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.513 h#p://den*stry3000.pi#.edu swelling, overall patient satisfaction, and maximum mouth opening after mandibular impacted third molar surgery. material and methods ethics approval of the study this randomized, single blind, clinical trial study included 134 patients, aged 18-40 years with impacted lower third molar. the study methods were approved by the ethics committee of kermanshah university of medical sciences with the rigestery id of ir.kums.rec.1400.801 . witnessed written consent was obtained from of all participants before enrolment in the study. .this study was registered at www.irct.ir with irct registration code: irct20191127045516n6. the inclusion criteria were: age of 18 -40 years, healthy medical assessment ( asa i or ii), diagnosis of bony impacted mandibular third molars requiring surgery with panoramic radiography , free of pericoronitis or infection at the time of operation. exclusion criteria included: concurrent lactating or pregnancy, systemic disease, allergy to the drugs , history of smoking or drinking alcohol, impaction according to pell & gregory [24] and winter [25] classifications with severe complexity and those who use the antibiotic and anti-inflammatory medication in the one week before surgery and surgery lasting more than 1 hour. participants, study design, and setting according to paiva-oliveira j, et al [15] the sample size was calculated using a standard deviation and mean of s1=1.29 , x!!!!1 = 1.05 for dexamethasone group and s2=2.24, 𝑋!! = 2.35 for ketorolac tromethamine group, with considering 𝛼 = 0.05 (power) 1β=90%, minimum sample size in each group was 62 persons. the sample volume was calculated with the following formula: total of 134 patient using a sequentially numbered, opaque sealed envelopes (snose) divided into two groups (a, b) with a 1:1 allocation ratio. each patient allocated to the groups was based on systematic random numbering; letter “a” was allocated to the first group, letter “b” was allocated to the second group . group “a” received 6 mg/1ml ampoule betamethasone la (alborz iran company) in masseter muscle and the group b received 30 mg/ml of ketorolac (alborz iran company) into the masseter muscle immediately after anesthesia. neither the surgeons nor the participants were blinded to use of the receiving drugs .postoperative amoxicillin 500 mg , metronidazole 250 and acetaminophen 325mg were prescribed for all patients in the study. the operations were performed by one surgeon in private office. two surgeries in both sides were carried out in each patient with a minimum interval of 15 days. using a solution of 2% lidocaine with 1:2 00,000 adrenaline (persocaine-e ; darou pakhsh, tehran, iran). inferior alveolar nerve block and long buccal nerve block were carried out. standard impacted third molar surgery were carried out with bone removal and irrigation with sodium chloride 0.9%. sutures were performed with 3.0 silk (braided silk; supa, tehran, iran). a gauze pack was held in the surgical site for 30 -45 minutes. the effect of the surgery on postoperative pain, swelling, general patient satisfaction and maximum mouth opening were measured on days 2 and 7 after operation by telephonic interview. postoperative pain scores were estimated using a visual analog scale(vas), 10 mm in length, ranging from 0 for “no pain” to 10 for “the worse unbearable pain” [1,15]. http://dentistry3000.pitt.edu/ comparison of the effect of ketorolac and betamethasone injec8on on post complica8ons of third molar surgery (a clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.513 h#p://den*stry3000.pi#.edu swelling scores was assessed by asking the patient to rate it on a visual analog scale of 0 to 5 [1,2628] .general patient satisfaction of the surgery was recorded from 0 to 10 by telephonic interview at 2 and 7 days after surgery [1]. the patients were asked to score their satisfaction from 0 to 10, where 0 represents no satisfaction and 10 representing complete satisfaction of the surgery. patients measured maximum distance inter upper central and lower central incisal with ruler for 2 and 7 days after operation [15,29]. before enrollment the study, these variables were explained to all patients by leaflet and how to report them to the operator, who called them through a phone at 2 and 7 days after operation. statistical analysis data analysis was carried out using spss version 18 statistical software (spss, inc, chicago, il). subject demographic data were analyzed by chi-square or independent samples t-test, as appropriate. to compare the paired results among groups and across time were used mann whitney test and wilcoxon, respectively. p value of less than 0.05 was considered significant. results 134 participants of both genders, between 18 and 39 years (mean 25.03±5.47 years) with impacted lower third molars were present in this study. 57of the participants (42.5%) were male and the rest (77 individuals (57.5 %)) were female. 68 of which (50.7%) were allocated to the group a (betamethasone) and 66 individuals (49.3) to the group b (ketorolac). there were no statistically significant differences in the distribution of the mean age (p=0.788) and gender (p=0.979) between the two groups. there wasn't a significant difference in difficulty level of impacted teeth between the two groups (table 1). the betamethasone group had significantly less pain and swelling scores on the second and seventh days after the surgery compared with the ketorolac group (p<0.001) (table 2). general patient satisfaction and maximum mouth opening on the second day were significantly higher in the betamethasone group than the ketorolac group. however, no significant difference was showed between the groups on 7th day (table 2). pain and swelling scores on the seventh day after the surgery were lower than the second day after surgery in both groups (p < 0.0001). general patient satisfaction and maximum mouth opening on the seventh day after the surgery were higher than the second day after surgery in both groups (p < 0.0001) (table 2). medication group p-value betamethasone ketorolac sex male 29(42.6%) 28(42.4) 0.979† female 39(57.4%) 38(57.6) difficulty level of impacted teeth minimally difficult 27(39.7%) 26(39.4%) 0.971† moderate 41(60.3%) 40(60.6%) age 25.16±5.83 24.89±5.11 0.778‡ mean±standard deviation and percentage (%) are presented for parametric and categorical data, respectively. †chi-square test was used. ‡independent samples t-test was used. table 1: demographic and difficulty level of impacted teeth data http://dentistry3000.pitt.edu/ comparison of the effect of ketorolac and betamethasone injec8on on post complica8ons of third molar surgery (a clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.513 h#p://den*stry3000.pi#.edu discussion the main findings of this study were that there was a statistically significant difference in variables of pain intensity and swelling on the second and seventh day between the two study groups, as the average of these variables was lower in the betamethasone group than in the ketorolac group, and there was a statistically significant difference in the maximum mouth opening rate variable. there was a difference in the patient's maximum mouth opening on the second day between the two study groups, as the mean of this variable in the betamethasone group was higher than in the ketorolac group. also, the average of the maximum mouth opening on the second day in both groups was less than 35 mm. this study shows that betamethasone performed significantly better in reducing swelling, pain, and maximum mouth opening and general satisfaction. moghadasi et al [30]., messer et al. [31] , and mahmoud et al.[16] reported a significant reduction in pain, swelling, and trismus by administering dexamethasone intramuscularly, which was according to the results of our study. in the clinical trial study by jose marques et al. ,[29] a dose of 12 mg long-acting betamethasone was administered submucosally after items medication group betamethasone ketorolac p value mean ±sd mean±sd pain vas day 2nd 2.75± 2.43 4.37±3.04 0.001† pain vas day 7th 0.44±1.04 1.50±2.06 <0.001† <0.001‡ <0.001‡ swelling vas day 2nd 2.16±1.50 3.36±1.39 <0.001† swelling vas day7th 0.29±0.52 0.67±0.93 0.005† <0.001‡ <0.001‡ general patient satisfaction day 2nd 8.91 ±1.05 8.20±2.01 .012† general patient satisfaction day 7th 9. 43±0.97 9.09±1.77 .178† <0.001‡ <0.001‡ maximum mouth opening day 2nd 29.12±7.72 25.94±8.73 .027† maximum mouth opening day 7th 38.37±5.05 38.85 ±8.73 .698† <0.001‡ <0.001‡ table 2: p values in comparing the pain, swelling, general pa8ent sa8sfac8on scores between each 3 groups †independent samples t-test was used ‡paired t-test was used http://dentistry3000.pitt.edu/ comparison of the effect of ketorolac and betamethasone injec8on on post complica8ons of third molar surgery (a clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.513 h#p://den*stry3000.pi#.edu surgery, and no significant difference was observed between the two study groups in terms of postoperative pain, facial swelling, and trismus, which was contrary to the results of our study. the clinical trial study by bertossi et al. [32] showed that the use of a 4 mg submucosal injection of betamethasone after surgery leads to a reduction in edema, trismus, and pain in the patients undergoing extraction of impacted third molar which was according to the results of our study. in a clinical trial study, chopra et al.[33] showed that ibuprofen is more effective than betamethasone in reducing pain and swelling, which was contrary to the results of our study. in paiva-oliveira [15] clinical trial study in 2016, compared 10 mg of oral ketorolac (1 hour before third molar surgery and then every 8 hours for two days) with 8 mg of oral dexamethasone (1 capsule 1 hour before third molar surgery) and concluded that there was no statistically significant difference between the two groups in terms of pain and edema variables. however, for mouth opening limitation, 24 hours and 7 days after surgery, the dexamethasone group had less limitation in mouth opening, and it behaved better than ketorolac for this variable in these periods, which was contrary to the results of our study. in martin's 2021 [34] clinical trial study, 8 mg single dose of oral dexamethasone was compared with 20 mg single dose of sublingual ketorolac tromethamine 1 hour before surgery. compared to ketorolac tromethamine, dexamethasone showed a significant reduction in pain level up to 72 hours, in swelling, and trismus up to 72 hours and 7 days later, and the clinical performance of dexamethasone in controlling pain, swelling, and trismus following mandibular third molar removal was superior to ketorolac tromethamine. it seems that the superiority of dexamethasone compared to ketorolac in this study is related to the characteristics of its broad antiinflammatory effect mechanism and its long half-life. therefore, it is a more accessible and more suitable option for preventive prescription in oral surgery. in a clinical trial study by meta et al., [35] postoperative pain and swelling after dental implants were compared in patients treated with nonsteroidal anti-inflammatory drugs (nsaids) versus nsaids and corticosteroids. this rct did not show a difference between patients treated with ketorolac-betamethasone versus ketorolac alone in terms of postoperative pain and swelling, which was against the results of our study. these controversies among the results of the studies may be related to various factors, including drugs, dosage, administration method, administration time, and method of measuring postoperative complications including swelling and pain, and the follow-up period of patients after surgery. one of the limitations of this study is the measurement of swelling and maximum mouth by using a telephone report from the patients. it would be better to evaluate the intensity of pain on the first day after surgery, which is most intense in the first 6 hours after surgery. conclusion long-acting corticosteroids can be more effective in reducing swelling, pain, and maximum mouth opening, and in increasing general satisfaction than nsaids. briefly, the present study shows that betamethasone has acted significantly better in reducing swelling, pain, limitation of maximum mouth opening, and general satisfaction. references 1. comparison of the effecgveness of dexamethasone injecgon into two different sites in prevengng the postoperagve complicagons aker mandibular third molar surgery: a randomized clinical trial. shirani m, hasanzade m, moadabi a, arar b. br j med med res 2016; 13(10), 1–11. hrp://dx.doi.org/10.9734/bjm mr/2016/23638. 2. clinical postoperagve findings aker removal of impacted mandibular third molars: predicgon of postoperagve facial swelling and pain based on preoperagve variables . yuasa h, sugiura m. bri+sh journal http://dentistry3000.pitt.edu/ comparison of the effect of ketorolac and betamethasone injec8on on post complica8ons of third molar surgery (a clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.513 h#p://den*stry3000.pi#.edu of oral and maxillofacial surgery 2004;42(3),209-14. hrps://doi.org/10.1016/j.bjoms. 2004.02.005. 3. psychological aspects of pain. gorczyca r, filip r, walczak e. ann agric environ med 2013, 20(1), 23–7. pmid: 25000837. 4. the use of corgcosteroids and nonsteroidal anginflammatory medicagon for the management of pain and inflammagon aker third molar surgery: a review of the literature. kim k, brar p, jakubowski j, kaltman s, lopez e.. oral surg, oral med oral pathol oral radiol endododed 2009,10(5), 630–40. hrps://doi.org/10.1016/j.tripleo .2008.11.005. 5. pimenta l, peterson lj, ellise e, hupp jr tm. contemporary oral & maxillofacial surgery 4th edigon. mosby, st. louis craniofacial and dental developmental defects: diagnosis and management 2003. 113–124. 6. submucosal dexamethasone injecgon improves quality of life measures aker third molar surgery: a comparagve study. majid ow, al-mashhadani ba. j oral maxillofac surg 2011,69(9),289–97. hrps://doi.org/10.1016/j.joms.2 011.01.037. 7. the impact of third molar symptoms, pain, and swelling on oral health-related quality of life. slade gd, foy sp, shugars da, phillips c, white rp. j oral maxillofac surg2004,62(9),11181124. hrps://doi.org/10.1016/j.joms.2 003.11.014. 8. complicagons of third molar surgery. bouloux gf, steed mb, perciaccante vj. oral and maxillofac surg clin north am 2007,19(1),117–28. hrps://doi.org/10.1016/j.coms. 2006.11.013 9. beneficial effect of methylprednisolone aker mandibular third molar surgery: a randomized, double-blind, placebo-controlled split-mouth trial. acham s, klampfl a, truschnegg a, kirmeier r, sandner-kiesling a, jakse n. clin oral invesgg, 2013,17(7)1693– 700. hrps://doi.org/10.1007/s00784012-0867-1. 10. randomized controlled trial to evaluate the efficacy of oral dexamethasone and intramuscular dexamethasone in mandibular third molar surgeries. sabhlok s, kenjale p, mony d, khatri i, kumar p. j clin diagn res 2015,9(11), zc48-5 hrps://doi.org/10.7860%2fjcdr %2f2015%2f13930.6813. 11. do corgcosteroids sgll have a role in the management of third molar surgery? advances in therapy. ngeow wc, lim d. springer healthcare; 2016,33(7)1105–1139. hrps://doi.org/10.1007/s12325016-0357-y. 12. betamethasone and methylprednisolone usage in lower third molar surgery : review literature. gozali p, kiaravornchareon s, wu m, wongsirichat n, suphangul s. m dent j 2015,35(3),273–80. 13. corgcosteroids reduce postoperagve morbidity aker third molar surgery: a systemagc review and metaanalysis. markiewicz mr,brady mf, ding el , dodson tb. j oral maxillofac surg 2008,66(9), 1881-94. hrps://doi.org/10.1016/j.joms.2 008.04.022. 14. analgesic efficacy of paracetamol vs ketorolac aker dental extracgons. rao td, kumar mps. res j pharm technol. 2018,11(8),3375-9. hrp://dx.doi.org/10.5958/0974360x.2018.00621.2 15. comparison of the anginflammatory effect of dexamethasone and ketorolac in the extracgons of third molars. paiva-oliveira j, bastos p, pontes e, silva j, delgado j, oshiro-filho n. oral maxillofac surg 2016,20(2), 123-33. hrps://doi.org/10.1007/s10006015-0533-2. 16. efficacy of sub-mucosal, intramasseteric and intra-muscular routes of dexamethasone administragon on post-operagve complicagons following impacted mandibular third molar surgeries, comparagve clinical trial. mahmoud n. egyp+an dental journal 2019,65(1), 113–33. hrps://dx.doi.org/10.21608/edj. 2015.71254. 17. comparison of efficacy of methylprednisolone injecgon into masseter muscle versus gluteal muscle for surgical removal of impacted lower third molar. selvaraj l, hanumantha rao s, lankupalli asmaxillofac oral surge. 2014,13(4), 495–8. hrps://doi.org/10.1007/s12663013-0562-z. 18. efficacy of methylprednisolone injected into the masseter muscle following the surgical extracgon of impacted lower third molars. vegas-bustamante e, micó-llorens j, gargallo-albiol j, satorres-nieto m, berini-aytés l, gay-escoda c. int j oral maxillofac surge2008,37(3),260– 3. hrps://doi.org/10.1016/j.ijom.2 007.07.018. 19. effect of submucosal injecgon of dexamethasone on http://dentistry3000.pitt.edu/ comparison of the effect of ketorolac and betamethasone injec8on on post complica8ons of third molar surgery (a clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.513 h#p://den*stry3000.pi#.edu postoperagve discomfort aker third molar surgery: a prospecgve study. grossi gb, maiorana c, garramone ra, borgonovo a, berera m, farronato d, et al. j oral maxillofac surg. 2007,65(11), 2218–26. hrps://doi.org/10.1016/j.joms.2 006.11.036. 20. comparison of efficacy of methylprednisolone injecgon into masseter muscle versus gluteal muscle for surgical removal of impacted lower third molar. selvaraj l, hanumantha rao s, lankupalli as. j maxillofacoral surg,2014,13(4), 495–8. hrps://doi.org/10.1007/s12663013-0562-z. 21. post-operagve pain and inflammatory reacgon reduced by injecgon of a corgcosteroid. a controlled trial in bilateral oral surgery. skjelbred p, loekken p, løkken p. eur j clin pharmacol ,1982,21(5), 391–6. hrps://doi.org/10.1007/bf0054 2325. 22. comparison of intramuscular ketorolac tromethamine and morphine sulfate for analgesia of pain aker major surgerypharmacother.yee jp, koshiver je, allbon c, brown cr. j hum pharmacol drug ther 1986,6(5), 253–61. hrps://doi.org/10.1002/j.18759114.1986.tb03485.x. 23. comparagve evaluagon of preempgve analgesic efficacy of intramuscular ketorolac versus tramadol following third molar surgery. shah a v., arun kumar k v., rai kk, rajesh kumar bp. j maxillofac oral surg 2013,12(2), 197–202. hrps://doi.org/10.1007/s12663012-0420-4. 24. how well do clinicians esgmate third molar extracgon difficulty? susarla sm, dodson tb. j oral maxillofa surg 2005,63(2), 1919. hrps://doi.org/10.1016/j.joms.2 004.05.220. 25. winter gb: principles of exodongas as applied to the impacted third molar 1ed. st. louis american medical books 1926. in contemporary oral and maxillofacial sugery. 2th edigon. edited by: peterson lj, ellis e, hupp jr, tucker mr. mosby, st louis;1993:225-260. 26. visual analogue scale assessment of postoperagve swelling: a study of clinical inflammatory variables subsequent to third-molar surgery. berge ti. acta odontol scand 1988,2 6(4), 233–40. hrps://doi.org/10.3109/000163 58809004772. 27. the use of a visual analogue scale in observer assessment of postoperagve swelling subsequent to third-molar surgery. berge ti. acta odontol scand1989,47(3),167–74. hrps://doi.org/10.3109/000163 58909007697. 28. primary and secondary closure of the surgical wound aker removal of impacted mandibular third molars: a comparagve study. pasqualini d, cocero n, castella a, mela l, bracco p. int j oral maxillofac surg 2005,34(1),52–7. hrps://doi.org/10.1016/j.ijom.2 004.01.023. 29. effect of the local administragon of betamethasone on pain, swelling and trismus aker impacted lower third molar extracgon. a randomized, triple blinded, controlled trial. marques j, pié-sánchez j, figueiredo r, valmasedacastellón e, gay-escoda c. med oral patol oral cir bucal 2014,19(1),e49-54. hrps://doi.org/10.4317/medora l.19280. 30. effect of preoperagve single dose dexamethasone injecgon in masseter muscle on postoperagve sequelae following lower impacted third molar surgery. moghadasi m, golestaneh a, ghodosi a, golestani sj isfahan dent sch 2021 apr 10,17(1), 87–94 .hrps://doi.org/10.18502/ijds.v1 7i1.5919. 31. the use of intraoral dexamethasone aker extracgon of mandibular third molars. messer ej, keller jj. oral surgery, oral med oral pathol1975,4(5),594–8. hrps://doi.org/10.1016/00304220(75)90369-2. 32. evaluagon of pain, swelling and trismus aker extracgon of impacted third molars relagve to the use of betamethasone in submucusal infiltragon. bertossi d, donadello d, ricciardi g, luciano u, zox f, nocini r, et al. j biol regul homeost agents 2019 20;33:9–17. pmid: 30966728. 33. a randomized, double-blind, placebo-controlled study comparing the efficacy and safety of paracetamol, serragopepgdase, ibuprofen and betamethasone using the dental impacgon pain model. chopra d, rehan hs, mehra p, kakkar ak. int j oral maxillofac surg 2009,38(4),350–5. hrps://doi.org/10.1016/j.ijom.2 008.12.013. 34. is dexamethasone superior to ketorolac in reducing pain, swelling and trismus following mandibular third molar removal? a split mouth tripleblind randomized clinical trial. margns-de-barros av, barros ami, de siqueira akc, lucena ee de s, de souza phs, araújo fa da http://dentistry3000.pitt.edu/ comparison of the effect of ketorolac and betamethasone injec8on on post complica8ons of third molar surgery (a clinical trial) vol 12 no 1 (2024) doi 10.5195/d3000.2024.513 h#p://den*stry3000.pi#.edu c. med oral patol oral cir bucal 2021,26(2),141–50. hrps://doi.org/10.4317/medora l.24088. 35. randomized controlled trial comparing the effects of 2 analgesic drug protocols in pagents who received 5 dental implants. meta if, bermolen m, macchi r, aguilar j. implant dent. 2017,26(3)3,412–6. 10.1097/id.0000000000000544. http://dentistry3000.pitt.edu/ comparative analysis of serum titanium level in patient with healthy dental implant and patients with peri-implantitisa cross sectional prospective study vol 9 no 1 (2024) doi 10.5195/d3000.2024.594 http://dentistry3000.pitt.edu comparative analysis of serum titanium level in patient with healthy dental implant and patients with peri-implantitisa cross sectional prospective study mahantesha s1, vibha shetty2, kranti k3, shobhasubbaiah4, greeshma c5, babashankar alva6. 1 dept of periodontology, faculty of dental sciences, ruas, bengaluru india. 2 dept of prosthodontics and implantology, faculty of dental sciences, ruas, bengaluru india 3 dept of periodontology, faculty of dental sciences, ruas, bengaluru india 4 dept of periodontology, oxford dental college, bengaluru india 5 dept of periodontology, faculty of dental sciences, ruas, bengaluru india 7 dept of prosthodontics and implantology, faculty of dental sciences, ruas, bengaluru, india abstract objectives: titanium dental implants can last for more than two decades in the oral environment. corrosion of the implant surface can release metallic particles or ions into surrounding tissue. the metallic constituents like titanium in human blood serum have not been fully studied. the study compares titanium serum levels before and after dental implant placement and compares levels in patients with healthy implants and those with per-implantitis. methods: the study comprised 2groups of group 1 patient observing implant surgery and group 2 patients with diagnosed peri-implantitis. each group comprised of 60 patients. serum titanium level was measured from blood obtained from group 1 at three different intervals (one month prior to implant surgery, 4th and 8th month after successful loading) and from group 2 during the course of peri-implantitis by inductively coupled plasma–mass spectroscopy. the statistical analysis was done for the obtained data. results: analysis showed a raised level of serum titanium at 4th month of post implant placement (2.39 mg/dl) and in patient with peri-implantitis(2.94 mg/dl) and both levels are significantly differ (anova test) from pre-surgical estimation of serum titanium level (1.79 mg/dl). conclusions: understanding the correlation between titanium corrosion and peri-implantitis is vital for enhancing the long-term success and safety of dental implants. additional research is required to investigate these links and potential strategies to protect the well-being of implant patients. keywords: dental implants; serum titanium; peri-implantitis; inductivelycoupled plasma– mass spectroscopy; titanium alloys. citation: mahantesha s, et al. (2024) comparative analysis of serum titanium level in patient with healthy dental implant and patients with peri-implantitisa cross sectional prospective study. dentistry 3000. 1:a001 doi:10.5195/d3000.2024.594 received: september 20, 2023 accepted: december 22, 2023 published: may 9, 2024 copyright: ©2024 mahantesha s, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: dentopathfider2009@gmail.com introduction titanium implants have gained substantial usage in dentistry to supplant natural teeth because of their high biocompatibility. titanium or titanium admixtures are the usual constituents of titanium implants, given their mechanical properties and biocompatibility. there is evidence indicating that titanium dental implants can endure exposure to the oral environment for more than two decades [1]. the biocompatibility exhibited by dental implants can be attributed to the formation of a titanium dioxide layer that prevents direct contact between the implant and the biological environment. this, in turn, reduces the potential for metal reactivity [2]. currently, scholars are focusing on diverse approaches to expedite the process of osseointegration of dental implants and to augment the surface area of implant-bone contact. to this end, the osseointegration surface areas are enlarged by creating irregularities on the surfaces of materials used for dental implants [3]. the examination of dental implants' interactions with biological tissues involves the use of a titanium dioxide (tio2) layer. when subjected to loading conditions, the tio2 layer is susceptible to damage during movement between the implant and bone tissue, resulting in implant http://dentistry3000.pitt.edu/ mailto:dentopathfider2009@gmail.com comparative analysis of serum titanium level in patient with healthy dental implant and patients with periimplantitisa cross sectional prospective study vol 9 no 1 (2024) doi 10.5195/d3000.2024.594 http://dentistry3000.pitt.edu corrosion and consequential weakening. furthermore, corrosion can trigger the release of minute metallic particles or ions into the surrounding living tissues [4]. a recent investigation revealed that the micromotion of the abutment under cyclic loading may produce wear particles of varying sizes between 2 and 80 nm in conical dental implant systems. wear debris comprises titanium particles that are recognized to elicit a macrophage response [5]. metal nanoparticles have been widely acknowledged for their ability to trigger an inflammatory response through their immunomodulatory potential. this potential is primarily exerted at the macrophage level and is characterized by an escalation in dna damage, protein carbonylation, lipid per-oxidation, oxidative stress, and a reduction in superoxide dismutase activity, total glutathione levels, and total antioxidant capacity catalase [3]. since ferguson’s seminal study, the generation of metal debris from joint replacement surgeries has been a significant concern within the field of orthopedic surgery. the surface oxide film layer of a metallic object implanted within the human body may undergo disruption or degradation over time due to spontaneous mechanical or electrochemical corrosion. this particular interaction can yield chemically reactive metallic byproducts, which may prompt the discharge of metal into the systemic circulation [6]. bianco et al. investigated the dissimilarity between the levels of titanium in the serum and urine of rabbits before and following implant insertion. their findings suggest that there was no noteworthy elevation observed [7]. a comprehensive examination of the metallic constituents in human blood serum has yet to be fully explored. it is uncertain whether the discharge of titanium or titanium particles from dental implants could have an impact locally or systemically. the present study aimed to compare the titanium serum level before and after the placement of dental implants and to compare the level of serum titanium in patients with healthy dental implants with per-implantitis. materials and methods study design a null hypothesis was developed for a prospective quasi-experimental study stating that there would be no alteration in the serum titanium level of patients before and after implant placement either in health or disease. this study was carried out in the department of periodontics from 2019 to 2022 and was permitted by the institutional ethical review committee [ec-19/12-f-fds]. the study design consisted of 2 groups of 60 patients each. group 1 comprising healthy patients seeking implant placement, group 2 included patients with peri-implantitis who had implants placed more than 6 months. sample size calculation the statistical software gpower (version 3.1) developed by franz faul at the university of kiel in germany was employed to determine the appropriate sample size for this study, with a type 1α error rate of 0.05 and a power of 95% with effect size of 0.5[7]. ultimately, a sample size of 53 was selected; however, in anticipation of potential sample attrition, the sample size was increased to 60. as per the sample size calculation each group of the study comprises of 60 patients contributing to total population of 120. inclusion criteria group 1(healthy patients) • partially edentulous patients. • periodontally healthy patients. • patients with appropriate inter-occlusal distance for the placement of the implants • patients with no history of metal allergy. group 2 (peri-implantitis) • post implant period should be more than 6 months but less than 2 years • implants having probing depths ≥ 5mm. http://dentistry3000.pitt.edu/ comparative analysis of serum titanium level in patient with healthy dental implant and patients with periimplantitisa cross sectional prospective study vol 9 no 1 (2024) doi 10.5195/d3000.2024.594 http://dentistry3000.pitt.edu • bleeding on probing and/or suppuration. • bone loss ≥ 2mm were considered in the periimplantitis group. exclusion criteria • patients using systemic or local antibiotics in the last 3 months. • immuno-compromised patients, who have received chemotherapy or radiotherapy. • pregnant and lactating women. method of assessment of periimplantitis the determination of peri-implantitis diagnosis was established through the evaluation of clinical and radiologic criteria, in accordance with existing recommendations. specifically, implants with probing depths equal to or greater than 5mm, accompanied by bleeding upon probing and/or suppuration, as well as bone loss of 2mm or more, were diagnosed as peri-implantitis (figure 1, 2). in order to determine the diagnoses, it was necessary to obtain a consensus from three reviewers (ms, vs, kk) who worked independently. furthermore, supplementary information regarding the subjects' age, gender, smoking habits, and diabetes status was also documented. methodology blood serum was collected from patients of group 1 at 3 different intervals, one month prior to implant placement, 4th month after surgical phase (loading of implant) and 8th month after surgical phase (loading of implant), and from the study group 2, at the course time of peri-implantitis. 2 ml of blood was withdrawn from the anterior cubital fossa of the patients. blood samples were stored at -20 c̊. after collection of the whole blood, it was left undisturbed in the vacutainer, allowing it to clot at room temperature. centrifugation was performed at 1000–2000 rpm for 20 min in a refrigerated centrifuge. the resultant serum samples were obtained and analysis for titanium was performed using inductively coupled plasma mass spectrometry (icp-ms), with a detection limit of 0.5 nanograms at the ramaiah advanced drug testing laboratory, bangalore. statistical analysis the normality of the obtained dataset was checked using the shapiro wilk test. after stating the descriptive analysis for all the groups, multiple group comparison was done with anova test. the level of significance was set at alpha value 0.05. all statistical analyzes were performed using spss software version 22 study groups sample size subgroup mean±sd 95% confidence interval mean coefficient of variation upper lower group 1 60 pre-surgical 1.736±0.318 1.817 1.655 0.183 at 4th month 2.390±0.208 2.443 2.337 0.087 at 8th month 2.357±0.148 2.395 2.320 0.063 group 2 60 periimplantitis 2.943±0.177 2.988 2.898 0.060 figure 1: clinical and radiological assessment of healthy implant patient [a] periodontal pocket depth less than 2 mm [b] absence of bone loss figure 2: clinical and radiological assessment of peri-implantitis patient [a] periodontal pocket depth more than 5 mm [b] radiographic bone loss more than 2 mm table (1). descriptive analysis of estimated serum titanium values in study groups http://dentistry3000.pitt.edu/ comparative analysis of serum titanium level in patient with healthy dental implant and patients with periimplantitisa cross sectional prospective study vol 9 no 1 (2024) doi 10.5195/d3000.2024.594 http://dentistry3000.pitt.edu (developed by ibm corporation, armonk, n.y., usa in 2013) results the present study included 120 patients, comprised of 53 females and 67 males. the age range was 23– 47 years with an average value of 40±8 years. in the current investigation, no statistically meaningful variance was observed with regard to the demographic characteristics of the participants enlisted for the research, suggesting the absence of any bias in the selection process based on age and gender. the analysis of aggregated data showed increasing trend line in serum titanium level in group 1 and group 2 patients (figure 3). patients with periimplantitis showed higher mean serum value (2.94±0.17) comparing to group 1 patients. in the group 1 patients highest mean serum titanium level (2.39±0.20) was observed at 4th month after loading procedure (table 1). the comparative analysis of subgroups in group 1 with repeated anova test showed a significant difference (p=.001) and subgroups comparison with post hoc analysis with paired t test showed significant result except between the serum titanium level at 4th month and at 8th month (table 2, 3). significant results were obtained when group 2 was submitted to paired analysis with group 1 subgroups by using unpaired t test (table 4). discussion: since approximately 1981, titanium has been utilized for the construction of dental implants. the primary alloys employed are commercially pure titanium (cpti) and ti-6al-4v, both of which exhibit clinical success rates of up to 99% after a decade. these alloys possess biocompatibility when in contact with bone and gingival tissues, and have the ability to undergo osseointegration [7]. the corrosion behavior of metal implants is a crucial determinant of their biocompatibility. this is due to the potential detrimental effects resulting from the release of metal ions during the corrosion process. the tissue in the immediate vicinity of the implant, as well as the systemic environment, can be affected by these factors, potentially leading to allergic reactions. the presence of an oxide layer on the implant's surface significantly influences the outcome of osseointegration. the utilization of dental implants may result in elevated levels of titanium in both the bloodstream and serum [8]. gopi g conducted an assessment on the liberation of titanium, aluminium, and vanadium from dental implants through a comparison of the serum concentrations of these ions prior to and following surgical procedures. notably, a marginal variation was observed in the postoperative levels of titanium (2.31mg/dl) in relation to the preoperative levels (2.28 mg/dl), without any statistical significance (p > 0.5). [7] the current investigation assessed significant differences in the concentrations of titanium in the bloodstream before (1.79 mg/dl) and after (2.39 mg/dl) the 4th month of the loading of the implant, and the findings demonstrated an absence of agreement with the study conducted by gopi et al. another study demonstrated a comparable lack of significance in the relationship between the average concentration of titanium in the serum at the beginning, after 8 weeks, and after 6 months, with values of 2.39 mg/dl, 2.35 mg/dl, and 2.38 mg/dl, respectively [9]. our study also found lack of significance in titanium level at pre-surgical phase and at 8th month of post loading of the implant. this signifies that serum group 1 f value p value effect size pre-surgical 227.399 < 0.0001* 0.794 at 4th month at 8th month figure 3: graph showing the trend line for increasing serum titanium level in study groups table (2). comparison of mean scores of subgroups by repeated anova test *p<0.05 significant http://dentistry3000.pitt.edu/ comparative analysis of serum titanium level in patient with healthy dental implant and patients with periimplantitisa cross sectional prospective study vol 9 no 1 (2024) doi 10.5195/d3000.2024.594 http://dentistry3000.pitt.edu titanium levels significantly raises immediately after loading the implant but with due time the concentration lowered down by the macrophages. the release of titanium particles from the surface of the implant has a detrimental impact on both the nearby and far-reaching tissue as it infiltrates the surrounding tissues and enters the bloodstream [10]. in the localized region of dental implant known as the peridontium, the presence of titanium particles can trigger an inflammatory condition called peri-implantitis, characterized by an escalation in inflammatory mediators such as macrophages, cytokines, tnf-alpha, and il-6. multiple investigations have been conducted in order to assess the concentration of titanium and inflammatory mediators in the serum; however, no substantial findings were attained [11, 12,13]. our research, on the other hand, revealed a statistically significant variance in the serum concentration of titanium between individuals with uncompromised dental implants and those experiencing peri-implantitis. previous research has demonstrated the existence of titanium particles in the tissues surrounding dental implants. nevertheless, no conclusive statistical proof has been presented to establish a connection between dissolved titanium and periimplantitis [14]. the current investigation assessed the levels of titanium in the serum of patients with both healthy implants and implants affected by peri-implantitis. the findings obtained from this study demonstrated statistical significance, which aligns with the research conducted by olmedo et al [10]. although the occurrence of inflammation is observed as a healing response promptly following the loading of an implant and is accompanied by heightened levels of titanium in the serum [9], a similar response can also manifest in cases of peri-implantitis. during periimplantitis, macrophages that are recruited engulf wearable titanium particles, resulting in an elevation of titanium levels in the serum [10, 12]. the potential of titanium particles that have undergone corrosion to induce an immune response may result in inflammation of the periodontium and the subsequent degradation of bone tissue. the primary focus during the management of peri-implantitis was previously centered on mitigating inflammation. however, the emphasis should be on reducing the corrosion of titanium implants. while the serum's titanium level does not reach toxic levels in instances of dental implants, it is important to note that these titanium particles have the potential to be transported through the bloodstream to various regions of the body, thereby inducing toxic consequences. the exposure of titanium in dental implants to fluoride ions can occur through mouth rinses, toothpastes, drinking water, or food. consequently, the utilization of fluoride as a potential confounding factor should be taken into consideration in forthcoming confirmatory investigations that aim to evaluate the connection between titanium corrosion and periimplantitis [16, 17]. in order to mitigate the leaching of titanium particles, an examination was conducted using an aqueous solution of lactic acid and phosphate-buffered saline. however, it was discovered that there was no discernible connection between the augmentation of surface roughness and the release of ions, both in experimental and biological circumstances [16]. paired subgroup difference of mean t value p value cl at 95% cohen’s d effect size pre-surgical vs 4th month -0.654 16.374 < 0.0001* 0.5635 and 0.7565 2.53 pre-surgical vs 8th month -0.622 16.301 < 0.0001* 0.5307 and 0.7093 2.57 4th month vs 8th month 0.033 1.405 0.496 0.0224 and 0.1024 0.23 table (3). subgroup comparison by post hoc analysis with paired t test *p<0.05 significant http://dentistry3000.pitt.edu/ comparative analysis of serum titanium level in patient with healthy dental implant and patients with periimplantitisa cross sectional prospective study vol 9 no 1 (2024) doi 10.5195/d3000.2024.594 http://dentistry3000.pitt.edu themedicinal capability of neutralizing antibodies against il1β, il6, or tnfα in the prevention of osteolysis caused by ti particles was also studied [13]. limitations of the study the current investigation utilizes the icp-ms technique to evaluate the serum titanium level which present in minute amounts. further investigations are required in order to assess the level of titanium and inflammatory components present in gingival tissues and blood serum, despite the observed significant correlation between healthy implants and those affected by periimplantitis. conclusion in summary, understanding the complex relationship between titanium corrosion and periimplantitis is crucial for improving the long-term success and safety of dental implants. further research is needed to explore these connections and potential mitigation strategies to ensure the continued well-being of dental implant patients. acknowledgment we gratefully thank the indian council of medical research for its financial support under the research grant number 5/4/2-15/2019-ncd conflict of interest the authors declare no competing interest. ethical approval the study was approved by institutional ethical review committee [ec-19/12-f-fds]. funding indian council of medical research grant number 5/4/2-15/2019-ncd-ii author’s contribution dr. mahantesha s – substantial contribution to concept and study design, critical review of the manuscript dr.vibha shettypatient selection and study design, analysis and interpretation of the data, critical review of the manuscript dr. kranti kanalysis and interpretation of the data, critical review of the manuscript dr.shobhasubbaiahfollowed laboratory investigation and data segregation, critical review of the manuscript dr. greeshma cdata analysis and critical review of the manuscript dr. babashankar alvacritical review of the manuscript for important contents references 1microbial biofilm decontamination on dental implant surfaces: a mini review. dhaliwal js, abdrahman na, ming lc, dhaliwal sks, knights j, albuquerque junior rf. front. cell. infect. microbiol. 2021, 11:736186. mean ± sd difference of mean t value p value cl at 95% cohen’s d effect size pre-surgical 1.736 ± 0.318 -1.207 25.646 < 0.0001* 1.119 and 1.300 4.84 peri-implantitis 2.943 ± 0.177 4th month 2.390 ± 0.208 -0.553 15.683 < 0.0001* 0.482 and 0.617 2.96 peri-implantitis 2.943 ± 0.177 8th month 2.358 ± 0.148 -0.586 19.622 < 0.0001* 0.533 and 0.646 3.78 peri-implantitis 2.943 ± 0.177 table (4). pair wise analysis of group 2 and group 1 by unpaired t test *p<0.05 significant http://dentistry3000.pitt.edu/ comparative analysis of serum titanium level in patient with healthy dental implant and patients with periimplantitisa cross sectional prospective study vol 9 no 1 (2024) doi 10.5195/d3000.2024.594 http://dentistry3000.pitt.edu 2immunological aspects of dental implant rejection. baseri m, radmand f, hamedi r, yousefi m, kafil hs. biomed res int. 2020 dec 9;2020:7279509 3metal nanoparticles released from dental implant surfaces: potential contribution to chronic inflammation and peri-implant bone loss. bressan e, ferroni l, gardin c, bellin g, sbricoli l, sivolella s, et al. materials (basel). 2019 jun 25;12(12):2036. 4evaluation of titanium ions levels in blood in patients with endosseous titanium dental implants using inductively coupled plasma mass spectrometry a retrospective study. vinayak r, rosh .r .m, praveen j. international journal of science and research 2019 vol 8(12), 115-123 5metal elements in tissue with dental periimplantitis: a pilot study. fretwurst t, buzanich g, nahles s, woelber jp, riesemeier h, nelson k. clin. oral impl. res. 2016 sep;27(9):1178-86. 6serum metal levels in maxillofacial reconstructive surgery patients: a pilot study. mercuri lg, miloro m, skipor ak, bijukumar d, sukotjo c, mathew mt. j oral maxillofac surg. 2018 oct;76(10):20742080. 7evaluation of serum metal ion levels in dental implant patients: a prospective study. gopi g, shanmugasundaram s, krishnakumar raja vb, afradh km. ann maxillofacsurg 2021;11:261-5. 8titanium release in serum of patients with different bone fixation implants and its interaction with serum biomolecules at physiological levels. nuevo-ordóñez y, montes-bayón m, blancogonzález e, paz-aparicio j, raimundez jd, tejerina jm, et al. anal. bioanal. chem. 2011, 401, 2747–2754 9analysis of serum metal ion levels in dental implant patients. saini, r. s., &kaur, k. (2022). international journal of health sciences, 6(s5), 5645–5649. 10exfoliative cytology and titanium dental implants: a pilot study. olmedo dg, nalli g, verdú s, paparella ml, cabrini rl. j periodontol. 2013 jan;84(1):78-83. 11evaluation of inflammatory cytokine and plasma titanium levels in dental implant treated patients. süleyman kiliç , hakki kazancioğlu , ümit küçüksezer , günnur deniz, gülsüm ak. curr res dent sci 2015; 24: 199205 12influence of fluoride, hydrogen peroxide and lactic acid on the corrosion resistance of commercially pure titanium. mabilleau g, bourdon s, jolyguillou ml, filmon r, baslé mf, chappard d. actabiomater 2006;2:121129. 13mechanism and prevention of titanium particle-induced inflammation and osteolysis. eger m, hiram-bab s, liron t, sterer n, carmi y, kohavi d, et al. front. immunol.2018; 9:2963. 14increased levels of dissolved titanium are associated with peri-implantitis a crosssectional study. safioti lm, kotsakis ga, pozhitkov ae, chung wo, daubert dm. j periodontol. 2017 may;88(5):436-442. metal concentrations in the serum and hair of patients with titanium alloy spinal implants. kasai y, iida r, uchida a. spine (phila pa 1976). 2003 jun 15;28(12):1320-6. 15titanium release from implants prepared with different surface roughness.wennerberg a, ide-ektessabi a, hatkamata s, sawase t, johansson c, albrektsson t, martinelli a, et al. clin oral implants res. 2004 oct;15(5):50512. 16the effect of oral topical fluorides on the surface of commercially pure titanium. siirila hs, kononen m. int j oral maxillofac implants 1991;6:50-54 http://dentistry3000.pitt.edu/ https://dentistry-ataunipress.org/en/evaluation-of-inflammatory-cytokine-and-plasma-titanium-levels-in-dental-implant-treated-patients-131275%5c https://dentistry-ataunipress.org/en/evaluation-of-inflammatory-cytokine-and-plasma-titanium-levels-in-dental-implant-treated-patients-131275%5c https://dentistry-ataunipress.org/en/evaluation-of-inflammatory-cytokine-and-plasma-titanium-levels-in-dental-implant-treated-patients-131275%5c https://dentistry-ataunipress.org/en/evaluation-of-inflammatory-cytokine-and-plasma-titanium-levels-in-dental-implant-treated-patients-131275%5c https://dentistry-ataunipress.org/en/evaluation-of-inflammatory-cytokine-and-plasma-titanium-levels-in-dental-implant-treated-patients-131275%5c 788 final influence of different temperatures on the polymeriza9on preand post-cured of various resin materials vol 13 no 1 (2025) doi 10.5195/d3000.2025.788 h#p://den*stry3000.pi#.edu influence of different temperatures on the polymerization preand post-cured of various resin materials fanar turki al-jadwaa1, mahammad munthir abdurazzaq2, emad farhan alkhalidi1 1 college of dentistry, university of mosul, mosul, iraq 2 college of dentistry, al-iraqia university, bagdad, iraq abstract objec&ve: to evaluate and compered the effect of different temperatures (5°c, 37°c and room temp. ±23°c) precured and post-cured for three universalchroma composite materials (hybrid-nano fillers, supra-nano, nano filler) on the polymerizahon degree and micro-hardness. materials: a seventy-five disc-samples-shaped were fabricated from (omnichroma, vinra aps, denfil n), for each test in different temperatures (5°c, 37°c and room temp. ±23°c) were light cured according to manufacture instruchon. the fourier trans-form infrared spectroscopy was used to the polymerizahon degree measured for each sample while the micro-hardness was measured by the using of vickers hardness test. data were analyzed using one-way-analysis of variance at level p < 0.05. results: the analysis showed that there was significant difference in the polymerizahon degree and in the micro-hardness of the samples fabricated at the different temperatures when heated preand post cured of all materials increase, in the polymerizahon degree and the micro-hardness of the samples. conclusions: increasing three universalchroma composite materials (hybrid-nano fillers, supra-nano, nano filler) temperature whether pre-cured and post-cured allows for maintaining or increasing polymerizahon degree and hardness of three universalchroma composite materials especially denwiln nanofiller composite. keywords: temperature, universalchroma composite, degree of polymerizahon, microhardness. cita:on: al-jadwaa ft, et al. (2025) influence of different temperatures on the polymeriza:on preand post-cured of various resin materials. den:stry 3000.1:a001 doi:10.5195/d3000.2025.788 received: december 4, 2024 accepted: february 8, 2025 published: february 12, 2025 copyright: ©2025 al-jadwaa ft, et al. this is an open access ar:cle licensed under a crea:ve commons ayribu:on work 4.0 united states license. email: fanarturki@uomosul.edu.iq introduction composite resins (crs) are aesthetic dental-restorative materials that can be placed directly with minimal loss of tooth structure. crs have become the most widely used posterior tooth fillings [1]. crs are composed of monomer converted to polymer during polymerization and can be studied with fourier transform infrared spectroscopy technique without changing typical clinical conditions [2,3]. the cr as a direct restoration can be usage facilitated via pre-heating but is still a controversy. cr restorations are technique sensitive and are typically placed and cured in 2mm layers [4,5]. temperature plays an important role in the polymerization process of crs [6]. when temperature is higher, it enhances monomer mobility, due to lowered viscosity, with increased degree of conversion [7,8]. hardness is a property of major importance for assessing the adequate setting and stability of restorative materials [9]. refrigerating crs appeared to decrease degree of conversion, requiring more time for curing [10,11]. higher degree of conversion improves fracture resistance [12]. heating crs allow for reduction of curing time and influence of different temperatures on the polymeriza9on preand post-cured of various resin materials vol 13 no 1 (2025) doi 10.5195/d3000.2025.788 h#p://den*stry3000.pi#.edu less thickness, which is desirable in deep restorations [13]. the objective of this study was to evaluate and compare the effect of different temperatures (5°c, 37°c and room temperature, ±23°c) in three preand post-cured universalchroma composite materials (hybrid-nano fillers, supra-nano, nano filler). materials and methods table 1 and figure 1 describe the composite resins used in the study. specimen fabrication and distribution seventy-five samples were prepared and distributed as follows: group 1: refrigerated samples before curing at 5°c for 24h. group 2: refrigerated samples after curing at 5°c for 24h. group 3: pre-heated samples before curing at 37°c for 24h group 4: heated samples after curing at 37°cfor 24h group 5: samples stored at room temperature after curing (±23°c for 24h). all specimens were prepared by condensing the crs into a polyethylene mold (2mm depth or height and 5mm in diameter) and then the materials were covered with cellulose strip matrix and cover glass slide. after that, specimens were cured via led-lcu according to manufacture instructions using a digital timer. the lcu tip was placed directly over each sample (without space between them) at the top surface after that was finished and polished (tdv, brazil) with a lowspeed handpiece. then, samples were put in a dark container in an incubator (fisher scientificisotemp/incubator/usa). ftir (fourier trans-form infrared spectroscopy) was used to determine the degree of conversion. the spectra of crs was recorded. the double vinyl bonds are shown by the intensity of the peak (1608-1637 cm-1) referring to the c=c, then degree of conversion (dc) was calculated using the following equation: dc = {(ao-at)/ao} *100. where ao = uncured resin material and at = cured resin-material. statistical analysis was done using anova. microhardness was measured with a vickers diamond indenter attached as figure 2a. a load was applied over the top of the polished surface of each specimen (300g) for 15 seconds. two point evenly spaced indentation measurements were made. there was no instance with less than 1 mm between indentations or to the margin of the discs. measurements were made as the diagonal lengths of the indentation mark made on the surface of each disc. average values were calculated. the resultant dimensions were measured with the aid of an optical microscope (300 x) (yamayo make tr-200) (figure 2b). statistical analysis was performed with spss 18 software (ibm analytics, armonk, ny, usa), with p < 0.05 considered statistically significant. influence of different temperatures on the polymeriza9on preand post-cured of various resin materials vol 13 no 1 (2025) doi 10.5195/d3000.2025.788 h#p://den*stry3000.pi#.edu figure 1. materials and fabricated discs used in the study. figure 2. (a) vickers diamond (b) indentations of vickers micro-hardness. a b influence of different temperatures on the polymeriza9on preand post-cured of various resin materials vol 13 no 1 (2025) doi 10.5195/d3000.2025.788 h#p://den*stry3000.pi#.edu material/ shade type manufacture composition vittra aps nique / universal chroma hybrid-nano fillers fgm, brazil matrix: mixture of methacrylate monomers, udma, tegdma, photo initiator compound (aps). fillers: 72-80% by weight, 52-60% by volume boron aluminum silicate glass. omnichroma/ universal chroma supra-nano tokuyama dental, japan matrix: udma, tegdma. fillers: 79% by weight uniform supra-nano spherical filler (sio2 zro2 260 nm). denfil n / universal chroma nanofiller vericom, korea matrix: bisgma, tegdma filler content: barium, aluminosilicate, fumed silica. filler: 80% by weight results degree of conversion (dc) descriptives of dc values of the three tested crs pre-cured and postcured at different temperatures (5°c, 37°c, and room temperature, ±23°c) are seen in table 2. the dc values for the disc table 1. materials used in the study. influence of different temperatures on the polymeriza9on preand post-cured of various resin materials vol 13 no 1 (2025) doi 10.5195/d3000.2025.788 h#p://den*stry3000.pi#.edu samples preand post-cured at 5°c were lower than those for disc samples preand post-cured at 37°c or at room temperature. analysis of variance anova showed that there were significant differences at 37°c. the dc increased pre-and post-cured at 37°c, while there were no significant differences between dc at 5°c (table 3). vickers micro-hardness (vh) vickers microhardness (vh) evaluations of the different tested composite materials before and after polymerization at different temperatures are shown in table 4. the vh values for the disc samples at 5°c were lower than those for specimens at 37°c. this was shown for the three cr types tested with 2 mm thickness, whether preor post-cured. analysis of variance anova showed that there were no significant differences at 37°c of vh of the three cr tested preand post-cured (p<0.05). the vh increased at 37°c, while there were no significant differences between vh of the three cr tested at 5°c, whether preand post-cured are presented in table 5. discussion the study demonstrated the influence of different temperatures on microhardness and dc on universal chroma cr materials. no clinically relevant differences were found in the temperatures and materials studied. higher dc and microhardness were reached on the top aspect of samples tested whether preor post-cured at 37°c when compared with crs cured at room temperature. these results may be because higher temperatures will allow for additional polymerization and increased degrees of conversion [14,15]. influence of different temperatures on the polymeriza9on preand post-cured of various resin materials vol 13 no 1 (2025) doi 10.5195/d3000.2025.788 h#p://den*stry3000.pi#.edu table 2. descriptives the degree of conversion. n mean std. deviation f pvalue degree_0f_ conversion at room temp. ±23°c dentfiln 5 46.8000 2.16795 38.160 0.000 omnichro ma 5 33.0000 5.70088 vittra 5 53.0000 2.00000 total 15 44.2667 9.30796 pre_cure_at_37°c dentfiln 5 54.6000 8.56154 0.175 0.842 omnichro ma 5 52.4000 2.30217 vittra 5 53.0000 5.70088 total 15 53.3333 5.71548 post_cure_at_37°c dentfiln 5 71.4000 .89443 89.274 0.000 omnichro ma 5 82.0000 2.00000 vittra 5 89.8000 4.08656 total 15 84.4000 9.87638 pre_cure_at_5°c dentfiln 5 34.2000 7.98123 18.635 0.000 omnichro ma 5 17.2000 1.78885 vittra 5 32.4000 1.81659 total 15 27.9333 9.07482 post_cure_at_5°c dentfiln 5 44.4000 3.64692 152.785 0.000 omnichro ma 5 46.0000 .70711 vittra 5 21.4000 2.19089 total 15 37.2667 11.85909 influence of different temperatures on the polymeriza9on preand post-cured of various resin materials vol 13 no 1 (2025) doi 10.5195/d3000.2025.788 h#p://den*stry3000.pi#.edu table 3. descriptives the vickers micro-hardness. n mean std. deviation f p-value vickers micro-hardness at room temp. ±23°c dentfiln 5 72.4200 .50695 2163.804 .0000 omnichroma 5 47.5400 1.37949 vittra 5 38.1200 .13038 total 15 52.6933 14.99788 pre_cure_at_37°c dentfiln 5 71.5600 .25100 2920.686 0.000 omnichroma 5 47.7800 .80436 vittra 5 50.2800 .40866 total 15 56.5400 11.05550 post_cure_at_37°c dentfiln 5 75.0400 .11402 93128.00 0.000 omnichroma 5 50.6400 .11402 vittra 5 51.4000 .07071 total 15 59.0267 11.72534 pre_cure_at_5°c dentfiln 5 40.7800 .16432 4797.748 0.000 omnichroma 5 33.1400 .08944 vittra 5 29.5400 .26077 total 15 34.4867 4.85399 post_cure_at_5°c dentfiln 5 45.8200 .13038 17154.27 3 0.000 omnichroma 5 37.8800 .04472 vittra 5 36.5400 .05477 total 15 40.0800 4.23998 influence of different temperatures on the polymeriza9on preand post-cured of various resin materials vol 13 no 1 (2025) doi 10.5195/d3000.2025.788 h#p://den*stry3000.pi#.edu table 4. vickers microhardness values of the study. n mean standard deviation standard error vickers microhardness at room temperature (±23°c) dentfiln 5 72.4200 .50695 .22672 omnichroma 5 47.5400 1.37949 .61693 vittra 5 38.1200 .13038 .05831 total 15 52.6933 14.99788 3.87244 pre-cured at 37°c dentfiln 5 71.5600 .25100 .11225 omnichroma 5 47.7800 .80436 .35972 vittra 5 50.2800 .40866 .18276 total 15 56.5400 11.05550 2.85452 post-cured at 37°c dentfiln 5 75.0400 .11402 .05099 omnichroma 5 50.6400 .11402 .05099 vittra 5 51.4000 .07071 .03162 total 15 59.0267 11.72534 3.02747 pre-cured at 5°c dentfiln 5 40.7800 .16432 .07348 omnichroma 5 33.1400 .08944 .04000 vittra 5 29.5400 .26077 .11662 total 15 34.4867 4.85399 1.25330 post cured at 5°c dentfiln 5 45.8200 .13038 .05831 omnichroma 5 37.8800 .04472 .02000 vittra 5 36.5400 .05477 .02449 total 15 40.0800 4.23998 1.09476 influence of different temperatures on the polymeriza9on preand post-cured of various resin materials vol 13 no 1 (2025) doi 10.5195/d3000.2025.788 h#p://den*stry3000.pi#.edu table 5. one-way anova for vickers microhardness. f p-value vickers micro-hardness at room temperature (±23°c) between groups 2163.804 0.000 within groups total pre-cured at 37°c between groups 2920.686 0.000 within groups total post-cured at 37°c between groups 93128.000 0.000 within groups total pre-cured at 5°c between groups 4797.748 0.000 within groups total post-cured at 5°c between groups 17154.273 0.000 within groups total improved flow ability of heated crs may result in better adaptation to internal walls of deep cavity preparation. also, curing at higher temperatures might have improved physiomechanical properties of the material [16], and higher microhardness values [15,17]. our tests were done using accurate (ftir) and accessible methods (vh) [18,19]. finally, elevating cr temperature during light curing is caused by both the heat generation from cr polymerization and from the light source. this may lead to pulp tissue damage. however, the blood circulation in influence of different temperatures on the polymeriza9on preand post-cured of various resin materials vol 13 no 1 (2025) doi 10.5195/d3000.2025.788 h#p://den*stry3000.pi#.edu the chamber of pulp may play a role in decreasing the temperature induced by the cr polymerization [20]. conclusions increasing the temperature of composite resins, whether pre or post-cured, allows for maintaining or increasing degree of polymerization and hardness. references 1siriboina sirisha, c vinay, rama krishna alla, ks uloopi, penmatsa chaitanya, nadella chandana. physicomechanical characteristics of ormocer and bulk fill composite resin restorative materials: an in-vitro study. journal of clinical and diagnostic research, journal of clinical and diagnostic research. 2023; vol-17(7): zc01zc04. doi:10.7860/jcdr/2023/6 2857.18107 2fanar t. h. al-jadwaa, moataz gh. s. al-shaekh, amer a. taqa. the effect of storage media on the degree of conversion of different types of composite resin. alrafidain dental journal, the 5th scientific conference of dentistry college. 2011; special issue :317-322. 3norbert kramer, ulrich lohbauer, franklin garciagodoy, roland frankenberger. light curing of resin-based composites in the led era. am j dent. 2008; 21:135-142. 4abdulwahhab zs, alkhalidi ef. effect of staining and bleaching on surface roughness of different nano hybrid resin composite materials. al–rafidain dent j. 2021;21(1):42-50. 5alshaafi mm. effects of different temperatures and storage time on the degree of conversion and microhardness of resinbased composites. j con temp dent pract 2016;17(3):217-223. 6calheiros fc, et al. effect of temperature on composite polymerization stress and degree of conversion. dent mater. dental.2014;02.024. http://dx.doi.org/10.1016/j 7sultan ma. the effect of various pre-cured temperature of different resin materials on the degree of conversion and microhardness of cured composite resin. al– rafidain dent j. 2014; 14(2):279-287. 8trujillo m, newman sm, stansbury jw. use of nearir to monitor the influence of external heating on dental composite photopolymerization. dent mater 2004;20(8): 766-777. 9 behnam bolhari, naghmeh meraji, pegah khazaee, sholeh ghabraei, sara valizadeh. do different time intervals in placement of restorative materials over calcium silicate cements, affect interface microhardness of different restorative materials. dentistry 3000. 2021; 1:a001 doi:10.5195/d3000.2021.1 59 10kitzmuller k, graf a,watts d, et al. setting kinetics and shrinkage of self-adhesive resin cements depend on cure-mode and temperature. dent mater. 2011; 27:544-51. 11international standard 4049. dentistry-polymerinfluence of different temperatures on the polymeriza9on preand post-cured of various resin materials vol 13 no 1 (2025) doi 10.5195/d3000.2025.788 h#p://den*stry3000.pi#.edu based filling, restorative and luting materials. geneva, switzerland: international organization for standardization;200. 12trujillo m, newman sm, stansbury jw. use of nearir to monitor the influence of external heating on dental composite photo polymerization. dent mater. 2004; 20:766-77. 13holmes rg, blalock js, rueggeberg fa. composite film thickness at various temperatures. j dent res. 2004;83 (speciel issue a). abstract 3265. 14watts dc, alnazzawi a. temperature-dependent polymerization shrinkage stress kinetics of resincomposites. dent mater 2014; 30(6):654-660. 15myoung uk jin, sung kyo kim. effect of pre-heating on some physical properties of composite resin. j kor acad cons dent. 2009; 34(1):30-37. 16kurachi c, tuboy a, magalhaes s. hardness evaluation of a dental composite polymerized with experimental ledbased devices. dent mater. 2001; 17:309-315. 17asmussen e. restorative resins: hardness and strength vs. quantity of remaining double bonds. scand j dent res. 1982; 90:484-489. 18acquaviva pa, cerutti f, adami g, gagliani m, et al. degree of conversion of three composite materials employed in the adhesive cementation of indirect restorations: a microraman analysis. j dent. 2009; 37(8):610-5.doi: 10.1016/j.jdent.2009.04.00 1. 19 farnaz farahat, abdolrahim davari, marzieh fadakarfard. effect of storage time and composite thickness on degree of conversion of bulk-fill and universal composites using ftir method. braz dent sci 2020; 23(2). doi:10.14295/bds.2020.v2 3i2.1915. 20fanar t. a.al-jadwaa. effect of different curing times of composite resin and glass ionomer liner use on the pulpal temperature. mdj. 2022; 1(18):1-7. the role of different factors in the development of cleft lip, palate, or both vol 12 no 1 (2024) doi 10.5195/d3000.2024.647 http://dentistry3000.pitt.edu the role of different factors in the development of cleft lip, palate, or both natheer ayed jassem1, ghufran a. hasan 2, mudher mb. alsunbuli3 1 consultant in oral and maxillofacial surgery in iraqi minister of health; doctor teacher in al-bayan university oral and maxillofacial department, college of dentistry, al-bayan university, baghdad /ira 2department of oral diagnosis, college of dentistry, baghdad university/baghdad/iraq; department of oral and maxillofacial surgery, college of dentistry, albayan university /baghdad/iraq; teacher, b.d.s. (bachelor of dental surgery), msc in oral medicine 3bds, fibms, oral and maxillofacial surgery department, college of dentistry. al-bayan university/baghdad/ iraq. abstract background: cleft lip and cleft palate refer to congenital malformations characterized by fissures or divisions in the upper lip, the palatal region of the mouth, or both. cleft lip and cleft palate occur due to incomplete closure of face tissues during fetal development. objective: to identify the primary elements that could contribute to the occurrence of cleft lip and palate. materials and methods: the sample included 1080 patients distributed between cleft lip only, cleft palate only, and cleft lip and palate. parents of participants answered questions related to their medical and exposure history. subjects were seen over 13 years, from 1 january 2010 to 31 december 2022. participants came to our hospital with ages from a few hours to 3 months. results: the distribution of cleft types in the sample was as follows: 11.6% had cleft lip alone, 29.5% had cleft palate exclusively, and 57.3% had both cleft lip and palate. the most significant number of patients, 28%, were found to be first-born individuals. the majority of dads who exhibited patience were aged 30 years or older, accounting for 65% of the sample. among these individuals, 8.6% had a concurrent systemic ailment, while 40% were identified as smokers. most moms were between the age range of 16 to 30 years (65.4%). incorporate additional factors into the analysis, including the presence of familial cleft history and psychiatric disorders. conclusion: we found a high frequency of consanguinity between the parents maternal psychology factors such as fear, hearing bad news or hearing loud sounds like gunshots (prevalent during civilian war in iraq between 2010-2014 years) among parents of children born with cleft lip and palate. keywords: alternative medicine, cytotoxicity, plant extract, aristolochia cymbifera, oral medicine. citation: jassem, na et al. (2024) the role of different factors in the development of cleft lip, palate, or both dentistry 3000. 1:a001 doi: 10.5195/d3000.2024.647 received: november 13, 2023 accepted: january 14, 2024 published: february 12, 2024 copyright: ©2024 jassem, na et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: natheer.ayed@albayan.edu.iq introduction cleft lip and cleft palate are among the most common birth disorders and are inheritable conditions that can be part of a syndrome (1-5). multiple genes and genetic mechanisms (6-11) as well as some teratogens and maternal illnesses have been associated with clefts (13-23). clefts can be associated with defects of the central nervous system, eyes, heart, muscleskeleton, genitalia, abdomen, ears, thorax, endocrine system and skin (24). the aim of this work was to identify risk factor for clefts relevant to the studied population. materials and methods the sample of this study included 1080 patients (592 males and 488 females) who http://dentistry3000.pitt.edu/ mailto:natheer.ayed@albayan.edu.iq the role of different factors in the development of cleft lip, palate, or both vol 12 no 1 (2024) doi 10.5195/d3000.2024.647 http://dentistry3000.pitt.edu came to al-wasity hospital as they are referred from different areas. we studied all clefts born between 1 january 2010 to 31 december 2022, and children were a few hours to 3 months of age. parents were asked to fill out a questionnaire and a clinical examination was done for recording the type of cleft. the comprehensive assessment of a patient often encompasses a clinical examination, consisting of both extraoral and intraoral examinations. following this examination, relevant information is recorded, and a case sheet is completed, which involves addressing key inquiries of utmost significance. the categorization of abnormalities includes cleft lip, full cleft palate, soft palate cleft, and cleft lip and palate. the inquiries pertain to details such as name, age, residence, and the number of siblings they have. additionally, there are queries on the health state of their father, including if he smokes, his educational background, the presence of chronic illnesses, and any medications he may be taking. for mother same question in addition to 1psychological problem during first 12 weeks like stress, fairness, and any violence 2if she affected by disease during first 12 weeks 3if she takes medication and type of these drugs 4any history of apportion. finally, we record family history of clefts. results table 1 shows the frequency of each cleft type by sex: female male type of cleft lip and palate 9 4 bilateral cleft lip 53 75 unilateral cleft lip 38 42 left unilateral cleft lip 15 33 right unilateral cleft lip 150 169 complete cleft palate 9 20 cleft soft palate 295 324 cleft lip and palate 90 120 bilateral cleft lip and palate 95 131 left unilateral cleft lip and palate 110 73 right unilateral cleft lip and palate http://dentistry3000.pitt.edu/ the role of different factors in the development of cleft lip, palate, or both vol 12 no 1 (2024) doi 10.5195/d3000.2024.647 http://dentistry3000.pitt.edu the father: among fathers of children born with clefts, approximately 65% (702 individuals) of fathers aged above 30 years age, 8.61% of these fathers (93 individuals) had been diagnosed with systemic diseases. the majority of these fathers (81%, 875 individuals) were employed in the free enterprise sector, while the remaining individuals worked in civilian and noncivilian occupations. the systemic diseases observed in these fathers include hypertension, diabetes, anemia, hormonal disorders, and others. additionally, 40.6% of these fathers are smokers, and 2% consume alcohol. mother : the study revealed that a significant proportion of mothers, namely 65.4% (n = 706), fell between the age range of 20 to 30 years. furthermore, a substantial majority of mothers, accounting for 90% (n = 972), were identified as housewives. among these housewives, 70% (n = 756) were discovered to possess a poor level of education. in the study, it was found that 80.6% (n=870) of women had normal births, whilst 19.4% (n=210) underwent cesarean operations. furthermore, it was found that 19.8% (n=216) of the surveyed mothers had a documented history of undergoing abortion procedures. among this group, 20% (n=216) had experienced repeated instances of abortion. consanguinity: the study revealed that the degree of consanguinity among the parents was determined to be 71% (n=767). the researchers acquired information about the occurrence of clefts within the nuclear family unit, which includes the father, mother, and siblings. the prevalence of clefts within this family was found to be 10%. there were two sets of male and female twins, all of them had cleft lip and palate at an identical location. all individuals with a familial predisposition to cleft lip and palate had full clefts, which were seen to be either unilateral or bilateral. during the first trimester of pregnancy, a significant proportion of mothers, namely 39.2%, experienced the presence of illnesses. the most frequent conditions were shown to be urinary tract infections, anemia, respiratory illnesses, and hypertension... during the first trimester of pregnancy, a significant proportion of mothers, namely 40% or 432 individuals, reported the use of various medicines including antibiotics, vitamins, insulin, anti-inflammatory drugs, steroids, and contraceptive pills, among others. psychological role in cleft development: 46.6% of mothers reported fear, hearing bad news or hearing loud sounds like gunshots. 6% of mothers had a history of physical trauma . most events were concentrated between the years 2010 to 2014 where there was military action in most of iraq. when taking the order of birth of the affected child, 32.12%, or 347 were first born. http://dentistry3000.pitt.edu/ the role of different factors in the development of cleft lip, palate, or both vol 12 no 1 (2024) doi 10.5195/d3000.2024.647 http://dentistry3000.pitt.edu table 2 shows the frequency of associations with other congenital anomalies: percentage system affected 3.8 41 craniofacial 1.57 17 central nervous system 0.64 7 ocular 2.6 28 cardiac 1.2 13 urogenital 3.61 39 musculoskeletal system 1.48 16 ear discussion in this research, the prevalence of cleft lip was seen to be lower (11.6%) compared to other types, which is consistent with the findings of previous studies such as kinaan (15%), alzubaidee and hammash (16.67%), and dawood (15%) (21-23). however, a significant difference was seen among the other investigations conducted by natsume and kawai (2013) (41.3%), manhal (2012) (10%), and al-zubaidee et al. (1998) (50%) in their respective findings. a previous study by manhal (24), al-zubaidee, and hammash (22) indicated that the frequency of unilateral clefts was greater than that of bilateral clefts.. the findings of this research also revealed a prevalence of left-sided clefts, which accounted for a greater proportion than right-sided clefts across all kinds of cleft lip instances. this phenomenon might perhaps be attributed to the rotation of the head towards the right during the embryonic development of the heart, resulting in the inferior positioning of the left side of the face. in the instance of cleft palate, the prevalence rate of 29.5% was observed, which was somewhat lower compared to the findings reported by sesgin and stark (33%), conway and wagner (32%), padilla and gonzalez (1986) (33.1%), dawood (1997) (33%), and alzubaidee et al. (2000) (38%). the occurrence of cleft of the soft palate was comparatively less frequent as compared to that of the hard palate. borcbakan (1969) documented a greater prevalence of clefts occurring in the soft palate compared to those occurring in the hard palate. cleft lip and palate, also known as orofacial clefts, are congenital conditions characterized by a separation or gap in the upper lip and/or the roof of the mouth. the aforementioned group constituted the majority of various forms previous investigations have shown the occurrence of cleft, with manhal (21-25) reporting a prevalence rate of 67%, dawood (23) reporting a rate of 52.14%, and al-zubaidee (22) reporting a rate of 45%. the research conducted in this study revealed a more significant occurrence of unilateral cleft lip and palate compared to bilateral cleft lip and palate, which aligns with the findings provided by dawood (23). however, these results differ from those http://dentistry3000.pitt.edu/ the role of different factors in the development of cleft lip, palate, or both vol 12 no 1 (2024) doi 10.5195/d3000.2024.647 http://dentistry3000.pitt.edu obtained by manhal (24). the individual in question, referred to as kinaan (21), is the subject of discussion. the examination revealed a lack of occurrences of median cleft lip and palate, a facial abnormality that is seldom seen and has been hardly documented in previous studies, including the research conducted by manhal (24). dawood (2019) discovered a specific form of median cleft in a subset of individuals, constituting around 1.07% of the population. when considering the impact of sex, it is seen that males are impacted to a greater extent than females, with a ratio of 1.2:1. specifically, a study conducted by manhal (24) found a slightly higher ratio of 1.27:1. the sexual dimorphism in the prevalence of clefts remains uncertain; nonetheless, it has been postulated that divergent morphogenic processes in facial development between males and females may underlie this disparity.. upon examination of the fathers' age, it was noted that there was a greater incidence of cleft in the older age cohort. nevertheless, jensen et al. (1988) documented a somewhat elevated incidence of cleft births in cases when the fathers were below the age of 24, which contrasts with the relationship between maternal age and cleft occurrence. specifically, it was found that approximately 65.4% of cleft patients were born to younger mothers aged between 14 and 30 years old. this finding aligns with the research conducted by manhal (24). according to slavkin (1992), research conducted by fraser and calnan (1961) discovered a positive correlation between the incidence of mouth clefts and advanced maternal age. this research reveals that a significant proportion of parents, namely 71%, exhibit a familial relationship. this finding aligns with the findings of dawood (23). the prevalence of family history, as reported by pietrzyk et al. (1985), was found to be consistent with the findings of the current study in 10% of cases. al-zubaidee et al. (2000)(22), on the other hand, found a lower rate of 6.8%. according to dawood (23), a study found that 39.2% of women experienced illnesses during the first trimester of their pregnancy. additionally, the study reported that 36.3% of moms had a condition during this period. several studies have identified certain diseases, like epilepsy (niebly, 12), diabetes (nada, 1975), and rubella (seaver and hoye, 1992), as potential causes of cleft lip and palate.. a study conducted by manhal (24) found that around 46.6% of moms experienced psychological distress. it is worth noting that this percentage falls within the range of 65% given by manhal. around 50.8% of children who get a diagnosis of either unilateral or bilateral cleft lip and palate need the use of a feeding plate, a medical intervention normally delivered by an orthodontist. in instances when there is a co-occurrence of defects in other organs, it has been shown that around 14.9% of patients exhibit conditions such as cranial abnormalities, intellectual disability, visual impairments, cardiac malformations, and urogenital abnormalities. the prevalence of related abnormalities in the musculoskeletal system and ear was found to be greater in the current research compared to a previous study conducted in iran. the previous study had a sample size of 1669 patients with cleft lip and palate and reported a prevalence rate of around 7.73% for associated http://dentistry3000.pitt.edu/ the role of different factors in the development of cleft lip, palate, or both vol 12 no 1 (2024) doi 10.5195/d3000.2024.647 http://dentistry3000.pitt.edu anomalies. however, in research conducted in france (25), it was shown that the proportion was lower than 36.7%. conclusions 1 the use of drugs during the first trimester of pregnancy has a crucial role in the occurrence of this particular kind of deformity. 2 in the sample, a positive consanguinity between the parents was observed in 71% of cases. 3 the research demonstrates the potential of psychological variables in influencing outcomes. disclosure of interest the authors declare that they have no competing interests. references 1etiology, prenatal diagnosis, obstetrical management and recurrence of orofacial clefts; wilkins-haug l. 2015 may 12; http://www.uptodate.com/ho me. 2epidemiology of oral clefts in a large south american sample; menegotta bg, salzano fm.; (1991) 28(4 :)373377 3plastic surgery cleft lip and palate and craniofacial anomalies; mccarthy jg, james w, littler j. (1990) vol. (4), w.; saunders company . 4influence of maternal anaemia during early pregnancy on the development of cleft palate; natsume n, sugimoto s , kawi i; br g maxillofac surg; . (1990) 37(4): 330-331. 5cleft lip and palate; crockett dj, et al... facial plastic surgery clinics of north america. 2014;22:573. 6cleft lip and cleft palate. centers for disease control and prevention; accessed, 2015 may 12; http://www.cdc.gov/ncbddd / birth defects /cleftlip.html. 7cleft lip and cleft palate. american speech-languagehearing; .2015; association .http //:www.asha.or g / public/speech/disorders /cleft lip / . 8incidence of cleft lip, cleft palate and cleft lip and palate among races; apostole pv. a review. cleft palate j(1987) (3 :)216-225 9orthodontics and pediatric dentistry colour guide; millett d, welbury r. (2000): toronto, churchill livingstone . 10facial clefts and craniosynostosis principles and management; turvey ta, vig kwl, fonsecarj, (1996), w.b.saunders company. 11present status of drugs as teratogen in man; wilson jg;1993; teratology; 7:3-16 12maternal, metabolic, endocrine, environmental influences in the etiology of cleft lip with or without cleft palate; niebyl r,blake , da,rocco le, et al..: .cleft palate j(1985):22:20-28. 13prenatal dexamethasone exposure in rats-effect of dose, age at exposure and druginduced hypophagia on malformations and fetal organ weight; laborde jb, hansen dk, young jf, sheehan dm ,holson rr; appl toxicol(1992) 9( 4 )1;554-545 14teratology in pediatric practice; seaver h, hoyme m: medical genetic;(1992)39(1):26-31 15environment and genetics in the etiology of cleft lip and palate with reference to the role of folic acid; bianchi http://dentistry3000.pitt.edu/ the role of different factors in the development of cleft lip, palate, or both vol 12 no 1 (2024) doi 10.5195/d3000.2024.647 http://dentistry3000.pitt.edu e,calzelar e,et al..:.epidemiol preven j;(2000)24(1):121-127. 16etiology in colour atlas of cleft lip surgery; maneksha m. (1986): etiology in colour atlas of cleft lip surgery .wolfe year book . 17cleft lip and palate genetic aspect; abdul-rahim kk : iraqi dental j;(1987)206-219 . 18-your baby's first year. cleft palate foundation. http://www.cleftline.org/paren tsindividuals/publications/bookle ts/. accessed may 13, 2015 . 19houston wjb, stephens cd ,tully wj. (1992): a textbook of orthodontics. 2nd ed. bristol wright 20in-utero cleft lip repair in mice; hallock gg; plast.reconst .surg;(1985)75:78 5-791. 21kinaan bk. (1990); baghdad university . iraqi-unpublished data .as cited by manhal , 1990 . ) 22cleft lip and palate in primary school's student of sadam city; al-zubaidee af,hammash mh; baghdad.jfac med baghdad (1998): – ;40(30):46-52 23genetic of cleft lip and palate and associated congenital anomalies; dawood t; (1997); genetic of cleft lip and palate and associated congenital anomalies .a thesis submitted to iraqi commission for medical specialization . 24a study of cleft lip and / or palate in iraqi infants; manhal sh.; 1990 : )a thesis submitted to the college of dentistry baghdad university in partial fulfillment of the degree of master of science of orthodontics . 25syndromes and anomalies associated with cleft; r. venkatesh; plast. surg. doi: 2009 oct; 42(suppl): s51–s55. indian j 10.4103/09700358.57187 http://dentistry3000.pitt.edu/ 873 final some proper)es of 3d printed acrylic resin material modified by an)fungal vi)s vinifera oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.873 h#p://den*stry3000.pi#.edu some properties of 3d printed acrylic resin material modified by antifungal vitis vinifera oil amal abdul latif rashid college of health and medical techniques, middle technical university, baghdad, iraq abstract objec&ve: the aim of the study was to study the consequences of different ra5o of vi5s vinifera oil on the surface hardness, surface roughness and color change of 3d printed resin material. material and methods: 80 samples have been intended according to ada specifica5on 40 samples for hardness and roughness test and 40 samples for color change test, these two groups subdivided into four groups based on vi5s vinifera oil concentra5on 1%, 2%, 3% and control group without adding oil (0%). for each group, 10 specimens were prepared from 3d printer acrylic resin denture base, hardness was tested by shore d tester, roughness test was done by tr 200 portable tester and color change test by spectrophotometer. results: the control group recorded the maximum mean value for hardness and 3% vi5s vinifera recorded the lowest value, while for roughness and color test the minimum value recorded by control and 3% vi5s vinifera oil showed the maximum value for them, for all test the differences between control and 1% were not significant, but there were significant differences with 2% and 3%. conclusion: the addi5on of 1% vi5s vinifera oil (an5fungal oil) showed no adverse effect in hardness, roughness and color change value of 3d printer acrylic resin in comparison to control group. keywords: vi5s vinifera oil, 3d printer acrylic resin, proper5es. cita:on: rashid aal. (2025) some proper:es of 3d printed acrylic resin material modified by an:fungal vi:s vinifera oil. den:stry 3000. 1:a001 doi:10.5195/d3000.2025.873 received: march 4, 2025 accepted: march 16, 2025 published: april 18, 2025 copyright: ©2025 rashid aal. this is an open access ar:cle licensed under a crea:ve commons avribu:on work 4.0 united states license. email: amal_den:st58@mtu.edu.iq introduction the non-metallic materials using in manufacturing of dentures have a long tradition in dentistry [1,2]. recent advancements in 3d printing had utilizing resin as a key component for creating objects through a layering process were the speed, flexibility, competitive and tangible design were some of its advantages [3,4]. 3d-printed resin exhibited inferior properties such as strength, hardness, increased candida adhesion and roughness of surface in in comparison to the heatpolymerized resin [5-7]. however, studies and a notable challenge demonstrated to advance their properties and susceptibility to fungal growth, compromising their integrity by incorporating different additives that will improve properties of 3d-printed materials. delves into the innovative integration of antifungal oil into 3d printer resin, focusing on the consequential evaluation of resin properties. discern the effects of this incorporation, providing valuable insights for applications that demand 3d-printed materials with heightened on the advancement of its properties. the 3d flexo denture base v2 3d printer resin is a specialized some proper)es of 3d printed acrylic resin material modified by an)fungal vi)s vinifera oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.873 h#p://den*stry3000.pi#.edu material designed for use in 3d printing applications, particularly in the production of denture bases. according to the manufactural of this material this resin is specifically formulated to provide the necessary properties and characteristics required for the fabrication of high-quality dentures using 3d printing technology. vitis vinifera seed extract encompass proanthocyanins, which have been assessed for different health conditions [8,9], have many medicinal uses and indications used to treat a range of health problems and to protect against bacterial infections [1012]. also, it minimizes degradation of collagen and thus could have the potential in holding up the periodontitis progression [13] and had most beneficial antifungal activity that prevent adhesion of c. albians to the denture base material [14,15]. no study concerning the addition of vitis vinifera oil to 3d printer acrylic has been done. taqa et al. [16] demonstrated the effect of incorporation of grape oil into cold acrylic material and showed that incorporation of (2.5%) oil into cold cured acrylic showed significant decrease in transverse strength in comparison with a control group. it also showed significant difference in lightness in different concentrations (1.5%, 2%, 2.5%). the 10% w/w of the same oil when incorporated to soft liner mainly improved the antifungal effect and tensile bond strength of the soft lining material that attached to the base of the denture material not compromising the surface roughness of the liner material [14]. hardness was one mechanical property which had the ability to resist to abrasion forces (scratching, and/or penetration), without deformation [17]. a rough surface exhibited most retention of microorganisms which compromised the longevity of this material [18]. color and transparency were aesthetic properties of 3d printed resin that can influence the visual appeal and functionality of the printed prosthesis [19]. this study specifically aims to evaluate the surface hardness, surface roughness and color change of 3d printed resin after incorporating with different concentration of antifungal oil (vitis vinifera) focusing on determining the optimal oil percentage without compromising these properties, hypothesizing an improvement in properties. despite potential benefits, there was a gap in research regarding this impact on properties of 3d resin. material and methods preparation of test specimens the design of specimens for surface hardness, surface roughness and color change were done according to specification for that test and materials. the design of surface hardness and surface roughness were the same which was rectangular shaped according to (ada no.12, 1999)[20] the dimensions was (65x10x2.5 mm) length, width and thickness subsequently as seen in figure 1a, while for color change the sample as disc, with dimensions of (50 ± 1mm in diameter and 0.5 ± 0.05 mm in thickness) according to ada specification no.12, 1999 [20] (figure 1b). (a) (b) figure 1. specimen design. a surface hardness, surface roughness; b color change. specimens grouping 80 samples were prepared for this study constructed from (3d flexo denture base v2 resin, germany) 40 samples for hardness and some proper)es of 3d printed acrylic resin material modified by an)fungal vi)s vinifera oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.873 h#p://den*stry3000.pi#.edu roughness test and 40 samples for color change test, these two groups subdivided into four groups depended on vitis vinifera oil concentration 1%, 2% , 3% and control group without adding oil (0%) for each group, 10 specimens were prepared (table 1). table 1. percentage of vitis vinifera oil mixed with 3d printed resin. software design the specimens were designed by using the chitubox software before printing. the sample data were saved as stl file (standard tessellation language) to measure the mechanical and physical property tests according to ada specification [20], with a rectangular 2.5× 10× 65 mm3 (±0.2 mm) for surface hardness and surface roughness while as disc its dimensions 50 ± 1mm in diameter and 0.5 ± 0.05 mm in thickness for color change, then stl file imported into the 3d printing software (phrozen 3d printer, taiwan) according to device instructions. printer processing the file was exported to 3d printer software (phrozen 3d printer, taiwan) (figure 2) and printed. after completed the printing (figure 3 a,b,c), when the samples were removed from the platform stage then the support structure was cut from the samples and the samples was washed by ethanol 99% (italy) in an ultrasonic device cleaner (bella gusto, china) (figure 4), for 2 minutes, to remove the excess monomer based on the instruction of manufactural then left in air for thirty minutes for drying prior of post curing. figure 2. printing procedure while cover of printer closed. figure 3(a, b, c) final printed specimens at the printer plate. figure 4. ultrasonic cleaner device. post-curing procedure after printing process each 3d printing resin group subjected to postcuring that they were placed in the uv curing chamber for 10 minutes according manufacturer instruction of resin. after completed post-curing stage, polishing disk dental burs are used to polish all specimens. all specimens prior to testing were put in an incubator temperature was 37⁰c for 48 hours as claimed by iso 20795-1 [21]. shore d hardness test the shore d hardness tester was used (china), according to iso7619 (astmd 2240). the device was vertically placed over the specimen, which was vitis vinifera oil % group 3d printed resin vitis vinifera oil (ml) control (0%) 100 ml 0 ml vitis vinifera oil (1%) 99 ml 1 ml vitis vinifera oil (2%) 98 ml 2 ml vitis vinifera oil (3%) 97 ml 3ml some proper)es of 3d printed acrylic resin material modified by an)fungal vi)s vinifera oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.873 h#p://den*stry3000.pi#.edu encouragement on horizontal, rigid base. the distance from the indenter of hardness tester and the specimen surface was around 5to 12mm. the contact period from the specimen surface to the indenter was six seconds. for each specimen, approximately five hardness measurements were obtained from the scale reading value, then the mean of these values was computed [22,23]. surface roughness test rectangular shaped specimen printed the dimensions was 65x10x2.5 mm length, width and thickness subsequently by printer. surface roughness test was performed using surface roughness profilometer tester (tr200) with 0.001 micrometer accuracy at the university of technology, materials engineering department. this tester contained a diamond sensitive needle (stylus) using to track the irregularities on the surface. three separated locations on the specimen’s surface were just touched by the stylus to have three readings for each sample, so according to profilometer instructions; the sample was located on a stable, rigid surface and the stylus should be allowed to contact the first point, then it was moved for 11 mm across the sample, the readings appeared on the digital scale in a spontaneous manner. later, a roughness values were determined by calculating the mean values of these reading in μm [24,25]. color change test an objective method used for evaluation the color change via measuring the amount of absorbed light percentage was performed by using uv –visible spectrophotometer [26]. specimens were placed on the outlet light of the device and subjected to light, absorbing light percentage was obtained at device screen. the instrument then auto zeroed with nothing in the sample across the working range to establish a baseline. spectrophotometer shimadzu used in the study measures the wavelength that range from 190 – 1100 nm; by measures intensity of light through a sample (i) and comparing with the intensity of light before passes through the sample (io). the absorbance (a) was expressed in a percentage (%) [27,28]. results surface hardness test the descriptive statistic as showed in table 2, showed that the hardness of 3d printer resin decreased significantly with the addition of vitis vinifera oil. the control group (without oil) recorded the maximum hardness value the average hardness was 33.1875. the average hardness decreased even further in groups d (3% oil), to 31 (minimum hardness value). table 3 showed post hoc analysis for action of vitis vinifera 1%, 2% and 3% concentration on hardness test of resin materials, the differences were significant between all groups excluding between: (control with1% oil) and (2% oil with 3% oil) the differences was not significant p > 0.05. surface roughness test table 4 showed the descriptive analysis of all groups. control group showed the minimum mean value for roughness test while the group 3% recorded the maximum mean value for roughness test. anova test showed the differences was significant among groups (p-value ≤ 0.0001). table 5 showed post hoc analysis for the action of vitis vinifera 1%, 2% and 3% concentration on roughness of resin materials, the differences were significant between all groups excluding between control with 1% oil and 2% oil with 3% oil (p > 0.05). color change test table 6 showed the descriptive analysis of all groups control group recorded the minimum mean value for color change while 3% oil recorded the maximum mean value for color change. some proper)es of 3d printed acrylic resin material modified by an)fungal vi)s vinifera oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.873 h#p://den*stry3000.pi#.edu groups n mean std. deviation std. error minimum maximum p-value anova test control 10 33.1875 1.28000 .45255 31.00 34.50 0. 001 1% 10 33.0625 1.76144 .62276 30.00 35.50 2% 10 31.1250 .95431 .33740 29.00 32.00 3% 10 31.0000 .80178 .28347 29.00 32.00 (i) groups (j) groups mean difference (i-j) std. error sig. 95% confidence interval lower bound upper bound control 1% .12500 .62623 .843 -1.1598 1.4098 2% *2.18750 .61623 .002 .9027 3.4723 3% *2.06250 .62523 .003 .8877 3.4463 1% 2% *2.06250 .62733 .003 .7777 3.3473 3% *1.93750 .61724 .005 .6527 3.2223 2% 3% -.12600 .63423 .844 -1.4098 1.1598 *. the mean difference was significant at the 0.05 level. groups n mean std. deviation std. error minimum maximum anova p-value control 10 .1931 .06501 .02298 .13 .29 ≤0.0001 1% 10 .3190 .13363 .04724 .13 .48 2% 10 .4863 .11480 .04059 .34 .63 3% 10 .5355 .18441 .06520 .28 .80 table 3. post hoc analysis for the action of visit vinifera with different concentration on hardness test. hardness test. table 4. descriptive and anova analysis for the action of vitis vinifera oil with different concentration on roughness test. hardness test. table 2. descriptive and anova analysis for the action of visit vinifera with different concentration on surface hardness test. some proper)es of 3d printed acrylic resin material modified by an)fungal vi)s vinifera oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.873 h#p://den*stry3000.pi#.edu (i) groups (j) groups mean difference (i-j) std. error sig. 95% confidence interval lower bound upper bound control 1% -.12588 .05580 .066 -.2607 .0089 2% *.34238.06580 .000 -.4772 -.2076 3% *.29313.05470 .000 -.4279 -.1583 1% 2% *.21650.06660 .003 -.3513 -.0817 3% *.16725.05670 .017 -.3020 -.0325 2% 3% .04925 .06670 .460 -.0855 .1840 *. the mean difference is significant at the 0.05 level. groups no. mean standard deviation standard error minimu m maximu m p-value control 10 1.434 .28943 0.10233 1.04 1.434 anova ≤0.0001 1% 10 1.4450 .11502 0.04066 1.34 1.68 2% 10 1.5325 .29198 0.10323 1.08 1.86 3% 10 1.6588 .03441 0.01217 1.62 1.70 table 5 showed post hoc analysis for the action of vitis vinifera 1%, 2% and 3% concentration on roughness of resin material. matreialmaterials.test hardness test. table 6. descriptive and anova analysis for the action of visit vinifera with different concentration on color changes test. materials.test hardness test. some proper)es of 3d printed acrylic resin material modified by an)fungal vi)s vinifera oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.873 h#p://den*stry3000.pi#.edu anova test analysis showed the differences were significant among all groups (p ≤ 0.0001). table 7 showed post hoc analysis for the action of vitis vinifera 1%, 2% and 3% concentration on color change of resin materials, the differences were significant between all groups except between: (1% oil with 2% oil) and (2% with 3% oil) the differences was not significant p > 0.05. (i) groups (j) group s mean differenc es (i-j) s.e pvalue 95% confidence interval lower bound upper bound control 1% 2.40875* .10607 .000 2.1894 2.6281 2% 2.32125* .10707 .000 2.1019 2.5406 3% 2.19500* .10617 .000 1.9757 2.4143 1% 2% -.08750 .10717 .421 -.3068 .1318 3% -.21375 .10708 .056 -.4331 .0056 2% 3% -.12625 .10618 .248 -.3456 .0931 *. the mean difference is significant at the 0.05 level. table 7. post hoc analysis for the action of grape oil with 1%,2%,3% concentration on color changes. test. materials.test hardness test. some proper)es of 3d printed acrylic resin material modified by an)fungal vi)s vinifera oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.873 h#p://den*stry3000.pi#.edu discussion 3-dimensional printing acrylic improved rapidly, by enhancing reliability, accuracy and carried to apply in the dentistry [29]. the aim of 3d was to produce rapid and functional materials. the 3d printed material was built up layer above layer while build plate was lifted to permit the next layer for curing [30]. many factors should be kept in control during the processing of 3d printing, the properties were affected via the depth of polymerization, thick layer, volume, direction and shrinkage of the light from the source [31,32]. 3d flexo denture base v2 resin was used in this study was a medical-grade resin designed specifically for printing dental prosthetic bases. it features unique properties that make it suitable for these applications. however, some disadvantages like poor mechanical properties of resin required strengthening acrylic resin materials properties [33] by adding various reinforcing materials such as fibers, nanoparticles [34-36]. recently extracts were used to improve the properties of acrylic material, in addition to its antibacterial and antifungal effects [37,38]. oils plant's extract had wide range for applications in dentistry so using to treat denture stomatitis because effect related to their composition. in this study vitis vinifera oil used to be incorporated into 3d acrylic resin due to documented and noticeable antimicrobial and antifungal effect in many previous studies [10-12,14,15]. this oil had antibacterial against many organisms including s. aureus, e. coli, k. pneumoniae and p. aeruginosa [39]. it derived from the seeds of grapes and has gained attention in various fields due to its unique properties. it is rich in antioxidants, vitamins and acids. this extract had more naturally occurring polyphenolic components that composed of free flavanols monomeric for example the (proanthocyanins) which have anti-inflammatory effect [11]. this study attempted to modify the properties of 3d printed acrylic material by addition of vitis vinifera oil, which had many applications in wide range felids in pharmaceuticals dentistry. the measurement of resin hardness played a crucial role in this study, allowing for the assessment of the impact of this oil proportions on 3d printer resin, many factors influencing the hardness of 3d printer resin. previous studies have indicated that the addition of certain additives could either increase or decrease the hardness of resin, depending on their chemical composition and interaction with the resin matrix. however, none of these studies specifically investigated the effects of vitis vinifera oil incorporation on resin hardness. therefore, the present research aims to fill this gap in this study by evaluating the hardness of 3d printer resin after incorporation with this oil. the standardized testing method using a shore d durometer provided accurate and reliable measurements [40], the addition of this oil to 3d printer resin affects the hardness of the resin. the higher the ratio of vitis vinifera oil in the resin, the control group recorded the lower the hardness of the resin in the group 1% the hardness was greater than groups 2% and 3%, and the hardness decreased statistically between them, but the statistically differences were no significant between group 1% and control group also in groups 2% and 3%, the hardness was not significant. the presence of this oil may introduce additional chemical bonds or alter the existing ones, leading to changes in the resin's mechanical properties, including hardness. there are several possible explanations for this some proper)es of 3d printed acrylic resin material modified by an)fungal vi)s vinifera oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.873 h#p://den*stry3000.pi#.edu decrease in hardness, vitis vinifera oil could act as a wetting agent, which might reduce the bonding between the molecules in the resin. another explanation is that the oil could be a polymerization inhibitor, which could reduce the formation of chemical bonds between the molecules of resin [41] and lead to form less hard resin. findings suggest that incorporating vitis vinifera oil could be a useful tool in the development of 3d printer resins with specific properties. for example, it could help to make 3d printer resins less hard, which might be beneficial in applications that require fracture resistance such as soft lining materials. the effects of this oil when incorporation into 3d printer resin on the hardness were a significant aspect to consider in resin formulation. this importance had provided an overview of the topic, discussed the mechanisms of oil incorporation, outlined the experimental design and testing procedure, and highlighted the significance of the findings. roughness test one of the important method useful to determine the mechanical properties of acrylic resin materials. roughness is necessary test in prevention microorganism to attached to denture resin base [42]. the results of this study showed that surface roughness increase when concentration of vitis vinifera oil increased. although the differences was no significant between control and 1% oil but there were significant differences between control and (2%,3%) oil the unnoticeable increasing of roughness with the oil groups might reveal that the oil coated particles of polymer and this particle coating would decrease the conversion of monomer into polymer that result in high amount of residual monomer, and this residual monomer affect adversely the mechanical properties especially the roughness property [43]. vitis vinifera oil also could act as a lubricant, which might reduce the friction between the molecules [14]. another possible reason for increasing roughness might related to flexibility in specimens with high concentration of the oil percentage [14,41]. these results agreed with previous work [14] that showed increasing roughness with 5% vitis vinifera oil groups incorporated to soft liner acrylic material. color stability was the most important clinical properties because the quality of appearance of prosthesis is very important, feature needed by patients therefore should satisfied their expectation [44]. color and transparency are aesthetic properties of 3d printer resin that can influence the visual appeal and functionality of the printed objects. resins are available in a wide range of colors, allowing for the creation of visually appealing objects both extrinsic and intrinsic factors was responsible for causing color change in acrylic resin material, the intrinsic factor include the discoloration of resin itself and changes in matrix that occur during aging process of material, while extrinsic include stain accumulation [45]. the addition of the oil to acrylic resin increased color test value however, the differences were no significant between: control and 1% oil and this slight increasing not induced change to the point causing degrading the organic matrix and cause staining the increasing in value of color change in the oil groups in comparison to control group might be due to reacted monomer, that coated with the oil they and could protect against light [46]. it is possible that the increasing in color test in group 2% and 3% oil could be because of the oil on the resin structure that could be because of the oil during the thermal some proper)es of 3d printed acrylic resin material modified by an)fungal vi)s vinifera oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.873 h#p://den*stry3000.pi#.edu processing because this oil when was added to the resin it interacts with the polymer matrix, affecting its crosslinking density and overall structure [41]. this result agreed with previous work [16] that showed significant differences in color change of grape oil (1.5%, 2%, 2.5) from standard group when incorporated to cold cured acrylic. conclusion the incorporation of vitis vinifera oil into 3d printer acrylic showed that the impact of 1% vitis vinifera oil had no effect on hardness, roughness and color change values. conflicts of interest the authors declare no competing interest. references 1. d. duraccio , mussano f. and faga m..biomaterials for dental implants: current and future trends journal of materials science.2015;50(14). doi:10.1007/s10853-015-90563. 2. azeem ul yaqin syed.acrylic denture base materials in book: advanced dental biomaterials .october 2020.publisher: elsevier (pp.79). 3. dimitrova m, vlahova a, kalachev y, zlatev s, kazakova r, capodiferro s. recent advances in 3d prinrng of polymers for applicaron in prosthodonrcs polymers 2023;15(23). 4. saba k.,vishalakshi n.,sreeparna d. and sanjana b. 3d prinrngan futurisrc art and science in denrstry pakistan. heart journal.2023;vol. 56 no. 1. 5. prpic´, v.; schauperl, z.; c´ arc´, a.; dulcˇic´, n.; cˇand imic´, s. comparison of mechanical properres of 3d-printed, cad/cam, and convenronal denture base materials. j. prosthodont. 2020; 29, 524–528. 6-perea-lowery l, gibreel m, valliuu p, lassila l. 3d-printed vs. heat-polymerizing and autopolymerizing denture base acrylic resins. materials. 2021; 14.5781. 7. al-dwairi zn, al haj ebrahim aa, baba nz. a comparison of the surface and mechanical properres of 3d printable denture-base resin material and convenronal polymethylmethacrylate (pmma). j prosthodont. 2023;32: 40–48. 8. harrison wein, nih research mauers is a weekly update of nih research highlights reviewed by nih’s experts. it's published by the office of communicarons and public liaison in the nih office of the director. 9. tocci n, d’auria fd, simonex g. a three-step culture system to increase the xanthone producron and anrfungal acrvity of hypericum perforatum subsp. angusrfolium in vitro roots. plant physiol biochem. 2012;57:54–58. 10. fujii h, sun b, nishioka h, et al. evaluaron of the safety and toxicity of the oligomerized polyphenol oligonol. food chem toxicol 2007;45(3):378–387. 11. mirkarimi m, eskandarion s, bargrizan m, et al. remineralizaron of arrficial caries in primary teeth by grape seed extract: an in vitro study. j dent res dent clin dent prospects 2013;7(4):206–210. 12. mutlu n. effects of grape seed oil nanoemulsion on physicochemical and anrbacterial properres of gelarn-sodium alginate film blends internaronal journal of biological macromolecules.2023; volume 237, 15. 13. parvar m, kandwal a, singh a. virs vinifera (grape) seed extract in periodontal health and disease: a review. j dent sci oral rehabil 2011;2:10–11. 14. neven s aref. an in-vitro assessment of surface roughness, tensile bond strength and anrfungal acrvity of grape seed extract modified so{ liner the journal of contemporary dental pracrce.2020; volume 21 issue 4. some proper)es of 3d printed acrylic resin material modified by an)fungal vi)s vinifera oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.873 h#p://den*stry3000.pi#.edu 15. osman rb, khoder g, fayed b, kedia ra, elkareimi y, alharbi n. influence of fabricaron technique on adhesion and biofilm formaron of candida albicans to convenronal, milled, and 3dprinted denture base resin materials: a compararve in vitro study. polymers (basel). 2023;15: 1836. 16. taqa a.a,saja a. a and ali m.m.the effect of addiron of castor oil, grape seed oil and fenugreek oil on some properres of cold cure acrylic resin denture base material. alrafidain dent j. 2015; 15(2): 412418. 17. fouda s.m. ,mohammed m , passent g., ellakany m., al ghamdi e. , soban q., akhtar k., ali m., al-harbi f. flexural properres, impact strength, and hardness of nanodiamondmodified pmma denture base resin int j biomater. 2022; 9.6583084. 18. kang sh, lee hj, hong sh, et al. influence of surface characterisrcs on the adhesion of candida albicans to various denture lining materials. acta odontol scand. 2013;71(1):241– 248. 19. espinar c, bona a, pérez m, tejada-casado m, pulgar r. the influence of prinrng angle on color and translucency of 3d printed resins for dental restorarons. dental materials 2023; volume 39, issue 4. pages 410-417. 20. ada. '' american dental standers insrtute'' american dental associaron specificaron no.12 for denture base polymer. chicago; council on dental materials and devices, 1999. 21. alshaikh, a.a.; khauar, a.; almindil, i.a.; alsaif, m.h.; akhtar, s.; khan, s.q.; gad, m.m. 3dprinted nanocomposite denturebase resins: effect of zro2 nanoparrcles on the mechanical and surface properres in vitro. nanomaterials. 2022;12, 2451. 22. alraziqi znr. water temperature effect on hardness and flexural strength of (pmma/tio2 nps) for dental applicarons. baghdad sci. j. 2022; 19(4):0922. 23. abdul larf rashid, a, kadhim gf. effect of temperature on surface hardness of different acrylic materials urlized for space maintainer. mustansiria dental journal.2023; 19(1), 107–116. 24. nabeel al-shakarchi m, himmadi hasan r. compararve evaluaron to surface roughness of modified heat cured acrylic resin by zinc oxide nanoparrcles and cad/cam denture base materials in vitro study. tikrit journal for dental sciences, 2023;11(2), 162–169. 25. turker s, sener i, akkus e, bugurman b. effect of staining solurons on the colour stability and surface properres of denture base material. balk j stom. 2012; 16:49-56. 26. albalasmeh aa, berhe aa, ghezzehei ta. 2013. a new method for rapid determinaron of carbohydrate and total carbon concentrarons using uv spectrophotometry. carbohydrate polymers, 97, 253261. 27. fouad m, moudhaffer m. 2016. effect of disinfecron on some properres of heat-vulcanized maxillofacial silicone elastomer reinforced by nano silicone dioxide. journal of baghdad college of den@stry, 28, 16-21. 28. zeynep y d,nuran d yand mustafak. evaluaron of color changed of acrylic resin material in the different solurons. asian journal of chemistry 2010;22(9):66696676. 29. alshamrani a.and ayman r. ellakwa, "effect of prinrng layer thickness and postprinrng condirons on the flexural strength and hardness of a 3dprinted resin," biomed research internaronal, 2022. 30. machado to, stubbs cj, chiaradia v, alraddadi ma, brandolese a, worch jc, dove ap. a renewably sourced, circular photopolymer resin for addirve manufacturing. nature. 2024;629(8014):1069-1074. some proper)es of 3d printed acrylic resin material modified by an)fungal vi)s vinifera oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.873 h#p://den*stry3000.pi#.edu 31. s. k. e.h. alwan. methods of addirve manufacturing for dental co-cr alloys: systemarc review. journal of techniques. 2022; vol. 4, pp. 80-85. 32.shendy, k., abou el fotouh, m., ali, t., m. abbas, s. e., abuelsadat, s. efficacy of two different layer heights on the accuracy of 3-d printed orthodonrc models. ain shams dental journal, 2024; 33(1): 5060. doi: 10.21608/asdj.2024.252684.119 6 33.elsisy, w., temirek, m., hamza, h., omar, r. compararve evaluaron of surface roughness and microbial adhesion of alkasite resin based composite versus bioacrve giomer a{er simulated toothbrushing: an in vitro study.. ain shams dental journal, 2024; 35(3): 162-171. doi: 10.21608/asdj.2024.311280.144 3 34.abdelghaffar, e. evaluaron of surface roughness and fracture resistance of maxillary convenronal & digital complete dentures: an invitro study. ain shams dental journal, 2024; 34(2): 42-48. doi: 10.21608/asdj.2024.277269.123 1 35. azuma a., akiba n. and minakuchi s. hydrophilic surface modificaron of acrylic denture base material by silica coarng and its influence on candida albicans adherence. j med dent sci .2012; 59: 1－7. 36. somani mv, khandelwal m, punia v, sharma v. the effect of incorporarng various reinforcement materials on flexural strength and impact strength of polymethylmethacrylate: a metaanalysis. j indian prosthodont soc. 2019; 19(2):101-112. 37. bhalodia nr, shukla vj. anrbacterial and anrfungal acrvires from leaf extracts of cassia fistula l.: an ethnomedicinal plant. j adv pharm technol res. 2011;2(2):104-9. 38. murugan t, wins ja, murugan m. anrmicrobial acrvity and phytochemical consrtuents of leaf extracts of cassia auriculata. indian j pharm sci. 2013; 75(1):122-5. 39. swamy mk, akhtar ms, sinniah ur. anrmicrobial properres of plant essenral oils against human pathogens and their mode of acron: an updated review. evid based complement alternat med. 2016; 3012462. doi: 10.1155/2016/3012462. 40. craig r.g; john m. powers; john c.w. “dental material properres and manipularon”, eight ediron, 2004, p.270-280. 41. shi j, yu j, pohorly je, jianmel yu, and kakuda y. polyphenolics in grape seeds-biochemistry and funcronality. j med food .2003;6(4):291–299. 42. kang, s. h., lee, h. j., hong, s. h., kim, k. h., & kwon, t. y. influence of surface characterisrcs on the adhesion of candida albicans to various denture lining materials. acta odontologica scandinavica.2012;71(1), 241– 248. 43. ivkovic n., božović d., risrc s.and mirjanić v. the residual monomer in dental acrylic resin and its adverse effect. contemporary materials.2013; iv(1):84-91. 44. alhotan a., elraggal a., yates j. and haider j. effect of different solurons on the colour stability of nanoparrcles or fibre reinforced pmma. polymers.2022; 14(1521):1-14 45. goiato m, santos d, souza j, moreno a, pesqueira a. chromarc stability of acrylic resins of arrficial eyes submiued to accelerated aging and polishing. j. appl. 2010;oral sci. 18(6). 46. harm n., taqa a., abbas w. and shuker a. the effect of thyme and nigella oil on some properres of acrylic resin denture base. al-rafidain dental journal.2010; 2, volume 10, issue 2, 2010, pages 205-213. cyclic fatigue resistance of edge file x7 and 2shape endodontic systems: in vitro study. vol 9 no 1 (2024) doi 10.5195/d3000.2024.569 http://dentistry3000.pitt.edu cyclic fatigue resistance of edge file x7 and 2shape endodontic systems: in vitro study. ahmed mustaf mohammed1, ashraf salim alchalabi2 1 university of mosul college of dentistry conservative dentistry department salah al-din governorate – iraq. 2 university of mosul college of dentistry conservative dentistry department ninawa governorate – iraq. abstract objectives: in order to test the resistance of rotary instruments to cyclic fatigue fracture and its importance in root canal preparationthe purpose of this study was to see how different tapers (4% and 6%) affected the cyclic fatigue resistance of the edge file x7 and 2shape endodontic systems at 45° and 60°canal curvature. methods: eighty niti files from two different systems (n= 40 each) were used; group 1: edge file x7, group 2: 2shape. each group was subdivided into four subgroups (n= 10 for each subgroup). the files were evaluated for cyclic fatigue resistance using a modified custom-made static model with 45°and 60° angles having a radius of 3mm and a diameter of 1.5 mm. each file was rotated in a continuous rotation motion at 300 rpm with 1.2 n.cm torque for ts1, 300 rpm with 2.5 n.cm torque for ts2, and edge file x7 was used at 300 rpm and 3 n.cm. until it fractured. the time it took to separate the files was noted. each file's number of cycles to failure was computed. a kolmogorov-smirnova normality test was done for all groups, and a two-way anova was used to statistically examine the data. results: the ranking of the groups from the highest to the lowest ncf was as follows: edgefile 4% 45 degrees (6594.00), edgefile 6% 45 degrees (6317.90), edgefile 4% 60 degrees (5682.00), edgefile 6% 60 degrees (2142.00), 2shape 4% 45 degrees (2517.00), 2shape 4% 60 degrees (535.90), 2shape 6% 45 degrees (474.50), and 2shape 6% 60 degrees (213.00). conclusions: edge file x7 showed better results than the 2shape system. edgefile x7 4% at 45 degrees showed the highest ncf. the 2shape system 6% at 60 degree that showed lowest ncf. as the canal curvature increased the ncf decreased. taper 4% is better than taper 6%. keywords: edgefile x7; 2shape; cyclic fatigue resistance; endodontic; canal curvature. citation: mohammed am, et al. (2024) cyclic fatigue resistance of edge file x7 and 2shape endodontic systems: in vitro study. dentistry 3000. 1:a001 doi:10.5195/d3000.2024.569 received: august 18, 2023 accepted: january 14, 2024 published: may 9, 2024 copyright: ©2024 mohammed am, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: ahmed.21dep21@student.uomosul.edu.iq introduction the primary goals of endodontic therapy are to properly prepare and clean root canal apparatus while retaining the original design and avoiding iatrogenic occurrences. manual root canal preparation has traditionally been done with reamers, and files. most hand preparation procedures, including ledging, zipping, apical blocking, and canal transit, are time-intensive and prone to iatrogenic mistakes. niti rotary devices have lately gained popularity[1]. niti endodontic rotary instruments may fracture as a result of severe torsion or cyclic fatigue [2]. cyclic failure is brought on by the instrument's continuous tension and compression load on the area of greatest root canal curvature. torsional failure happens when the instrument shank continues to rotate while the file tip binds into the canal, increasing the torque and exceeding the metal's plastic limit. during the production of files, supplementary components like the degree of rotation, cross-sectional layout, metallurgical characteristics, and thermomechanical techniques are all variables that can be considered [3]. niti endodontic rotary files' cyclic fatigue resistance is influenced by a number of variables, for example: canal radius and angle of curvature [4], irrigation solutions [5], number of sterilization cycles [6] and the alloy of niti [7]. several thermally treated niti alloys have been developed by manufacturers to improve their resistance to cyclic fatigue [8]. furthermore, several investigations have shown that the root canal morphology has concealed curvature; this type of curvature increases the bending stresses in niti rotary files [9]. 2shape (micro-mega, besancon, france) are rotated continuously in a sequence that has been heat treated using t-wire technology. the http://dentistry3000.pitt.edu/ cyclic fatigue resistance of edge file x7 and 2shape endodontic systems: in vitro study. vol 9 no 1 (2024) doi 10.5195/d3000.2024.569 http://dentistry3000.pitt.edu company claims that the flexibility of the instruments promotes user comfort and makes it easier to negotiate great curves with the instruments, which recover their original shape after each usage. the asymmetrical cross-section of the file, which has two primary and one secondary cutting edge, provides greater cleaning of the root canal walls; these edges increase the cutting efficiency of the file and improve debris removal [10]. edgefile x7 instruments are produced using a proprietary process known as firewire, which combines heat treatment and cryogenic applications to create a unique crystalline matrix of the heat-treated firewire alloy, increasing the file’s flexibility and fracture resistance while reducing the shape memory effect inherent in niti instruments. the file’s cross-section is triangular, and the helix angle is changeable [11]. the purpose of this study was to see how different tapers (4% and 6%) affected the cyclic fatigue resistance of the edge file x7 and 2shape endodontic systems at 45° and 60°canal curvature. materials and methods this study involved 80 new instruments: 40 instruments from the 2shape system (20 instruments for ts1 4% and 20 instruments for ts2 6%) (ts; micro-mega, besancon, france) and 40 instruments from edge file x7 (edge endo; albuquerque, new mexico, united states) (20 instruments for 4% and 20 instruments for 6%). sample grouping the systems used in this study are divided into eight groups as follow: group 1: edge 4% 45 degrees. group 2: edge 4% 60 degrees. group 3: edge 6% 45 degrees. group 4: edge 6% 60 degrees. group 5: 2shape 4% 45 degrees. group 6: 2shape 4% 60 degrees. group 7: 2shape 6% 45 degrees. group 8: 2shape 6% 60 degrees. canal curvature drawing criteria pruett's approach from 1997 uses two factors to define the geometry of any root canal curvature: angle of curvature and radius of curvature [12]. to describe these measurements, first line is established along the coronal portion of the canal's long axis and second line is established along the apical portion of the canal's long axis. when the canal start to deviate there will be a beginning point position and a finishing point position for the canal curvature. a circle with tangents at positions represents the curved section of the canal at these locations. the angle of curvature is the number of degrees on the arc of the circle between these two points. the radius of this circle is equal to the radius of curvature [13]. preparation of artificial canals. the artificial canals are prepared on a stainless-steel metal block with dimensions of 50mm length x 30mm height x 5mm thickness by a computer numerically controlled milling machine (cnc machine, taiwan) in mosul city with two types of canal curvature: 45° and 60° (as shown in figure 1: a and b). cyclic fatigue resistance test the cyclic fatigue test was carried out in a custom-made device modified from the device that was used by [14], consisting of an endo-motor handpiece attached to a table vice vertically (as shown in figure 2) and a stainless-steel metal block containing artificial canals 45° and 60° with a radius of curvature of 3mm and a diameter of 1.5 mm [15]. the curved segment of the canal was 3.5 mm in length. the stainless-steel artificial canal was fixed to a table vice horizontally. each instrument was introduced into the canal until the tip of the instrument was 5 mm away from the curve apically, as figure 1: a. schematic drawing of types of canal curvature drawn by autocad 2006 program according to the method proposed by [13]. b. artificial canals. http://dentistry3000.pitt.edu/ cyclic fatigue resistance of edge file x7 and 2shape endodontic systems: in vitro study. vol 9 no 1 (2024) doi 10.5195/d3000.2024.569 http://dentistry3000.pitt.edu stated by [16], and the limit of penetration still contained the tip of the instrument inside the simulated canals; all instruments didn’t reach the artificial canal exit. to standardize the instrument penetration depth, a red line was painted for all canals. this red line will keep the tip of the file at the same distance from the canal curvature apically for all canals and prevent over-penetration of the files inside the canals, so this will affect the results of the test. a t-type spirit level bubble was glued to the head of the handpiece by fix all 808 universal fast adhesive glue in order to ensure the centralization of the file inside the artificial canal, prevent titling in any unwanted direction, and deliver the rotation of the file inside the canal without pressure. to stop the instruments from slipping out of the artificial canal, see the instrument when it breaks, remove the broken instrument with ease, and prevent the loss of the shattered pieces, the artificial canal was protected with a glass plate [17]. counting the number of cycles to failure. instruments rotated freely without pressure as stated by speed and torque that was modified as said by [18] for 2shape ts1 used at 300 rpm and 1.2 n.cm, for 2shape ts2 used at 300 rpm and 2.5 n.cm as stated by [19] and for edge file x7 used at 300 rpm and 3 n.cm as used by [20]. using an electric endo-motor e-connect (changzhou sifary medical, china) until a fracture occurred, at which point the time to fracture was recorded in seconds and video recording was established. synthetic oil (wd-40 company, milton keynes, uk) was utilized as a lubricant to lessen friction as the file made contact with the artificial canal walls [21]. number of cycles to failure (ncf) was calculated using the method below: ncf = revolutions per minute (rpm) × time to fracture (s)/60, as stated by [22]. statistical analysis the obtained data were analyzed with spss version 26 (ibm-spss inc., chicago, il, usa). the normal distribution of the variables was tested using the kolmogorov-smirnov test. a one-way analysis of variance test was used. data are given as the mean and (± standard deviation). a p value less than 0.05 was considered statistically significant. results test of normality of all instruments at the start of the statistical analysis, the ncf results for all instruments were assessed using the generally used normality test (kolmogorovsmirnova) to determine if the data were normally distributed. as shown in the table below, this test revealed that the data were normally distributed. for all of the files, the kolmogorov-smirnova test value is bigger than the alpha value (p > 0.05) as showed by table (1). mean values and (± standard deviation) of number of cycles to failure for all group using two-way anova test. the report of two-way anova test shows the interaction between the effect of canal curvature and two taper used, this report distinguishes between the eight group of the study. regarding the means that took different letters, this indicates they are statistically different from each other. the results for the number of cycles to failure for each group are presented in table 2. there were statistically significant differences among the groups (p < 0.01). figure 2: custom-made device of cyclic fatigue test http://dentistry3000.pitt.edu/ cyclic fatigue resistance of edge file x7 and 2shape endodontic systems: in vitro study. vol 9 no 1 (2024) doi 10.5195/d3000.2024.569 http://dentistry3000.pitt.edu the ranking of the groups from the highest to the lowest ncf was as follows: edgefile 4% 45 degrees (6594.00), edgefile 6% 45 degrees (6317.90), edgefile 4% 60 degrees (5682.00), edgefile 6% 60 degrees (2142.00), 2shape 4% 45 degrees (2517.00), 2shape 4% 60 degrees (535.90), 2shape 6% 45 degrees (474.50), and 2shape 6% 60 degrees (213.00). in both artificial canals, the smaller file taper resulted in significantly higher cyclic fatigue resistance when compared with that of the larger file taper (p < 0.01). discussion this study employed artificial canals with a 45° and 60° curvature angle and a 3.5-mm curvature radius with 1.5mm diameter [15], since most cyclic fatigue experiments are based on these parameters, to find out how the instruments' cyclic fatigue resistance is affected by canal curvature, making comparisons easier with other studies [23-26]. instead of using extracted teeth, stainless steel artificial canals were utilized in the study to standardize the test and exclude any other potential confounders brought on by causes of file separation other than cycle fatigue [27]. the block was made of stainless steel, and the canals were milled inside to prevent canal wear from occurring after continuous usage and maintain the same trajectory for all files. furthermore, in order to fit the various diameters and tapers of all files and allow them to freely spin inside the canals, the depth of the artificial canals was machined to 2 mm [28]. the stainless-steel testing block was covered with a glass top cover to enable visualization of the file as it worked in the canal and the moment when the instrument broke. it also helped keep the oil inside the canal for a longer period of time, preventing the file from deviating out of the canal space and preventing the loss of the broken fragments [17]. as said by azim, tarrosh [16] all instruments were used inside the artificial canal until the tip of the instrument was 5 mm away from the curve and a red line was marked at all canals. t-type spirit level bubble was connected to the head of the handpiece in order to ensure the centralization of the file inside the artificial canal, prevent tilting to any unwanted direction and deliver the rotation of the file inside the canal without pressure [29]. the effect of the niti alloy on the cyclic fatigue resistance of endodontic rotary files has also been studied previously, and most researchers believe that the martensitic phase of the niti alloy is the crystalline structure that is most resistant to cyclic fatigue [7]. endodontic instruments are thermo-mechanically processed to improve the systems statistic df sig. group 1: edge 4% 45 degrees 0.153 10 0.200 group 2: edge 4% 60 degrees 0.239 10 0.112 group 3: edge 6% 45 degrees 0.143 10 0.220 group 4: edge 6% 60 degrees 0.134 10 0.200 group 5: 2shape 4% 45 degrees 0.185 10 0.210 group 6: 2shape 4% 60 degrees 0.214 10 0.185 group 7: 2shape 6% 45 degrees 0.131 10 0.195 group 8: 2shape 6% 60 degrees 0.264 10 0.059 p > 0.05 normally distributed table (1). kolmogorov-smirnova test of normality (n= 80) http://dentistry3000.pitt.edu/ cyclic fatigue resistance of edge file x7 and 2shape endodontic systems: in vitro study. vol 9 no 1 (2024) doi 10.5195/d3000.2024.569 http://dentistry3000.pitt.edu microstructure and transformation behavior of niti alloys [30]. in this study two different alloy used fire wire (edge file x7) and t wire (2shape). the current study revealed that the fire wire (edge file x7) is better than t wire (2shape), because the fire wire treated using a special thermomechanical procedure. firewire looks to have cm-like features with the capacity to precurve the file and retain the bend [31]. t-wire treatment features revealed that 2shape files were lower resistance to cyclic fatigue and flexibility than cm wire [32]. this might be attributed to the different metallurgical properties and thermal treatments of the niti alloy during manufacture [33]. the endodontic rotary systems of this study was chosen because it offered an identical apical diameter but different cross-sectional designs, niti alloy crystal structures, and taper. the flexibility and cyclic fatigue resistance of niti endodontic files were also impacted by the metallurgical properties and thermal treatments of the niti alloy [34]. gambarini, gerosa [35] discovered that 25.04 niti endodontic rotary files had much greater cyclic fatigue resistance in comparison between 20.06 and 25.06. these findings support those of the current investigation and emphasize the importance of the taper above the apical diameter. increasing the mass of the instrument based on an increase in the taper and/or apical diameter negatively affected the instrument's resistance to cyclic fatigue, influencing their flexibility and causing excessive root canal dentine removal, apical transport, root perforations, and fractures [36, 37]. increasing the apical diameter and taper of niti endodontic rotary files reduced their cyclic fatigue resistance [47]. regarding the influence of the angle of canal curvature, both edge file x7 system and 2shape system instruments had dramatically reduced ncf as the angle of canal curvature increased. as canal curvature increased, the instruments received larger compressive and tensile stresses and hence fatigued sooner [38]. the cyclic fatigue resistance of niti instruments was evaluated using various angles of curvature at 45°, 60°, and 90° [39]. as stated by a previous research [40], the radius of curvature has a considerable impact on the fatigue life of niti rotary instruments. that is, when the radius of curvature grows, ncf increases dramatically. the multivariate linear regression model revealed that increasing the radius of curvature from 2 to 8 mm improves cycle fatigue resistance. the current study's findings did not support the null hypothesis since there was a difference in the number of cycles to failure between the two types of endodontic files, with edgefile x7 having a greater mean ncf compared to 2shape instruments. groups mean (± standard deviation) group 1: edgefile 4% 45 degrees a 6594.00 ± 3.46 group 2: edgefile 4% 60 degrees c 5682.00 ± 3.47 group 3: edgefile 6% 45 degrees b 6317.90 ± 4.62 group 4: edgefile 6% 60 degrees e 2142.00 ± 2.58 group 5: 2shape 4% 45 degrees d 2517.00 ± 3.46 group 6: 2shape 4% 60 degrees g 535.90 ± 7.65 group 7: 2shape 6% 45 degrees h 474.50 ± 3.48 group 8: 2shape 6% 60 degrees j 213.00 ± 2.26 table (2). mean values (± standard deviation) of number of cycles to failure for each group. a, c, b, e, d, g, h, j different letters means that they were statistically different. http://dentistry3000.pitt.edu/ cyclic fatigue resistance of edge file x7 and 2shape endodontic systems: in vitro study. vol 9 no 1 (2024) doi 10.5195/d3000.2024.569 http://dentistry3000.pitt.edu this might be attributable to a variety of variables affecting rotary file cyclic fatigue resistance, including manufacturing process, metallurgy design, helix angle, cross-sectional design, and heat treatment performed to the niti endodontic instrument [41]. according the results obtained by this study, among the eight groups the edgefile x7 4% at 45 degrees has the highest mean of ncf edgefile x7 4% at 45 degrees (6594.00) . this may be a result of a proprietary technique called firewire used during its manufacturing process [11]. this might be explained by the peculiar matrix of the heat-treated fire-wire alloy, which claims to deliver high instrument strength with greater flexibility[29]. edge file x7 demonstrated great flexibility as a result of its surface electropolishing, and heat treatment [42]. the edge file x7 includes a triangular cross-section and changeable helix angle, which are said to give enough strength while remaining flexible to boost cyclic fatigue resistance [11]. in contrast, a triple helix having two primary and one secondary cutting edges is employed in the production of 2shape instruments with an asymmetrical cross-section [43]. regarding to 2shape system based on the results of the present study this system shows lower ncf and time to fracture in comparison with edge file x7 system in all tested canals, but when compared the taper 4% with taper 6% of the 2shape system taper 4% shows higher ncf and time to fracture at all canal curvature. it has been stated that the tiny core diameter increases the instrument's cyclic fatigue resistance [35, 44]. the 2shape instruments showed lower ncf among the eight groups as 2shape 6% at 60 degrees (213.00), 2shape system that made from heattreated t-wire technology .the 2shape system had the lowest ncf and time to fracture because it is subjected to a revolutionary thermomechanical treatment (t-wire technology) that, according to the manufacturer, boosts its cyclic fatigue resistance by 40% over traditional niti alloys, when compared to conventional niti systems, the t-wire performed better, but when compared to other thermosmechanically treated systems, it performed similarly or lower. the current results are consistent with the higher cyclic fatigue resistance percentages suggested by the manufacturers, demonstrating that the difference in cyclic fatigue resistance is related to various thermomechanical treatments [45]. this is consistent with previous studies on the 2shape system [43, 46, 47]. conclusion within the limitation of this study, it is concluded that the cyclic fatigue resistance of the edge file x7 showed better results than the 2shape system. edgefile x7 4% at 45 degree showed the highest ncf. the 2shape system 6% at 60 degree that showed lowest ncf. as the canal curvature increased the ncf decreased. taper 4% is better than taper 6%. acknowledgments authors wish to acknowledge university of mosulcollege of dentistry, for providing the necessary support to conduct this study. references 1. comparative evaluation of instrumentation timing and cleaning efficacy in extracted primary molars using manual and niti rotary technique–invitro study. mehlawat, r., r. kapoor, k. gandhi, d. kumar, r. malhotra, and s. ahuja. j oral biol craniofac res. 2019. 9(2): p. 151155. 2. cyclic fatigue resistance of reciproc blue and reciproc files in an s-shaped canal. topçuoğlu, h.s. and g. topçuoğlu. j endod. 2017. 43(10): p. 1679-1682. 3. torsional resistance of proglider, hyflex edm, and one g glide path instruments. inan, u. and c. keskin. j endod. 2019. 45(10): p. 1253-1257. 4. cyclic fatigue resistance of new reciprocating files (reciproc blue, waveone gold, and smarttrack) in two different curved canals. topçuoğlu, h.s., s. demirbuga, s. düzgün, and g. topçuoğlu. j investig clin dent. 2018. 9(3): p. e12344. http://dentistry3000.pitt.edu/ cyclic fatigue resistance of edge file x7 and 2shape endodontic systems: in vitro study. vol 9 no 1 (2024) doi 10.5195/d3000.2024.569 http://dentistry3000.pitt.edu 5. effect of temperature of sodium hypochlorite on cyclic fatigue resistance of heat-treated reciprocating files. keles, a., e.u. ozyurek, m.o. uyanik, and e. nagas. j endod. 2019. 45(2): p. 205-208. 6. novel electronic device to quantify the cyclic fatigue resistance of endodontic reciprocating files after using and sterilization. zubizarreta-macho, á., ó. alonsoezpeleta, a. albaladejo martínez, v. faus matoses, j. caviedes brucheli, r. agustín-panadero, j. mena álvarez, and f. vizmanos martínezberganza. appl sci. 2020. 10(14): p. 4962. 7. the influence of niti alloy on the cyclic fatigue resistance of endodontic files. ruiz-sánchez, c., v. faus-llácer, i. faus-matoses, á. zubizarreta-macho, s. sauro, and v. faus-matoses. j clin med. 2020. 9(11): p. 1-16. 8. cyclic fatigue resistance of heat-treated nickel-titanium instruments. tanomaru-filho, m., c. galletti espir, a. carolina venção, n. macedo-serrano, j. camilo-pinto, and j. guerreiro-tanomaru. iran endod j. 2018. 13(3): p. 312-317. 9. symmetry of root and root canal morphology of mandibular incisors: a cone-beam computed tomography study in vivo. valentiobino, f., d. di nardo, l. quero, g. miccoli, g. gambarini, l. testarelli, and m. galli. j clin exp dent. 2019. 11(6): p. e527-e533. 10. shaping ability of 2shape and neoniti rotary instruments in preparation of curved canals using micro-computed tomography. faisal, i., r. saif, m. alsulaiman, and z.s. natto. bmc oral health. 2021. 21(1): p. 595. 11. in vivo evaluation of operative torque generated by two nickel-titanium rotary instruments during root canal preparation. gambarini, g., m. galli, m. seracchiani, d. di nardo, m.a. versiani, l. piasecki, and l. testarelli. eur j dent. 2019. 13(4): p. 556-562. 12. role of the crystallographic phase of niti rotary instruments in determining their torsional resistance during different bending conditions. zanza, a., m. seracchiani, r. reda, d. di nardo, g. gambarini, and l. testarelli. mater 2021. 14(21): p. 6324. 13. cyclic fatigue testing of nickel-titanium endodontic instruments. pruett, j.p., d.j. clement, and d.l. carnes, jr. j endod. 1997. 23(2): p. 77-85. 14. comparison of cyclic fatigue resistance of five nickel titanium rotary file systems with different manufacturing techniques. aminsobhani, m., n. meraji, and e. sadri. j dent 2015. 12(9): p. 636-46. 15. comparison of cyclic fatigue resistance of nickel-titanium coronal flaring instruments. capar, i.d., h. ertas, and h. arslan. j endod. 2014. 40(8): p. 1182-5. 16. comparison between singlefile rotary systems: part 2-the effect of length of the instrument subjected to cyclic loading on cyclic fatigue resistance. azim, a.a., m. tarrosh, k.a. azim, and l. piasecki. j endod. 2018. 44(12): p. 1837-1842. 17. comparison of cyclic fatigue resistance of novel nickel-titanium rotary instruments. capar, i.d., h. ertas, and h. arslan. aust endod j. 2015. 41(1): p. 24-28. 18. comparison of canal transportation and centering ability of three different endodontic rotary systems (an invitro study). kadir, s.k. erbil dent j. 2021. 4(2): p. 175181. 19. cyclic fatigue resistance of various nickel-titanium rotary endodontic instruments at body temperature: an in vitro study. saeed, d.h. and f.a. rafea. journal of duhok university. 2019: p. 61-66. 20. comparative evaluation of resistance to cyclic fatigue of three rotary endodontic ni-ti instruments. intzes, l., z.-d. tzima, and c. gogos. balkan journal of dental medicine. 2019. 23(1): p. 3639. 21. effect of movement kinematics on the cyclic fatigue resistance of nickel-titanium instruments. karataş, e., h. arslan, m. büker, f. seçkin, and i.d. çapar. int endod j. 2016. 49(4): p. 361-4. 22. mechanical and metallurgical evaluation of 3 different nickel-titanium rotary http://dentistry3000.pitt.edu/ cyclic fatigue resistance of edge file x7 and 2shape endodontic systems: in vitro study. vol 9 no 1 (2024) doi 10.5195/d3000.2024.569 http://dentistry3000.pitt.edu instruments: an in vitro and in laboratory study. zanza, a., p. russo, r. reda, p. di matteo, o. donfrancesco, p. ausiello, and l. testarelli. bioengineering (basel). 2022. 9(5): p. 221. 23. cyclic fatigue resistance of 2shape, twisted file and endosequence xpress nickeltitanium rotary files at intracanal temperature. uslu, g., t. özyürek, m. gündoğar, and k. yılmaz. j dent res dent clin dent prospects. 2018. 12(4): p. 283-287. 24. cyclic fatigue resistance of waveone gold, protaper next and 2shape nickel titanium rotary instruments using a reliable method for measuring temperature. olcay, k., t.f. eyuboglu, and e. erkan. niger j clin pract. 2019. 22(10): p. 13351340. 25. comparison of the cyclic fatigue resistance of vdw.rotate, trunatomy, 2shape, and hyflex cm nickel-titanium rotary files at body temperature. gündoğar, m., g. uslu, t. özyürek, and g. plotino. restor dent endod. 2020. 45(3): p. e37. 26. the effect of multiple autoclave sterilization on the cyclic fatigue of three heat-treated nickel-titanium rotary files: edgefile x7, vortex blue, and trushape. sharroufna, r. and m. mashyakhy. biomed res int. 2020. 2020(2020): p. 1-6. 27. cyclic fatigue resistance: comparison of af f-one and one curve rotary instruments with hyflex edm onefile in root canal therapy. ghahramani, y., m. sedighshams, m. atbaei, and s. asar. adv appl nanobio-technol. 2022. 3(1): p. 14-17. 28. the effect of autoclave sterilization on cyclic fatigue resistance of two ni-ti systems (an in-vitro study). fiad, a., m. el faramawy, and s. fahmy. egypt dentl j. 2021. 67(3): p. 2743-2747. 29. the effect of different curvature levels on cyclic fatigue of three single file niti instruments (a comparative study). altaay, a. and b. shukri. int. medical j. 2020. 25(5): p. 2387-2394. 30. cyclic fatigue resistance of p ro t aper n ext nickel-titanium rotary files. elnaghy, a.m. intl endod j. 2014. 47(11): p. 1034-1039. 31. cyclic and torsional fatigue resistance of heat treated and conventional super elastic nickeltitanium files. todd, s.j., s.j. sidow, j.m. dutner, p.m. parker, d. dickinson, and m.d. uniformed services univ of the health sciences bethesda. uniformed services university of the health sciences, bethesda, maryland 2017. 20(8): p. 14. 32. cyclic fatigue resistance of nickel-titanium rotary instruments according to the angle of file access and radius of root canal. pedullà, e., g.r.m. la rosa, c. virgillito, e. rapisarda, h.c. kim, and l. generali. j endod. 2020. 46(3): p. 431-436. 1. 33. evaluation of the cyclic fatigue resistance of 2shape, protaper gold and vortex blue rotary niti files in curved canals: a comparative in vitro study. isaac, s., s. el mankabadi, g. ahmed, and h. elasfouri. endo ept 2020. 14(4): p. 297-304. 34. effect of temperature on the cyclic fatigue resistance of thermally treated reciprocating instruments. klymus, m.e., m.p. alcalde, r.r. vivan, m.v.r. só, b.c. de vasconselos, and m.a.h. duarte. clin oral investig. 2019. 23(7): p. 3047-3052. 35. mechanical properties of a new and improved nickel-titanium alloy for endodontic use: an evaluation of file flexibility. gambarini, g., r. gerosa, m. de luca, m. garala, and l. testarelli. oral surg oral med oral pathol oral radiol endod. 2008. 105(6): p. 798800. 36. cyclic fatigue resistance and three-dimensional analysis of instruments from two nickel– titanium rotary systems. grande, n.m., g. plotino, r. pecci, r. bedini, v.a. malagnino, and f. somma. int endod j. 2006. 39(10): p. 755-763. 37. effect of clinical use on the cyclic fatigue resistance of protaper nickel-titanium rotary instruments. ounsi, h.f., z. salameh, t. al-shalan, m. ferrari, s. grandini, d.h. pashley, and f.r. tay. j endod. 2007. 33(6): p. 737-741. 38. evaluation of cyclic fatigue of hyflex edm, twisted files, and protaper gold manufactured with http://dentistry3000.pitt.edu/ cyclic fatigue resistance of edge file x7 and 2shape endodontic systems: in vitro study. vol 9 no 1 (2024) doi 10.5195/d3000.2024.569 http://dentistry3000.pitt.edu different processes: an in vitro study. khandagale, p.d., p.p. shetty, s.d. makandar, p.a. bapna, m.i. karobari, a. marya, p. messina, and g.a. scardina. int j dent. 2021. 2021(2021): p. 1-6. 39. cyclic fatigue resistance of oneshape and waveone instruments using different angles of curvature. elsaka, s.e. and a.m. elnaghy. dent mater j. 2015. 34(3): p. 358-63. 40. cyclic fatigue resistance of rotary niti instruments produced with four different manufacturing methods. keskin, n.b. and u. inan. microsc res tech. 2019. 82(10): p. 1642-1648. 41. cyclic fatigue resistance of novel genius and edgefile nickeltitanium reciprocating instruments. bueno, c.r.e., m.t.s. cury, a.m.v. vasques, g. sivieri-araújo, r.c. jacinto, j.e. gomes-filho, l.t.a. cintra, and e. dezan-júnior. braz oral res. 2019. 33(028): p. 1-9. 42. cyclic fatigue resistance of protaper gold, edgefile, oneshape and protaper universal. khalil, w.a.b., k. merdad, t.s. abu-haimed, m. howait, and l. alsofi. egypt dent j. 2018. 64(1): p. 589-596. 43. cyclic fatigue resistances of hyflex edm, waveone gold, reciproc blue and 2shape niti rotary files in different artificial canals. özyürek, t., m. gündoğar, g. uslu, k. yılmaz, s. staffoli, g. nm, g. plotino, and a. polimeni. odontology. 2018. 106(4): p. 408-413. 44. factors influencing defects of rotary nickel-titanium endodontic instruments after clinical use. parashos, p., i. gordon, and h.h. messer. j endod. 2004. 30(10): p. 722-5. 45. evaluation of the cyclic fatigue resistance of 2shape, protaper gold and vortex blue rotary niti files in curved canals: a comparative in vitro study. el mankabadi, s.i., g.m. ahmed, and h.a. elasfouri. endod prac today. 2020. 14(4): p. 297-304. 46. cyclic fatigue resistance of one curve, 2shape, profile vortex, vortex blue, and race nickeltitanium rotary instruments in single and double curvature canals. elnaghy, a.m. and s.e. elsaka. j endod. 2018. 44(11): p. 1725-1730. 47. cyclic fatigue resistance of reduced-taper nickel-titanium (niti) instruments in doubled-curved (sshaped) canals at body temperature. uslu, g., m. gundogar, t. özyurek, and g. plotino. j dent res dent clin dent prospects. 2020. 14(2): p. 111. http://dentistry3000.pitt.edu/ 835 final molecular detec,on of tetracycline-resistant streptococcus viridans bacteria using the rpoa gene vol 13 no 1 (2025) doi 10.5195/d3000.2025.835 h#p://den*stry3000.pi#.edu molecular detection of tetracycline-resistant streptococcus viridans bacteria using the rpoa gene aseel jalil ibrahim al-karawi1, abdulhameed salim hameed2, sally talib da'aj3, hadeel j. ibrahim4 1university of bilad alrafidain, diyala, iraq 2ministry of health, diyala, iraq 3university of bilad alrafidain, diyala, iraq 4general directorate of education, diyala, iraq abstract objec&ve: the aim of this study was to established diagnos4c approaches to dental caries using polymerase chain reac4on technology. methods: pcr series. 60 samples of oral bacteria were collected between 8/6/2023 and 12/1/2023, where 27 teeth showed a growth rate for bacterial culture (45%). the bacterial isolates under study were characterized. the sensi4vity of the bacterial isolates of streptococcus viridans under study to eight an4bio4cs was tested. the results of the current study showed that the resistance rates were as follows: 71.4% for tetracycline, 57% for augmen4n, 57% for ciprofloxacin, 28% for clindamycin, 57% for erythromycin, 57% for doxycycline, and 28% for doxycycline for clarithromycin. results: the minimum inhibitory concentra4on (mic) for the bacterial isolates under study was determined for the tetracycline an4bio4c. s. viridans isolates showed resistance to this an4bio4c through a sensi4vity test using the disk method, as the mic value. for the isolates ranged from (1024-8) micrograms/m l. the percentage of s. viridans isolates producing virulence factors was as follows: protease enzyme 42%, and membrane protease. bioac4ve 71%, bacteriocin 14%, hemolycin 57%, dnaase 71%, capsule 28%, and lipase 28%. conclusion: the total dna of all bacterial isolates under study was extracted, axer which a polymerase chain reac4on (pcr) was performed for the s. viridans resistant to tetracycline and with an mic value higher than 64 µg/ml using specialized primers targe4ng the specific sequence of the tetm gene with a size of 1,862bp. when the amplified products were migrated on an agarose gel, one band appeared in all tracks in the gel at the same level for all samples. the results showed that the presence of the tetm gene was 100%. keywords: streptococcus viridans, rpoa, tetm, sequencing, pcr cita9on: al-karawi aji, et al. (2025) molecular detec9on of tetracycline-resistant streptococcus viridans bacteria using the rpoa gene. den9stry 3000. 1:a001 doi:10.5195/d3000.2025.835 received: january 26, 2025 accepted: march 30, 2025 published: april 1, 2025 copyright: ©2025 al-karawi aji, et al. this is an open access ar9cle licensed under a crea9ve commons ayribu9on work 4.0 united states license. email: aseeljaleel68@gmail.com introduction streptococcus is a gram-positive coccus that includes several species, as they share common characteristics, as they are facultative anaerobic and spherical in shape [1]. some of them are obligate anaerobic and some are facultative anaerobic that ferment carbohydrates and grow with the availability of co2 and are negative to produce oxidase and catalase enzymes. the growth requirements of this species are complex as they require culture media rich in blood, such as blood agar [2]. the pathogenicity of the streptococcus genus is linked to its ability to produce several virulence factors, which include the production of hemolytic toxins such as hemolysin, in addition to its production of intestinal toxins that cause food poisoning. it also possesses the ability to produce many extracellular enzymes such as lipase, proteinase, and streptokinase, which help the bacteria in tissue invasion and spread of infections. most of its types are surrounded by a capsule and grow at a temperature of 37°c, molecular detec,on of tetracycline-resistant streptococcus viridans bacteria using the rpoa gene vol 13 no 1 (2025) doi 10.5195/d3000.2025.835 h#p://den*stry3000.pi#.edu which is the optimum temperature for their growth, and growth is weak in ordinary solid or liquid media [3]. s. viridans are gram positive, facultative anaerobic, and negative for oxidase and catalase tests. their growth is inhibited by optochin and their colonies do not dissolve in bile salts. unlike pneumococci, some streptococci viridans, such as s. mutans, accumulate large polysaccharides, such as dextran or levans of sucrose, and contribute to the development of tooth decay [4]. some types of streptomyces may enter the bloodstream by tooth brushing or during dental work [5]. when these organisms enter the bloodstream, they will form colonies and form biofilms, causing chronic endocarditis. resistance to tetracycline and its other derivatives is widespread in spherical gram-positive bacteria, and the mechanisms of resistance against the tetracycline family are either by changing the target site for the action of the antibiotic or reducing the permeability of the antigen through the outer membrane, or through genetic expression of the transposon gene containing it [6]. there is an interest in diyala governorate on methods to detect resistance of streptococcus viridans bacteria to tetracycline antibiotics among hospitalized patients. the aim of this study was to isolate s. viridans and characterize them phenotypically, biochemically and by molecular methods using 16srrna and rpoa. material and methods isolation and diagnosis of the bacteria under study oral samples were cultured on blood agar and on mitis salivarius agar medium and incubated in an anaerobic growth cylinder candle jar for 48 hours at a temperature of 37°c, and then the isolates were morphologically characterized based on the shape of the colony [7], and microscopically, based on the shape, color, cell clusters, and type of pigmentation [8]. in addition, biochemical characterization, based on the ability of the bacteria to produce enzymes such as oxidase [9] and catalase [10] was performed. finally, we tested the solubility in bile salts [11] and tested sensitivity to optogen [12]. sample collection 60 oral samples were collected from patients at the specialized dental center in baquba, diyala, iraq and from outpatient dental clinics, under the supervision of specialized dentists, during the period between august and december 2017. the samples were taken using sterile cotton swabs inside a closed carrier medium for the purpose of avoiding external contamination as well as to prevent contamination of samples during their transport to the laboratory. detection of virulence factors various methods were utilized for determination of virulent factor: the ability of bacterial isolates to produce hemolysin [13] lipase [11], protease [14], dnase [15] enzymes, biofilm production [16], composition of the portfolio [17], and the ability to produce bacteriocin [18]. antibiotic susceptibility testing the sensitivity of the s. viridans isolates under study to eight antibiotics was tested using the disk diffusion method on muellerhinton agar medium [19,20]. antibiotics used in this study were doxycycline (30 µg), erythromycin (15 µg), clindamycin (15 µg), clarithromycin (15 µg), ciprofloxacin (5 µg), bacitracin (0.04 micrograms), tetracycline (30 micrograms), and augmentin (30 micrograms). extraction of total dna and pcr amplification total dna extraction and pcr amplification of the tetracyclineresistant bacterial isolates under study was done using geneaid. genomic dna was purified and the rpoa and tetm genes (445 base pairs) were amplified using specific primers: rpoa f(cac agt tcc agg tgt tcg) and r(tgc tga molecular detec,on of tetracycline-resistant streptococcus viridans bacteria using the rpoa gene vol 13 no 1 (2025) doi 10.5195/d3000.2025.835 h#p://den*stry3000.pi#.edu aag ccc taa agcat) and tetm f (agttttagctcatgttgatg) and r(tccgactatttgggacgacgg), respectively. for the pcr master mix, geneaid, accupower®profi taq pcr premix was placed in stander accupower®profi taq pcr premix) that contained all other components. to execute the pcr reaction, taq dna polymerase, dntp, tris-hcl, ph 9, kcl mgcl2 stabilizer, and tacking dye were used (table 1). table 1. components for the polymerase chain reaction (pcr). pcr tubes were prepared according to manufacture instructions. they were transferred into a spin vortex centrifuge for 3 minutes at 3000 rpm and then placed in the pcr thermocycler. pcr amplification conditions for rpoa were denaturation at 950c for 5 minutes followed by 30 cycles of annealing at 500c for 30 seconds, extension at 72°c for 30 seconds, and a final extension cycle at 72°c for 5 minutes. for the tetm gene, the reaction conditions were denaturation at 950c for 5 minutes, followed by 30 cycles, annealing at 640c for 30 seconds, extension at 72°c for 30 seconds, and a final extension cycle at 72°c for 5 minutes. pcr products were resolved in 1% agarose gel and visualized by uv gel documentation and the image was captured by digital camera (canon, usa) [21]. results and discussion isolation and identification of streptococcus spp the results showed that 27 samples, representing 45% of the total 60, had streptococcus. isolation rate of s. viridans bacteria in the current study was 25.9%. these results were inconsistent with previous work [22,23], which showed the isolation rate of these bacteria as 11% and 5%, respectively, and more like others [24-26] that found percentages of 43%, 19.6%, or 24.5%. the reason for the difference in the percentage of isolation of hemolytic streptococcus in the current study in comparison with previous studies is possibly due to differences in the source of isolation, geographical location, ages, or nutritional and health habits. volume components 5 master mix pcr solution 3 dna template 2 forward primer 2 revers primer 13 pcr water 25 total volume molecular detec,on of tetracycline-resistant streptococcus viridans bacteria using the rpoa gene vol 13 no 1 (2025) doi 10.5195/d3000.2025.835 h#p://den*stry3000.pi#.edu 445 b.p figure 1. electrophoresis of amplification products of rpoa (445 base pairs) in an agarose gel. detection of streptococcus viridans using the diagnostic rpoa gene rpoa was detected in all samples (figure 1). antibiotic susceptibility testing for streptococcus viridans the sensitivity of streptococcus viridans isolates to eight antibiotics (doxycycline, tetracycline, augmentin, ciprofloxacin, clindamycin, bacitracin, clarithromycin, and erythromycin) was tested. the results of the current study showed that the rate of resistance of viridans to the tetracycline antibiotic was 71.4%, which is inconsistent with previous results [27,28], that showed the rate of resistance of s. viridans bacteria to this antibiotic was 2.7% or 15.8%. the reason for this resistance is suggested as the inability of the antibiotic to reach inhibitory concentrations into the bacterial cell because of the presence of plasmids that encode efflux pumps, which are responsible for either increasing the transfer of the antigen outside the bacterial cell or reducing its permeability [4]. the tetracycline antagonist inhibits protein synthesis by binding to the 30s ribosomal unit. as a result of this inhibition, it prevents the binding of amionacyl-trna to the special site on the mrna. the resistance rate of ciprofloxacin for s. viridans isolates was 57%, which is like results of the resistance of s. mutans defined as 75% [29]. the antibiotic effect of ciprofloxacin is bactericidal by inhibiting the dna synthesis process by stopping the action of the enzyme dna gyrase [30]. as for the antibiotic erythromycin, the resistance rate of the isolates under study was 57%. the reason for the resistance of bacterial isolates to the anti-erythromycin is likely due either to the presence of plasmids that carry genes responsible for this resistance or to a change in the target site, or to add a methyl group to adenine to rrna [31,32]. resistance of the bacterial isolates of s. viridans to the antibiotic clindamycin was 28%. previous data showed resistance of s. pyogenes bacteria to this antibiotic was 1.6% [33]. clindamycin is a macrolide, and bacteria become resistant to macrolides by acquiring the mef (a) gene. this gene encodes the 5 4 3 2 1 m b.p 2000 1000 500 100 molecular detec,on of tetracycline-resistant streptococcus viridans bacteria using the rpoa gene vol 13 no 1 (2025) doi 10.5195/d3000.2025.835 h#p://den*stry3000.pi#.edu synthesis of efflux pump protein synthesis, which works on pumping 4-15 rings of the macrolide group [34].). as for the resistance of bacterial isolates to the antibiotic augmentin, it reached 57% for s. viridans, which is different than previous work that showed a resistance rate of s. pyogenes of 6.6% [22]. the resistance of s. viridans isolates to doxicyclin was 57%, which was different to s. mutans isolates in previous work that were sensitive at 100% level [29]. for clarithromycin, the resistance of s. viridans was 71%. these results were close to the results reached earlier for s. viridans [35]. the increase in resistance resulting from misuse of antibiotics leads to the transfer of this resistance through genetic factors such as transposons or plasmids, or because of a change in the permeability of the bacterial cell wall [36]. the results of the study showed that all isolates were 100% resistant to bacitracin. these results were close to results reached earlier, where the resistance of s. mutans bacteria to this antibiotic was 100% [37]. detection of the tetm gene using pcr technology five bacterial isolates under study, belonging to the s. viridans bacterium, were tested [38]. the results of the current study showed that all isolates had the tetm gene that encodes resistance to tetracycline in s. viridans. the results of our study were inconsistent with previous work, which indicated s. viridans resistance of 43% [39]. figure 2. electrophoresis of the amplification products of the tetm gene in an agarose gel at a concentration of 1% and a voltage of 100 v for an hour and using a dna ladder (100bp-2000bp), as this appears in the m path and starts from 100 base pairs, and the gene packages are 1,862 base pairs long in s. viridans bacteria. conflicts of interest the authors declare no competing interest. references 1. brooks gf, caroll kc, butel js, mores sa, maietzner ta. (2010). jawets, melnik and adelberg's medical microbiology. 25th ed. the mcgraw-hill companies. 2. murray pr, rosenthl ks, pfaller ma. (2013). medical microbiology seventh ediion (textbook). philadelphia, pa. 1,862 bp b p 2000 1000 500 100 molecular detec,on of tetracycline-resistant streptococcus viridans bacteria using the rpoa gene vol 13 no 1 (2025) doi 10.5195/d3000.2025.835 h#p://den*stry3000.pi#.edu 3. ryan kj, ray cg, champoux jj, drew wi, neidhardt fc, plorde jj. (2004). sherris medical microbiology: an introducion to infecious diseases. 4th ediion. mcgraw-hill company. 4. jawetz e, melnick ja, adelberg ea. (2016). review of medical microbiology. 27th ed. mcgrawhill educaion, inc: 851pp. 5. wescombe pa, heng nck, burton jp, chilcoj cn, tagg jr. (2009). streptococcal bacteriocins and the case for streptococcus salivarius as model oral probioics. future microbial. 4:819–835. 6. reyes j, hidalgo m, d1i'az l, rinco'n s, moreno j, vanegas n, castaneda e, arias ca. (2007). internaional j. of infect. dis. 11:329-336. 7. holt jg, krieg nr, sneath pha, staley jt, williams st. (1994). bergey's manual of determinaive bacteriology. 9th ed. williams and wilkins: balimore, maryland; pp. 20 & 527-558. 8. yoo s, kim p, hwang h, kim k, choe s, min b, kook j. (2005). idenificaion of non-mutans streptococci organisms in dental plaques recovering on miis salivarius bacitracin agar medium. microbial. 43(2): 204-208. 9. koneman ew, allen sd, janda wm, schreckenberger pc, winn wcj. (1992). color atlas and textbook of diagnosic microbiology. (4th) ed. j.b. lippincoj company. philadelphia. 10. tadesse a, alem m. (2006). medical bacteriology. ephti. gondar university. 11. collee jg, fraser ag, marmion bp simmons a. (1996). mackie and mccartney pracical medical microbiology. 14th ed. churchill livingston. p.173-174. 12. messmer to, sampson js, sinson a, wong b, carlone gm, facklam rr. (2004). comparison four polymerase chain reacion assays for specificity in the idenificaion of s. pneumoniae. diagn. microbiol. infect. dis., 49: 249-254. 13. atlas rm, brown ae, parks lc. (1995) laboratory manual experimental microbiology. 1th ed. inc. biofilm formaion and determinaion of chemical inhibiion. msc thesis. college of science. university of baghdad. 14. straus dc, mapngly sj, milligan tw, doran ti nealon tj. (1980). protease producion by clinical isolates of type iii group b streptococci. j. clin. microb. 12: 421-425. 15. jeffries cd, holtman df, guse dg. rapid method for determining the acivity of microorganisms on nucleic acids. j bacteriol. 1957;73(4):590-1. 16. mathur t, singhal s, khan s, upadhyay dj, fatma t, rajan a. (2006). detecion of biofilm formaion among the clinical isolates of staphylococci: an evaluaion of three different screening methods. indian j. med. microbiol. 24(1): 25-29. 17. stukus pe. (1997). invesigaing microbiology: a laboratory manual for general microbiology. harcout brace and companies. 18. al-qasab abdul-jabbar’emer, al-khafaji zahra mahmoud. (1992). effect of different condiions on the inhibitory efficacy of intesinal lactobacilli in the direcion of intesinal bacteria causing diarrhea. faculty of agricultural sciences. folder 123 (7), 26-18. 19. clsi (2010) methods for diluion animicrobial suscepibility tests for bacteria that grow aerobically; approved standard— ninth ediion. 32(8). clsi, wayne, pennsylvania, usa. 20. clsi (2017) methods for diluion animicrobial suscepibility tests for bacteria that grow aerobically; approved standard— ninth ediion. 32(8). clsi, wayne, pennsylvania, usa. 21. park hk, yoon jw, shin jw, kim jy, kim w. (2010). rpoa is a useful gene for idenificaion and classificaion of streptococcus pneumoniae from the closely related viridans group streptococci. fems microbiology lejers, 305(1), 58-64. 22. tamimi zainab amer hatem (2013). bacteriological and hereditary study of streptococcus pyogenes isolated from paients with tonsilliis in the city of muqdadiya, master thesis, college of educaion for pure sciences, university of diyala. molecular detec,on of tetracycline-resistant streptococcus viridans bacteria using the rpoa gene vol 13 no 1 (2025) doi 10.5195/d3000.2025.835 h#p://den*stry3000.pi#.edu 23. srinivasan r, karaoz u, volegova m, mackichan j, katomaeda m, miller s, lynch sv. (2015). use of16s rrna gene for idenificaion of a broad range of clinically relevant bacterial pathogens. 10(2). 24. al-saeed, muhammad sabry abdel razzaq. (1997). plasmid transcriptome of aerobic respiratory bacteria. doctoral thesis. college of science. baghdad university. 25. al-mahdawi, abbas yassin hassan. (2005). study of the effect of pomegranate peel extract on bacteria isolated from tonsilliis paients in diyala governorate and some of their immunological features. master’s thesis. faculty of educaion. diyala university. 26. lhja m, raisanen s, jokineno k, stenfors, l. (1997). direct microscopy of effusions obtained from peritonsillar abscesses as a complement to bacterial culturing. j. laryngology. otology, 111:392395. 27. brenciani a, tiberi e, tili e, mingoia m, palmieri c, varaldo pe, giovanep e. (2013). geneic determinants and elements associated with anibioic resistance in viridans group streptococci. journal of animicrobial chemotherapy, 69(5), 1197-1204. 28. thummeepak r, leerach n, kunthalert d, tangchaisuriya u, thanwisai a, sijhisak s. (2015). high prevalence of muli-drugresistant streptococcus pneumoniae among healthy children in thailand. journal infecion and public health, 8(3), 274-281. 29. jebar ms. (2011). virulence factors of streptococcus mutans isolated from pregnant women with acute vaginiis. 24(7), 27-34. 30. akinjogunla oj, eghafona no, enabulele io. (2011). revalence, haemolyic aciviies and flourokuinolones suscepibility profiles of moraxella catarhalis, streptococcus pneumonia and haemophilus influenza associated with acute oiis media. in nature and science, 9(6):85-92. 31. iroha ir, chibuko nm, moses ib, ejikeugwu pc, ugbo en, nwakaeze ea, agumah nb. (2015). anibioic suscepibility pajerns of streptococcus pneumoniae isolated from the nasopharyngeal mucosa of children in enugu metropolis, nigeria. int. j. curr. microbiol app. sci, 4(9), 1-9. 32. zmantar t, kouidhi b, miladi h, bakhrouf a. (2011). detecion of macrolide and disinfectant resistance genes in clinical staphylococcus aureus and coagulase-negaive staphylococci. bmc res. 4(453): 2-9. 33. megged o, assous m, weinberg g, chlesinger y. (2013). inducible clindamycin resistance in beta-hemolyic streptococci and streptococcus pneumoniae. imaj. 34. sutcliffe j, grebe t, kamradt at, wondrack lt. (1996). detecion of erythromycin-resistant determinants by pcr. animicro. ag. and chemoth. 40 (11): 2562– 2566. 35. al-marzoqi ah. (2009). animicrobial suscepibiliies among respiratory isolates of haemophilus influenza, methicillin-resistant staphylococcus aureus (mrsa) and streptococcus pneumoniae in hillah infants. journal of alqadisiyah for pure science (quarterly), 14(1), 23-32. 36. sharat k. (2004). group b streptococcus infecion. medicine. 16(3):1425. 37. turki sumaya hassan (2018). a study on streptococcus mutans, isolated from tooth decay, and comparing the effect of some plant extracts and animicrobial agents on these bacteria, master thesis, faculty of science, diyala university. 38. doherty n, trzcinski k, pickerill p, zawadzki p, dowson cg. (2000). geneic diversity of the tet (m) gene in tetracycline-resistant clonal lineages of streptococcus pneumoniae. animicrobial agents and chemotherapy, 44(11), 29792984. 39. sun jq, li l, zhao k, zhang lf, ji ht, he yx. (2017). molecular epidemiology of tetracycline resistance among viridians group streptococci isolated from various clinical specimens. biomedical research (0970938x), 28(2). microsoft word 380 2023.docx the effec'veness of turmeric gel in the healing of donor site for free gingival gra8 vol 11 no 1 (2023) doi 10.5195/d3000.2023.380 h#p://den*stry3000.pi#.edu the effectiveness of turmeric gel in the healing of donor site for free gingival graft zuhir alasfar1, rowaida saymeh1, mays khazem2 1faculty of dentistry, damascus university, syria 2faculty of pharmacy, damascus university, syria abstract background: prepara'on of turmeric extract gel with a concentra'on of 10% and comparing it with non-eugenol gingival dressing (coe-paktm) in the healing and reepithelializa'on of palatal donor site a8er free gingival gra8, and reduc'on of postopera've pain. materials and methods: this study was conducted on twenty-two pa'ents with lack of aiached gingival 'ssue for various reasons and received so8 'ssue augmenta'on via free gingival gra8s. par'cipants were randomly divided into two equal groups: group i (test) was treated with 10% turmeric gel while group ii (control) was treated with (coe-paktm). both pain level index which assessed by using the visual analogue scale (vas) and analgesic consump'on index were recorded for 7 days a8er surgery. the healing index, the color match index, and the re-epithelializa'on index were assessed on days 7, 14, 21, and 28. finally, wound size index was measured via periodontal probe at baseline and days 7, 14, 21, 28, and a8er 2 months. results: group i showed significant pain reduc'on measured by vas and analgesic consump'on on day 3, 4, 5, 6, 7 (p<0.05) with no significant difference on the first two days (p>0.05). group i demonstrated significant accelera'on in the ini'al healing process during the 1st week (p=0.002) and 2nd week (p=0.01) with no significant difference between both groups in the 3rd and 4th weeks (p>0.05). group i showed sta's'cally higher vas for the color match on day 7, 14, 21, and 28. no sta's'cal difference was noted between the groups a8er two months. there was no sta's'cal difference between groups i and ii in the re-epithelializa'on and wound size index at any of the follow-up periods (p>0.05). conclusion: 10% turmeric extract gel showed relief of post-opera've pain, accelera'on of the healing process, and beier outcomes in terms of the color match for the palatal wound a8er free gingival gra8ing procedure comparing to the coe-paktm periodontal dressing. keywords: curcumin; free gingival gra8; periodontal dressing; donor site cita9on: alasfar, z et al. (2023) the effec9veness of turmeric gel in the healing of donor site for free gingival gram den9stry 3000. 1:a001 doi:10.5195/d3000.2023.380 received: august 8, 2022 accepted: march 11, 2023 published: april 7, 2023 copyright: ©2023 alasfar, z et al. this is an open access ar9cle licensed under a crea9ve commons avribu9on work 4.0 united states license. email: zuhiralasfar@gmail.com introduction free gingival graft (fgg) has been used for treating gingival recession and is considered the most common technique for soft tissue augmentation around teeth and dental implants [1]. however, postoperative discomfort associated with the presence of two surgical sites, one of which is usually the palate, limits the usage of this technique and requires alternative treatments with less associated morbidity [2]. bjorn, one of the pioneers in the field, described fgg in 1963 [3] as a technique that impedes the recession progress, facilitates plaque control, and reduces or eliminates root sensitivity [4, 5]. miller has also mentioned the ability the effec'veness of turmeric gel in the healing of donor site for free gingival gra8 vol 11 no 1 (2023) doi 10.5195/d3000.2023.380 h#p://den*stry3000.pi#.edu of fgg to achieve complete root coverage in a one-stage procedure in 89.9% of sites [6]. however, postoperative pain and the long healing time required for the donor site are undesirable complications for both clinician and patient [7]. traditional dressings are used as a physical barrier that protects wounds from oral environment without promoting cellular activities or biological sequence during the healing process [8]. therefore, researchers have been interested in using natural substances, including turmeric and its extracts, as dressings to reduce postoperative pain and inflammation [9]. turmeric belongs to the zingiberaceae family of plants. it is native to tropical asia [10]. it consists of a group of three curcuminoids: curcumin (77%), demetossicurcumin (17%), and bisdemetossicurcumina (6%) [11]. curcuminoids are known as phenolic compounds that have shown a great variety of biological functions, such as: antioxidant, antiinflammatory, antimicrobial, and anticancer effects [12]. consequently, there has been an increasing interest in curcuminoids due to their medicinal use. however, the curcuminoids have a weak pharmacokinetics, such as its low plasma concentration, limited tissue distribution, high rate of metabolism and rapid systematic clearance [10, 13, 14], especially the orally administered form [14]. this has underlined the need for modifications to current formulations [13]. many studies evaluated the safety of the curcuminoids [14] and they have concluded that standardized powder and extract forms of turmeric and curcuminoids are safe for human use, even at high doses. turmeric is considered a gras (generally recognized as safe) by the u.s. food and drug administration (fda) [16]. baum's study showed that the oral intake of 1 4 g/day of curcuminoids for 6 months was bioavailable and did not show any major toxicity or severe adverse effects [15]. in periodontal surgery, curcuminoids have been used in adjunctive therapy along with the mechanical phase to treat gingivitis and periodontitis and that is due to their anti-inflammatory properties as well as their ability to accelerate the healing process [9, 17, 18]. such substance has also been used on aphthous lesions and lichen planus, as it possesses analgesic effect [19]. this study aimed to compare the therapeutic effects of an oral gel prepared from a dried ethanolic turmeric extract with a 10% concentration comparing to the conventional gingival dressing (coepak) on the donor site following gingival grafting procedures. material and methods study design this study was a 2-arm, parallelgroup, randomized controlled clinical trial with a 1:1 allocation ratio. the research gel obtained approval from the ethical committee of damascus university, syria (no 471/m 12/5/2021). this study received approval from the institutional review board (irb) of damascus university (no 3150 7/9/2020). this study was registered at the isrctn database (identifier: isrctn14159005), and it was funded by damascus university, postgraduate research budget (ref: 88032002507den). sample size calculation a pilot study was conducted on 8 patients to determine the sample size. based on the pilot study results the effect size was 1,73 (standard deviation = 0,57); the sample size was calculated using g.power 3.1.9.2 program with 95% power at the 5% significance level, the confidence level was determined by 95% and two independent means as the statistical test. the number of the study sample was 20 patients, but to avoid any dropouts which may occur during the study, the number of patients was increased to 22 participants. the sample size was determined based on the null hypothesis, which states that the 10% turmeric gel has no effect on the healing and re-epithelialization of the donor site for free gingival graft and reduces postoperative pain. the effec'veness of turmeric gel in the healing of donor site for free gingival gra8 vol 11 no 1 (2023) doi 10.5195/d3000.2023.380 h#p://den*stry3000.pi#.edu participants, eligibility criteria, and setting twenty-two patients were included in this study. the patients were recruited from the periodontology department at damascus university in syria between august and december 2021. the inclusion criteria in this study were the following: patients 18 years of age or older, good oral hygiene, no systematic disease, and have less than 1 mm attached gingiva width in the mandibular anterior region. the exclusion criteria were: pregnant or lactating women, smokers, patients undergoing active orthodontic treatment, gingival or periodontal diseases, systemic conditions, uncontrolled plaque accumulation, or those taking medications that impede the healing process. the surgical procedure, benefits, and possible side effects were explained to all patients, and written informed consent was obtained. randomization and allocation concealment. patients were randomly distributed into 2 groups: group i (was treated with 10% turmeric gel), or group ii (was treated with non-eugenol gingival dressing). the allocation sequence (allocation ratio of 1:1) was done using a box containing 22 opaque sealed envelopes, eleven of which were containing a piece of paper identified with "control group" and eleven were containing a piece of paper marked with "experiment group". each patient was instructed to choose one envelop from the box; the patient was put into one of the two groups based on their chosen envelop. the envelope was opened immediately at the end of the surgical procedure. follow-up measurements were done by another clinician who was blind to the current study. interventions in the experimental group, donor sites were treated with 10% curcuma longa extract gel following the surgical procedure of fgg, whereas the control group was treated using non-eugenol periodontal dressing (coe-pak). turmeric extract preparation the whole turmeric plants were obtained from the local market. the botanical classification was done at the department of pharmacognosy at faculty of pharmacy, damascus university; then turmeric rhizomes washed, and air-dried for one week. turmeric roots were manually crushed, smashed into little pieces to increase the extraction outcome, and then soaked in 100% ethanol with a proportion of 1:20 at ambient temperature for 72 hours. the extraction liquid was filtered using a filter paper then the liquid was desiccated using a rotary evaporator at 50o c until the ethanol was vaporized. the calibration for curcuminoids was done by a team of pharmacists from damascus university by using high-performance thin layer chromatography (hptlc), and it was between a range from 46% similar to other studies [42,43] gel preparation every 100 grams (g) of turmeric gel contains 10 (g) of dried ethanolic turmeric extract which was added to 3 g of 24-hour-soaked carbopol 934, then 3 milliliters (ml) of triethanolamine and 70 ml of polyethylene glycol 400 were added, with the help of an electric stirrer, then 0.02 g of ethylenediamine tetraacetic acid (edta), 0.02 g methylparaben, and 0.18 g propylparaben were added to the mixture. finally, sorbitol 70% was added to refine the taste. the components were weighed by an electronic scale (precisa company/ uk). sterilization was done by 25-kilo grays of gamma-ray at the atomic energy commission of syria. gel administration group i patients were instructed to apply a thin layer of the turmeric gel four times per day and wear the stent and avoid eating and drinking for 30 minutes to allow the concentration of curcuminoids to increase to a level of clinical manifestation. pre-surgical procedures intra-oral examination and periodontal evaluation were conducted for each patient. all patients underwent mechanical periodontal treatment and received the effec'veness of turmeric gel in the healing of donor site for free gingival gra8 vol 11 no 1 (2023) doi 10.5195/d3000.2023.380 h#p://den*stry3000.pi#.edu oral hygiene instructions. after 2 weeks, oral hygiene and periodontal health were re-evaluated. only patients with good compliance were included. a maxillary alginate impression was taken during the same re-evaluation session to fabricate a 1-mm acrylic stent for post-operative use in order to hold the materials in close contact with the surgical site. the surgical procedure patients in both groups underwent the same surgical procedures. local anesthesia was administrated in both donor and recipient sites using 2% lidocaine with 1:80,000 epinephrine. the recipient site was prepared by performing a horizontal incision along the mucogingival line and two vertical releasing incisions were made from the lateral ends of the horizontal incision to the alveolar mucosa. a split-thickness flap was made without disturbing the underlying periosteum, and the flap was made with sufficient depth to avoid graft movement. the recipient site was rinsed with saline and covered with saline-moistened gauze until the graft was transferred. a #15 surgical blade was used to harvest the graft from the palate, between the distal aspects of the canines and the first molars, leaving 3 mm of undisturbed tissue from the gingival margin. after harvesting the graft, a saline-moistened gauze was applied to the palatal site to achieve hemostasis. the graft was immediately transferred to the recipient site and secured to the adjacent intact attached gingiva using two 5-0 nylon sutures. furthermore, a periosteal suture was used to stabilize the graft without penetrating it. gentle pressure was applied for two minutes on the graft with a salinemoistened gauze. care was taken to avoid mobilizing the graft as to not impede the healing process. the periodontal dressing was used in donor and recipient sites in the control group (group ii) (figure 1). in the experimental group (group i), patients were treated with 10% turmeric extract gel which was applied to donor and recipient sites and patients were instructed to apply the gel four times per day for 14 days (figure 2). post-surgical care diclofenac potassium 50 mg tablets (cataflam, novartisò) were described only if needed and patients were instructed to count the number of pills taken for indirect pain measurement via mean consumption of analgesics. patients were recommended to follow a soft diet and to avoid brushing and flossing in the surgical area for 14 days, after which sutures were removed. a 0.12% chlorhexidine rinse was prescribed twice daily to limit bacterial growth. photographs were taken for the palatal wound on day 7, 14, 21, 28, and finally after two months, after surgery for evaluation of the healing of the palatal wound. outcomes the primary outcome pain level via vas: patients were asked to record palatal pain values every day for the first 7 days figure 1. palatal wound healing for group ii: (a) day 0, (b) day 7, (c) day 14, (d) day 21, (e) day 28, and (f) after two months figure 2. palatal wound healing for group i: (a) day 0, (b) day 7, (c) day 14, (d) day 21, (e) day 28, and (f) after two months the effec'veness of turmeric gel in the healing of donor site for free gingival gra8 vol 11 no 1 (2023) doi 10.5195/d3000.2023.380 h#p://den*stry3000.pi#.edu according to the visual analogue scale (vas) of yale university, which defines 0 as no pain and 10 as extreme pain [20]. consumption of analgesics: the indirect assessment for pain index was done by mean consumption of analgesics for 7 days after surgery, recorded in milligrams [21]. the secondary outcome the wound healing index: this index evaluates healing process based on bleeding, pus, redness, granulation formation, and the amount of exposed connective tissue. healing assessments were done once per week for a total of 4 assessments and were based on a scale of 1 (very poor) to 5 (excellent) healing [22]. color match index: the color of the palatal wound was evaluated by comparing it with the palatal mucosa of the opposite side by using the objective vas of yale university. where 0 indicates no color match and 10 indicates an excellent color match with the adjacent tissues. the assessment process was done on day 7, day 14, day 21, day 28, and after 2 months [23]. re-epithelization index: using 1% toluidine blue, was applied to the donor site every week for the first four weeks following surgery. the navy-blue staining was considered incomplete epithelization, whereas light blue staining was considered complete epithelization. results were graded on a scale of i to iii, where (i) is re-epithelialization of less than half of the surgical site, (ii) is reepithelialization of more than half of the surgical site, and (iii) is complete re-epithelialization [24]. wound size index: wound size was measured vía periodontal pobre (unc 15). a transparent film was placed on the wound area, and the margin of the wound was traced with a pen, then the length and width of the wound were meagered using unc-15 probe. the measuring process was done on surgery day and days 7, 14, 21, and 28 [25]. statistical analysis the statistical data analysis was performed using spss software version 25.0 (spss, inc., chicago, usa). the statistical significance level was determined at p<0.05. mannwhitney u was applied for the healing index, the independent samples test was used to compare mean differences between the two groups in terms of vas for pain level, analgesic consumption, color match, and wound size index, and chi-square for the re-epithelization index. results consort guideline shows the flow and distribution of the participants (figure 3). a total of 22 patients (6 males, 16 females) were included in the study and divided into two groups of 11 participants. during the followup phase, 2 patients were lost (one patient from each group). the statistical analysis was only applied to patients who were present for both the examination and the follow-up. group i consisted of 80% female and 20% male, while group ii was 70% female and 30% male with no statistical difference. the age of patients ranged between 20 and 52 years of age. the mean age was 38 ± 8.95 for group i and 37 ±9.39 for group ii with no statistical difference. the effec'veness of turmeric gel in the healing of donor site for free gingival gra8 vol 11 no 1 (2023) doi 10.5195/d3000.2023.380 h#p://den*stry3000.pi#.edu the primary outcome pain level via vas: the mean of vas values was significantly lower in group i than in group ii on days 3-7 (p<0.05), whereas the difference was not significant on days 1 and 2, where the highest mean was recorded in group ii (p=0.112 and p=0.107 respectively) (table 1). postoperative analgesic consumption (in mg): on day 1, and 2 the highest median values for total analgesic consumption were recorded in group ii; however, this difference did not reach the level of statistical significance. on days 3-7, there was a statistically significant difference, where the group i showed a noticeable reduction in analgesic consumption (p<0.05) (table 2). the secondary outcome wound healing index: mean values were significantly higher in group i than in group ii for the first week (p=0.002), and second week (p=0.01), whereas the difference was not significant in the third (p=0.328) and fourth weeks (p=1) (table 3). color match index: there was a significant difference with higher figure 3. consort flow diagram. the effec'veness of turmeric gel in the healing of donor site for free gingival gra8 vol 11 no 1 (2023) doi 10.5195/d3000.2023.380 h#p://den*stry3000.pi#.edu matching in group 1 on day 7, 14, 21, and 28 where the highest median and range values were recorded in group i, compared to group ii; however, there was no significant difference between the groups after two months where both groups recorded complete color matching (table 4). re-epithelization index: there was no significant difference in the donor site re-epithelization between group i and ii at any of the follow-up appointments (p>0.05). on week 1, re-epithelization appeared to be less than half of the surgical site for both groups (p=1). on week 2, 60% of group i demonstrated reepithelialization of more than half of the surgical site, whereas only 40% was seen in group ii. on week 3, complete re-epithelialization was seen in 50% of group i and 40% of group ii. on week 4, complete palatal re-epithelization was seen in all patients (p=1) (table 5). wound size index: the results of the study demonstrated that the difference between groups did not reach the level of statistical significance, although the highest mean value was recorded in group ii at all of the follow-up periods (table 6). discussion previous studies aimed to expedite the healing process through the use of many biological materials (a-prf) [26], ozone therapy [27], hyaluronic acid [28], especially following mucogingival surgery. since each material has its pros and cons, this study is fundamental in the search for the ideal material. this study is the first to use 10% turmeric extract gel in terms of the healing of second intention wounds. such concentration is considered to be the maximum possible concentration in the gel form and is sufficient to show clinical results. on the other hand, raghava et al, meghana et al. and jalaluddin et al studies have used 1% extract [9, 17, 18]. a large number of studies have revealed the benefits of turmeric extract as antioxidants [29] and antiinflammatory agents [30]. however, very limited research is available to test turmeric’s effectiveness in improving the healing process and reducing postoperative pain following mucogingival surgeries. turmeric extract has poor oral bioavailability and was therefore applied topically to the palatal surgical site. post-operative pain is considered a natural phenomenon. it gradually reduces as the healing progress [31]. because postoperative pain of the donor site is associated with the wound depth rather than surface area, the thickness of the graft was standardized as 1-1.5 mm. thicker grafts could cause necrosis as a result of ischemia, while a graft that is shallower would have a low resistance to functional forces [32]. pain values were recorded daily for the first 7 postoperative days according to vas values between 0 and 10. on days 1 and 2, there was no significant difference in the highest median of the vas score in each group. although the highest value for vas pain score was noted in group ii (table 1). this could be due to the fact that the effectiveness of curcuminoids relies on their concentration in the wound site. this concentration may take multiple days to build up, hence the lack of difference in vas score between groups i and ii. the range and mean values of the vas score decreased significantly in group i on days 3 to 7 when compared to group ii. this could be explained by the fact that curcuminoids reduce pain through several mechanisms: first, curcuminoids can prevent the synthesis of prostaglandin e2 (pge2) [33] which is an important mediator produced in injured nerves, and facilitate the synthesis of pain-related molecules [34]. second, curcuminoids can stimulate corticosteroid secretion from the adrenal gland by inhibiting potassium channels (i.e. btrek-1) and depleting neurotransmitter substances at the nerve endings [33]. third, the curcumin molecule has a vanilloid moiety which causes the desensitization of the transient receptor potential cation channel (trpv1) which plays a critical role in nociceptive neural detection and transmission [15]. the effec'veness of turmeric gel in the healing of donor site for free gingival gra8 vol 11 no 1 (2023) doi 10.5195/d3000.2023.380 h#p://den*stry3000.pi#.edu in this study pain index was measured both directly via vas and indirectly via consumption of analgesics; thus patients were instructed to take diclofenac potassium 50 mg orally only if needed. no other medications were prescribed even antibiotics. infections after mucogingival surgery usually are less than 5%, if the patient maintains good oral hygiene and this low-risk infection does not justify the preventive use of antibiotics. the maximum level of analgesic consumption was in the initial healing phase (first and second day) with no satistical difference between the study groups. group i showed a dramatically significant decrease compared with group ii in the amount of analgesic use from day 3 to day 7, and that reflects the powerful analgesic and antiinflammatory effect of curcuminoids. such a result agrees with the findings of the pain index measured via vas score. curcuminoids are characterized by having in addition to their anti-inflammatory effects, high tolerance and fewer side effects compared with noncorticosetiroid anti-inflammatory drugs, especially in long-term treatment [35]. although all the criteria used to evaluate the pain level are subjective but it is the most wildly used for this purpose in other studies and it has proven a good efficiency in the evaluation result treatments [36]. the healing progress was recorded on days 7, 14, 21, and 28 following the surgery in accordance to the landry, et al. index, which consists of 5 degrees. group i exhibited a significant difference in the rate of healing in weeks 1 and 2 when compared to group ii (p < 0.05). this result can be explained by the results of the in vetro study by rujirachotiwat and suttamanatwong which reveal the ability of curcumin in increasing the expression of rna-m for growth factors (egfr, kgf-1) and collagen type-1 gene (col1) in term of healing [37]. iova et al also demonstrated the antioxidant properties of curcumin, as it decreases the free radical damage in periodontal tissue which improves the healing process [38]. since uncontrolled inflammation hinders the healing process, curcuminoids act through several mechanisms to modify the inflammatory process by disturbing the calcium channels in the cell membranes which play an important role in the activation of lymphocytes and inflammatory cells. curcuminoids also reduce the number of molecules involved in inflammation such as tumor necrosis factor-alpha (tnf-a), interleukin-1 (il-1), cyclooxygenase-2 (cox-2), leukotrienes, nitric oxide, and lipoxygenase [29, 30]. even though median values in group i were higher than in group ii, our study showed that there is no statistical difference between the two groups on days 21 and 28 (table 3). this can be explained that the gel was applied only for 14 days leading to a decrease in concentrations of the extract in the surgical site, in addition to the completion of the healing on day 28. no studies were found on the effect of curcumin or turmeric extract on the healing of intraoral secondary intention wounds. the re-epithelialization test was scored at 1, 2, 3, and 4 weeks following surgery using 1% toluidine blue colorant. there was no statistical difference between both groups at any of the follow-up periods. it is worth noting that the percentage of re-epithelialization was higher in group i compared to group ii in all of the follow-up periods (table 5). many studies have confirmed that epithelization was not complete during week 1 in (coe-pak) groups [39] and complete epithelization was scored in the fourth week for all individuals [26, 40]. no studies were found on the effect of topical curcumin or turmeric extract on the re-epithelialization of intraoral surgical sites. table 5 shows a higher percentage in the re-epithelialization area at all follow-up periods with no satistical difference for group i and that can be explained by the ability of curcuminoids to improve collagen deposition and accelerate the reepithelialization [29] by stimulating the formation of transforming growth factor-beta (tgf-b) that accelerates angiogenesis and the remodeling process [41]. color matching index: as healing of fgg takes place by secondary intention, the color change in donor the effec'veness of turmeric gel in the healing of donor site for free gingival gra8 vol 11 no 1 (2023) doi 10.5195/d3000.2023.380 h#p://den*stry3000.pi#.edu site is observed. color matching was assessed on day 7, day 14, day 21, day 28, and after 2 months via vas. there is a significantly higher score in group i compared with group ii in days 7, 14, 21, and 28. the color matching between the palatal donor site and the adjacent normal tissue would reflect the degree of reepithelization and wound-healing so maybe the curcuminoids have an antiinflammatory effect more than accelerating the epithelial creeping of palatal wound.ace between the stud there is no statistical difference between the study groups after three months as all patients achieved a complete palatal color match. wound size was measured in this study by periodontal prob (unc 15). the evaluation was taken on surgery day, the seventh day, the fourteenth day, the twenty-fist day, and the wenty-eighth day and that was when complete epithelial healing was recorded for all patients. all groups showed a significant reduction in wound size from day 0 to day 42; however, no significant difference between groups was detected in wound size in each time interval. the measuring process was only done on width and length which does not provide accurate measurement due to the irregular healing pattern of the wound. there were no side effects observed on any patient in the experimental group. although more clinical studies are needed on turmeric extract in the field of dentistry and particularly in periodontology, this study has shown its effectiveness in accelerating surgical site healing, as well as postoperative pain control. p value 95% cl of the difference (lower bound upper bound) range min max median time 0.207 ** -2.881 0.681 5 8 5.6 group (i) day 1 5 9 7 group (ii) 0.150 ** -3.597 0.597 4 8 5.3 group (i) day 2 4 10 6.8 group (ii) 0.000 * -4.771 -1.829 2 7 3.7 group (i) day 3 4 9 6,3 group (ii) 0.000 * -5.043 -2.957 2 6 3.4 group (i) day 4 3 8 5.9 group (ii) 0.000 * -5.262 -2.338 2 6 3.2 group (i) day 5 5 8 5,7 group (ii) 0.000 * -5.445 -2.955 1 6 2.8 group (i) day 6 3 8 5,2 group (ii) 0.000 * -5.201 -2.599 0 5 1.9 group (i) day7 1 7 4.8 group (ii) table 1. median and range vas score for postoperative pain from day 1 to day 7. significance level: p ≤ 0.05; * = significant; ** = nonsignificant. the effec'veness of turmeric gel in the healing of donor site for free gingival gra8 vol 11 no 1 (2023) doi 10.5195/d3000.2023.380 h#p://den*stry3000.pi#.edu p value 95% cl of the difference (lower bound upper bound) max min median time 0.492 ** 0.600 -1.200 4 2 160 group (i) day 1 4 3 180 group (ii) 0.292 ** 0.374 -1.174 4 2 150 group (i) day 2 4 3 165 group (ii) 0.021 * -0140 -1.860 3 1 100 group (i) day 3 4 2 150 group (ii) 0.012 * -0.320 -2.273 3 1 80 group (i) day 4 3 2 145 group (ii) 0.009 * -0.400 -2.400 3 0 80 group (i) day 5 3 1 120 group (ii) 0.040 * -0.045 -1.755 2 0 50 group (i) day 6 2 1 105 group (ii) 0.037 * 0.350 -1.993 2 0 45 group (i) day7 2 0 95 group (ii) time median p value week 1 group (i) 2.4 0.002 * group (ii) 1.4 week 2 group (i) 3.6 0.01 * group (ii) 2.8 week 3 group (i) 4.6 0.328 ** group (ii) 4.3 week 4 group (i) 5 1 ** group (ii) 4.9 table 2. median and range values of analgesic consumption (mg) daily. significance level: p ≤ 0.05; * = significant; ** = nonsignificant. table 3. median score for healing index. significance level: p ≤ 0.05; * = significant; ** = nonsignificant. the effec'veness of turmeric gel in the healing of donor site for free gingival gra8 vol 11 no 1 (2023) doi 10.5195/d3000.2023.380 h#p://den*stry3000.pi#.edu p value max min median time 0.041 * 5 2 3.3 group (i) week 1 4 0 1.5 group (ii) 0.037 * 6 3 4.8 group (i) week 2 5 2 3.2 group (ii) 0.032 * 8 5 6.9 group (i) week 3 7 4 5.1 group (ii) 0.047 * 10 6 8.1 group (i) week 4 9 5 6.9 group (ii) 0.660 ** 10 10 10 group (i) after 2 months 10 10 10 group (ii) time grades percentage p value week 1 group (i) g (i) 100% 1 ** group (ii) g (i) 100% week 2 group (i) g (i) 40% 0.65 ** g (ii) 60% group (ii) g (i) 60% g (ii) 40% week 3 group (i) g (ii) 50% 0.074 ** g (iii) 50% group (ii) g (ii) 60% g (iii) 40% week 4 group (i) g (iii) 100% 1 ** group (ii) g (iii) 100% p value 95% cl of the difference (lower bound upper bound) std. error std. dev. mean time 1 ** 151.37 117.53 9.23 32.46 132.0 group (i) day 0 150.36 98.83 9.73 36.62 125.0 group (ii) table 4. median and range values of color match measured by the visual analogue scale score. significance level: p ≤ 0.05; * = significant; ** = nonsignificant. table 5. median and percentage of re-epithelialization from week 1 to week 4. g (i): re-epithelialization of less than 50% of the wound area g(ii): re-epithelialization of more than 50% of the wound area g (iii): complete re-epithelialization significance level: p ≤ 0.05; * = significant; ** = nonsignificant. table 6. mean values for the wound size index (mm) at all time points. the effec'veness of turmeric gel in the healing of donor site for free gingival gra8 vol 11 no 1 (2023) doi 10.5195/d3000.2023.380 h#p://den*stry3000.pi#.edu 0.227 ** 135.24 97.48 8.34 37.21 102.4 group (i) day 7 134.62 84.95 8.41 35.78 108.7 group (ii) 0.06 ** 117.41 74.38 10.84 29.72 82.5 group (i) day 14 114.73 68.46 9.19 33.67 88.3 group (ii) 0.782 ** 93.18 56.27 10.51 31.84 65.6 group (i) day 21 91.48 53.92 9.39 34.54 74.9 group (ii) 0.595 ** 67.52 35.61 8.46 33.63 49.7 group (i) day 28 63.83 32,74 8.52 35,27 57.2 group (ii) conclusion topical application of 10% curcuma longa extract gel on palatal wound following fgg harvesting could effectively promote palatal wound healing index. in addition to reducing postoperative pain, it improves the color match index for donor site following fgg harvesting, thereby 10% turmeric extract gel is a viable alternative to traditional gingival dressing (coe-pak), as it is easily applied and haas better biological effects. references 1. surgical techniques to increase the apicocoronal dimension of the a5ached gingiva: a 1-year comparison between the free gingival gra= and the modified apically reposi?oned flap. carnio, j., p.m. camargo, and p.q. pirih. int j periodon6cs restora6ve dent, 2015. 35(4): p. 571-8. 2. clinical and histological evaluation of two dressing materials in the healing of palatal wounds. shanmugam, m., et al. journal of indian society of periodontology, 2010. 14(4): p. 241. 3. free transplantation of gingival propria. bjorn, h. odontol revy, 1963. 14: p. 323. 4. dimensional changes in free gingival grafts: scalpel versus er: yag laser—a preliminary study. türer, ç.c., et al. lasers in medical science, 2015. 30(2): p. 543-548. 5. root coverage grafting for regeneration and aesthetics. miller jr, p.d. periodontology 2000, 1993. 1(1): p. 118-127. 6. root coverage using the free soft tissue autograft following citric acid application. iii. a successful and predictalbe procedure in areas of deep-wide recession. miller jr, p. int. j. periodont. rest. dent, 1985. 5: p. 14-36. 7. postoperative complications following gingival augmentation procedures. griffin, t.j., et al. journal of periodontology, 2006. 77(12): p. 2070-2079. 8. effect of propolis extract in combination with eugenol-free dressing (coe-paktm) on pain and wound healing after crownlengthening: a randomized clinical trial. askari, m., et al. journal of dentistry, 2017. 18(3): p. 173. 9. comparison of effect of curcumin gel and noneugenol periodontal dressing in tissue response, early wound healing, and pain assessment following periodontal flap surgery in chronic periodontitis patients. meghana, m.v.s., et al. journal of indian society of periodontology, 2020. 24(1): p. 54. 10. curcumin: biological, pharmaceutical, nutraceutical, and analytical aspects. kotha, r.r. and d.l. luthria. molecules, 2019. 24(16): p. 2930. 11. safety and efficacy of turmeric extract, turmeric oil, turmeric oleoresin and turmeric tincture from curcuma longa l. rhizome when used as sensory additives in feed for all animal species. significance level: p ≤ 0.05; * = significant; ** = nonsignificant. the effec'veness of turmeric gel in the healing of donor site for free gingival gra8 vol 11 no 1 (2023) doi 10.5195/d3000.2023.380 h#p://den*stry3000.pi#.edu additives, e.p.o., et al. efsa journal, 2020. 18(6): p. e06146. 12. therapeutic effects of turmeric in several diseases: an overview. hay, e., et al. chemico-biological interactions, 2019. 310 : p. 108729. 13. bioavailability of curcumin: problems and promises. anand, p., et al. molecular pharmaceutics, 2007. 4(6): p. 807-818. 14. turmeric (curcuma longa) and its major constituent (curcumin) as nontoxic and safe substances. soleimani, v., a. sahebkar, and h. hosseinzadeh. phytotherapy research, 2018. 32(6): p. 985995. 15. "six-month randomized, placebo-controlled, doubleblind, pilot clinical trial of curcumin in patients with alzheimer disease." baum, larry, et al. journal of clinical psychopharmacology 28.1 (2008): 110-113. 16. safety evaluation of certain food additives and contaminants. organization, w.h. vol. 68. 2008: world health organization. 17. efficacy of curcumin as an adjunct to scaling and root planing in chronic periodontitis patients: a randomized controlled clinical trial. raghava, k.v., et al. the journal of contemporary dental practice, 2019. 20(7): p. 842-846. 18. antimicrobial activity of curcuma longa l. extract on periodontal pathogens. jalaluddin, m., et al. journal of pharmacy & bioallied sciences, 2019. 11(suppl 2): p. s203. 19. curcumin, a turmeric extract, for oral lichen planus: a systematic review. white, c.m ., k. chamberlin, and e. eisenberg. oral diseases, 2019. 25(3): p. 720-725. 20. the clinical evaluation of the effects of low-level laser therapy on the donor and recipient sites of the free gingival graft: a case series. lafzi, a., et al. journal of lasers in medical sciences, 2019. 10(4): p. 355. 21. patient morbidity and root coverage outcome after subepithelial connective tissue and de-epithelialized grafts: a comparative randomizedcontrolled clinical trial. zucchelli, g., et al. journal of clinical periodontology, 2010. 37(8): p. 728-738. 22. the clinical evaluation of platelet-rich plasma on free gingival graft's donor site wound healing. samani, m.k., et al. european journal of dentistry, 2017. 11(04): p. 447-454. 23. effect of topically applied hyaluronic acid on pain and palatal epithelial wound healing: an examiner-masked, randomized, controlled clinical trial. yıldırım, s., et al. journal of periodontology, 2018. 89 )1(: p. 36-45. 24. toluidine blue: a review of its chemistry and clinical utility. sridharan, g. and a.a. shankar. journal of oral and maxillofacial pathology: jomfp, 2012. 16(2): p. 251. 25. contents: measure: a proposed assessment framework for developing best practice recommendations for wound assessment. keast, d.h., et al. wound repair and regeneration, 2004. 12: p. s1s17. 26. effect of a-prf application on palatal wound healing after free gingival graft harvesting: a prospective randomized study. sousa, f., et al. european journal of dentistry, 2020. 14(01): p. 063-069. 27. effects of laser photobiomodulation and ozone therapy on palatal epithelial wound healing and patient morbidity. isler, s.c., et al. photomedicine and laser surgery, 2018. 36 )11(: p. 571580. 28. clinical comparison of mebo and hyaluronic acid gel in the management of pain after free gingival graft harvesting: a randomized clinical trial. hassan, a., et al. international journal of dentistry, 2021. 2021. 29. curcumin as a wound healing agent. akbik, d., et al. life sciences, 2014. 116(1): p. 1-7. 30. efficacy of turmeric extracts and curcumin for alleviating the symptoms of joint arthritis: a systematic review and metaanalysis of randomized clinical trials. daily, j.w., m. yang, and s. park. journal of medicinal food, 2016. 19(8): p. 717-729. 31. post–operative complications of periodontal surgery. kirmani, m., et al. int j contemp med res, 2016. 3: p. 1285-6. 32. periodontal soft tissue non–root coverage procedures: a consensus report from the aap regeneration workshop. scheyer, e.t., et al. journal of periodontology, 2015. 86: p. s73-s76. 33. curcumin treatment attenuates pain and enhances functional recovery after incision. sahbaie, p., et al. anesthesia & analgesia , 2014. 118)6(: p. 1336-1344. 34. does cox2-dependent pge2 play a role in neuropathic pain? ma, w. and r. quirion. neuroscience letters, 2008. 437(3): p. 165-169. 35. analgesic efficacy and safety of curcuminoids in clinical practice: a systematic review and metathe effec'veness of turmeric gel in the healing of donor site for free gingival gra8 vol 11 no 1 (2023) doi 10.5195/d3000.2023.380 h#p://den*stry3000.pi#.edu analysis of randomized controlled trials. sahebkar, a. and y. henrotin. pain medicine, 2016. 17(6): p. 1192-1202. 36. concordance among different pain scales in patients with dental pain. dos santos calderon, p., et al. journal of orofacial pain , 2012. 26)2(: p. 126. 37. curcumin promotes collagen type i, keratinocyte growth factor-1, and epidermal growth factor receptor expressions in the in vitro wound healing model of human gingival fibroblasts. rujirachotiwat, a. and s. suttamanatwong. european journal of dentistry, 2021. 15(01): p. 063-070. 38. the antioxidant effect of curcumin and rutin on oxidative stress biomarkers in experimentally induced periodontitis in hyperglycemic wistar rats. iova, g.m., et al. molecules, 2021. 26(5): p. 1332. 39. clinical comparison of plateletrich fibrin and a gelatin sponge in the management of palatal wounds after epithelialized free gingival graft harvest: a randomized clinical trial. femminella, b., et al. journal of periodontology, 2016. 87(2): p. 103-11 3. 40. clinical study of laser biomodulation (650λ) after free gingival grafts. vieira, j., et al. j oral laser appl, 2010. 10: p. 159-63. 41. a review of therapeutic potentials of turmeric (curcuma longa) and its active constituent, curcumin, on inflammatory disorders, pain, and their related patents. razavi, b.m., m. ghasemzadeh rahbardar, and h. hosseinzadeh. phytotherapy research, 2021. 35(12): p. 64896513. 42. hydrotropic extraction of curcuminoids from turmeric. dandekar dv, gaikar v. separation science and technology. 2003;38(5):11851215. 43. extraction and purification of curcuminoids from turmeric (curcuma longa l.). kulkarni s, maske k, budre m, mahajan r. international journal of pharmacology and pharmaceutical technology. 2012;1(2):81-84. 812 final impact of rubbing and wai2ng technique of a universal adhesive system on shear bond strength of orthodon2c brackets: a compara2ve analysis vol 13 no 1 (2025) doi 10.5195/d3000.2025.812 h#p://den*stry3000.pi#.edu impact of rubbing and waiting technique of a universal adhesive system on shear bond strength of orthodontic brackets: a comparative analysis zahraa mohammed al-fadhily1*, selma merza hasan1,2, rhaf essam naji2, hayder sahib kandwh3 1college of dentistry, university of kufa, najaf, iraq 2college of dentistry, ahl al bayt university, iraq 3al-sajaad general hospital, iraq abstract background: one important factor in orthodon0cs to consider is the bracket's bonding strength to the teeth substrate. this study's objec0ve is to see how the rubbing and wai0ng approach of a universal adhesive system affects the shear bond strength and adhesive remnant index (ari) of orthodon0c brackets. materials and methods: this was an experimental study (in vitro). fify newly extracted human maxillary first premolars were divided into five groups of 10 teeth each. the universal adhesive was applied on the buccal surface with 20 seconds of rubbing followed by 0, 5, 10, 15, or 20 seconds of wai0ng protocol according to their group. the teeth were set into blocks of acrylic resin, and the shear bond strength was tested. aferwards, ari was assessed. sta0s0cal analyses were performed using the one-way analysis of variance and tukey hsd post hoc. in addi0on to the kruskal-wallis test. results: one-way anova showed significant differences (p=0.000) between the groups. the maximum shear bond strength was for group 5 (18.93 ± 2.82), with significant differences from group 1 (p= 0.000) and group 2 (p= 0.006). the minimum bond strength was for group 1 (11.09 ± 1.50), with significant differences from all the other groups. ari scores revealed no significant differences between the groups (p= 0.406). conclusion: by allowing for adequate solvent evapora0on and monomer infiltra0on—both of which are essen0al for the clinical outcome of orthodon0c treatment—extending the wai0ng period improves the shear bond strength. the ari was not significantly impacted by wai0ng 0me. keywords: orthodon0cs; shear bond strength; universal adhesive. cita9on: al-fadhily zm, et al. (2025) impact of rubbing and wai9ng technique of a universal adhesive system on shear bond strength of orthodon9c brackets: a compara9ve analysis. den9stry 3000.1:a001 doi:10/5195/d3000.2025.812 received: december 27, 2024 accepted: february 9, 2025 published: february 14, 2025 copyright: ©2025 al-fadhily zm, et al. this is an open access ar9cle licensed under a crea9ve commons a[ribu9on work 4.0 united states license. email: zahraam.al-fadhily@uokufa.edu.iq introduction fixed appliances are among the most important orthodontic tools used to give patients more aesthetically beautiful and appealing smiles, which enhances their function and self-image [1]. in fixed orthodontic mechanics, orthodontic brackets are the most crucial passive components that transfer the force generated by the force elements to the teeth [2]. one key objective of fixed orthodontic therapy is creating a suitable bond between orthodontic brackets and tooth surfaces. the bond must be sufficiently robust to withstand the forces that arise during treatment and permit bracket take-off after therapy without causing harm to the tooth enamel [3]. orthodontic bond strength depends on a variety of factors. regarding the materials, details are provided about the enamel etching technique, bracket layout, and kind of adhesive agent; regarding the teeth, details are provided on the type of tooth and fluorosis; regarding the surroundings, details are provided about the moisture, saliva, and blood contamination [4-6]. fixed appliance attachments should be firmly bonded to enamel surfaces to resist various events in impact of rubbing and wai2ng technique of a universal adhesive system on shear bond strength of orthodon2c brackets: a compara2ve analysis vol 13 no 1 (2025) doi 10.5195/d3000.2025.812 h#p://den*stry3000.pi#.edu the oral environments. frequent debonding of orthodontic brackets leads to therapy interruption and lengthens treatment duration which causes discomfort for orthodontists and their patients [7]. over the years, research has been done on direct bracket bonding to dental enamel. in an effort to enhance and achieve sufficient bond strength in orthodontics, evaluations of bonding systems and various enamel surface preparation techniques have been carried out [8-10]. adhesive systems referred to as "universal" or "multimode" were introduced into the market. these materials exhibited a less aggressive ph level of 2 to 3 and accomplished adhesion through the use of functional monomers [11]. these substances may be applied to the substrate with or without prior etching [12,13]. universal adhesives differ from standard one-step self-adhesives as they contain functional phosphate and/or carboxylate monomers. several of these functional monomers can initiate chemical bonding with calcium in hydroxyapatites [14,15]. the universal adhesive was successfully employed for bonding orthodontic brackets, especially with etching mode [16-18]. this study aims to evaluate the impact of the rubbing and waiting technique of a universal adhesive system on the shear bond strength and the adhesive remnant index of orthodontic brackets. material and methods specimen collection and preparation for this experimental study, 50 newly extracted human maxillary first premolars were collected from three orthodontic clinics. collected from healthy 15–30-year-old donors their teeth needed to be extracted as a part of orthodontic treatment plans. donors provided written agreements for the use of extracted data. the teeth sample had to meet certain requirements to be included: no apparent decalcification, cavities, cracks, hypoplastic areas, or restorations (the teeth were checked using a light curing unit) [19]. the teeth were cleared of debris and preserved in a closed container with normal saline at room temperature (220±3). the normal saline was regularly replaced to prevent bacterial development until the bonding procedure. before the bonding procedure, all teeth' buccal surfaces were cleaned and given a 20-second polish with non-fluoridated pumice. before mounting, teeth were irrigated with a water spray for ten seconds and then dried with three air syringes for ten seconds. mounting technique to improve the teeth's retention inside the acrylic blocks, a big fissure bur was utilized to create retentive grooves that serrated every tooth. afterward, the teeth were positioned vertically in a selfcure acrylic mounting, exposing the crown and directing the buccal surface to run parallel to the surveyor analyzing rod. this resulted in the application of force at a 900 angle to the bracket-tooth contact. specimen grouping and bonding technique the buccal surfaces of the mounted specimens were etched for 20 seconds using phosphoric acid gel (37%) (ortho-technology; fl/usa) followed by water rinsing and drying [20]. following that, the teeth were divided into five groups randomly according to the bonding technique with ten teeth per group (figure 1). group 1: the adhesive (3m™ scotchbond™ universal adhesive) was applied on the buccal surface with 20 seconds of rubbing, air drying for five seconds, and 10 seconds of curing utilizing led curing light valo ortho (ultradent, ut). this protocol was according to the manufacturer's instructions. group 2: the adhesive was put on the buccal surface with 20 seconds of rubbing, waiting for 5 seconds, 5 impact of rubbing and wai2ng technique of a universal adhesive system on shear bond strength of orthodon2c brackets: a compara2ve analysis vol 13 no 1 (2025) doi 10.5195/d3000.2025.812 h#p://den*stry3000.pi#.edu seconds of air drying, and 10 seconds of curing. group 3: the adhesive was put on the buccal surface with 20 seconds of rubbing, waiting for 10 seconds, 5 seconds of air drying, and 10 seconds of curing. group 4: the adhesive was put on the buccal surface with 20 seconds of rubbing, waiting for 15 seconds, 5 seconds of air drying, and 10 seconds of curing. group 5: the adhesive was put on the buccal surface with 20 seconds of rubbing, waiting for 20 seconds, 5 seconds of air drying, and 10 seconds of curing. following that, 50 maxillary first premolar stainless-steel brackets (dentarum company, ispringen, germany) were attached to the teeth using orthodontic adhesive paste (transbond xt, 3m). the bracket was then carefully positioned on the tooth using the least force possible and pushed firmly to ensure precise positioning. next, use a sharp scalar to remove any leftover adhesive paste that may have flown out from behind the bracket. figure 1. a diagram displaying the specimen grouping and sample size. shear bond strength experiment a universal testing machine (model 4411; instron, usa) was employed to measure the force required to debond the brackets (figure 2). the preparation of the specimens involved positioning them in acrylic resin with brackets orthogonal to the shear blade. all five experimental groups underwent a shear bond strength test, wherein the active shear blade tip was positioned on the upper part of the bracket base (figure 3). the debonding force was applied to the bracket-tooth interface using a blade operating at a crosshead velocity of 0.5 mm/minute [21]. the measurements were obtained in kilogram-force (kg), converted to newton (n), and the shear bond strength was estimated by dividing the amount of force by the bracket surface area. the bracket base's average surface area was 14 mm2, which allowed for the measurement of the shear bond strength in mpa. figure 2. universal tes;ng machine. figure 3. shear bond experiment. adhesive remnant index evaluation after the debonding process, each tooth surface and the bracket base were evaluated by a stereomicroscope (olympus, tokyo, japan) with an x20 magnification for evaluation of the adhesive remnant index (ari) (figure 4), based on the ari scores (table 1) [17]. impact of rubbing and wai2ng technique of a universal adhesive system on shear bond strength of orthodon2c brackets: a compara2ve analysis vol 13 no 1 (2025) doi 10.5195/d3000.2025.812 h#p://den*stry3000.pi#.edu figure 4. microscopic evalua;on of the adhesive remnant index. score 0: indicates that no adhesive remains attached to the enamel. score 1: less than ½ of the adhesive remains attached to the enamel. score 2: more than ½ of the adhesive remains attached to the enamel. score 3: the whole adhesive remains attached to the enamel. statistical analyses the statistical analyses were conducted utilizing spss version 26. mean, minimum, maximum, standard deviation, and standard error were employed to evaluate the data. shapiro-wilk and levene's tests, respectively, were used to screen the data for homogeneity and normal distribution. inferential statistics utilize a 95% confidence interval. a p-value was deemed statistically significant if it was less than 0.05. as the data of shear bond revealed a normal distribution according to the shapiro-wilk test, one-way anova and tukey hsd post hoc multi-comparison test were used to test if there were significant differences between groups. since the data was not continuous and did not follow a normal distribution, the ari scores were examined by applying the kruskalwallis test. results the results of descriptive statistics included min, max, m, sd, se, and shapiro-wilk test values of shear bond strength for all studied groups demonstrated in table (2). the data were considered to satisfy the normality criteria within the 5% significance level based on the shapiro-wilk test, which found that the p value for all groups was greater than the significance level of 0.05. levene's test indicated that the data were homogeneous. one-way anova revealed significant differences (p=0.000) between the groups (table 3). the highest shear bond strength was for group 5 (18.93 ± 2.82), with significant differences from group 1 (p= 0.000) and group 2 (p= 0.006). the lowest bond strength was for group 1 (11.09 ± 1.50), with significant differences from all the other groups. group 2 differs considerably from all other groups, but not significantly from group 3 (p= 0.195) (table 4). the ari score variation for each of the five groups is shown in table (5). the statistical examination of the ari scores with the kruskal-wallis test showed no significant differences between the groups (p= 0.406). table 1: scores for the adhesive remnant index. impact of rubbing and wai2ng technique of a universal adhesive system on shear bond strength of orthodon2c brackets: a compara2ve analysis vol 13 no 1 (2025) doi 10.5195/d3000.2025.812 h#p://den*stry3000.pi#.edu table 2. descriptive data of shear bond strength (mpa) for all groups. table 3. one-way anova test of shear bond strength of all groups. groups n min max mean sd se shapirowilk test (p-value) group 1 10 8.99 13.30 11.09 1.50 0.47 0.487 group 2 10 11.99 17.80 15.16 1.96 0.62 0.426 group 3 10 14.00 22.00 17.44 2.33 0.73 0.891 group 4 10 15.30 24.00 18.75 2.67 0.84 0.491 group 5 10 14.56 23.80 18.93 2.82 0.89 0.718 sum of squares df mean square f p-value between groups 426.836 4 106.709 19.959 0.000 within groups 240.593 45 5.347 total 667.428 49 impact of rubbing and wai2ng technique of a universal adhesive system on shear bond strength of orthodon2c brackets: a compara2ve analysis vol 13 no 1 (2025) doi 10.5195/d3000.2025.812 h#p://den*stry3000.pi#.edu table 4. tukey hsd post hoc multiple comparison test of shear bond strength of all groups. groups mean difference se sig. 95% confidence interval lower bound upper bound group 1 group 2 -4.067* 1.03 0.003 -7.00 -1.12 group 3 -6.351* 1.03 0.000 -9.28 -3.41 group 4 -7.662* 1.03 0.000 -10.60 -4.72 group 5 -7.841* 1.03 0.000 -10.77 -4.90 group 2 group 1 4.067* 1.03 0.003 1.12 7.00 group 3 -2.284 1.03 0.195 -5.22 0.65 group 4 -3.595* 1.03 0.010 -6.53 -0.65 group 5 -3.774* 1.03 0.006 -6.71 -0.83 group 3 group 1 6.351* 1.03 0.000 3.41 9.28 group 2 2.284 1.03 0.195 -0.65 5.22 group 4 -1.311 1.03 0.712 -4.24 1.62 group 5 -1.490 1.03 0.605 -4.42 1.44 group 4 group 1 7.662* 1.03 0.000 4.72 10.60 group 2 3.595* 1.03 0.010 0.65 6.53 group 3 1.311 1.03 0.712 -1.62 4.24 group 5 -0.179 1.03 1.000 -3.11 2.75 group 5 group 1 7.841* 1.03 0.000 4.90 10.77 group 2 3.774* 1.03 0.006 0.83 6.71 group 3 1.490 1.03 0.605 -1.44 4.42 group 4 0.179 1.03 1.000 -2.75 3.11 impact of rubbing and wai2ng technique of a universal adhesive system on shear bond strength of orthodon2c brackets: a compara2ve analysis vol 13 no 1 (2025) doi 10.5195/d3000.2025.812 h#p://den*stry3000.pi#.edu table 5. scores for each group of the adhesive remnant index (ari). discussion one advantage of the self-etching adhesive system is that it requires fewer stages and lowers the possibility of mistakes during the adhesive method [22]. the methacrylate phosphoric acid esters found in these adhesive systems are typically produced from phosphoric acid and demineralize the surface of the tooth by removing calcium ions [23,24]. one important factor to consider while evaluating an adhesive's efficacy is its bond strength. the adhesive strength between the two substrates is assessed using the shear bond strength test. the load that a material can withstand in a direction parallel to its face is measured by the shear bond strength test [25]. the adhesion strength can be affected by: the chemical makeup of the adhesive, light-curing apparatus, acid content, and variations in the experimental methodology [2628]. even though two-step complete etch bonding techniques can yield high initial resin–dentin bond strength values, they still undergo substantial decline after in vitro aging [29]. because of the hydrophilic nature of the polymer inside the hybrid layer that promotes water sorption and increases permeability, it is vulnerable to hydrolytic deterioration [30,31]. with time, the adhesive-formed hybrid layer could deteriorate because of an increase in water absorption and a loss of collagen fiber cross bands, weakening the bond between the surfaces. this time frame could range from six months to three to five years [26,32]. in addition, research revealed that insufficient resin monomer infiltration resulted in a significant number of exposed collagen fibrils at the hybrid layer [29]. the dentin-restorative material's connection is weakened when the collagen fibrils are targeted by cysteine cathepsins and matrix metalloproteinases (mmps) [33]. current studies established and highlighted the role of mmps and cysteine cathepsins on the degradation of dentin bonding after using both total-etch bonding systems and self-etch [34-36]. in order to overcome the complex nature of dentin, researchers have proposed a number of modifications to the recommended application techniques that should make the application easier to apply and maximize infiltration. some of these modifications include longer light exposure times for bonding resins [37], multiple adhesive coatings with delayed polymerization, adhesive rubbing, and increased application times for primers and bonding resins [38, 39]. it doesn't seem to be entirely true that self-etch bonding solutions can penetrate along all demineralized dentin [34,35]. if the bonding resin is applied for a longer period of time, there will be a longer period of uninterrupted waiting before light curing. it was groups score 0 score 1 score 2 score 3 p-value group 1 2 4 2 2 0.406 group 2 2 5 3 0 group 3 1 4 5 0 group 4 0 6 3 1 group 5 1 6 3 0 impact of rubbing and wai2ng technique of a universal adhesive system on shear bond strength of orthodon2c brackets: a compara2ve analysis vol 13 no 1 (2025) doi 10.5195/d3000.2025.812 h#p://den*stry3000.pi#.edu anticipated that this amount of waiting time would help with the evaporation of leftover solvents and unbound water [37]. additionally, this approach change would facilitate the creation of a strongly cross-linked polymer and aid in the decrease of nanopores. as a consequence, the resin– dentin hybrid layer that is produced would be of higher quality, stronger bonds, and more resistant to deterioration [36]. the active application of the substance by rubbing has already been discussed concerning the application of resin to dentin and enamel. the main objectives are to enhance the adhesive-substrate contact surface and promote solvent evaporation [27,40]. the result showed highest shear bond strength was for group 5 in which the waiting time extended to 20 seconds with significant differences from other groups with less waiting time. the lowest bond strength was for group 1 which included waiting for 5 seconds only. so, the result declared that increased waiting time of bonding before curing can increase the bonding strength. waiting time permits for solvent evaporation and better infiltration of monomer. different solvents, like: ethanol, water, or acetone, are included in the universal adhesive system to aid in the adhesive's transfer to the enamel. to prevent residual solvent from weakening the adhesive layer and interfering with the polymerization process, proper solvent evaporation is essential. if there is not enough waiting time before light curing, the residual solvent may get trapped in the adhesive layer, which can cause voids and weaker bonding. depth of monomer penetration is more likely when the adhesive has enough time to interact with the enamel. additionally, it provides time for the adhesive to level out and create a consistent layer, which is essential for guaranteeing proper mechanical stability and polymerization. it has been shown in several earlier investigations that the quick curing of bonding resin, without a waiting interval for the resin monomers to permeate into the etched dentin formed by total-etch bonding methods, is only partially effective. similarly, it doesn't seem to be entirely true that self-etch bonding techniques can penetrate along all demineralized dentin [34]. marchesi et al. (2014) found that a short waiting period between the application of adhesive and curing could weaken the binding. for sufficient solvent evaporation and adhesive contact with the substrate, the authors advised waiting for at least 10 seconds [35]. studies reveal that using a rubbing motion when applying adhesive enhances the orthodontic brackets' shear bond strength (sbs). rubbing improves micromechanical retention by facilitating the entry of sticky monomers into the enamel. furthermore, improved contact with the smear layer is achieved through active application, especially in self-etch systems, which is essential for forging a robust adhesive interface [13]. in contrast to passive applications, active adhesive system applications greatly increased binding strengths, according to a study by papadogiannis et al. (2019). the results of the investigation demonstrated a correlation between improved adhesive penetration and higher sbs, which strengthened the binding between the enamel surface and orthodontic brackets [41]. according to a study by hanabusa et al. (2012), the most dependable orthodontic bonding procedure produced the highest sbs values when a universal adhesive system was applied using both active application and a waiting period prior to curing [13]. regarding the adhesive remnant system (ari), the result cleared that increasing the waiting time had no effect on the ari scores and impact of rubbing and wai2ng technique of a universal adhesive system on shear bond strength of orthodon2c brackets: a compara2ve analysis vol 13 no 1 (2025) doi 10.5195/d3000.2025.812 h#p://den*stry3000.pi#.edu the groups did not differ significantly from one another. this result was approved by the quantities of adhesive left on the enamel surfaces did not significantly increase with longer application times or more agitation of the self-etching primer, according to protásio et al. (2016). these findings, along with those of a different study, imply that longer application times and more agitation should not result in a higher risk of enamel fracture or require more time for tooth cleanup following deboning [24,42]. conclusion increasing the waiting time enhances the shear bond strength by allowing for proper solvent evaporation and monomer infiltration, which is critical for clinical success in orthodontic treatment. waiting time did not produce a significant effect on the ari. practical implications in orthodontics combining rubbing and waiting techniques can be a standard practice in orthodontics, particularly when using universal adhesive systems. orthodontic brackets need to withstand substantial forces, and a strong, durable bond is crucial for treatment efficacy and the longevity of the bond. availability of data this article contains the data used to support the study's conclusions. conflicts of interest according to the authors, there are no conflicts of interest. references 1. de almeida, a.b., i.c.g. leite, and g.a. da silva, brazilian adolescents' perception of the orthodontic appliance: a qualitative study. american journal of orthodontics and dentofacial orthopedics, 2019. 155(4): p. 490-497. 2. çiçek, o. and n. özkalaycı, orthodontic brackets; part ii. j. int. dent. sci, 2018. 4: p. 134-140. 3. trakinienė, g., et al., impact of fluorosis on the tensile bond strength of metal brackets and the prevalence of enamel microcracks. scientific reports, 2019. 9(1): p. 5957. 4. alzainal, a.h., et al., orthodontic bonding: review of the literature. international journal of dentistry, 2020. 2020(1): p. 8874909. 5. bakhadher, w., et al., factors affecting the shear bond strength of orthodontic brackets– a review of in vitro studies. acta medica, 2015. 58(2): p. 43-48. 6. khan, h., et al., bracket failure in orthodontic patients: the incidence and the influence of different factors. biomed research international, 2022. 2022(1): p. 5128870. 7. salama, f., et al., shear bond strength of new and rebonded orthodontic brackets to the enamel surfaces. journal of orthodontic science, 2018. 7(1): p. 12. 8. elnafar, a.a., m.k. alam, and r. hasan, the impact of surface preparation on shear bond strength of metallic orthodontic brackets bonded with a resin-modified glass ionomer cement. journal of orthodontics, 2014. 41(3): p. 201207. 9. pereira, t.b.j., et al., effects of enamel deproteinization on bracket bonding with conventional and resin-modified glass ionomer cements. the european journal of orthodontics, 2013. 35(4): p. 442446. 10. pithon, m.m., et al., evaluation of the shear bond strength of two composites bonded to conditioned surface with self-etching primer. dental press journal of orthodontics, 2011. 16: p. 94-99. 11. van meerbeek, b., et al., state of the art of self-etch adhesives. dental materials, 2011. 27(1): p. 17-28. 12. chen, c., et al., bonding of universal adhesives to dentine– old wine in new bottles? journal of dentistry, 2015. 43(5): p. 525536. 13. hanabusa, m., et al., bonding effectiveness of a new ‘multi-mode’adhesive to enamel and dentine. journal of dentistry, 2012. 40(6): p. 475-484. impact of rubbing and wai2ng technique of a universal adhesive system on shear bond strength of orthodon2c brackets: a compara2ve analysis vol 13 no 1 (2025) doi 10.5195/d3000.2025.812 h#p://den*stry3000.pi#.edu 14. yoshida, y., et al., adhesion to and decalcification of hydroxyapatite by carboxylic acids. journal of dental research, 2001. 80(6): p. 1565-1569. 15. yoshihara, k., et al., adhesive interfacial interaction affected by different carbonchain monomers. dental materials, 2013. 29(8): p. 888897. 16. proença, m.a.m., et al., shear strength of brackets bonded with universal adhesive containing 10-mdp after 20,000 thermal cycles. international journal of dentistry, 2020. 2020(1): p. 4265601. 17. shafiei, f., et al., comparison of shear bond strength of orthodontic brackets bonded with a universal adhesive using different etching methods. dental press journal of orthodontics, 2019. 24: p. 33. e133. e8. 18. goracci, c., et al., bracket bonding to all-ceramic materials with universal adhesives. materials, 2022. 15(3): p. 1245. 19. shaik, j.a., et al., in vitro evaluation of shear bond strength of orthodontic brackets bonded with different adhesives. contemporary clinical dentistry, 2018. 9(2): p. 289-292. 20. alavi, s. and f. gharibpour, effect of universal adhesive on shear bond strength of metal bracket to enamel and dental amalgam. pakistan journal of medical and health sciences, 2021. 15: p. 1765-72. 21. bucur, s.m., d. cocoș, and a. saghin, bond strength of three adhesive systems used for bonding orthodontic brackets. romanian journal of oral rehabilitation, 2020. 12(1): p. 162-7. 22. pashley, d.h., et al., state of the art etch-and-rinse adhesives. dental materials, 2011. 27(1): p. 1-16. 23. ostby, a.w., et al., influence of self-etchant application time on bracket shear bond strength. the angle orthodontist, 2007. 77(5): p. 885889. 24. protásio, m.f., et al., effects of application mode of selfetching primer on shear bond strength of orthodontic brackets. revista portuguesa de estomatologia, medicina dentária e cirurgia maxilofacial, 2016. 57(1): p. 9-13. 25. yesilyurt, c. and b. bulucu, bond strength of total-etch and self-etch dentin adhesive systems on peripheral and central dentinal tissue: a microtensile bond strength test. j contemp dent pract, 2006. 7(2): p. 26-36. 26. triani, f., et al., universal adhesives: evaluation of the relationship between bond strength and application strategies—a systematic review and meta-analyses. coatings, 2022. 12(10): p. 1501. 27. kharouf, n., et al., does etching of the enamel with the rubbing technique promote the bond strength of a universal adhesive system? the journal of contemporary dental practice, 2020. 21(10). 28. reicheneder, c.a., et al., shear and tensile bond strength comparison of various contemporary orthodontic adhesive systems: an in-vitro study. american journal of orthodontics and dentofacial orthopedics, 2009. 135(4): p. 422. e1-422. e6. 29. breschi, l., et al., dental adhesion review: aging and stability of the bonded interface. dental materials, 2008. 24(1): p. 90-101. 30. king, n.m., et al., effect of resin hydrophilicity on watervapour permeability of dental adhesive films. european journal of oral sciences, 2005. 113(5): p. 436-442. 31. ito, s., et al., water sorption/solubility of self-etching dentin bonding agents. dental materials, 2010. 26(7): p. 617626. 32. sirisha, k., et al., validity of bond strength tests: a critical review-part ii. journal of conservative dentistry and endodontics, 2014. 17(5): p. 420426. 33. carrilho, m., et al., in vivo preservation of the hybrid layer by chlorhexidine. journal of dental research, 2007. 86(6): p. 529-533. impact of rubbing and wai2ng technique of a universal adhesive system on shear bond strength of orthodon2c brackets: a compara2ve analysis vol 13 no 1 (2025) doi 10.5195/d3000.2025.812 h#p://den*stry3000.pi#.edu 34. mazzoni, a., et al., effects of etch-and-rinse and self-etch adhesives on dentin mmp-2 and mmp-9. journal of dental research, 2013. 92(1): p. 82-86. 35. marchesi, g., et al., adhesive performance of a multimode adhesive system: 1-year in vitro study. journal of dentistry, 2014. 42(5): p. 603-612. 36. el-malky, w. and k. abdelaziz, the effect of precuring waiting time of different bonding resins on micro-tensile bond strength to dentin. tanta dental journal, 2015. 12(2): p. 99110. 37. el-din, a., effect of changing application times on adhesive systems bond strengths. american journal of dentistry, 2002. 15(5): p. 321-324. 38. de carvalho cardoso, p., et al., effect of prolonged application times on resin-dentin bond strengths. journal of adhesive dentistry, 2005. 7(2). 39. dal-bianco, k., et al., effects of moisture degree and rubbing action on the immediate resin– dentin bond strength. dental materials, 2006. 22(12): p. 11501156. 40. caneppele, t.m.f., et al., effects of surface hydration state and application method on the bond strength of self-etching adhesives to cut enamel. journal of adhesive dentistry, 2012. 14(1). 41. papadogiannis, d., et al., universal adhesives: setting characteristics and reactivity with dentin. materials, 2019. 12(10): p. 1720. 42. iijima, m., et al., effects of application time and agitation for bonding orthodontic brackets with two self-etching primer systems. dental materials journal, 2009. 28(1): p. 89-95. 712 2024 the combined effect of two natural extracts on enamel remineraliza;on (in vitro study) vol 13 no 2 (2024) doi 10.5195/d3000.2024.712 h#p://den*stry3000.pi#.edu the combined effect of two natural extracts on enamel remineralization (in vitro study) shatha a. abbas1, alhan a. qasim1, rusul j. hadi1, sarah m. khammas3, asmaa m. khammas1 1college of den*stry, university of baghdad, baghdad, iraq 2department of den*stry, al hikma university college, baghdad, iraq abstract objec&ve: dental caries is a chronic infec/ous disease that is prevalent worldwide in all age groups. numerous a9empts have been made to develop conserva/ve approaches to halt caries progression and restore enamel defects. this study aimed to inves/gate the effect of applying grape seed extract and chicken eggshell extract on the microhardness of demineralized enamel in permanent teeth. methods: forty-eight sound upper first premolars were used. following demineraliza/on with the demineralizing solu/on for 96 hours, they were distributed into four groups consistent with the treatment agent used: group a was treated with casein phosphopep/de amorphous calcium phosphate (as a control group), group b was treated with grape seed extract, group c was treated with chicken eggshell extract solu/on, and group d was treated with grape seed extract followed by chicken eggshell extract solu/on. vickers microhardness measurements were performed on sound enamel, aner demineraliza/on and remineraliza/on. results: paired t-test, one-way anova, and tukey’s hsd test were used for sta/s/cal analysis. enamel microhardness was significantly reduced following demineraliza/on (p=0.000) and significantly increased aner remineraliza/on, with group d showing the highest values (mean microhardness=218.99). conclusion: grape seed extract and chicken eggshell extract solu/ons have a synergis/c effect on enamel remineraliza/on which was interpreted from the increase in surface microhardness values. keywords: caseins, eggshell, enamel, grape seed extract, tooth remineraliza/on. cita:on: abbas s, et al. (2024) the combined effect of two natural extracts on enamel remineraliza:on (in vitro study). den:stry 3000. doi:10.5195/d3000.2024.712 received: august 21, 2024 accepted: september 1, 2024 published: october 3, 2024 copyright: ©2024 abbas s, et al. this is an open access ar:cle licensed under a crea:ve commons auribu:on work 4.0 united states license. email: shatha.a@codental.uobaghdad.edu.iq introduction dental caries is a dynamic process that is influenced by the balance of pathological and defensive mechanisms [1]. the remineralization of dental tissues is characterized by the re-deposition of minerals, particularly calcium, and phosphate, which are supplied by therapeutic agents into the gaps between hydroxyapatite crystals of demineralized enamel, resulting in mineral gain [2]. casein phosphopeptide amorphous calcium phosphate (cpp-acp) is a supplement for calcium and phosphate ions derived from milk protein [3]. the anti-cariogenic mechanisms have been suggested to keep the enamel minerals calcium and phosphate supersaturated in the dental plaque, thereby interrupting biofilm formation, hindering bacterial adhesion on the enamel surface, and buffering ph reduction in the oral environment [4]. thousands of years ago, therapeutic agents were prepared from natural products that have been considered promising sources for new medicines, particularly in oral diseases, such as dental caries [5]. chicken eggshell powder (cesp) has various medical applications owing to its elevated calcium concentration (approximately 93%) and increased bioavailability [6]. cesp can be used as a coating material in dental implants [7] and as a remineralizing agent against the combined effect of two natural extracts on enamel remineraliza;on (in vitro study) vol 13 no 2 (2024) doi 10.5195/d3000.2024.712 h#p://den*stry3000.pi#.edu dental caries and enamel erosion with promising results [8-10]. grape seed extract (gse) showed a noticeable effect on the prevention and restoration of demineralized teeth by stabilizing the collagen matrix and inhibiting its breakdown. it has several beneficial properties, but high levels of proanthocyanidins are thought to be responsible for its role in caries prevention [11]. previous studies used the two extracts separately for enamel remineralization. this study aimed to evaluate the combined effect of cesp and gse on the microhardness of demineralized enamel. the null hypothesis states that there is no synergistic effect between cesp and gse in remineralization of enamel. material and methods tooth preparation upper first premolars extracted from humans were used, any teeth that had cracks or dental caries, as shown by a magnifying lens, were excluded. rubber cup and non-fluoridated pumice were used to polish the samples, which were then stored in deionized water containing 0.1% thymol [12]. each tooth was coated with nail varnish (acid-resistant), except for a window of 2 × 2 mm (in dimension) left in the middle of the buccal surface. these windows were flattened with sof-lex disks (3m espe, usa) in an advanced manner [13]. sample the sample size was calculated using g power 3.0.10 program [14]. alpha error of probability=0.05 two-sided was used and 95% was the power of the study [15]. the sample size was 48 teeth (each group consisted of 12 samples). the samples were randomly distributed into four groups after demineralization. treatment with different agents was conducted for 14 days. group a: treatment with cpp-acp cream (gc tooth mousse, gc co, usa) for 3 min daily, then the cream was wiped off with a cotton piece [16] (as control). group b: treatment with grape seed extract solution for 2 min daily [17]. group c: treatment with cesp solution for 5 min daily. group d: treatment with grape seed extract solution for 2 min, then with cesp solution for 5 min. following the treatment procedure, the samples were washed with deionized water (2 min for each separately) daily and stored until the next day. preparation of grape seed extract solution gse powder (bulk supplements, usa) was dissolved in a phosphate buffer solution (0.025 m kh2po4, ph=7.4) to obtain a concentration of 6.5% [18]. preparation of cesp solution the calcination protocol of chicken eggshells was performed according to the world property intellectual organization [19]. a gram of the resultant powder was dissolved with 20 ml of acetic acid (concentration =4%), and only the clear solution was used as a remineralizing agent [20]. demineralizing protocol the following concentrations were used to prepare demineralizing solution: acetic acid (0.05 m), sodium dihydrogen orthophosphate dehydrate (2.2 mm), and calcium chloride (2.2 mm). the solution ph was 4.4 (modified by the addition of 1m potassium hydroxide). each tooth was immersed separately for four consecutive days (96 h) to stimulate enamel demineralization the combined effect of two natural extracts on enamel remineraliza;on (in vitro study) vol 13 no 2 (2024) doi 10.5195/d3000.2024.712 h#p://den*stry3000.pi#.edu [21]. the solution was changed every day. finally, the samples were washed thoroughly and kept in deionized water. measurement of enamel microhardness the microhardness was measured using a digital vickers microhardness tester (fischer technology, inc., usa) for sound enamel, following demineralization, and after treatment with the selected agent. the load was 500g for 30 sec. for each reading, the tooth was subjected to three indentations, and the average was calculated to denote its hardness value. statistical analysis spss-26 program was used for analysis. one-way anova, paired t-test, and tukey’s hsd test were used for statistical analysis. results baseline surface microhardness (smh) was not significantly different among the groups (p=0.11, f-value=2.14), as shown in figure (1). following demineralization, smh significantly decreased in all samples (p=0.000). table 1 describes the difference among the groups at the sound and demineralization stages. following treatment with the remineralizing agents, enamel smh significantly increased (p=0.000, f=203.87). smh increased compared with that after demineralization (p=0.000) (table 2). tukey's test revealed that group d had the highest smh value followed by group c (with a non-significant difference between them p=0.94) then groups a and b (table 3). discussion dental tissue remineralization is the preferred preventive measure for dental caries [22]. many studies have focused on the use of natural products for disease management [5,23]. the present study selected gse and cesp because they are available and economical and have advantageous properties. baseline smh was not significantly different among the groups, indicating a relative approximation in the mechanical appropriates of the samples. following demineralization, enamel microhardness significantly reduced. initial enamel lesions have fewer minerals, resulting in a reduced microhardness value [8]. following remineralization, enamel microhardness significantly increased in all samples. group d showed the highest smh indicating that the combination gse and cesp had a synergistic effect on remineralizing the enamel surface. a possible explanation is that gallic acid, a major constituent of gse [24], promotes the deposition of cesp minerals, primarily on the enamel surface. this is consistent with a preceding study which concluded that gse could enhance enamel remineralization by increasing smh [25]. mirkarimi et al. (2013) reported the remineralizing property of gse by forming insoluble deposits on enamel by using a scanning electron microscope and reported elevated microhardness value following demineralization [26]. yassen and safy (2018) compared gse with sodium fluoride (naf) by vmh and found a non-significant difference between them in dentin remineralization [27]. amin et al. (2019) found that gse could increase smh but the value is lower than that achieved by naf [28]. the current study demonstrated the gse remineralizing efficacy as evident by a significant increase in smh of group b following demineralization. furthermore, cesp has a high calcium concentration and elevated ph [29], causing a thorough blockage of surface pores and a net increase in enamel the combined effect of two natural extracts on enamel remineraliza;on (in vitro study) vol 13 no 2 (2024) doi 10.5195/d3000.2024.712 h#p://den*stry3000.pi#.edu microhardness8. preceding studies stated that cesp could increase smh [9] and decrease the surface roughness of enamel affected by erosive lesion [20]. cesp also increases smh and decreases lesion depth of demineralized enamel [10], in addition to increasing the ca/p ratio and acid resistance of enamel [30]. this finding can clarify the increase in the enamel microhardness of both groups (d & c with a nonsignificant difference between them) following exposure to ceps solution, and the value is higher than those of other groups. no previous study used the same combination to compare with this result. ccp-acp has remineralizing properties, which have been proven previously. the cpp/acp paste treatment of the enamel surface results in filling the interprism voids and, to some extent, covering the enamel prisms with a layer-like structure [16], thereby increasing the smh of group a samples. the demineralization procedure used in the current study induced the loss of minerals from the subsurface layer along with preserving calcium and phosphate in the superficial layer [31]. vmh measurement is a quick, nondestructive method for the assessment of surface layer changes in demineralization and remineralization [32]. flat and polished samples were used for standardization and elimination of naturally occurring variances between teeth that result in different reactions to acid dissolution [33]. finally, consistent with the obtained outcomes, the null hypothesis was rejected, and the presence of a synergistic effect between gse and cesp in enamel remineralization was confirmed. conclusion gse and cesp had a positive role in enamel remineralization. the combination improved the mineral gain and enhanced the mechanical properties of the enamel surface following demineralization, as shown by the increased smh values. the findings of this study are limited because it was conducted in vitro. future clinical studies should verify the outcome presented. additionally, different application techniques, time intervals, and variables are recommended to investigate the combined effect of these extracts. ethical approval this study was approved by the research ethics committee of the college of dentistry, university of baghdad (reference number: 721, date 28-12-2022). conflicts of interest the authors declare no competing interest. references 1. obaid sf, munther s, abbas mh. relakonship between salivary levels of protein carbonyl and total ankoxidant capacity and prevalence of dental caries among type 1 diabekc children: an analykcal cross-seckonal study. dental hypotheses. 2023;14(2):59. doi: 10.4103/denthyp.denthyp_44_23. 2. arifa mk, ephraim r, rajamani t. recent advances in dental hard kssue remineralizakon: a review of literature. internakonal journal of clinical pediatric denkstry. 2019;12(2):139. doi: 10.5005/jpjournals-10005-1603 3. aoar ij, ghaib nh. the effect of some remineralizakon materials and resin infiltrakon (icon) on the depth of white spot lesions. j bagh coll denkstry. 2018;30(2). hops://doi.org/10.12816/0049755 4. reema sd, lahiri pk, roy ss. review of casein phosphopepkdesamorphous calcium phosphate. chin j dent res. 2014;17(1):7-14. pmid: 25028684 the combined effect of two natural extracts on enamel remineraliza;on (in vitro study) vol 13 no 2 (2024) doi 10.5195/d3000.2024.712 h#p://den*stry3000.pi#.edu 5. turki oh, jafar zj. whitening effect of juglans regia dry husk extract on primary and permanent teeth. internakonal journal of biomaterials. 2023;2023. 6. park jw, bae sr, suh jy, lee dh, kim sh, kim h, et al. evaluakon of bone healing with eggshell-derived bone gras subsktutes in rat calvaria: a pilot study. journal of biomedical materials research part a. 2008;87(1):203-14. 7. kadhim dr, hamad ti, fatalla aa. use of eggshells as bone grass around commercially pure ktanium implant screws coated with nano calcium sulfate. internakonal journal of biomaterials. 2022;2022. hops://doi.org/10.1155/2022/872 2283 8. haghgoo r, mehran m, ahmadvand m, ahmadvand mj. remineralizakon effect of eggshell versus nano-hydroxyapakte on caries-like lesions in permanent teeth (in vitro). journal of internakonal oral health. 2016;8(4):435. 9. yaberi m, haghgoo r. a comparakve study of the effect of nanohydroxyapakte and eggshell on erosive lesions of the enamel of permanent teeth following sos drink exposure: a randomized clinical trial. journal of internakonal oral health. 2018;10(4):176. doi:10.4103/jioh.jioh_84_18 10. mohamed eo, sharaf aa, talaat dm, nagui da. remineralizakon effect of eggshell powder and novamine on inikal caries-like lesions in young permanent teeth (in-vitro study). alexandria dental journal. 2020;45(2):52-9. 11. delimont nm, carlson bn. prevenkon of dental caries by grape seed extract supplementakon: a systemakc review. nutrikon and health. 2020;26(1):43-52. doi: 10.1177/0260106019887890 12. hassan nm, jafar zj, abdul lakf mh. nano-hydroxyapakte preparakon for the remineralizakon of primary tooth enamel surface subjected to liquid medicakon: an observakonal study. health science reports. 2023;6(4):e1188. 13. yahya y, alrawi n. remineralizing efficacy of biomimekc self-assembling pepkde on arkficially induced enamel lesions (in vitro study). j res med dent sci,. 2020;8 (7): 40712. 14. faul, f., erdfelder, e., buchner, a., & lang, a.-g. stakskcal power analyses using g*power 3.1: tests for correlakon and regression analyses. behavior research methods. 2009;41: 1149-1160. 15. cohen j. stakskcal power analysis for the behavioral sciences. 1988 (2nd ed) (hillsdale, nj:erlbaum). 16. poggio c, lombardini m, vigorelli p, ceci m. analysis of denkn/enamel remineralizakon by a cpp-acp paste: afm and sem study. scanning: the journal of scanning microscopies. 2013;35(6):366-74. 17. tang cf, fang m, liu rr, dou q, chai zg, xiao yh, chen jh. the role of grape seed extract in the remineralizakon of demineralized denkne: micromorphological and physical analyses. archives of oral biology. 2013 dec 1;58(12):176976. doi: 10.1016/j.archoralbio.2013.0 9.007 18. saragih d, herda e, triaminingsih s, editors. the influence of topical applicakon of grapeseed extract gel on enamel surface hardness aser demineralizakon. journal of physics: conference series; 2017: iop publishing. 19. suzuki f, wang p-y., weatherspoon j, mead l. method of producing eggshell powder. patents: us 20060062857 a1. 2004. 20. feroz s, moeen f, haq sn. proteckve effect of chicken egg shell powder solukon (cesp) on arkficially induced dental erosion: an in vitro atomic force microscope study. internakonal journal of dental sciences and research. 2017;5(3):49-55. doi:10.12691/ijdsr-5-3-2 21. iohagarun a, verma s, lalloo r, king n, wefel j, nair r. effects of fluoridated milk on arkficial enamel carious lesions: a ph cycling study. journal of denkstry. 2011;39(12):817-24. the combined effect of two natural extracts on enamel remineraliza;on (in vitro study) vol 13 no 2 (2024) doi 10.5195/d3000.2024.712 h#p://den*stry3000.pi#.edu 22. leal sc, dame-teixeira n, barbosa cdb, kominami paa, raposo f, nakagawa emt, et al. minimum intervenkon oral care: defining the future of caries management. brazilian oral research. 2022;36. doi: 10.1590/18073107bor-2022.vol36.0135. 23. obeid rs, khalaf ms. impact of different types of storage media on enamel surface roughness and granularity distribukon of avulsed teeth (in vitro study). j bagh coll denkstry. 2022;34(4):34-43. 24. cheng l, li j, hao y, zhou x. effect of compounds of galla chinensis on remineralizakon of enamel surface in vitro. archives of oral biology. 2010;55(6):43540. doi: 10.1016/j.archoralbio.20 10.03.014. 25. rubel m, prashant gm, naveen kumar pg, sushanth vh, imranulla m, potlia i, mallick s. effect of grape seed extract on remineralizakon of arkficial caries: an in-vitro study. asian j pharm clin res. 2016;9(5):174-6. 26. mirkarimi m, eskandarion s, bargrizan m, delazar a, kharazifard mj. remineralizakon of arkficial caries in primary teeth by grape seed extract: an in vitro study 2013;7(4):206-10. 27. yassen aa, safy rk. grape seed extract and denkn remineralizakon. egypkan dental journal. 2018;64:1719-26. 28. amin ra, awad sm, abd el saoar ea. evaluakon of remineralizakon of grape seed extract versus sodium fluoride on demineralized primary anterior teeth: an in-vitro study. egypkan dental journal. 2019;65: 1977-84. 29. tangboriboon n, kunanuruksapong r, sirivat a. preparakon and properkes of calcium oxide from eggshells via calcinakon. materials sciencepoland. 2012;30(4):313-22. hops://doi.org/10.2478/s13536012-0055-7. 30. elshik ae, etman wm, genaid tm. resistance of chicken eggshell powder-treated demineralized enamel to further acidic challenges. tanta dental journal. 2020;17(2):38. 31. magalhães ac, moron b, comar l, wiegand a, buchalla w, buzalaf mar. comparison of crossseckonal hardness and transverse microradiography of arkficial carious enamel lesions induced by different demineralizing solukons and gels. caries research. 2009;43(6):474-83. doi: 10.1159/000264685 32. ren y-f, zhao q, malmstrom h, barnes v, xu t. assessing fluoride treatment and resistance of dental enamel to sos drink erosion in vitro: applicakons of focus variakon 3d scanning microscopy and stylus profilometry. journal of denkstry. 2009;37(3):167-76. 33. hemingway c, parker d, addy m, barbour m. erosion of enamel by non-carbonated sos drinks with and without toothbrushing abrasion. briksh dental journal. 2006;201(7):447-50. pmid: 17031352. the combined effect of two natural extracts on enamel remineraliza;on (in vitro study) vol 13 no 2 (2024) doi 10.5195/d3000.2024.712 h#p://den*stry3000.pi#.edu figure 1. baseline enamel microhardness. table 1. microhardness at baseline and a=er demineraliza@on. groups baseline mean ± sd after demineralization mean ± sd mean difference ± se 95% confidence interval of the difference t-test correlation pvalue lower upper a 342.18 ± 0.54 175.62 ± 4.83 166.56 ± 1.42 163.43 169.68 117.23 -0.1040.000 b 343.03 ± 2.85 174.50 ± 3.34 168.53 ± 0.93 166.47 170.58 180.46 0.464 0.000 c 343.75 ± 1.31 176.22 ± 1.75 167.53± 0.52 166.37 168.68 319.89 0.326 0.000 d 342.51 ± 0.52 176.31 ± 3.24 166.20 ±0.98 164.03 168.37 168.41 -0.2480.000 the combined effect of two natural extracts on enamel remineraliza;on (in vitro study) vol 13 no 2 (2024) doi 10.5195/d3000.2024.712 h#p://den*stry3000.pi#.edu table 2. microhardness a=er demineraliza@on and a=er remineraliza@on . study group demineraliza@on mean ± sd remineraliza@on mean ± sd mean difference ± se 95% confidence interval of the difference t-test correla@on p-value lower upper a 175.62 ± 4.83 198.68 ± 4.09 -23.05±1.92 -27.28 -18.82 -12.003-.1040.000 b 174.50 ± 3.34 180.94 ± 2.95 -6.44±1.25 -9.20 -3.68 -5.1380.053 0.000 c 176.22 ± 1.75 217.84 ± 2.19 -41.62±0.90 -43.60 -39.64 -46.239-0.2380.000 d 176.31 ± 3.24 218.99 ± 6.80 -42.68±1.18 -45.28 -40.07 -35.992 0.904 0.000 table 3. post hoc test (tukey hsd) for groups microhardness at the remineraliza@on stage. groups mean difference ± se p-value 95% confidence interval lower limit upper limit a & b 17.73 ± 1.78 0.000 12.9660 22.5073 a& c 19.16 ± 1.78 0.000 23.9373 14.3960 a& d 20.31 ± 1.78 0.000 25.0806 15.5394 b & c 36.90 ± 1.78 0.000 41.7640 32.1327 b & d 38.04 ± 1.78 0.000 42.8173 33.2760 c & d 1.14 ± 1.78 0.918 5.1940 3.6273 777 final evalua&on of postopera&ve pain intensity following the use of three different canal prepara&on systems: a randomized clinical trial vol 13 no 1 (2025) doi 10.5195/d3000.2025.777 h#p://den*stry3000.pi#.edu evaluation of postoperative pain intensity following the use of three different canal preparation systems: a randomized clinical trial salma issa daheem ashur university, college of dentistry, iraq abstract objec&ve: the objec)ve of this study was to compare the intensity of postopera)ve pain a8er hand protaper prepara)on, wave one gold and a hyflex edm instrument. materials and methods: thirty pa)ent were randomly divided into three groups. endodon)c treatment was performed for premolar and molar root canals, and they were filled with guha-percha and an epoxy resin-based root canal sealer using a lateral condensa)on technique. teeth were restored using a resin composite material. a single operator performed the treatments in a single visit for asymptoma)c molars, and the pa)ents were asked to record their pain severity during a 12, 24, 36, and 72 h follow-up period using a visual analog scale (vas). one-way anova and tukey’s mul)ple comparison test were used for sta)s)cal comparisons. results: up to 12 hours, postopera)ve pain was significantly higher in the group treated by hyflex edm (p < 0.01). no differences were seen from 36 hours to 7 days. conclusion: for all three techniques, higher pain intensity was seen at 12 hours and then the pain steadily decreased. no significant difference was noted in quality of life, sugges)ng the filling systems or techniques has a similar effect. keywords: asymptoma)c molars; endodon)c treatment; postopera)ve pain; wave one gold; hyflex edm; hand protaper. cita9on: daheem si. (2025) evalua9on of postopera9ve pain intensity following the use of three different canal prepara9on systems: a randomized clinical trial. den9stry 3000.1:a001 doi:10.5195/d3000.2025.777 received: november 21, 2024 accepted: december 19, 2024 published: february 12, 2025 copyright: ©2025 daheem si. this is an open access ar9cle licensed under a crea9ve commons awribu9on work 4.0 united states license. email: salmaalabudi22@gmail.com introduction postoperative pain, which is a complex and multi-factorial process, may develop even following an ideal root canal treatment. during root canal preparation, apical extrusion of debris, irrigants, and/or bacteria can occur, potentially leading to such complications as post operative pain, flare-ups, or even treatment failure [1]. apical extrusion has been reported as the main cause of pain after completion of endodontic treatment [2]. which factors increase the number of extruded debris remains controversial; studies have demonstrated associations with the type of file motion, to working length, crosssection, tip, taper, flexibility, heat treatment, and number of files used [2]. during canal instrumentation, dentine chips, pulp tissue fragments, necrotic tissue, bacteria, and intracranial irritants may frequently be extruded into the apical foramen, causing post-operative pain. additionally, it has been proposed that the reciprocating motion itself may contribute to the packing of the debris into the irregularities of the root canal space, raising the possibility of post-operative discomfort. employing nickel–titanium (niti) shaping instruments in rotary or reciprocating motion reduces cycle fatigue and improves root canal centering ability. because varying amounts of irritants are extruded from the root canal area, different nickel–titanium (ni-ti) evalua&on of postopera&ve pain intensity following the use of three different canal prepara&on systems: a randomized clinical trial vol 13 no 1 (2025) doi 10.5195/d3000.2025.777 h#p://den*stry3000.pi#.edu rotational systems may produce varied patterns of neurogenic inflammatory response in the periodontal ligament [3]. there are numerous potential causes of post endodontic discomfort. during chemo mechanical preparation, the extrusion of pulp tissue, microbes, and irrigants to the periapical tissues may result in inflammation. in recent years, numerous systems with novel designs have been introduced because of significant advancements in rotating instrumentation and metallurgy. despite, all preparation methods and tools currently in use are still connected to some degree of extrusion of debris, which can result in post endodontic discomfort [4]. frequency of postoperative endodontic pain is common, with a reported frequency ranging between 25% and 40%. the hyflex edm (coltene/whaledent, switzerland) niti gpf is a gpf file system produced using an innovative manufacturing process called electrical discharge machine with a controlled memory (cm) wire. in edm, instead of conventional grinding, electric discharges are used to shape the file via melting and vaporization of the material [5]. this method creates a cratered surface, which further increases the file’s fatigue resistance and lifetime. the hyflex edm gpf consists of a single file with a tip size of 10 and a 5% taper. the cross-section of the hyflex edm gpf varies along its length, being quadratic at the tip, trapezoidal in the middle, and triangular at the shaft. the flexibility of the hyflex edm gpf confers this instrument the ability to maintain the apical canal curvature despite its greater taper [6]. protaper (universal) files are shaped as convex, triangular, and cross-sectional, with a guiding tip, and a factually variable helical angle and slope. initially, handused files with universal design were a revolutionary advancement in endodontics but mechanical preparations became faster with rotary instruments, and it is easier to maintain original anatomy, centered position, and taper of the root canals [6]. thus, improved and better forms of biomechanical preparation are being developed, which include the coronal to apical approach techniques, having advantages such as less debris extrusion and elimination of coronal interferences. protaper rotary system was started with protaper universal, then improved to protaper gold and one of the recent refinements is protaper next files with m-wire technology, imparting improved flexibility and less cyclic fatigue both protaper universal and protaper gold have the same convex triangular crosssections, but protaper next have off-centered, rectangular crosssections. the purpose of the present study was to evaluate the incidence, intensity and prediction of postoperative pain after glide path preparation performed with hand protaper instruments, wave one gold and a hyflex edm [7]. material and methods a total of 30 patients were selected from the outpatients of our dental clinic. patients were selected with asymptomatic nonvital lower first molars with three separate canals and without periapical lesion and patients with medically compromised patients were excluded the study included teeth diagnosed with symptomatic/asymptomatic irreversible pulpitis or apical periodontitis was diagnosed according to the absence of clinical symptoms, responsiveness to pulp sensibility tests and the presence of a periapical radiolucency the clinical diagnosis of symptomatic irreversible evalua&on of postopera&ve pain intensity following the use of three different canal prepara&on systems: a randomized clinical trial vol 13 no 1 (2025) doi 10.5195/d3000.2025.777 h#p://den*stry3000.pi#.edu pulpitis was based on positive pulp sensibility test result, extensive restorations or fractures exposing the pulp and x-ray. symptomatic apical periodontitis was diagnosed according to the presence of painful response to biting / percussion / palpation, spontaneous pain and peri radicular radio graphical features varying from a normal periapical structure to a periapical radiolucency, whereas asymptomatic apical periodontitis. other exclusion criteria were the following: patients younger than 18 years, patients with vertical root fractures, excessive periodontal disease, teeth that need periodontal surgery prior to coronal restorations due to marginal deficiency. inclusion criteria for patients were as follows: age between 18-45 years, both males and females, medically-free patients, mandibular posterior teeth (premolars and/ or molars) with vital or non-vital pulp without periapical pathosis, symptomatic or asymptomatic cases, and positive patients’ acceptance for participation in the study. grouping the samples group i: hand protaper (n=10) after preparing the glide path with 15 k file, the coronal portion of canals were prepared using hand protaper sx, s1, s2 (shaping files), f1–f5 (finishing files) within 0.25 tip size and 8% taper. and obturated by ah plus sealer. group 2: hyflex edm (n=10) teeth and obturated by ah plus sealer. group3: (n=10) prepared by wave one gold and obturated by ah plus sealer., the root canals were prepared with primary wave one gold file (0.25 tip size and 7%taper) till full working length. teeth were prepared by pro taper, wave one gold, hyflex edm according to manufacture instructions. the master apical file and irrigation was done between each file using plastic syringe with side perforated 27-g needle containing 2.5% sodium hypochlorite and another one containing 17% edta solution. master apical file was done by 35 size k file. after instrumentation each canal was flushed with saline and then dried by paper point. all teeth obturation was made by lateral condensation technique where ah plus sealer was introduced using lentulo spiral and master cone was introduced, and the accessory cones were added after it. in group 3, waveone (dentsply maillefer) was used for cleaning and shaping root canals. each tooth was prepared at 0.5 mm from the apex. primary (red; size 25, 0.08) or large (black; size 40, 0.08) files were used according to root canal diameter. irrigation and final irrigation were performed using protocols like those used in patients in groups 1, 2 and 3. the single-cone technique was used to introduce to seal the root canal by means of a matching gutta-percha cone (wave-one) in brushing motion. accessory guttapercha cones [25] were used if needed employing the noncompaction method coronal restorations were performed using total-etch adhesive system (single bond, 3m espe), according to the manufacturer’s instructions. root canal orifices were sealed using a flowable composite resin (filtek ultimate flowable, 3m espe) as the base material. remaining coronal restorations were performed using composite resin (filtek ultimate, 3m espe) restoration performing fixed restoration depending on the prosthetic plan. post-clinical procedures patients are asked to evaluate the pain level & although the patients are not prescribed an analgesics, they can take if needed. assessment of post-operative pain by using the visual analogue scale (vas) described by pinkham et al. the vas consists of a list of adjectives describing different levels of pain intensity with scores assigned to each of the levels of pain intensity (table 1). the (vds) was translated into colloquial arabic. evalua&on of postopera&ve pain intensity following the use of three different canal prepara&on systems: a randomized clinical trial vol 13 no 1 (2025) doi 10.5195/d3000.2025.777 h#p://den*stry3000.pi#.edu table 1. description of levels of pain intensity. 0 1 2 3 4 5 no pain slight pain moderate pain strong pain severe pain maximum pain 0= no pain 1= moderate pain relieved by analgesics 2= slight pain/discomfort 3= moderate pain not completely relieved by analgesics 4= severe pain not completely relieved by analgesics 5= severe pain/swelling not relieved by analgesics and required an unscheduled visit statistical analysis the data collected was statistically analyzed using statistical software. kruskal wallis anova nonparametric test was carried out to determine the comparison between the three groups at different time intervals and mann whitney u test was used to do the intra group comparison at significant difference of pvalue <0.05 results statistical analysis indicated significant difference in pain intensity between 6, 12, 24, 36, and 72 hours and 7 days among the groups (p = 0.001). when pairwise comparisons of 6-, 12-, 18-, and 24-hour pain intensity values were compared, group 3 (waveone) group presented the highest pain intensity values, followed by patients treated using group 2 (p = 0.001) and one shape (p = 0.001). pain intensity values for patients treated using group 1 (p < 0.01) were significantly higher than those of patients treated using one wave (p = 0.001). none of the patients reported any postoperative pain at 1-week follow-up. two patients from the hyflex edm, three patients from the one wave, and six patients from the hand protaper group used analgesics (naproxen sodium) to reduce the pain which represents the number of debris extruded and the actual instrumentation time (in seconds) for all groups. there was no significant difference between the hyflex edm and one wave groups (p > 0.05), again, there was no significant difference between all (p > 0.05) after 1 week. the highest mean value of pain score was found in hand protaper have the least mean value of pain scores was found in (hyflex edm) (p =0.33 ± 0.32). table 1. mean preoperative and six postoperative vas scores with median, minimum and maximum values of nrs scores and standard deviation (sd) at different time points in the tested groups. group time mea n ± sd rang e (minmax) hand protape r preop 2.42 ± 1.20 1 5 12h 3.00 ± 0.72 1 4 24h 1.20 ± 0.98 0 3 36h 1.00 ± 0.30 0 1 wave one gold preop 2.10 ± 1.20 1 5 12h 1.30 ± 0.40 3 4 24h 1.00 ± 0.25 0 3 36h 0.10 ± 0.00 0 1 hyflex edm preop 2.25 ± 0.30 1 5 12h 0.42 ± 0.00 0 2 evalua&on of postopera&ve pain intensity following the use of three different canal prepara&on systems: a randomized clinical trial vol 13 no 1 (2025) doi 10.5195/d3000.2025.777 h#p://den*stry3000.pi#.edu 24h 0.11 ± 0.00 0 1 36h 0.01 ± 0.00 0 1 all groups 1 wee k 0.00 ± 0.00 0 0 table 2. protaper hand and wave one gold. group times sd pvalue hand protaper 12 hr 0.72 0.33 24 hr 0.98 36 hr 0.30 wave one gold 12 hr 0.40 24 hr 0.25 36 hr 0.00 table 3. protaper hand and hyflex edm. group times sd pvalue hand protaper 12 hr 0.72 0.272 24 hr 0.98 36 hr 0.30 hyflex edm 12 hr 0.42 24 hr 0.11 36 hr 0.00 discussion persistent pain after root canal treatment is a common occurrence, with a frequency of 5.4%. it can be classified as either or both odontogenic and nonodontogenic in etiology [9]. while odontogenic origin might be the root canal-treated tooth or the adjacent tooth, non-odontogenic origin was shown to be temporomandibular disorder pain or dentoalveolar pain disorder [10]. to eliminate the risk of persistent pain, which would affect the results of this study, follow-ups were continued up to 1 month to ascertain the complete relief of pain. several factors such as age, gender, pulpal and periradicular status, tooth type, preoperative pain, and technical aspects affect postoperative dental pain. of these factors, only technical aspects, including instrumentation technique, file characteristics, and irrigation and obturation protocols could be controlled by the clinician. these technical aspects, also referred to as operatordependent factors, are the main causes of non-biologic (chemical and mechanical) or biologic (bacterial) injuries during root canal preparation [11]. therefore, in the present study, to limit the effect of variables in the procedure and prevent unwanted interaction with the apical tissues, no chemical solvents were used during the removal of the previous root canal filling. although pain is subjective, biological and clinical factors are often responsible for its initiation [12]. in our study, the total amount of medication intake did not differ between the three instrumentation motions. however, there were some conflicting studies in the 24-hour. various instrumentation systems were associated with some degree of postoperative pain [13]. three clinical trials reported similarity regarding the intensity of postoperative pain between regarding the intensity of postoperative pain between hand protaper file groups; waveone and hyflex edm. some studies have concluded that variables such as gender, age, tooth type and preoperative pain are significant factors for the development of postoperative evalua&on of postopera&ve pain intensity following the use of three different canal prepara&on systems: a randomized clinical trial vol 13 no 1 (2025) doi 10.5195/d3000.2025.777 h#p://den*stry3000.pi#.edu pain. these findings are in accordance with the literature that reported that greater postoperative pain incidence is significantly linked with the presence of preoperative pain [14]. today, preoperative pain is considered as a significant factor for the prediction of postoperative pain. therefore, patients whose chief complaint is endodontic pain could be warned about probable postoperative pain and possible need for analgesic intake. in the present study subjective nature of the pain evaluation method could be considered as a limitation. the visual analogue scale was used to assess pain levels as it is a basic method with greater reliability, validity and sensitivity than descriptive scales [15]. pain was followed up to 72 hours after the completion of root canal treatment as the incidence and intensity of pain were the greatest in the first 24 hours and then decreased substantially after 48 hours. as the follow up period of postoperative pain included the first 48 to 72 hours after treatment in several clinical studies [16]. another limitation was the inability of blinding the operators regarding the groups; however, assignment of the patients to the experiment groups was performed after working length determination just prior to the root canal preparation step to minimize a possible selection bias. the systems associated with the most apical extrusion has been reported as the main cause of pain after endodontic treatment. debris extrusion was those that required longer instrumentation times, indicating a possible correlation as demonstrated by simple linear regression that the longer the instrumentation time, the greater the number of debris extruded [16]. likewise, dincer et al. [17] demonstrated that the protaper system extruded more debris and required longer instrumentation times compared to the wog system, as did ehsani et al. [18], who observed greater extrusion of debris with those systems in which instrumentation took longer. the authors believe this might be explained because using a greater number of files naturally requires a longer instrumentation time, which means more time spent cutting dentin and, consequently, greater formation of debris, which may eventually be extruded through the apex. thus, when working with this type of system, the use of irrigation protocols capable of removing debris from the canal isthmus is paramount. however, when the reciprocating systems included in this study were compared to the other system (which, although rotary, is also a single-file system), the number of extruded debris was found to be similar. gummadi et al. [19] observed greater debris extrusion with the waveone system when compared to the one shape single-file rotary system; however, they analyzed the first generation of this reciprocating system, while the present study used the later waveone gold iteration. the findings of this experiment demonstrate that instrumentation kinematics play a relevant role the number of debris extruded, but that even rotary systems which employ a single file to simplify preparation reduce the risk of debris extrusion compared to multiple-file systems. this can be explained by the fact that using a greater number of instruments can generate a greater number of debris. despite being mentioned by other authors such as amaral et al [20]. as a possible interfering factor in the extrusion of debris, instrument taper was not relevant in the present study. in our experiment, #25 rotary instruments but with smaller tapers generated more debris evalua&on of postopera&ve pain intensity following the use of three different canal prepara&on systems: a randomized clinical trial vol 13 no 1 (2025) doi 10.5195/d3000.2025.777 h#p://den*stry3000.pi#.edu than reciprocating instruments with the same diameter but a relatively larger taper, a finding also reported by dincer et al. [22]. the systems evaluated in this study all have different crosssections, but again, this was not a determining factor in the amount of debris extrusion observed. among the rotary systems analyzed, ptn uses an m-wire alloy, while the hcm system employs a memory niti wire and would thus theoretically be capable of greater canal-centering ability, with less deviation, thus allowing more conservative preparations. nevertheless, this potential advantage was not associated with any difference in the number of debris extruded through the apical foramen of curved canals between the two systems. the greater flexibility of these systems is probably associated with greater canalcentering ability but has no bearing on the number of debris extruded through the apical foramen. the extrusion of microorganisms, material and dentinal debris may cause periapical inflammation. caviedes-bucheli et al. [21] evaluated the expression of substance p along with calcium gene related p. manual protaper techniques compared to instrumentation procedures that include a rotating force, push-andpull canal enlargement actions, such as filing, generate a greater number of apical debris. this led to the formation of a hypothesis that engine-driven rotary devices using the balanced force technique would generate less debris than hand-filing operations, hence lowering the risk of periradicular tissue irritation and postoperative sequelae [22]. similar studies show that using the rotation technique for canal preparation causes less posttreatment pain than k-files [23]. however, kashefinejad et al. [24] discovered that all patients included in the study were successfully treated in a single visit. therefore, the findings of the present study cannot be applied or interpreted for multiple-visit treatments, which warrants for further randomized clinical trials. conclusion within the parameters and limitations of this randomized clinical trial, the following conclusions can be drawn. the intensity of post-operative pain varied between the three groups tested. no significant difference was noted between the parameters of quality of life assessed, suggesting the filing systems or techniques had a similar effect. conflicts of interest the author declares that there is no competing interest. references 1. nixdorf dr, moana-filho ej, law as, mcguire la, hodges js, john mt. frequency of persistent tooth pain aqer root canal therapy: a systemarc review and metaanalysis. j endod 2010;36:224– 230. 2. vijayranvk, khetarpal a, vats a, ahlawat m, singhal n, harshita. comparison of the incidence of postoperarve pain in single sisng root canal treatment aqer using two reciprocarng systems and two conrnuous rotary systems: an in vivo study. j conserv dent 2023; 26(1):12-19 3. arslan h, doğanay e, alsancak m, çapar i d, karataş e, gündüz h a. comparison of apically extruded debris aqer root canal instrumentaron using reciproc instruments with various kinemarcs. int endod j 2016;49:307-10. 4. spohr, a.r.; sarkis-onofre, r.; pereira-cenci, t.; pappen, f.g.; evalua&on of postopera&ve pain intensity following the use of three different canal prepara&on systems: a randomized clinical trial vol 13 no 1 (2025) doi 10.5195/d3000.2025.777 h#p://den*stry3000.pi#.edu dornelles morgental, r. a systemarc review: effect of hand, rotary and reciprocarng instrumentaron on endodonrc postoperarve pain. g. ital. endod. 2019, 33, 24–34. 5. kirchhoff al, fariniuk lf, mello i. apical extrusion of debris in flat-oval root canals aqer using different instrumentaron systems. j endod 2015;41:237-41. 6. herlakian d, cunha rs, ehrhardt ic, zuolo ml, kishen a, da silveira bueno ce. comparison of the incidence of postoperarve pain aqer using 2 reciprocarng systems and a conrnuous rotary system: a prospecrve randomized clinical trial. j endod 2016; 42(2):171–6. 7. mollashahi n f, saberi e a, havaei s r, saber m. comparison of postoperarve pain aqer root canal prepararon with two reciprocarng and rotary single-file systems: a randomized clinical trial. iran endod j 2017;12:15-9. 30. 8. jain n, pawar a m, naganath m, gupta a, daryani h. incidence and severity of postoperarve pain aqer canal instrumentaron with reciprocarng system, conrnuous rotary single file system, versus saf system. endo pract (lond engl) 2016;10:153-60. 9. adiguzel m, tufenkci p, pamukcu ii. comparison of postoperarve pain intensity following the use of three different instrumentaron techniques: a randomized clinical trial. j dent res dent clin dent prospects 2019; 13(2):133–40. 10. alani ma, al-huwaizi h. evaluaron of apically extruded debris and irrigants during root canal prepararon using different rotary instrumentaron systems: an in vitro compararve study. int j med res health sci 2019; 8(2):21– 6. 11. uslu g, özyürek t, yılmaz k, gündoğar m, plorno g. apically extruded debris during root canal instrumentaron with reciproc blue, hyflex edm, and xp-endo shaper nickel-rtanium files. j endod 2018;44. 12. neelakantan p, sharma s. pain aqer single-visit root canal treatment with two single-file systems based on different kinemarcs-a prospecrve randomized mulrcenter clinical study. clin oral invest. 19(9):2211–(2015)7. 17. 13. saber s, nagy m, schäfer e. compararve evaluaron of the shaping ability of waveone, reciproc and oneshape single-file systems in severely curved root canals of extracted teeth. int endod j. 48(1)(2015):109–1856– 9.1. 14. amaral ap, limongi pboc, fontana ce, et al. debris apically extruded by two reciprocarng systems: a compararve quanrtarve study. eur j dent 2019;13:625 15.tavares sg, fontana ce, marrn as de, et al. in vivo evaluaron of painful symptomatology aqer endodonrc treatment performed using two different irrigaron needle inserron depths. eur j dent 2020;14:274-280. 16. pedullà e, lo savio f, boninelli s, et al. torsional and cyclic fargue resistance of a new nickel-rtanium instrument manufactured by electrical discharge machining. j endod 2016;42:156-9. 17. capar id, arslan h, akcay m, ertas h. an in vitro comparison of apically extruded debris and instrumentaron rmes with protaper universal, protaper next, twisted file adaprve, and hyflex instruments. j endod. 2014 oct;40(10):1638-41. 18. ehsani m, robab f, azadeh h. comparison of apical extrusion of debris by using single-file, full evalua&on of postopera&ve pain intensity following the use of three different canal prepara&on systems: a randomized clinical trial vol 13 no 1 (2025) doi 10.5195/d3000.2025.777 h#p://den*stry3000.pi#.edu sequence rotary and reciprocarng systems. journal of denrstry, tehran university of medical sciences, tehran, iran (2016; vol. 13, no. 6). 19. gummadi ac, guddar ak. apical extrusion of debris following the use of single-file rotary/reciprocarng systems, combined with syringe or ultrasonically-facilitated canal irrigaron, j conserv dent. 2019 jul-aug; 22(4): 351–355. 20-amaral ap, limongi pboc, fontana ce, et al. debris apically extruded by two reciprocarng systems: a compararve quanrtarve study. eur j dent 2. 2019;13:625-8. 21. caviedes-bucheli j, moreno jo, carreno cp, et al. the effect of singlefilereciprocarng systems on substance and calcitonin generelatedpeprde expression in human periodontal ligament. int endod j 2013; 46 :419. 22. del fabbro m, afrashtehfar ki, corbella s, elkabbaney a, et al. . in vivo and in vitro effecrveness of rotary nickel-rtanium vs manual stainless-steel instruments for root canal therapy: systemarc review and metaanalysis. j evid based dent pract. 18(1): 59-69. 23. duque ja, duarte m. compararve effecrveness of new mechanical irrigant agitarng devices for debris removal from the canal and isthmus of mesial roots of mandibular molars. j endod 2017;43:326-331. 23. caviedes-bucheli j, muñoz hr, azuero-holguín mm, ulate e. neuropeprdes in dental pulp: the silent protagonists. j endod. 2013;34:773–788. 24. kashefinejad m, harandi a, eram s, bijani a. comparison of single visit post endodonrc pain using two rotary and hand k-file instruments: a randomized clinical trial. j dent (tehran). 2016 jan; 13(1): 10–17. 813 final periodontal and biochemical correla1ons during alendronate treatment in postmenopausal women: a longitudinal study vol 13 no 1 (2025) doi 10.5195/d3000.2025.813 h#p://den*stry3000.pi#.edu periodontal and biochemical correlations during alendronate treatment in postmenopausal women: a longitudinal study aseel jalil ibrahim al-karawi1, hajer ali ahmed hentati 2 1ibn el jazzar faculty of medicine, university of sousse, tunisia, and ministry of health, diyala. iraq 2faculty of dental medicine, laboratory of oral health and maxillofacial rehabilitation (lr12es11), university of monastir, monastir, tunisia abstract objec&ve: postmenopausal osteoporosis (op) is a prevalent condi6on o7en treated with bisphosphonates like alendronate, which may influence periodontal health. in this study, we aimed to evaluate the rela6onship between clinical periodontal parameters [probing pocket depth (ppd), clinical adachment level (cal)], biochemical markers [osteoprotegerin (opg), and calcium (ca)] and alendronate therapy. methods: this case-control study was carried out on 60 females aged from 55 to 65. all par6cipants were divided into two groups equally, group a that included healthy postmenopausal females with healthy periodon6um and group b included pa6ents with osteoporosis. pa6ents were followed up for one year through for three consecu6ve periods (0, 6, and 12 months) a7er receiving alendronate (aln) treatment. pa6ents with osteoporosis or aln were further subdivided equally based on gingivi6s and periodon66s. blood samples were gathered for quan6ta6ve propor6ons of opg through enzyme–linked immune sorbent assay (elisa). addi6onally, calcium was analyzed using full automated calcium detector device. the clinical periodontal parameters (ppd, and cal) were reported for all teeth except for third molars and kept in specifically designed case sheet documents following assortment of serum. results: there was a significant decrease in ppd and cal a7er 12 months of aln treatment. however, there was significant increase in serum opg and calcium level at each consecu6ve recall follow up in comparison to no aln treatment. addi6onally, our study revealed a posi6ve correla6on between opg and calcium at base line in pa6ents with osteoporosis (p<0.001). conclusion: probing pocket depth, clinical adachment level and osteoprotegerin and calcium serum levels improved significantly with alendronate therapy. keywords: postmenopausal, osteoporosis, alendronate, periodontal clinical parameters, calcium, bone remodeling markers, osteoprotegrin. cita9on: al-karawi aji, et al. (2025) periodontal and biochemical correla9ons during alendronate treatment in postmenopausal women: a longitudinal study. den9stry 3000. 1:a001 doi:10.5195/d3000.2025.813 received: december 28, 2024 accepted: february 7, 2025 published: february 12, 2025 copyright: ©2025 al-karawi aji, et al. this is an open access ar9cle licensed under a crea9ve commons ayribu9on work 4.0 united states license. email: aseeljaleel68@gmail.com introduction the literature has shown that postmenopausal women with osteoporosis or osteopenia may experience more significant clinical attachment loss compared to those with normal bone mineral density [1]. this finding suggests a possible interaction between osteoporosis and periodontal health, emphasizing the importance of investigating and appropriately managing both conditions in this patient group. an association between chronic periodontitis and osteoporosis was also found, especially among postmenopausal women [2]. this finding highlights the complex link between periodontal health and bone mineral density, suggesting that these conditions may influence each other's development [3]. research has indicated that combining oral treatments and drug therapy for osteoporosis may help reduce the risk of tooth loss compared to oral care alone. alendronate (aln), an aminobisphosphonate, is an individual of the second generations of bisphosphonates which are chemical analogs of pyrophosphate (a result of human metabolism) established to be capable of inhibition of bone periodontal and biochemical correla1ons during alendronate treatment in postmenopausal women: a longitudinal study vol 13 no 1 (2025) doi 10.5195/d3000.2025.813 h#p://den*stry3000.pi#.edu resorption by osteoclast through modulation of bone mineralization. various studies confirmed that the systemic use of aln in humans and some animal models reduced bone loss and increased bone density. it was also proved that treatment with aln in postmenopausal women with osteoporosis carries a considerable improvement in bone mass [4]. osteoprotegrin (opg) and receptor activator of nuclear factor kappa-b ligand (rankl) act as negative and positive regulators in osteoclastogenesis and bone resorption. opg is a soluble molecule that inhibits osteoclast differentiation. it binds to rankl and inhibits its interaction with rank leading to neutralization and inhibition of oc formation (4), as well as provoking apoptosis of matured oc [5]. evidence obtained from experimental studies advocated that increase bone resorption after menopause may be due to excessive activation of osteoclast (oc) by increase proinflammatory cytokines production such as tnf-α due to decrease estrogenic secretion, which considered as critical factor in the pathogenesis and development of pd as well as osteoporosis [6]. calcium is the major component of the bone, where it is present at more than 99% as calciumphosphate complexes, and provides the skeleton strength and structure, making the bone a metabolic reservoir to maintain the intraand extra-cellular calcium pool. the remaining part is present in blood, in extracellular fluids, in muscles and other tissues, where it is responsible for mediating muscle contraction, vascular contraction and vasodilatation, nerve impulse transmission, and intra and extracellular signaling [7]. therefore, the aim of the study was to evaluate the effects on clinical attachment level (cal), probing pocket depth (ppd), osteoprotegerin (opg), and calcium (ca) with alendronate therapy. material and methods this case-control study was carried out on 60 females aged from age from 55 to 65. all participants were divided into two groups equally where control group included 30 systemically healthy postmenopausal females with healthy periodontium and case group included 30 postmenopausal females. patients were checked at three times readings: (1) first newly diagnosed with osteoporosis and periodontitis (op group) and baseline data was recorded, (2) after six months of receiving alendronate treatment (aln6 group), (3) after 12 months of aln treatment (aln12 group). sample collection occurred from 11th november 2022 until 6th may 2024. the samples were collected from patients treated at the department of rheumatology of baqubah teaching hospital in baqubah city. each subject was informed about the aims and protocol of the study, and they were permitted to accept or reject to participate in the study. exclusion criteria females with systemic conditions or medication intake affecting bmd or pd severity, postmenopausal women, those with recent disorders, those treated with nsaids, hrt, or other bone metabolism-modifying treatments, smoking females, those with edentulous, systemic lupus, rheumatoid arthritis, periodontal therapy, alcohol use, and neoplastic diseases. inclusion criteria postmenopausal women without osteoporosis, with good general health, are considered a control group. postmenopausal women with osteoporosis, and chronic periodontitis at least 4 sites with deep periodontal pockets depth of ≥4 mm and 1-2 mm or more of clinical attachment loss (cal) periodontal and biochemical correla1ons during alendronate treatment in postmenopausal women: a longitudinal study vol 13 no 1 (2025) doi 10.5195/d3000.2025.813 h#p://den*stry3000.pi#.edu identification of op all participants included in this study were scanned by densitometry (central dexa stratos 3d). depending on this scan woman were diagnosed as op, osteopenia or normal healthy (non–osteopenia, non osteoporotic). dexa scan type (stratos 3d) used to obtain t-score at spine (l1-l4), neck, g.t, and inter of femur (total hip). bmd rate is compared with the mean of a healthy young people expressed in the number of standard deviations (sd), a name defines as a t-score. t-score of -2.5 or fewer was diagnosed as op, where as a tscore of -1 to -2.5 was diagnosed as osteopenia. according to the who definition, osteoporosis is present when the t-score is at least 2.5 sd. collection of serum sample venous blood samples of 5 milliliters (ml) were drawn from all subjects in a disposable plane tube; the samples were placed in a cooling package for inhibition of microorganism growth. each donor number and group was written on each corresponding tube, and then the blood was allowed to clot at 37c for 15-20 minutes, and then centrifuged at 3000 rpm for approximately 10-15 minutes then serum were obtained. the laboratory tests were done in the laboratory of baqubah teaching hospital. assessment of probing pocket depth (ppd) it is defined as the millimeters distance from gingival margin to the most apical penetration of periodontal probe inserted into the gingival crevice or periodontal pocket without pressure or force. the considered locations were buccal, palatal/lingual, mesio buccal and disto buccal line angles [8]. assessment of clinical attachment loss (cal) it is the distance from the cej to the location of the inserted probe tip (bottom of gingival crevice or periodontal pocket), the level of the cej could be determined by feeling it with probe. the measurements were made at four surfaces of each tooth except 3rd molars by using marquis periodontal probe. the distance was measured indirectly by subtracting the distance from the gingival margin to the cej from ppd. in some cases, when there was gingival recession, loss of attachment was measured either directly or by adding the distance from the gingival margin to the cej to the ppd. tooth expulsion may occur due to tooth demolition, crown coverage, extended fillings, bad carious teeth, or heavy calculus covering teeth. biochemical assays the supernatant was separated using micropipette into two eppendorf tubes and stored at -20 ͦ c in deep freeze for later analysis by enzyme linked immunosorbent assay (elisa) kit for quantitative determination of opg, and calcium detected using full automated device. statistical analysis statistical analysis was performed with ibm® spss® (ver. 26. spss inc., ibm corporation, armonk, ny, usa). descriptive data were presented by mean, standard deviation, simple linear regression graph. pearson correlation analysis was used to describe the association between numerical variables within each group. shapiro-wilks test was used to evaluate the normality of the distribution of data. anova test used to evaluate the difference between k related mean with tukey hsd, independent t test used to compare means between two groups, dun test for multiple comparisons with control. receiving operating characteristic curve (roc) for diagnostic periodontal and biochemical correla1ons during alendronate treatment in postmenopausal women: a longitudinal study vol 13 no 1 (2025) doi 10.5195/d3000.2025.813 h#p://den*stry3000.pi#.edu capability as for discrimination or differentiation is varied. results in the present study, it was found that there was a statistically significant difference between both groups regarding age while there was insignificant difference between both groups regarding menopause duration, and bmi (table 1). table 1. demographic data of the study groups. ppd and cal findings in the present study, it was found that ppd was decreased in aln12 group compared to aln6 and op groups (p=0.000). additionally, cal demonstrated declining in op in comparison to aln6 and aln12 groups (p=0.000) (table 2). table 2. ppd and cal in pd. serum biomarkers in the present study, it was found that there was a statistically significant difference between case and control groups regarding opg levels where mean opg in control group was higher than in op. furthermore, mean opg in remaining subgroups (aln6, aln12) was increased after six and one year of aln treatment as control op p-value mean ±sd mean ±sd age 48.512 3.656 50.512 4.583 0.07 years since menopause 7.067 3.258 8.533 2.532 0.06 bmi 33.656 3.549 31.657 4.543 0.065 variable mean ±sd p-value ppd (opp) 7.393 0.515 0.000 ppd (alnp6) 6.327 0.489 ppd (alnp12) 5.307 0.438 cal (opp) 7.947 0.673 0.000 cal (alnp6) 7.133 0.719 cal (alnp12) 6.087 0.595 periodontal and biochemical correla1ons during alendronate treatment in postmenopausal women: a longitudinal study vol 13 no 1 (2025) doi 10.5195/d3000.2025.813 h#p://den*stry3000.pi#.edu compared to control group (6.967, 11.253 and 3.417, respectively). for serum calcium, it was found that there was a statistically significant difference between cases and controls regarding ca level where mean level in control group was higher than in op (9.613 and 7.027, respectively). after six months of treatment, there was a statistically significant difference among groups where mean of aln6 levels were still lower than control group (8.260 and 9.613, respectively). after 12 months, there was a statistically significant difference among groups where mean calcium levels in aln12 groups were higher than control group (10.253 and 9.613, respectively) (table 3). table 3. biomarker comparisons. serum osteoprotegrin (opg) in the present study, it was found that serum opg concentration increased from baseline opg (op) until 6 months (aln6) then after 12 months (aln12) of treatment with significant difference. additionally, mean of opg concentration in periodontitis state (pd) among study groups (op, aln6, and aln12) with highly significant difference between them (p=0.000) (table 4). opg calcium mean ±sd mean ±sd control 3.417 0.432 9.613 0.637 op 1.533 0.522 7.027 0.219 aln6 6.967 0.694 8.260 0.422 aln12 11.253 1.120 10.253 0.300 p-value 0.000 0.000 periodontal and biochemical correla1ons during alendronate treatment in postmenopausal women: a longitudinal study vol 13 no 1 (2025) doi 10.5195/d3000.2025.813 h#p://den*stry3000.pi#.edu table 4. opg among people with periodontitis. serum calcium levels there was a significant difference among groups with calcium levels increased from baseline to 12 months (table 5). table 5. calcium in periodontitis. correlation between biomarkers and ppd and cal there were not significant correlations except for after 12 months in periodontitis patients (table 6). time pd mean ±sd opg (op) 1.533 0.522 opg (aln6) 6.967 0.694 opg (aln12) 11.253 1.120 p-value 0.000 time pd mean ±sd op 7.027 0.219 aln6 8.260 0.422 aln12 10.253 0.300 p-value 0.000 periodontal and biochemical correla1ons during alendronate treatment in postmenopausal women: a longitudinal study vol 13 no 1 (2025) doi 10.5195/d3000.2025.813 h#p://den*stry3000.pi#.edu table 6. correlation between biomarkers with ppd and cal. variable opg0 opg6 opg12 ca0 ca6 ca12 r p r p r p r p r p r p op 0.328 0.233 0.306 0.267 aln6 0.050 0.860 0.130 0.644 aln12 0.381 0.162 -0.030 0.915 op 0.125 0.658 -0.160 0.570 aln6 0.286 0.302 0.386 0.155 aln12 -0.315 0.253 0.465 0.081 correlation between biomarkers and time there was a positive correlation between opg and calcium at baseline (table 7 and figure 1). table 7. correlation between biomarkers among study groups and time. pd-group ca0 ca6 ca12 r p r p r p pd op 0.743 0.002 aln6 0.110 0.697 aln12 0.091 0.747 periodontal and biochemical correla1ons during alendronate treatment in postmenopausal women: a longitudinal study vol 13 no 1 (2025) doi 10.5195/d3000.2025.813 h#p://den*stry3000.pi#.edu `figure 1. correlation between opg and calcium in patients with periodontitis. discussion osteoporosis is one of the risk factors that have been implicated in the progression of periodontal disease [9]. several studies showed that there is a relationship between oral and systemic bone loss as well as an association of osteoporosis with periodontal diseases [10,11]. estrogen hormone plays vital protective effect as anti-resorptive agent on alveolar bone, since it improves opg production and decrease rankl expression, therefore estrogen deficiency after menopause considered as a key feature for development of osteoporosis, progression of periodontitis, alveolar bone resorption, and osteoporotic changes in the jaw [12]. in the present study, it was found that there was there was a significant difference between both groups regarding age while there was insignificant difference between both groups regarding menopause duration, and bmi. the development of op is predisposed by the apparent degradation of the components, organization, and functions of bone because of age progression. age, in conjunction with intrinsic and extrinsic factors, accelerates the loss of bone mass because of uneven bone remodeling. consequently, bone resorption exceeds bone creation, a phenomenon that is particularly evident in women after menopause, when estrogen is no longer produced [13]. there are several studies that proved the correlation between age progression and op [14-16]. in line with our results, rocha et al. revealed that there was insignificant difference between case and control group regarding age where the mean age in case group was 57.8 ± 2.9 while the mean age in control group was 58.0 ± 2.8 [17]. additionally, laza et al. included 64 patients in the study who were divided equally into two groups patients who did not follow systemic therapy with bisphosphonates and patients who followed systemic therapy with bisphosphonates (alendronate 70mg/week, for 12 months) [18]. numerous studies have demonstrated a positive relationship between bmi and bmd. walsh and colleagues reported a significant correlation between bmi and bone mineral density (bmd) and proposed that potential mechanisms may include periodontal and biochemical correla1ons during alendronate treatment in postmenopausal women: a longitudinal study vol 13 no 1 (2025) doi 10.5195/d3000.2025.813 h#p://den*stry3000.pi#.edu increased loading and heightened aromatase activity [19]. another study revealed that each unit increase in bmi was linked to a 0.0082 g/cm2 increase in bmd (p < 0.001) [20]. elderly women were more vulnerable to op due to menopausal transition, in which there is dropping in estrogen hormone that led to acceleration of bone resorption [21]. in contrast, kerksick et al. found that women with adequate calcium and vitamin d intake experienced minimal differences in bone health, regardless of menopause duration [22]. in the present study, it was found that there was a highly significant difference among study groups (op, aln6, and aln12) regarding mean of opg concentration in periodontitis state (pd). lopez et al. suggested that early differences in periodontal disease severity, such as the presence of bone loss in periodontitis, may not be fully reflected in opg levels at baseline [23]. kumar et al. reported that periodontitis patients may have slightly lower opg levels at baseline, because of more pronounced bone resorption associated with chronic inflammation in deeper periodontal pockets [24]. peacock et al. observed that baseline opg levels might differ based on systemic factors, such as osteoporosis or age, which could skew initial readings. opg levels significantly increased in periodontitis patients after treatment with bisphosphonates, with the greatest improvements seen within the first six months and continued stabilization after twelve months [25]. sedghizadeh et al. confirmed that alendronate treatment significantly elevates opg levels, particularly in patients with bone loss, as it helps to balance the rankl/opg ratio, favoring bone preservation [26]. özden et al. showed that there was a significant increase in serum level of opg after 3-6 months of bp treatment in patients with postmenopausal op and periodontal disease [27]. conversely, verde et al. revealed that there was no significant difference in opg level in gcf after bps treatment [28]. according to clinical observation in postmenopausal women and experimental animal studies, estrogen deficiency is a critical factor in the development of op and low alveolar bone density. this is because estrogen regulates the construction of opg and rankl by pdl cells, which are characterized by estrogen receptors. consequently, estrogen can serve as a critical anti-resorptive agent in the alveolar bone [29]. aln has been revealed as modulators of oc function and bone metabolism as a result it may well inhibit the development of op. additionally it is able to induce the production of mediators that inhibit osteoclastogenesis and influences the rankl/opg system by increasing opg and declining rankl production. it was also able to maintain early osteoblastogenesis and reduce in vitro prostaglandin synthesis. moreover, it diminishes rankl secretion by gingival fibroblast, thus reduce the risk of periodontitis [30]. lopez et al. suggested that periodontal disease, particularly in its more advanced stages (such as periodontitis), could lead to mineralization imbalances, which may explain lower calcium concentrations [23]. peacock et al. found that bisphosphonate therapy, such as alendronate, increases calcium levels by stabilizing bone mineralization, which can be observed as an elevation in systemic calcium concentrations after six months of treatment [16]. lopez et al. found that systemic therapy with bisphosphonates significantly reduces pocket depth by stabilizing the inflammatory process, allowing for periodontal tissues to heal and regenerate [23]. the significant reduction in cal over the course of treatment in the periodontitis periodontal and biochemical correla1ons during alendronate treatment in postmenopausal women: a longitudinal study vol 13 no 1 (2025) doi 10.5195/d3000.2025.813 h#p://den*stry3000.pi#.edu group demonstrates the beneficial effects of alendronate therapy, which helps to stabilize the periodontal attachment apparatus by addressing both bone resorption and soft tissue inflammation. in line with our results, laza et al., özden et al., and dutra et al. noted that the ppd decrease was more significant for group underwent systemic alendronate therapy. cal values followed the same trends as ppd [18,27,31]. the prevalence of pd may be overestimated by the examination of the cal parameter, as the loss of attachment fibers suggests the cumulative effect of pathological periodontal tissue destruction, as well as protective and destructive immunological processes, as well as noninflammatory causes such as traumatic brushing and nail biting [32]. it was confirmed that the periodontal damage might be drastically affected by the systemic bone loss in op [33]. numerous studies verified significant relationship between the two conditions [33-35]. shen et al. established greater clinical attachment loss and gingival recession in osteoporotic women as compared to non-osteoporotic one [36]. however, verde et al. revealed that there were no statistical differences in ppd after bps treatment of postmenopausal women with op and periodontal disease [28]. the contrary among studies can be justified by although op does not initiate pd, it may influence disease pattern through reducing bone mass and density, moreover emerging evidence reported that op increases susceptibility for pd through the effect of periodontal inflammation that mediated destruction of periodontal tissue due to unbalanced expression of inflammatory cytokines [13]. randomized clinical trials have shown a significant increase in bone mineral density in postmenopausal women diagnosed with osteoporosis after taking alendronate [37]. in addition, the literature shows that both systemic and local administration of alendronate reduced alveolar bone loss in periodontal flap surgical procedures [38]. bisphosphonates' main effect on the body is their ability to inhibit osteoclast activity. they act both directly by promoting apoptosis and reducing the recruitment of these cells and indirectly by blocking the secretion of prostaglandins and interleukins by osteoclasts, thus limiting their stimulation [39]. this improvement can be partially attributed to the scaling and debridement procedures of the root surfaces and the oral hygiene instructions provided to the patients in both groups, both at the beginning and throughout the study [40,41]. rocha et al. support the efficacy of alendronate therapy in improving periodontal status in postmenopausal patients diagnosed with osteoporosis and periodontitis. research to date has demonstrated the ability of alendronate to reduce bone loss in experimentally induced and naturally occurring animal models of periodontitis [17]. human clinical trials investigating the effect of bisphosphonate therapy on periodontal parameters have reported significant improvements in periodontal clinical status following bisphosphonate treatment. for example, in a matched case control study of 40 patients with type 2 diabetes and stabilized periodontitis, six months of treatment with alendronate 10 mg/day resulted in notable improvements in alveolar bone height and a significant reduction in gingival bleeding [17]. in contrast to the results obtained in our research, some previous studies have shown that using bisphosphonates to treat periodontal disease may not provide significant benefits. for example, in a retrospective cohort study of a small number of patients with periodontitis that investigated the effect of antiresorptive periodontal and biochemical correla1ons during alendronate treatment in postmenopausal women: a longitudinal study vol 13 no 1 (2025) doi 10.5195/d3000.2025.813 h#p://den*stry3000.pi#.edu therapy on alveolar bone loss, the oral medication groups did not show significant improvements in maintaining alveolar bone levels. the results indicate that opg was the most effective as it demonstrated the highest ability to differentiate between the two groups. despite its relatively low sensitivity, opg still showed significant discriminatory power suggesting that, although opg is not highly sensitive or specific, it may still provide valuable information in the context of the study, potentially acting as an early indicator or a supplementary diagnostic tool. calcium low auc and sensitivity suggest that calcium levels do not significantly contribute to differentiating between the study and control groups and it may not be a useful diagnostic marker in this context. therefore, opg is promising biomarkers for differentiation between study and control groups. the findings of this study underscore the complex interplay between osteoporosis and periodontal disease, highlighting the role of biochemical markers as potential indicators of disease progression and treatment outcomes. alendronate treatment appears to beneficially influence serum levels of opg, and calcium, contributing to improved clinical periodontal parameters. while opg shows promise as a biomarker for monitoring bone turnover, the overall improvement in t-scores suggests that achieving optimal bone density may require extended treatment duration or combination therapies. conflicts of interest the authors declare no competing interest. references 1. penoni d, fidalgo t, torres s, varela v, masterson d, leão a, et al. bone density and clinical periodontal atachment in postmenopausal women: a systemauc review and metaanalysis. journal of dental research. 2017;96:261-9. 2. sharma n, reche a. unraveling the relauonship between osteoporosis, treatment modaliues, and oral health: a comprehensive review. cureus. 2023;15. 3. passos j, vianna m, gomes-filho i, cruz ssd, barreto ml, adan l, et al. osteoporosis/osteopenia as an independent factor associated with periodonuus in postmenopausal women: a case– control study. osteoporosis internauonal. 2013;24:1275-83. 4. ebeuno fh, sun s, cherian p, roshandel s, neighbors jd, hu e, et al. bisphosphonates: the role of chemistry in understanding their biological acuons and structureacuvity relauonships, and new direcuons for their therapeuuc use. bone. 2022;156:116289. 5. liu w, zhang x. receptor acuvator of nuclear factor-κb ligand (rankl)/rank/osteoprotegerin system in bone and other ussues. molecular medicine reports. 2015;11:3212-8. 6. mohammed an. effect of alendronate treatment on salivary levels of osteoprotegrin and tnfα in postmenopausal woman with osteoporosis and periodontal diseases. 2021. 7. rizzoli r, biver e, brennansperanza tc. nutriuonal intake and bone health. the lancet diabetes & endocrinology. 2021;9:606-21. 8. atassi f, newman h, bulman j. probe une diameter and probing depth. journal of clinical periodontology. 1992;19:301-4. 9. yu b, wang cy. osteoporosis and periodontal diseases–an update on their associauon and mechanisuc links. periodontology 2000. 2022;89:99-113. 10. bernal m, elenkova m, evensky j, stein sh. periodontal disease and osteoporosis-shared risk factors periodontal and biochemical correla1ons during alendronate treatment in postmenopausal women: a longitudinal study vol 13 no 1 (2025) doi 10.5195/d3000.2025.813 h#p://den*stry3000.pi#.edu and potenuauon of pathogenic mechanisms. current oral health reports. 2018;5:26-32. 11. wang c-w, mccauley lk. osteoporosis and periodonuus. current osteoporosis reports. 2016;14:284-91. 12. jayusman pa, nasruddin ns, baharin b, ibrahim ni, ahmad hairi h, shuid an. overview on postmenopausal osteoporosis and periodonuus: the therapeuuc potenual of phytoestrogens against alveolar bone loss. fronuers in pharmacology. 2023;14:1120457. 13. zioupos p, kirchner ho, peterlik h. ageing bone fractures: the case of a ducule to britle transiuon that shizs with age. bone. 2020;131:115176. 14. guan x, guan y, shi c, zhu x, he y, wei z, et al. estrogen deficiency aggravates apical periodonuus by regulaung nlrp3/caspase-1/il-1β axis. american journal of translauonal research. 2020;12:660. 15. weaver cm, gordon cm, janz kf, kalkwarf h, lappe jm, lewis r, et al. the nauonal osteoporosis foundauon’s posiuon statement on peak bone mass development and lifestyle factors: a systemauc review and implementauon recommendauons. osteoporosis internauonal. 2016;27:1281-386. 16. formosa m, christou m, mäkiue o. bone fragility and osteoporosis in children and young adults. journal of endocrinological invesugauon. 2024;47:285-98. 17. rocha ml, malacara jm, sánchez-marin fj, de la torre cjv, fajardo me. effect of alendronate on periodontal disease in postmenopausal women: a randomized placebo-controlled trial. journal of periodontology. 2004;75:1579-85. 18. laza g-m, martu m-a, sufaru ig, martu s, pasarin l, martu i. invesugaung the impact of systemic alendronate on periodontal parameters in osteoporosis and periodonuus pauents. an intervenuonal and prospecuve study. romanian journal of oral rehabilitauon. 2024;16. 19. walsh js, vilaca t. obesity, type 2 diabetes and bone in adults. calcified ussue internauonal. 2017;100:528-35. 20. lloyd jt, alley de, hawkes wg, hochberg mc, waldstein sr, orwig dl. body mass index is posiuvely associated with bone mineral density in us older adults. archives of osteoporosis. 2014;9:1-8. 21. borrelli j. the relauonship of peak bone mass, aging, and bone loss to osteoporosis and fragility fractures. arthroplasty for the treatment of fractures in the older pauent: indicauons and current techniques. 2018:3-17. 22. kerksick cm, roberts md, campbell bi, galbreath mm, taylor lw, wilborn cd, et al. differenual impact of calcium and vitamin d on body composiuon changes in postmenopausal women following a restricted energy diet and exercise program. nutrients. 2020;12:713. 23. lópez roldán a, garcía giménez jl, alpiste illueca f. impact of periodontal treatment on the rankl/opg rauo in crevicular fluid. plos one. 2020;15:e0227757. 24. kumar ig, raghunath n, kiran h. rank-rankl-opg: a current trends in orthodonuc tooth movement and its role in accelerated orthodonucs. int j appl dent sci. 2022;8:630-5. 25. minisola s, peacock m, fukumoto s, cipriani c, pepe j, tella sh, et al. tumour-induced osteomalacia. nature reviews disease primers. 2017;3:1-15. 26. sedghizadeh pp, sun s, jones ac, sodagar e, cherian p, chen c, et al. bisphosphonates in denustry: historical perspecuves, adverse periodontal and biochemical correla1ons during alendronate treatment in postmenopausal women: a longitudinal study vol 13 no 1 (2025) doi 10.5195/d3000.2025.813 h#p://den*stry3000.pi#.edu effects, and novel applicauons. bone. 2021;147:115933. 27. özden fo, sakallioğlu ee, demir e, bilgici b, tunçel ök, gökosmanoğlu f, et al. effect of bisphosphonate as an adjunct treatment for chronic periodontitis on gingival crevicuar fluid levels of nuclear factor-κb ligand (rankl) and osteoprotegerin in postmenopausal osteoporosis. journal of oral science. 2017;59:14755. 28. verde me, bermejo d, gruppi a, grenón m. effect of bisphosphonates on the levels of rankl and opg in gingival crevicular fluid of patients with periodontal disease and postmenopausal osteoporosis. acta odontológica latinoamericana. 2015;28:215-21. 29. rossetti br, lemos af, so bb, weissheimer t, martins md, so mvr. effects of estrogen deficiency on the progression of apical periodontitis. a systematic review of preclinical studies. archives of oral biology. 2022;142:105496. 30. huang l, cai p, bian m, yu j, xiao l, lu s, et al. injectable and high-strength plga/cpc loaded aln/mgo bone cement for bone regeneration by facilitating osteogenesis and inhibiting osteoclastogenesis in osteoporotic bone defects. materials today bio. 2024:101092. 31. dutra bc, oliveira amsd, oliveira pad, manzi fr, cortelli sc, cota lodm, et al. effect of 1% sodium alendronate in the non-surgical treatment of periodontal intraosseous defects: a 6month clinical trial. journal of applied oral science. 2017;25:310-7. 32. hugoson a, norderyd o. has the prevalence of periodontitis changed during the last 30 years? journal of clinical periodontology. 2008;35:33845. 33. shum i, leung pc, kwok a, corbet ef, orwoll es, phipps kr, et al. periodontal conditions in elderly men with and without osteoporosis or osteopenia. journal of periodontology. 2010;81:1396-402. 34. brennan rm, genco rj, hovey km, trevisan m, wactawski-wende j. clinical attachment loss, systemic bone density, and subgingival calculus in postmenopausal women. journal of periodontology. 2007;78:2104-11. 35. nackaerts o, gijbels f, sanna a-m, jacobs r. is there a relation between local bone quality as assessed on panoramic radiographs and alveolar bone level? clinical oral investigations. 2008;12:31-5. 36. shen e-c, gau c-h, hsieh yd, chang c-y, fu e. periodontal status in post-menopausal osteoporosis: a preliminary clinical study in taiwanese women. journal chinese medical association. 2004;67:389-93. 37. wang y-k, qin s-q, ma t, song w, jiang r-q, guo j-b, et al. effects of teriparatide versus alendronate for treatment of postmenopausal osteoporosis: a meta-analysis of randomized controlled trials. medicine. 2017;96:e6970. 38. kellesarian s, abduljabbar t, vohra f, malignaggi v, malmstrom h, romanos g, et al. role of local alendronate delivery on the osseointegration of implants: a systematic review and meta-analysis. international journal of oral and maxillofacial surgery. 2017;46:91221. 39. pauli ma, bordignon nct, martini gr, minamisako mc, gondak r. prevalence of dental alterations in patients under bisphosphonates therapy: a systematic review. oral and maxillofacial surgery. 2023;27:399409. 40. sl t. osteoclasts: what do they do and how do they do it? am j pathol. 2007;170:427-35. 41. tipton d, seshul b, dabbous mk. effect of bisphosphonates on human gingival fibroblast production of mediators of osteoclastogenesis: rankl, osteoprotegerin and interleukin-6. journal of periodontal research. 2011;46:39-47. 723 2024 tea tree oil effect on dimensional change and detail reproduc9on of addi9on silicon impression material vol 13 no 2 (2024) doi 10.5195/d3000.2024.723 h#p://den*stry3000.pi#.edu tea tree oil effect on dimensional change and detail reproduction of addition silicon impression material samir samier hammed, aseel mohammed al-khafaji college of den*stry, university of baghdad, baghdad, iraq abstract objec&ve: impression materials are thought to be the one of the primary sources of cross infec&on between pa&ents and den&sts. however, it was discovered that disinfec&on of the impression is not conducted rou&nely in ordinary dental treatment. disinfec&on of addi&on silicon impression, whether by immersion or spray should not produce dimensional or detail errors. the purpose of this study was to examine the immersion of addi&on silicon impression material in tea tree oil and its influence on dimensional stability and detail reproduc&on of the addi&on silicon imprint material. materials and methods: this study employed a total of 120 heavyand light-body addi&on silicon impression material specimens. the specimens were randomly sorted and immersed into six groups. these test groups where four concentra&ons of tto (0.25%, 0.5%, 0.75%, and 1%), and the other two groups were dis&lled water (nega&ve control) and 2% glutaraldehyde (posi&ve control). all specimens were immersed for 10 minutes in the tes&ng solu&ons. each of the six groups had 20 specimens separated into two subgroups. results: there were no sta&s&cally significant differences in linear dimensional changes and detail reproduc&on among all test groups. conclusion: the addi&on silicon impression material may be safely submerged in tto for 10 minutes to disinfect it without impairing its dimensional correctness or detail replica&on of the impression. keywords: addi%on silicon, tea tree oil (tto), dimensional stability, detail reproduc%on cita:on: hammed ss, et al. (2024) tea tree oil effect on dimensional change and detail reproduc:on of addi:on silicon impression material. den:stry 3000. 2:a001 doi:10.5195/d3000.2024.723 received: september 1, 2024 accepted: september 14, 2024 published: october 7, 2024 copyright: ©2024 hammed ss, et al. this is an open access ar:cle licensed under a crea:ve commons auribu:on work 4.0 united states license. email: samer.samir2301m@codental.uobaghdad.edu.iq introduction dental impressions infected with potentially harmful germs come into touch with the patient's blood and saliva [1,2]. this might lead to cross-infections among dentists, dental assistants, and laboratory technicians [3]. impression materials are thought to be the primary cause of cross infection between patients and dentists. an infection prevention program for dental impressions is necessary because a variety of viruses, fungi, and bacteria found in the oral environment have been associated to devastating and lifethreatening disorders.[4] disinfection of impressions is considered mandatory [5], and to reduce cross-contamination between patients and dental personnel in dental offices and laboratories, the american dental association (ada) recommends disinfecting dental impressions as soon as they are removed from the patient's mouth [6]. heat and chemicals are utilized to sterilize the various impression materials. the chemical procedure is the most often used method, which includes spraying or immersing a chemical disinfectant [6]. the immersion approach ensures that the disinfecting solution reaches all surfaces of the imprint material and tray [1]. an important consideration in disinfecting an impression material is the effect of the disinfectant on the properties of the impression material, particularly impression-like addition silicon, where the exact fit of the dental prosthesis significantly influences the outcome of prosthodontics work, which is dependent on the tea tree oil effect on dimensional change and detail reproduc9on of addi9on silicon impression material vol 13 no 2 (2024) doi 10.5195/d3000.2024.723 h#p://den*stry3000.pi#.edu accurate recording of fine intraoral details. as a result, the basic requirement for many dental and maxillofacial rehabilitation treatments is to have a proper negative reproduction of the appropriate site that should not be affected by disinfection [1,2]. addition silicones are available in four consistencies: low, medium, heavy, and putty [7]. the polymer (polymethyl-hydro siloxane) in the base paste of this impression class contains more than three and up to ten pendant or terminal hydrosilane groups per molecule. filler is also included in the base [8]. vinyl terminal groups, filler, and dimethyl siloxane polymer make up the accelerator (catalyst) and base paste. an additional component of the accelerator is a complicated chemical termed a karstedt-type platinum catalyst, which is made of platinum and 1,3-divinyl tetramethyl disiloxan. the addition reaction, in contrast to the condensation type, rarely yields a low-molecular-weight byproduct; but, in the presence of -oh groups, a secondary reaction involving the generation of hydrogen gas may take place [9]. chlorhexidine, hydrogen peroxide, alcohol, sodium hypochlorite, and glutaraldehyde are among the disinfectants that are most frequently used [10]. it is imperative to select a disinfectant with strong antibacterial qualities that does not compromise the impression's surface features or dimensional stability, as there is no one disinfectant that works for all impression materials [11]. glutaraldehyde is an oily, colorless liquid with an overpowering odor. in addition, the lipid surfactant layer disintegrates and damages the airway epithelium, capillaries in the pulmonary system, and alveolar septum. atelectasis, interstitial inflammation, and the emergence of hyaline membrane are other pathogenic indicators [11]. natural or herbal derivatives have long been used in dentistry and medicine, and because of their antibacterial activity, biocompatibility, lack of germ resistance, antioxidant, antiinflammatory, easy availability, and affordable qualities, they are becoming even more well-liked [12]. tea tree oil (tto) is generated from melaleuca alternifolia, a native australian plant, whose leaves could be steam-distilled to yield oil as shown in figure 1. tto is composed of a mixture of compounds, primarily alcohols of monoterpene and sesquiterpene hydrocarbons. numerous investigations revealed that tto possessed antiseptic, antibacterial [11], anti-inflammatory, and antifungal qualities, especially those that were anti-candida [13]. additionally, phytoconstituents were compounds present in natural agents with a particular target impact for the treatment and prevention of diseases connected to biofilms [14]. m. alternifolia is the source of αterpineol [15]. against candida albicans, these phytoconstituents demonstrated antifungal action [16]. figure 1. tea tree oil shrub (melaleuca alternifolia). because of tea tree oil valuable properties like antibacterial, antifungal and antiviral activities and its availability and biocompatibility, and since there is no previous study on the effect of tto on dental impression material, this study was conducted to evaluate the effect on linear dimensional change and detail reproduction of addition silicon impression material after immersion in solutions with different tto concentrations. the null hypothesis predicted that the properties of the additional tea tree oil effect on dimensional change and detail reproduc9on of addi9on silicon impression material vol 13 no 2 (2024) doi 10.5195/d3000.2024.723 h#p://den*stry3000.pi#.edu silicon impression material would not be significantly impacted, whereas the alternative hypothesis proposed that the properties of the additional silicon impression material will be significantly impacted by the immersion of silicon impression material in tea tree oil solutions. material and methods addition silicone impression specimens were prepared, according to the iso 4823:2015 standard for evaluating elastomeric impression materials. the test specimens and procedures were prepared at 23 ± 2 ºc and with a relative humidity of 50% ± 10%. the equipment was conditioned at this temperature and humidity for at least 10 hours before testing [17]. the testing solutions, distilled water, tto concentrations (0.25%, 0.50%, 0.75%, and 1%) and 2% glutaraldehyde were also prepared under the same conditions. in a moisture-resistant container, the addition silicon impression was conditioned at a temperature of 23 ± 2 ºc. according to the ada specification no. 19 standard for evaluating elastomeric impression materials, an equipment was created, and 3d printed with the software package 3d max 2022. the equipment is made up of four pieces. the first is a ruled test block that has five v-shaped lines engraved into it. three vertical lines (a 50 µm, b 20 µm, and c 75 µm) and two horizontal lines (d1 and d2) have a depth of 75 µm. the length of the vertical lines between d1 and d2 is 25 mm, which is utilized to calculate the linear dimensional change. the second component is a ring mold that serves as a mold for a dental impression, while the third component is a riser as shown in figures 2 and 3. figure 2. testing block. figure 3. testing apparatus. measures addition silicon impression material (3m, usa) was prepared according to ada specification no. 19 [17] and used as the test impression material. before impression making, the test block was immersed in a water bath at 35°c for 15 minutes to ensure that the block temperature standardizes to intra-oral temperature as the ada standards recommend. the ring was cleaned with solvent alcohol (hema pharmaceuticals, tamil, india) and dried after soaking in cotton (jayamari enterprises, tamil, india). the ring was placed on the test block after lubricating with petroleum jelly (bharat pharmaceuticals, tamil, india). impressions were made in two steps, using a putty wash technique. first, the square sheet of plastic wrap was placed over the impression surface of the test tea tree oil effect on dimensional change and detail reproduc9on of addi9on silicon impression material vol 13 no 2 (2024) doi 10.5195/d3000.2024.723 h#p://den*stry3000.pi#.edu block, and then the impression ring was placed over the test block, mixing addition silicon putty by hand until gets mixture that has no streaks in it. when the putty was thoroughly mixed, it was placed over the impression ring, put the glass slab over it to produce a flat impression surface, and waited until it was completely set (3 to 5 minutes). then the impression mold with addition silicon putty was removed, and then the square sheet of plastic wrap was replaced with addition silicon light body mixed with an auto-mixing gun (china) [18], as shown in figure 4. the ring was filled with putty and light body addition silicon impression material to achieve a three mm thickness. a glass slab (star labs, star, india) was placed over the mold. figure 4. addition silicon impression specimen image after transferred from camera to personal computer. specimen grouping a total number of 120 impression specimens, which were divided into six test groups of 20 specimens each were used. the testing groups were: group 1: 20 addition silicon specimens were immersed for 10 minutes in 0.25% tto. group 2: 20 addition silicon specimens were immersed for 10 minutes in 0.5% tto. group 3: 20 addition silicon specimens were immersed for 10 minutes in 0.75% tto. group 4: 20 addition silicon specimens were immersed for 10 minutes in 1% tto. group 5: 20 addition silicon specimens were immersed for 10 minutes in distilled water (negative control). group 6: 20 addition silicon specimens were immersed for 10 minutes in 2% glutaraldehyde as advised by the ada (positive control). preparation of tto solutions tea tree pure essential oil (now foods, bloomingdale, il 60108, usa), as shown in figure 5, solution (tto) was prepared by combining it (according to the specimen groups) with 1% tween 80 as an emulsifier using a magnetic stirrer and distilled water. figure 5. organic essential tea tree oil (melaleuca alternifolia). tto solution was prepared in the chemistry lab, department of dentistry, university of karbala. tea tree oil, tween 80® and distilled water were used to prepare the solution. a 1:1 ratio of tea tree and tween 80 was used, and a series of tea tree concentrations (0.25%, 0.5%, 0.75%, and 1%) was prepared using magnetic stirrer device under 300 round per minute (rpm) (stuarthotplate & stirrers) at room temperature (28 ±5 ºc). at first, tween 80 surfactant was added to the container and then tea tree oil was added and let them steer for a couple of minutes in a 100 ml glass container. distilled water was added drop by tea tree oil effect on dimensional change and detail reproduc9on of addi9on silicon impression material vol 13 no 2 (2024) doi 10.5195/d3000.2024.723 h#p://den*stry3000.pi#.edu drop until 100ml volume was reached, and then let to be stirred for about 10 minutes. prepared tea tree oil solutions were stored at room temperature (28 ±5 ºc). specimens were conditioned in a water bath at 35°c for 10 minutes to mimic mouth temperature during impression taking. samples were immersed in the prepared glass containers of each group for 10 minutes, then removed and rinsed by distilled water and stored at room temperature. after 24 hours, these specimens were tested for detail reproduction and linear dimensional change. linear dimensional change evaluation digital imaging was recommended by oliveira et al. (2021) [19]. this approach did not significantly differ from measurements taken under a microscope, which was a common method used in research. there are benefits of using software to measure distance instead of a microscope, which include lower operating costs, the flexibility to store the photos for the measurements whenever the operator wants, and shorter average measurement times. this approach was used in this investigation to calculate the distance on the specimen between d1 and d2. after capturing the test specimens by camera after disinfection, the line b between the line d1 and the line d2 was measured by using image j 1.53k (national institutes of health, united states of america) (oliveira et al., 2021), as shown in figure 6. for each impression specimen, three measurements were taken at different times and according to world health organization (who) for interexaminer evaluation. we waited 30 minutes between the measurements, and the means of those readings were computed. ten measurements of the test block were taken in total, with the mean length of test block being 25.002 mm. this value served as the initial (before treatment) measure for each specimen (l1) [20]. the following calculation was used to determine the linear dimensional change percentage for each specimen in accordance with iso 4823:2015: δl = 100 × [ (l1 − l2 )/ l1] [21], where l1 represents the length of the line b as calculated on the test block, while l2 represents the length of the line b as calculated on the impression specimen after figure 6. evaluation of dimensional change of the addition silicon impression specimen by using the imagej software. detail reproduction evaluation elastomeric imprint materials used to make precision castings must meet the iso 4823:2015 standard of replicating fine detail to a depth of 20 µm or less, which is an accurate and comparable process. in this investigation, an iso 4823:2015 standard apparatus was employed to restrict the variances caused by uncontrollable circumstances, resulting in a more accurate and comparable assessment of the impression material's ability to recreate surface detail. the image was used to quantify linear dimensional change, where two observers viewed line b to determine the surface detail score using a ranking system developed by owen. this system aimed to evaluate how well line b was replicated in the specimens. the specimens were ranked with four scores. tea tree oil effect on dimensional change and detail reproduc9on of addi9on silicon impression material vol 13 no 2 (2024) doi 10.5195/d3000.2024.723 h#p://den*stry3000.pi#.edu • score 1: the entire line is sharply and clearly replicated between the markings (d1 and d2). • score 2: the line is distinct for more than half of its length, with less than half being obscure. • score 3: the line is hidden for more than 50% of its length, obscure for less than half of its length, or completely visible but rough and imperfect. • score 4: disparate in length and pitted, rough, or blemished. after disinfection, the 20 μm line between markings (d1 and d2) remained unaltered independent from the disinfectant type. statistical analysis individual linear dimensional change readings, as well as values for detail reproduction, were measured and then summarized. means and standard deviations for every group were computed utilizing spss program (ibm spss version 21) as shown in tables 1 and 2. the results were compared between the groups using oneway anova and kruskal-wallis. pvalues less than 0.05 were considered significant. results immersion of addition silicon impression material in different concentrations of tea tree oil for 10 minutes showed no changes in linear dimension and detail reproduction in comparison to controls. the line with 20μm depth was sharply visible on the impression surface of the specimens following disinfection with tto and 2% glutaraldehyde, and it showed that it was also identically sharply visible on the surfaces of all control addition silicon specimens. discussion because of the growing number of infectious diseases that pose a considerable risk not only to clinical workers but also to dental lab workers, impression disinfection is one of the most essential and frequently discussed topics. healthcare providers are accountable for maintaining hygienic standards in health care facilities, such as disinfecting instruments, aids, and dental impressions. in addition to the fundamental antibacterial efficacy of the disinfectants, it is required to analyze their effect on the properties of dental impression materials. ideal disinfectants for dental impressions should not only be effective against a wide range of germs and viruses, but they should also have no negative effects on the materials' qualities or structure. the null hypothesis for this study predicted that disinfectants have no influence upon the dimensional stability, accuracy, or quality of the impression material surfaces. data have shown that 67% of materials sent to dental laboratories are infected by various microorganisms [22]. infectious agents are transferred from saliva and blood to the casts via dental impressions [23]. among the various methods of sterilization and disinfection, chemical disinfection is the ideal choice for disinfecting dental impressions, because impressions cannot be disinfected by using heat [24]. there are basically three methods of chemical disinfection: a) spray disinfection, b) immersion disinfection, and c) mixing the disinfectant in the gypsum before tea tree oil effect on dimensional change and detail reproduc9on of addi9on silicon impression material vol 13 no 2 (2024) doi 10.5195/d3000.2024.723 h#p://den*stry3000.pi#.edu pouring the model [24]. immersion disinfection of impression is considered the best reliable method, because this method ensures that all the surfaces of the impression and tray will be in direct contact with the disinfectant solution [17]. for that reason, the immersion method was chosen for this study. tween 80 was used with tto as an emulsifier agent to create microemulsions that increase the activity of oil. hence, it had no antibacterial characteristics. it might be utilized to make synthetic dilutable u-type microemulsion, while surfactants could improve membrane fluidity and cell permeability. chemical cleaning involves immersing in solutions with antibacterial, antifungal, and solvent characteristics. these solutions can be used alone or in conjunction with mechanic or ultrasonic cleaners [25]. extracts possess advantages over produced products because of their non-toxic nature and antimicrobial tolerance [26]. tea tree oil was chosen as a disinfectant for addition silicon impression in this study because it is a natural product that has antimicrobial properties and is available, has low cost, is biocompatible, and has no harmful effects on operator’s skin or respiratory system. linear dimensional change test one of the most essential aspects about impression materials is its dimensional stability, which influences the accuracy of impressions and castings. the bulk of the dimensional shrinkage of the silicon impression material is caused by continuous polymerization during the first three minutes after removing the imprint from the mouth. this study evaluated the linear dimensional change using a wellestablished method to test elastomeric impression in vitro, using standardized parameters by iso:4823:2015 to test elastomer impression material in vitro [18]. despite the non-significant result regarding impression dimensional change, the specimens immersed in tto tended to be more dimensionally stable than the control ones. this may be attributed to the fact that the size of the impression material changes because of the disinfection treatment. this can be caused by a chemical interaction table 1. descriptive statistics for linear dimensional change. test-groups n mean stddeviation std-error minimum maximum control 10 1 0 0 1 1 0.25%tto 10 1 0 0 1 1 0.5%tto 10 1 0 0 1 1 0.75%tto 10 1 0 0 1 1 1%tto 10 1 0 0 1 1 2%glutraldehyde 10 1 0 0 1 1 tea tree oil effect on dimensional change and detail reproduc9on of addi9on silicon impression material vol 13 no 2 (2024) doi 10.5195/d3000.2024.723 h#p://den*stry3000.pi#.edu between the setting substance and the disinfectant [27]. this change in size effect might also be attributed to the impression material's continuous polymerization or the evaporation of its volatile components [28,29]. detailed reproduction test detailed reproduction iso 4823:2015 [18] specifies that elastomeric impression materials employed in precision castings shall replicate fine detail to 20 μm or less, ensuring an accurate and comparable approach. in this investigation, an iso 4823:2015 standard apparatus was used to limit the variations associated with uncontrolled factors; hence, the capacity of the imprint material to replicate surface detail was evaluated in a more exact and comparable manner. all specimens in all six groups maintained the crisp and distinct 20 μm line. the null hypothesis was accepted because of the outcomes of this investigation, which showed that impression specimens immersion in tto offered detailed reproduction equivalent to the control specimens. this work was also consistent with vrbova et al.'s [30] investigation, which employed the use of scan electron microscopy (sem), light microscopy, and micro computed tomography to examine dimensional changes. it was observed that the elastomeric impression materials are very resistant to disinfectants. each reproduced a 20 μm line [30]. conclusion within the parameters of this study, it was shown that addition silicone impression material can be safely disinfected by immersion in tto for ten minutes without affecting the details of the reproduction and dimensional accuracy of the impressions. references 1. chidambaranathan, ahila s., and muthukumar balasubramanium. "comprehensive review and comparison of the disinfection techniques currently available in the literature." journal of prosthodontics 28.2 (2019): e849-e856. 2. hussian, a.m. and jassim, r.k. "effect of sodium fluoride addition as a disinfectant on some properties of alginate impression material". journal of baghdad college of dentistry, 27(1), pp.70– 76(2015). doi:10.12816/001526 3. al-azawi, r.a. and al-naqash, w.a. (2016). the effect of silver-zinc zeolite incorporation on some properties of condensation silicone impression material. journal of baghdad college of dentistry, 28(4), pp.22–27. doi:10.12816/0033206. 4. khalaf, hanan abdul-rahman, and mithaq radhi mohammed. "effect of disinfectant agents on certain physical and mechanical properties of type iv dental stone." journal of baghdad college of dentistry 26.1 (2014): 24-31. 5. matalon, shlomo, et al. "do dental impression materials play a role in cross contamination?" quintessence international 42.10 (2011). 6. hardan l, bourgi r, cuevassuárez ce, lukomskaszymanska m, cornejo-ríos e, tosco v, monterubbianesi r, mancino s, eid a, mancino d, et al. disinfection procedures and their effect on the tea tree oil effect on dimensional change and detail reproduc9on of addi9on silicon impression material vol 13 no 2 (2024) doi 10.5195/d3000.2024.723 h#p://den*stry3000.pi#.edu microorganism colonization of dental impression materials: a systematic review and metaanalysis of in vitro studies. bioengineering. 2022; 9(3):123. https://doi.org/10.3390/bioen gineering9030123 7. kumari, nirmala; nandeeshwar, d. b.1. the dimensional accuracy of polyvinyl siloxane impression materials using two different impression techniques: an in vitro study. the journal of indian prosthodontic society 15(3):p 211-217, jul–sep 2015. | doi: 10.4103/09724052.158074 8. pande, neelam a., and r. d. parkhedkar. "an evaluation of dimensional accuracy of onestep and two-step impression technique using addition silicone impression material: an in vitro study." the journal of indian prosthodontic society 13 (2013): 254-259. 9. warrick, e. l., et al. "silicone elastomer developments 1967–1977." rubber chemistry and technology 52.3 (1979): 437-525. 10. kotsiomiti e, tzialla a, hatjivasiliou k: accuracy and stability of impression materials subjected to chemical disinfection – a literature review. j. oral rehabil. 2008; 35: 291–299. 11. saboo k, gemnani rr, sharma s, kumar s, acharya s. chemical pneumonitis and reactive airways dysfunction syndrome after fumigation exposure to dimethanol and glutaraldehyde: a silent menace unmasked. cureus. 2023 aug 4;15(8):e42939. doi: 10.7759/cureus.42939. pmid: 37667710; pmcid: pmc10475320. 12. al-badr rj, al-huwaizi hf. effect of tea tree, thymus vulgaris and nigella sativa oils on the elimination of enterococcus faecalis (in vitro study). j bagh coll dent [internet]. 2017 mar. 13 [cited 2024 jun. 21];29(1):55-62. 13. mehta p, shah r, lohidasan s, mahadik kr. pharmacokinetic profile of phytoconstituent(s) isolated from medicinal plantsa comprehensive review. j tradit complement med. 2015; 5(4):207_227. 14. bakhtiari s, jafari s, taheri jb, kashi t, namazi z, iman m, et al. the effects of cinnamaldehyde (cinnamon derivatives) and nystatin on candida albicans and candida glabrata. open access maced j med sci. 2019; 7(7): 1067– 1070. 15. mahalakshmi, a. s., et al. "comparative evaluation of the effect of electrolyzed oxidizing water on surface detail reproduction, dimensional stability and surface texture of poly vinyl siloxane impressions." the journal of indian prosthodontic society 19.1 (2019): 33-41. 16. cox sd, mann j, markham jl, bell hc, gustafson je, warmington jr, et al. the mode of antimicrobial action of the essential oil of melaleuca alternifolia (tea tree oil). j appl micro. 2000; 88(1):170-175. 17. ada. revised american dental association specification no. 19 for non-aqueous, elastomeric dental impression materials. the journal of the american dental association, (2004)94(4), pp.733–741. doi:10.14219/jada.archive.197 7.0334. tea tree oil effect on dimensional change and detail reproduc9on of addi9on silicon impression material vol 13 no 2 (2024) doi 10.5195/d3000.2024.723 h#p://den*stry3000.pi#.edu 18. iso (2015). 4823:2015 dentistry elastomeric impression materials. iso. 19. oliveira, adriano relvas barreira de, et al. "dimensional change of impression materials for dental prothesis using different measuring methods." matéria (rio de janeiro) 26.02 (2021): e12957. 20. elkholy, sahar, and w. lofty. "sacred lotus as an impression disinfectant and its effect on the dimensional stability of an elastomeric impression material." acta sci dent sci 2 (2018): 39-44. 21. stoeva v, bozhkova t, atanasowski a, et al.: study of knowledge of hand disinfection and dental impressions in everyday practice among dental students during a pandemic by coronavirus disease 2019. open access maced. j. med. sci. 2021; 9: 138–142 22. alzain, sahar. "effect of chemical, microwave irradiation, steam autoclave, ultraviolet light radiation, ozone and electrolyzed oxidizing water disinfection on properties of impression materials: a systematic review and meta-analysis study." the saudi dental journal 32.4 (2020): 161-170. 23. hummudi, israa m., and nidhal s. mansoor. "determination of surface roughness and accuracy of alginate impression material immersion." journal of baghdad college of dentistry 34.3 (2022): 50-57. 24. jasim, zaid m., and shorouq m. abass. "the effect of hypochlorous acid disinfectant on the reproduction of details and surface hardness of type iii dental stone." cureus 14.11 (2022). 25. noori, zahraa s., aseel m. alkhafaji, and fatemeh dabaghi. "effect of tea tree oil on candida adherence and surface roughness of heat cure acrylic resin." journal of baghdad college of dentistry 35.4 (2023): 46-54. 26. yahya khalid, and aseel mohammed al-khafaji. "the impact of tea tree oil on bacillus subtilis and the surface roughness of type iii dental stone." journal of international dental and medical research (2024). 27. sakaguchi rl, powers jm: craig’s restorative dental materials. 13th ed. 2012; 293. 28. walker mp, rondeau m, petrie c, et al.: surface quality and long-term dimensional stability of current elastomeric impression materials after disinfection. j. prosthodont. 2007; 16(5): 343–351 29. de groot, anton c., and erich schmidt. "tea tree oil: contact allergy and chemical composition." contact dermatitis 75.3 (2016): 129143. 30. vrbova, radka, et al. "the effect of disinfectants on the accuracy, quality and surface structure of impression materials and gypsum casts: a comparative study using light microscopy, scanning electron microscopy and micro computed tomography." dental materials journal 39.3 (2020): 500-508. tea tree oil effect on dimensional change and detail reproduc9on of addi9on silicon impression material vol 13 no 2 (2024) doi 10.5195/d3000.2024.723 h#p://den*stry3000.pi#.edu table 2. descriptive statistics for detail reproduction. test groups n mean std-deviation std-error minimum maximum control 10 -0.5754 0.4168 0.1318 -1.379 -0.023 0.25%tto 10 -0.5622 0.2699 0.08534 -0.895 -0.055 0.5%tto 10 -0.5348 0.3405 0.1077 -0.995 0.107 0.75%tto 10 -0.5344 0.4439 0.1404 -1.351 0.207 1%tto 10 -0.4966 0.04957 0.01568 -0.595 -0.439 2%glutraldehyde 10 -0.5007 0.05807 0.01836 -0.595 -0.435 736 2024 the prevalence of par.al edentulism and associated factors: a cross-sec.onal study vol 13 no 2 (2024) doi 10.5195/d3000.2024.736 h#p://den*stry3000.pi#.edu the prevalence of partial edentulism and associated factors: a cross-sectional study ruaa hameed karadi, ghufran dhari awad, zainab mahmood mansi, ahmed abed marzook dijlah university college, baghdad, iraq abstract objec&ve: the current study aimed to determine the prevalence of par5al edentulism according to kennedy’s classifica5on and its associa5on to arch, age and gender. methods: data were collected from 358 pa5ents aged 18 to 80 years. selected pa5ents were grouped according to gender and age (<45, =>45). intra oral examina5on for each pa5ent was done. the palern of par5al edentulism according to kennedy’s classifica5on was determined. sta5s5cal analysis was done using the spss v26 sta5s5cal program. chi-square test was used to test for differences in frequency at p-value<0.05. results and conclusion: the rate of class iii palern in maxillary arch was significantly higher than that of mandibular arch. class i palern in mandibular arch was significantly higher than that of maxillary arch (25.1% vs 14%, p=0.001). no differences in frequency were found between class ii and iv palern and affected arch. class iv was the least frequent in both dental arches. frequency of class i and class ii par5al edentulism increased with age, while class iii was more frequent at young age. class iv palern was more frequent among males than females. keywords: par5al edentulism, kennedy classifica5on, gender differences, sex differences, dental arches. cita;on: karadi rh, et al. (2024) the prevalence of par;al edentulism and associated factors: a crosssec;onal study received: september 21, 2024 accepted: september 30, 2024 published: october 8, 2024 copyright: ©2024 karadi rh, et al. this is an open access ar;cle licensed under a crea;ve commons axribu;on work 4.0 united states license. email: drzainabmansi2009@gmail.com introduction the physiological, social, and psychological aspects of the quality of life associated to oral health are greatly affected by tooth loss. it causes a variety of anatomical, aesthetic, and biomechanical problems that compromise a person's general health and quality of life [1]. in several countries during the past few decades, the prevalence of tooth loss has significantly decreased [2-4]. the decrease in the number of edentulous people is an indicator that oral health in the population is improving [5,6], and it also indicates that the health care system's preventive measures were effective [2,7]. when some natural teeth are lost, but not all of them, then partial edentulism or a gap or space in the dental arch that can be replaced by one or more teeth is established. caries, periodontal disease, trauma, impactions, supernumerary teeth, neoplastic and cystic lesions are the most common causes [8,9]. there are numerous classifications for partial edentulism, but the most popular ones are cummer, neurohr, kennedy, applegates, skinner, and bailyn [10,11]. kennedy's classification for partially edentulous arches is currently regarded as the most frequently accepted classification. kennedy's classification provides quick visualization, prosthesis support identification, and evaluation of removable partial denture design elements [12-14]. kennedy classified all partially edentulous arches into four major types. they are: class i, posterior the prevalence of par.al edentulism and associated factors: a cross-sec.onal study vol 13 no 2 (2024) doi 10.5195/d3000.2024.736 h#p://den*stry3000.pi#.edu to the remaining natural teeth, bilateral edentulous regions; class ii, a single edentulous region that is posterior to the natural teeth that are still present; class iii, an area that is unilaterally edentulous but still has natural teeth on each side of it and, class iv, a single, bilateral (across the midline) edentulous area that is anterior to the remaining natural teeth [15]. class iii has been reported as the most common pattern in maxillary arch, while class iv the least common [16]. also, kennedy type iii was the most common kind of classification in both the maxilla and the mandible, whereas kennedy type iv was found to be the least common in the maxilla and in the mandible [17]. class i and class ii tend to increase with age while class iii and class iv tend to decrease [16,18]. most authors have concluded that there are no significant differences in partial edentulism by gender [19]. this study's objective was to establish the frequency of partially edentulous patients within iraqi population by age, gender, and arches. material and methods this cross-sectional study was conducted at dijlah university dental hospital, iraq, baghdad, where patients treated at the prosthodontics department to restore missing teeth with removable partial denture were selected. this study extended from october 2021 to may 2022. each adult patient having partially edentulous spaces was interviewed and data on age, gender, number of teeth, site of missing teeth, and pattern of partial edentulism according to kennedy’s classification were collected. the study excluded patients who had only missing third molars, unerupted or congenitally missing teeth, retained roots, and mobile teeth that needed to be extracted. in cases of patients having complete edentulism were also not included. ethical consideration the dijlah university dentistry department's ethics and research committee thoroughly evaluated and approved the project. each patient was given a written consent after being fully informed of the study's objectives and receiving assurances regarding the confidentiality of the data that would be collected, which would be anonymous and utilized only for the current study. statistical analysis the statistical program spss v26 was used to analyze data. chisquare was used for all comparisons and a p-value < 0.05 was regarded as the discrimination point for significance. results according to the exclusion criteria, a total of 400 patients were examined and only 358 included in the study. among them, 38.5% were young adults (aged less than 45 years), 45% were males, 77.9% had maxillary involvement, 65.4% had mandibular involvement, and 43.3% had both arches were involved. totally dentate maxillary arches were found in 22.1% of cases, while 14%, 22.6%, 38%, and 3.4% of cases were class i, class ii, class iii and class iv partially edentulous, respectively. regarding the mandibular arch, 34.6% of cases were totally dentate, while 25.1%, 22.6%, 15.1%, and 2.5 % of cases were class i, class ii, class iii and class iv partially edentulous, respectively. the prevalence of par.al edentulism and associated factors: a cross-sec.onal study vol 13 no 2 (2024) doi 10.5195/d3000.2024.736 h#p://den*stry3000.pi#.edu n % age <45 year 138 38.5% ≥45 year 220 61.5% sex male 161 45% female 197 55% maxilla dentate 79 22.1% class i 50 14% class ii 81 22.6% class iii 136 38% class iv 12 3.4% mandible dentate 124 34.6% class i 90 25.1% class ii 81 22.6% class iii 54 15.1% class iv 9 2.5% jaw maxilla 279 77.9% mandible 234 65.4% both 155 43.3% at least class class i 50 14% class ii 112 31.3% class iii 175 48.9% class iv 21 5.9% table 2 displays the frequency of kennedy's classes in the maxillary and mandibular arches. frequency of class i pattern in the mandible was significantly higher than that on the maxillary arch (25.1% vs 14%, p=0.001), while the rate of class iii pattern in the maxillary arch was significantly higher than that of the mandibular arch (38% vs 15.1%, p=0.001). no differences were found between class ii and iv patterns and affected arches (p>0.05). table 1. distribution of studied cases according to the studied characteristics. the prevalence of par.al edentulism and associated factors: a cross-sec.onal study vol 13 no 2 (2024) doi 10.5195/d3000.2024.736 h#p://den*stry3000.pi#.edu mandible maxilla p-value n % n % class i 90 25.1% 50 14% 0.001 class ii 81 22.6% 81 22.6% 1 class iii 54 15.1% 136 38% 0.001 class iv 9 2.5% 12 3.4% 0.506 class iii was more prevalent among younger people (51.4%), which is significantly higher than the frequency found at the old age group (48.6%) (table 3). classes i and ii had the highest incidence among older people (78% and 77.7%, respectively), and both were significantly higher than the frequency found in younger people (22% and 22.3%, respectively). it was found that bounded saddles changed into free end saddles as people aged. class iv was the least frequent pattern, but more cases were found among younger people (57.1%) in comparison to the old age group (42.9%). <45 year =>45 year p-value n % n % class class i 11 22% 39 78% 0.01 class ii 25 22.3% 87 77.7% 0.001 class iii 90 51.4% 85 48.6% 0.001 class iv 12 57.1% 9 42.9% 0.071 frequency of various kennedy classes according to gender is shown in table 4. no significant differences were noticed between class i, ii or iii by gender (p>0.05), but the rate of class iv among males (76.2%) was significantly higher than that of females (23.8%) (p=0.033). table 2. frequency of various kennedy's classes in the maxillary and mandibular arches. table 3. frequency of kennedy’s classes according to age. the prevalence of par.al edentulism and associated factors: a cross-sec.onal study vol 13 no 2 (2024) doi 10.5195/d3000.2024.736 h#p://den*stry3000.pi#.edu male female p-value n % n % class class i 23 46% 27 54% 0.875 class ii 43 38.4% 69 61.6% 0.091 class iii 79 45.1% 96 54.9% 0.949 class iv 16 76.2% 5 23.8% 0.033 discussion removable partial dentures (rpds) should be classified in order to facilitate identification and enhance training. kennedy's classification was employed in this study because it provides a simple method to visualize the partially edentulous arch, enables a logical approach to design problems, and is therefore a logical method of classification [18,20]. in the present study, the high percentage of class iii partial edentulism was in accordance with various studies that found kennedy class iii is the most prevalent pattern of partial edentulism [15,21-25]. the result can be explained by the higher involvement of the maxillary arch, which recorded higher frequency of class iii partial edentulism compared with the mandibular arch, due to the relative early loss of premolars and molars [26-28]. the low prevalence of kennedy class iv can be attributed to the lower chance of tooth loss in the anterior region compared to the molar region. fixed restorations are preferred for the treatment of such edentulism. patients probably give more care and attention to anterior teeth [15,29,30]. in this study, class iii partial edentulism and maxillary arch were significantly correlated. previous work [18,22,31] found that kennedy's class iii was common in the maxillary arch. this study showed no association between class ii and maxillary arch, and this is in disagreement with previous reported data [32], which showed a higher incidence of class ii in the maxillary arch, and this may reflect differences in personal habits of oral hygiene. in this study, there was a significant correlation between kennedy's class i and mandibular arch. this result is in line with previous studies [29,33], in which the mandible was the arch with the highest prevalence of kennedy's class i occurrence. kennedy class i seems to be more commonly treated by rpds [22,24]. the early eruption of mandibular teeth in the oral cavity, which predisposes them to greater caries rates and increased chances of tooth extraction, may be the cause of the higher incidence of class i in the mandibular arch [18]. the study found that young group had more class iii partial edentulism. older age groups had more distal extension (class i, followed by class ii). higher rates of class iii in younger age groups may be attributed to early first molar loss caused by caries. agerelated tooth loss causes the preexisting saddle to be extended, which results in classes i and ii table 4. frequency of kennedy classes according to gender. the prevalence of par.al edentulism and associated factors: a cross-sec.onal study vol 13 no 2 (2024) doi 10.5195/d3000.2024.736 h#p://den*stry3000.pi#.edu [16]. these results were in accordance with previous data from saudi arabia [23,24], which showed higher occurrence of free extended spaces among older people. these studies reported a higher frequency of class iii and class i partial edentulism among younger and older people, respectively. several studies showed a high tendency for class i and class ii increases with age [15,16,18,25,34]. the data showed no evidence of a significant relationship between kennedy classes and gender. regarding class iv, it was more common among males and this may be due to maxillary central incisors trauma caused by physical injuries, such as car and bike accidents, or due to male violent behavior compared with females. conclusion kennedy's class iii is the most common pattern in the maxillary arch, whereas kennedy's class i is more common in the mandibular arch. age had a significant influence on different frequencies of kennedy classes, while gender had a significant effect on the prevalence of kennedy class iv only. it was clear that as people aged, their saddles would change from being bounded to being free. conflicts of interest the authors declare that they have no conflicts of interest. funding this research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors. references 1. al moaleem m. paherns of parial edentulism and its relaion to khat chewing in jazan populaion–a survey study. journal of clinical and diagnosic research: jcdr. 2017 mar;11(3):zc55. 2. madhankumar s, mohamed k, natarajan s, kumar va, athiban i, padmanabhan tv. prevalence of parial edentulousness among the paients reporing to the department of prosthodonics sri ramachandra university chennai, india: an epidemiological study. journal of pharmacy & bioallied sciences. 2015 aug;7(suppl 2):s643. 3. vadavadagi sv, srinivasa h, goutham gb, hajira n, lahari m, reddy gp. parial edentulism and its associaion with socio-demographic variables among subjects ahending dental teaching insituions, india. journal of internaional oral health: jioh. 2015;7(suppl 2):60. 4. mayunga gm, lutula ps, sekele ib, bolenge i, kumpanya n, nyengele k. impact of the edentulousness on the quality of life related to the oral health of the congolese. odontostomatologie tropicale. 2015 jun 1;38(150):31-6. 5. eustaquio-raga mv, almerichsilla jm. factors associated with edentulousness in an elderly populaion in valencia (spain). gaceta sanitaria. 2013;27:123-7. 6. bertossi d, rosseho a, piubelli c, rossini n, zanop g, rodella lf, bissolop g, collep g, chiarini l. evaluaion of quality of life in paients with total or parial edentulism treated with computer-assisted implantology. minerva stomatologica. 2013;8:1. 7. dolan ta, gilbert gh, duncan rp, foerster u. risk indicators of edentulism, parial tooth loss and prostheic status among black and white middle-aged and older adults. community denistry and oral epidemiology. 2001 oct;29(5):329-40. 8. mathur mn, nath s. tooth mortalityan analysis of extracion cases. j indian dent assoc. 1968; 40: 213-15. 9. burt ba. tooth loss. in: burt ba (editor), denistry, dental pracice, and the community, 6 h ed. missouri; elsevier saunders; 2005. p. 223. the prevalence of par.al edentulism and associated factors: a cross-sec.onal study vol 13 no 2 (2024) doi 10.5195/d3000.2024.736 h#p://den*stry3000.pi#.edu 10. kennedy e. classificaion. in: essenials of removable parial denture prosthesis. 2nd ed. philadelphia: wb saunders company; 1960. p. 9 25. 11. nallaswamy d. textbook of prosthodonics. jp medical ltd; 2017 sep 30. 12. mcgarry tj, nimmo a, skiba jf, ahlstrom rh, smith cr, koumjian jh, arbree ns. classificaion system for parial edentulism. journal of prosthodonics. 2002 sep;11(3):181-93. 13. bharathi m, babu kr, reddy g, gupta n, misuriya a, vinod v. parial edentulism based on kennedy's classifcaion: an epidemiological study. the journal of contemporary dental pracice. 2014 mar 1;15(2):229-31. 14. basnyat sk, sapkota b, shrestha s. epidemiological survey on edentulousness in elderly nepalese populaion. kathmandu university medical journal. 2014;12(4):259-63. 15. abdel-rahman hk, tahir cd, saleh mm. incidence of parial edentulism and its relaion with age and gender. zanco journal of medical sciences (zanco j med sci). 2013 aug 1;17(2):463-70. 16. zaigham am, muneer mu. pahern of parial edentulism and its associaion with age and gender. pakistan oral & dental journal. 2010 jun 1;30(1). 17. charyeva oo, altynbekov kd, nysanova bz. kennedy classificaion and treatment opions: a study of parially edentulous paients being treated in a specialized prostheic clinic. journal of prosthodonics: implant, estheic and reconstrucive denistry. 2012 apr;21(3):17780. 18. sadig wm, idowu at. removable parial denture design: a study of a selected populaion in saudi arabia. j contemp dent pract. 2002 nov 15;3(4):40-53. 19. jeyapalan v, krishnan cs. parial edentulism and its correlaion to age, gender, socio-economic status and incidence of various kennedy’s classes–a literature review. journal of clinical and diagnosic research: jcdr. 2015 jun;9(6):ze14. 20. kuzmanovic dv, payne ag, purton dg. distal implants to modify the kennedy classificaion of a removable parial denture: a clinical report. the journal of prostheic denistry. 2004 jul 1;92(1):8-11. 21. araby ya, almutairy as, alotaibi fm. pahern of parial edentulism in correlaion to age and gender among a selected saudi populaion. internaional journal of dental sciences and research. 2017 feb 23;5(1):1-4. 22. curis da, curis ta, wagnild gw, finzen fc. incidence of various classes of removable parial dentures. the journal of prostheic denistry. 1992 may 1;67(5):664-7. 23. fayad mi, baig mn, alrawaili am. prevalence and pahern of parial edentulism among dental paients ahending college of denistry, aljouf university, saudi arabia. journal of internaional society of prevenive & community denistry. 2016 dec;6(suppl 3):s187. 24. dentures and selecion of major connectors and direct/indirect retainers. turkish journal of medical sciences. 2001;31(5):445-9. 25. prabhu n, kumar s, d’souza m, hegde v. parial edentulousness in a rural populaion based on kennedy’s classificaion: an epidemiological study. j indian prosthodont soc. 2009;9(1):18–23. 26. lin hc, corbet ef, lo ec, zhang hg. tooth loss, occluding pairs, and prostheic status of chinese adults. journal of dental research. 2001 may;80(5):1491-5. 27. witler dj, eiteren vp, kayser af. signs and symptoms of mandibular shortened dental arches. j oral rehabil. 1988; 15: 413-420. the prevalence of par.al edentulism and associated factors: a cross-sec.onal study vol 13 no 2 (2024) doi 10.5195/d3000.2024.736 h#p://den*stry3000.pi#.edu 28. hussain fm. survey of removable parial denture wearers in relaion to age, sex and kennedy classificaion of edentulous areas. iraqi dent j. 2001; 27: 243-254. 29. gad mm, abualsaud r, althobity am, al-abidi ks, khan sq, abdel-halim ms, al-harbi fa, el zayat m, fouda sm. prevalence of parial edentulism and rpd design in paients treated at college of denistry, imam abdulrahman bin faisal university, saudi arabia. the saudi dental journal. 2020 feb 1;32(2):749. 30. al-angari n, algarni s, andijani a, alqahtani a. various classes of removable parial dentures: a study of prevalence among paients ahending a dental and educaional insitute in riyadh, saudi arabia. the saudi dental journal. 2021 nov 1;33(7):656-60. 31. al-jammali zm, denisrty aa, falih m, dawood m, yosif m. frequency of occurrence of different kennedy classified cases in college of denistry (iraq). prof.(dr) rk sharma. 2020 oct;20(4):41631. 32. miran fa. incidence of different types of removable parial dentures in sulaimania. jodr. 2018;5(1):70-. 33. khalil a, hussain u, iqbal r, ali w. paherns of parial edentulism among paients reporing to department of prosthodonics, khyber college of denistry, peshawar. journal of khyber college of denistry. 2013 jun 30;3(2):42-5. 34. devishree ra, sangeetha s, jain ar. prevalence of parial edentulism according to kennedy’s classificaion based on age, gender, and arch. drug invent today. 2018 jan 1;10(1):108-110. smart phone applica1on reminder for pa1ents with fixed orthodon1c appliance to improve oral hygiene vol 12 no 1 (2024) doi 10.5195/d3000.2024.699 h#p://den*stry3000.pi#.edu smart phone application reminder for patients with fixed orthodontic appliance to improve oral hygiene ali abdulkadhum jassim1, mehdi abdulhadi mehdi alrubayee2, shakeel kazmi3 1postgraduate student, department of orthodontics, college of dentistry, university of baghdad, baghdad, iraq 2assistant professor of orthodontics, department of orthodontics, college of dentistry, university of baghdad, baghdad, iraq 3associate professor of orthodontics, department of orthodontics, college of dentistry, university of shaheed zulfiqar ali bhutto, islamabad abstract introduction: in light of the extensive use of mobile phones and their associated soaware applica1ons (apps), as well as the undisputed role of reminders and apps in behavioral interven1ons, the goal of this study was to evaluate the usefulness of an applica1on for smartphones (brush dj) to enhance oral hygiene compliance in individuals with fixed orthodon1c therapy. materials and methods: in this prospec1ve study, 82 pa1ents between the ages of 18 and 22 were divided into four groups, two of which (the experimental group and the control group) had just begun fixed orthodon1c treatment (called new) and the other two (the experimental group and the control group) were have passed fiah months from the beginning of the treatment (called old). pa1ents in the interven1on group were asked to use the brush dj smartphone app aaer gepng tradi1onal oral hygiene training, compared to pa1ents in the control group who got conven1onal oral hygiene instruc1on. at baseline (t0), 4 weeks, 8 weeks, and 12 weeks following the start of the study, the plaque index (pi) and marginal bleeding index (mbi) were measured. only pa1ents with the soaware completed a ques1onnaire to determine how frequently and how long they brushed their teeth each day. results: both new and old orthodon1c cases in the interven1on groups showed improvements in pi, whereas the parameter of bleeding showed no significant differences. during the follow-up period, applica1on usage was associated with a higher brushing frequency and dura1on. conclusion: apps might significantly improve the oral hygiene compliance of orthodon1c pa1ents by ac1ng as reminders and mo1vators. keywords: fixed orthodon1c treatment; oral hygiene; smartphone apps; reminders cita:on: jassim, aa et al. (2024) smart phone applica:on reminder for pa:ents with fixed orthodon:c appliance to improve oral hygiene den:stry 3000. 1:a001 doi:10.5195/d3000.2024.699 received: march 31, 2024 accepted: april 5, 2024 published: october 21, 2024 copyright: ©2024 jassim, aa et al. this is an open access ar:cle licensed under a crea:ve commons awribu:on work 4.0 united states license. email: almiahyali99@gmail.com introduction the foundation of excellent oral health is good oral hygiene, particularly in patients with fixed orthodontic appliance where it might be difficult to maintain regular oral hygiene [1,2]. after the initial bonding, previous studies have showed a quick fall in oral hygiene, followed by an improvement by the fifth month of therapy [3]. but other studies have also shown that oral hygiene issues are most severe at the conclusion of orthodontic therapy, highlighting the difficulty in maintaining sustained and acceptable oral hygiene during orthodontic treatment course [4]. lack of basic oral hygiene practices causes dental plaque to build up over time, which raises the risk of overgrowth of cariogenic bacteria such as streptococcus mutans [5,6]. despite having functional and http://dentistry3000.pitt.edu/ smart phone applica1on reminder for pa1ents with fixed orthodon1c appliance to improve oral hygiene vol 12 no 1 (2024) doi 10.5195/d3000.2024.699 h#p://den*stry3000.pi#.edu cosmetic advantages, fixed orthodontic therapy is linked to a higher risk of dental caries, particularly white-spot lesions, whose incidence is estimated to be between 50 and 90% [7,8]. after treatment, these lesions may become aesthetically problematic and progress to become more extensive [9,10]. these undesirable side effects may result in disappointing outcomes or premature termination of orthodontic treatment. according to the majority of orthodontists, 5 to10% of patients discontinue orthodontic treatment early as a result of poor dental hygiene [11,12]. while fluoride administration (e.g. fluoride mouth rinses) and effective plaque removal from all tooth surfaces are crucial in preventing dental caries [13], individuals undergoing fixed orthodontic treatment encounter challenges in maintaining dental hygiene due to the impediment posed by the orthodontic brackets [14,15]. inappropriate oral health and periodontal awareness and knowledge in the iraqi population in particular is present. this emphasizes the need for intervention strategies to improve oral hygiene practice in young people with such appliances in iraq [16]. active reminders have been reported to improve dental and medical appointment attendance, adherence to medication schedules and positive behavior changes [17]. individuals who received motivational text messages reported an increase in tooth brushing habits after 3, 6 and 9 weeks [18]. using a reminder system to establish the habit of brushing is applicable according to a study that showed that the habit is formed after 66 days [19]. the usage of smartphones nowadays opens up new possibilities for creating oral health interventions. this widespread use, particularly among young persons, may guarantee that adolescents wearing fixed orthodontic equipment have full access to care [20]. smartphones have certain specific characteristics that make them appropriate for behavioral interventions, including: (1) their portability and high level of popularity among users, particularly teenagers and young adults. (2) mobile apps (apps) are a more practical and cost-effective form of intervention. (3) the distribution of health-related information and the delivery of behavioral therapies are made easier by cellphones' capacity to connect in a convenient manner [21]. considering the widespread use of smartphones and apps in diverse fields by the younger generation, as well as the undeniable role of reminders and apps in behavioral interventions, use of smartphone apps for this purpose is understandable. hence, in this study, we tested a mobile health technology, the brush dj app (https://www.nhs.uk/appslibrary/brush-dj/), to see if it improved oral hygiene measures of orthodontic patients. material and methods subjects and settings the study was conducted in the orthodontic clinic at al-shamyia specialized center in thi-qar. participants for this study included patients with fixed appliance orthodontic treatment according to the following criteria. inclusion criteria: i. pa`ents with ages between 18 and 22 years who received their orthodon`c appliances with the star`ng of the study and pa`ents that passed their fibh month of orthodon`c treatment. ii. pa`ents who had smartphones with enough storage to download the sobware program. iii. all anterior teeth were present without missing any tooth and cases with lateral incisor agenesis were avoided. iv. subjects should talk and read arabic language. exclusion criteria: http://dentistry3000.pitt.edu/ https://www.nhs.uk/apps-library/brush-dj/ https://www.nhs.uk/apps-library/brush-dj/ smart phone applica1on reminder for pa1ents with fixed orthodon1c appliance to improve oral hygiene vol 12 no 1 (2024) doi 10.5195/d3000.2024.699 h#p://den*stry3000.pi#.edu i. pa`ents with mental and physical disabili`es, as well as craniofacial problems. ii. pa`ents with dysplasia of the enamel or den`n. iii. pa`ents who are receiving an`plaque drugs, such as an`bio`cs or an`bacterial mouth rinses. iv. pa`ents suffering from periodontal disease. v. pa`ents needing first premolar extrac`ons. intervention period the sample was divided into four groups, two groups were experimental and control starting orthodontic treatment with the starting of the study (called new), and the other two, also experimental and control, had passed their fifth month from the starting of the orthodontic treatment (called old). only the new groups were recalled after one week after insertion of the appliances for recording the scores to give time for plaque formation and gingival irritation. all groups received oral hygiene instructions, toothpaste (sensodyne), oral hygiene kit at the baseline (t0) and only the experimental group participants were instructed to download the application (brush dj). the oral hygiene habits were recorded in the experimental groups (new and old) only, using a questionnaire before and after the end of the study. the study outcomes were the amount of plaque and the total number of gingival bleeding sites in the incisors, canines and first premolars of the maxilla and mandible. al-anezi and harradine plaque was used to measure the amount of plaque on the buccal surfaces [22]. gingival bleeding was assessed using the marginal bleeding index. statistical analysis the statistical package for social sciences software (spss inc., version 26, chicago, illinois, usa) was used to carry out the statistical procedures. data were analyzed through parametric tests including: 1. descriptive statistics: • mean • standard devia`on (sd) • tables and figures 2. inferential statistics: • paired sample t-test. in the statistical evaluation, the following levels of significance were used according to probability value (p-value): p ˃ 0.05 non-significant (ns) 0.05 ≥ p ˃ 0.01 * significant 0.01 ≥ p ˃ 0.001 ** highly significant results eighty-two fixed orthodontic patients were enrolled in this study with ages ranging from 18 to 22 years. fortyseven patients were females and 35 males. forty patients (20:20) were in the new groups (experimental and control) and 42 patients (21:21) in the old groups (experimental and control). follow up of plaque index new experimental group the plaque index was assessed after 4 weeks, 8 weeks, and 12 weeks, and compared to that at baseline. the mean plaque index of the new experimental group was significantly reduced from 0.39 at baseline to 0.30 (p= 0.037) after 4 weeks, then to 0.27 (p= 0.018) after 8 weeks, and to 0.24 (p= 0.022) after 12 weeks of orthodontic treatment. new control group the comparison of the mean plaque index of the new control group at baseline, after 4 weeks, 8 weeks, and 12 weeks of treatment showed no significant differences in the mean plaque index (p ≥ 0.05) throughout the study period. comparison between new experimental and control groups the comparison between the new experimental and control groups according to the percentage of change in plaque index revealed that the percentage reduction in the plaque index of the new experimental group was significantly higher after 8 weeks and 12 weeks of http://dentistry3000.pitt.edu/ smart phone applica1on reminder for pa1ents with fixed orthodon1c appliance to improve oral hygiene vol 12 no 1 (2024) doi 10.5195/d3000.2024.699 h#p://den*stry3000.pi#.edu treatment compared with the new control group (-47 vs -5.9, p= 0.014 after 8 weeks, and -50.5 vs -6.08, p= 0.008 after 12 weeks) (figure 1 and table 1). old experimental group in this group, the mean plaque index significantly reduced from 1.01 at baseline to 0.81 (p= 0.005) after 4 weeks, and to 0.78 (p= 0.001) after 8 weeks, then to 0.67 (p= 0.001) after 12 weeks of orthodontic treatment. old control group in this group, the mean plaque index after 4 weeks, 8 weeks, and 12 weeks of orthodontic treatment was not significantly different (p ≥ 0.05) than that at baseline. comparison between old experimental and control groups -60.00 -40.00 -20.00 0.00 20.00 40.00 60.00 baseline 4 weeks 8 weeks 12 weeks new experimental group new control group % plaque index change study groups t-test pvalue* new experimental mean ± sd new control mean ± sd after 4 weeks 31.2 ± 18.4 21.1 ± 6.62 0.584 0.563 after 8 weeks 47.3 ± 22.2 5.9 ± 2.35 2.606 0.014 after 12 weeks 50.5 ± 23.6 6.08 ± 3.62 2.817 0.008 figure 1. percentage of change in plaque index of the new experimental and control groups throughout the study period. table 1: comparison between new experimental and control groups by percentage change in the plaque index throughout the study period (red colour indicates statistical significant differences). http://dentistry3000.pitt.edu/ smart phone applica1on reminder for pa1ents with fixed orthodon1c appliance to improve oral hygiene vol 12 no 1 (2024) doi 10.5195/d3000.2024.699 h#p://den*stry3000.pi#.edu in comparison between the old experimental and control groups according to percentage of change in plaque index, after 8 weeks and 12 weeks the old experimental group had a significant higher percentage reduction in the plaque index than the old control group (-29 vs -3.89, p= 0.047 and -37.3 vs -13.42, p= 0.0039, respectively) (figure 2 and table 2). -60.00 -40.00 -20.00 0.00 20.00 40.00 60.00 baseline 4 weeks 8 weeks 12 weeks old experimental group old control group % plaque index change study groups t-test pvalue* old experimental mean ± sd old control mean ± sd after 4 weeks 19.3 ± 5.71 1.80 ± 0.88 1.296 0.203 after 8 weeks -29.0 ± 9.15 3.89 ± 1.63 2.040 0.047 after 12 weeks 37.3 ± 18.7 13.42 ± 4.05 2.063 0.039 figure 2. percentage of change in plaque index of the old experimental and control groups throughout the study period. table 2: comparison between old experimental and control groups by percentage change in the plaque index throughout the study period (red colour indicates statistical significant differences). http://dentistry3000.pitt.edu/ smart phone applica1on reminder for pa1ents with fixed orthodon1c appliance to improve oral hygiene vol 12 no 1 (2024) doi 10.5195/d3000.2024.699 h#p://den*stry3000.pi#.edu follow up of gingival bleeding index new experimental group regarding the comparison in the mean gingival bleeding index of the new experimental group, no significant difference (p ≥ 0.05) was obtained in the mean gingival bleeding over the course of this study. new control group in this group, the mean bleeding index significantly increased after 4 weeks compared to that at baseline (0.035 vs 0.018, p= 0.01). no significant difference (p ≥ 0.05) was detected after 8 weeks and after 12 weeks of treatment. comparison between new experimental and control groups the comparison between the new experimental and control groups according to the percentage of change in bleeding revealed that there was no significant difference (p ≥ 0.05) in the degree of bleeding reduction between the two groups (figure 3 and table 3). -60.00 -40.00 -20.00 0.00 20.00 40.00 60.00 baseline 4 weeks 8 weeks 12 weeks new experimental group new control group % bleeding index change study groups t-test pvalue* new experimental mean ± sd new control mean ± sd figure 3. percentage of change in bleeding index of the new experimental and control groups throughout the study period. table 3: comparison between new experimental and control groups by percentage change in the bleeding index throughout the study period. http://dentistry3000.pitt.edu/ smart phone applica1on reminder for pa1ents with fixed orthodon1c appliance to improve oral hygiene vol 12 no 1 (2024) doi 10.5195/d3000.2024.699 h#p://den*stry3000.pi#.edu old experimental group the mean bleeding index of this group significantly decreased within time from 0.063 at baseline to 0.041 (p= 0.011) after 4 weeks, and to 0.031 (p= 0.001) after 8 weeks, then to 0.029 (p= 0.001) after 12 weeks of orthodontic treatment. old control group in this group, the mean bleeding index was not significantly different (p ≥ 0.05) after 4 weeks, 8 weeks, and 12 weeks than that at baseline. comparison between old experimental and control groups in comparison between the old experimental and control groups according to the percentage of change in bleeding index, no significant difference was seen in the degree of bleeding reduction between the two groups (figure 4 and table 4). -60.00 -40.00 -20.00 0.00 20.00 40.00 60.00 baseline 4 weeks 8 weeks 12 weeks old experimental group old control group after 4 weeks 26.6 ± 9.81 23.33 ± 9.144 1.242 0.309 after 8 weeks 18.3 ± 8.62 16.66 ± 5.27 1.361 0.207 after 12 weeks 38.6 ± 19.81 36.6 ± 17.12 1.423 0.189 figure 4. percentage of change in bleeding index of the old experimental and control groups throughout the study period. http://dentistry3000.pitt.edu/ smart phone applica1on reminder for pa1ents with fixed orthodon1c appliance to improve oral hygiene vol 12 no 1 (2024) doi 10.5195/d3000.2024.699 h#p://den*stry3000.pi#.edu questionnaire new experimental groups the results of the questionnaire before and after the intervention were summarized in table 5. the mean score of the oral hygiene questionnaire of patients in new experimental group significantly increased after using the smartphone application (p = 0.003) (table 6). questions before intervention after intervention do you brush your teeth? 1) yes 75% (15) 75% (15) 2) occasionally or never 25% (5) 25% (5) how many times do you brush your teeth every day? 1) brush not every day 25% (5) 5% (1) 2) brush once a day 35% (7) 15% (3) 3) brush twice a day or more 40% (8) 80% (16) do you use toothpaste when brushing your teeth? 1) yes 95% (19) 100% (20) 2) no 5% (1) 0% 3) i don’t know 0% 0% do you use fluoride toothpaste when brushing your teeth? 1) yes 5% (1) 10% (2) 2) no 20% (4) 35% (7) 3) i don’t know 75% (15) 55% (11) do you use floss? 1) no 60% (12) 30% (6) 2) occasionally 20% (4) 35% (7) 3) once a week 10% (2) 25% (5) 4) daily use 10% (2) 10% (2) how often do you change your toothbrush? % bleeding index change study groups t-test pvalue* old experimental mean ± sd old control mean ± sd after 4 weeks 21.1 ± 11.03 16.88 ± 7.43 1.437 0.187 after 8 weeks -24.4 ± 10.55 22.2 ± 11.67 1.074 0.379 after 12 weeks 36.70 ± 17.84 33.24 ± 15.04 1.256 0.201 table 4: comparison between new experimental and control groups by percentage change in the plaque index throughout the study period. table 5: questionnaire data for new experimental group. http://dentistry3000.pitt.edu/ smart phone applica1on reminder for pa1ents with fixed orthodon1c appliance to improve oral hygiene vol 12 no 1 (2024) doi 10.5195/d3000.2024.699 h#p://den*stry3000.pi#.edu 1) every 3 months 45% (9) 50% (10) 2) every 6 months 35% (7) 50% (10) 3) every year 20% (4) 0% 4) more than year 0% 0% how long do you take for oral hygiene? 1) about one minute 65% (13) 5% (1) 2) about two minutes 0% 55% (11) 3) more than two minutes 15% (3) 20% (4) 4) i don’t know 20% (4) 20% (4) are you interesting in orthodontic treatment? 1) yes 100% (20) 100% (20) 2) no 0% 0% are you interesting to be compliant in oral hygiene control during and after orthodontic treatment? 1) yes 65% (13) 80% (16) 2) no 0% 0% 3) i don’t know 35% (7) 20% (4) old experimental the results of the questionnaire before and after the intervention were summarized in table 7. the mean score of the oral hygiene questionnaire of patients in the old experimental group significantly increased after using the smartphone application (p = 0.001) as shown in table 8. questions before intervention after intervention do you brush your teeth? 1) yes 90.5% (19) 100% (21) 2) occasionally or never 9.5% (2) 0% how many times do you brush your teeth every day? 1) brush not every day 0% 0% 2) brush once a day 23.8% (5) 9.5% (2) 3) brush twice a day or more 76.2% (16) 90.5% (19) do you use toothpaste when brushing your teeth? new experimental group questionnaire score mean ± sd paired t-test pvalue* before application 4.50 ± 1.39 3.470 0.003 after application 5.25 ± 1.29 table 6: comparison in oral hygiene scores of the new experimental group before and after using the smartphone application (red colour indicates statistical significant difference). table 7: questionnaire data for old experimental group. http://dentistry3000.pitt.edu/ smart phone applica1on reminder for pa1ents with fixed orthodon1c appliance to improve oral hygiene vol 12 no 1 (2024) doi 10.5195/d3000.2024.699 h#p://den*stry3000.pi#.edu 1) yes 100% (21) 100% (21) 2) no 0% 0% 3) i don’t know 0% 0% do you use fluoride toothpaste when brushing your teeth? 1) yes 19% (4) 33.3% (7) 2) no 14.3% (3) 14.3% (3) 3) i don’t know 66.7% (14) 52.4% (11) do you use floss? 1) no 42.9% (9) 52.4% (11) 2) occasionally 42.9% (9) 33.3% (7) 3) once a week 9.5% (2) 4.8% (1) 4) daily use 4.8% (1) 9.5% (2) how often do you change your toothbrush? 1) every 3 months 38.1% (8) 71.4% (15) 2) every 6 months 52.4% (11) 28.2% (16) 3) every year 9.5% (2) 0% 4) more than year 0% 0% how long do you take for oral hygiene? 1) about one minute 57.1% (12) 33.3 (7) 2) about two minutes 33.3% (7) 47.6% (10) 3) more than two minutes 9.5% (2) 19% (4) 4) i don’t know 0% 0% are you interesting in orthodontic treatment? 1) yes 100% (21) 90.5% (19) 2) no 0% 9.5% (2) are you interesting to be compliant in oral hygiene control during and after orthodontic treatment? 1) yes 52.4% (11) 76.2% (16) 2) no 0% 0% 3) i don’t know 47.6% (10) 23.8% (5) discussion plaque index new groups in this study, the new experimental group showed a significant reduction in plaque index (pi) from baseline to 12 weeks after orthodontic treatment, aligning with similar old experimental group questionnaire score mean ± sd paired t-test pvalue* before application 4.95 ± 1.39 4.483 0.001 after application 5.90 ± 1.29 table 8: comparison in the scores of oral hygiene questionnaire of the old experimental groups before and after using the smartphone application (red colour indicates statistical significant difference). http://dentistry3000.pitt.edu/ smart phone applica1on reminder for pa1ents with fixed orthodon1c appliance to improve oral hygiene vol 12 no 1 (2024) doi 10.5195/d3000.2024.699 h#p://den*stry3000.pi#.edu studies using smartphone apps as instructional tools [23]. notably, significant differences in percentage of change of pi between the two new groups (experimental and control) emerged after 8 and 12 weeks, but not at the 4-week follow-up. this initial one-month period after braces installation is challenging for patients, and it is crucial to establish good oral habits during this time [24,25]. old groups both groups (experimental and control) that had orthodontic devices for five months experienced reduced plaque values after 12 weeks, consistent with other studies that observed an initial decline in oral hygiene followed by improvement by the fifth month of treatment [3]. the lack of significant differences at the 4week follow-up between the two old groups could be due to the difficulty of changing oral habits within the first month, as forming new habits can take varying durations [26,27]. bleeding index new groups regarding bleeding scores, the experimental and control new groups generally showed no significant differences throughout the study, except for the control group, which saw an significant increase after 4 weeks. this aligns with a suggestion that patients with fixed orthodontic appliances may experience slight increases in oral parameters early on [28,29]. a study by hadzic et al. (2022) noted that the bleeding index increased but could be mitigated by intensive oral hygiene practices [30]. old groups in the groups with 5 months of orthodontic bonding, the experimental group showed a significant reduction in bleeding scores throughout the study, while the control group did not exhibit statistical differences. these results are similar to a study by eppright et al. (2014), which used text messages as reminders to improve oral conditions and found improvements in bleeding and plaque indices over time in the reminder group [31]. however, in contrast to their findings, there were no significant differences between the two old groups regarding the percentage of change in plaque values in this study. questionnaire brushing frequency most of the respondents brush their teeth before intervention in experimental groups both new group and the group that have passed 5 months of bonding the orthodontic device with only 40% of the new group and 76.2% of the other group brush twice or more a day. in a comparison with other studies, it was found that most of the participants brush twice a day and others brush trice [32,33]. in contrast to pandey et al. (2016) study who found that 63% of the sample brushed once daily while 26% brushed twice daily, and 11% brushed thrice a day [34]. after the intervention by using the brush dj reminder it was found an increase in the time of brushing a day (80% for the new group and 90.5% for other group) twice or more daily. these results are strongly consistent with results of other research by farhadifard, et al. (2020) who found that brushing frequency and duration were correlated positively with app usage during the follow-up period [35]. as the majority of the patients practice the brushing twice daily which is what is recommended during patient education, it is an indication that they took the instruction and the app seriously and practice them in a good manner [33]. dental floss the majority of the respondents doesn't floss their teeth before and after the intervention this may attributed to the difficulty for flossing the teeth. these results are in agreement with pinto et al. (2017) who found fewer patients reporting daily use as the duration of orthodontic treatment increased [36]. evidence suggests that the compliance rate for daily flossing is low, particularly among subjects wearing orthodontic appliances since the appliances make flossing more challenging [36,37]. fluoridated toothpaste http://dentistry3000.pitt.edu/ smart phone applica1on reminder for pa1ents with fixed orthodon1c appliance to improve oral hygiene vol 12 no 1 (2024) doi 10.5195/d3000.2024.699 h#p://den*stry3000.pi#.edu in this questionnaire the participants were asked for using toothpastes with fluoride, the majority of the percent respond for ''i don’t know''. while after intervention for both experimental groups, the percentage decreased and slightly increased for whom used fluoridated paste. this change it may be attributed to the patient’s education about reading the component of the toothpaste. toothbrush replacement before the intervention, less than half of the sample in both groups changed their toothbrush every 3 months as recommended by the american dental association. after the intervention, only one person in the new group followed this recommendation, making it 50%. in the group that had passed 5 months of treatment, there was a notable increase in patients (71.4%) choosing to change their toothbrushes every 3 months. this shift may be attributed to the complexity of orthodontic treatment as it progresses, making plaque removal more challenging, leading to increased brushing frequency and earlier damage to toothbrush bristles [38]. brushing duration in accordance with most of the respondents, the app encouraged them to wash their teeth for longer time. according to a us survey, individuals wash their teeth for an average of 46 seconds [39]. however, studies have shown that brushing for two minutes eliminates 26% more plaque than brushing for 45 seconds [40]. the increased brushing time reported by app users is encouraging, and it seems that the usage of music encourages this longer brushing period. this results are in agreement with underwood et al (2015) as they reported for 88% of respondents that the app motivated them to brush their teeth for longer duration [40]. conclusion the utilization of a smartphone application as an adjunctive tool for enhancing oral hygiene among orthodontic patients has yielded highly promising results. these findings suggest that smartphone applications can serve as valuable resources for orthodontic patients in maintaining their oral health during the course of treatment. data availability the data that support the findings of this study are available from the corresponding author upon reasonable request. conflict of interest all the authors declare no commercial or financial conflict of interest. references 1. zachrisson bu. oral hygiene for orthodon7c pa7ents: current concepts and prac7cal advice. am j orthod. 1974 nov;66(5):487-97. doi: 10.1016/0002-9416(74)90110-9. pmid: 4621247. 2. bollen am, cunha-cruz j, bakko dw, huang gj, hujoel pp. the effects of orthodon7c therapy on periodontal health: a systema7c review of controlled evidence. j am dent assoc. 2008 apr;139(4):413-22. doi: 10.14219/jada.archive.2008.018 4. pmid: 18385025. 3. al-jewair ts, suri s and tompson bd. predictors of adolescent compliance with oral hygiene instruc7ons during two-arch mul7bracket fixed orthodon7c treatment. the angle orthodon7st, 2011,81 (3), 525531. 4. cantekin k, celikoglu m, karadas m, yildirim h and erdem a. effects of orthodon7c treatment with fixed appliances on oral health status: a comprehensive study. journal of dental science, 2011,6 (4), 235-238. 5. rosenbloom rg, tinanoff n. salivary streptococcus mutans levels in pa7ents before, during, and aeer orthodon7c treatment. am j orthod dentofacial orthop. 1991 jul;100(1):35-7. doi: 10.1016/0889-5406(91)70046-y. pmid: 2069145. 6. lara-carrillo e, mon7el-bas7da nm, sánchez-pérez l, alanístavira j. effect of orthodon7c treatment on saliva, plaque and the levels of streptococcus mutans and lactobacillus. med oral patol oral cir bucal. 2010 nov 1;15(6):e924-9. doi: 10.4317/medoral.15.e924. pmid: 20383105. 7. boersma jg, van der veen mh, lagerweij md, bokhout b, prahlandersen b. caries prevalence measured with qlf aeer treatment with fixed orthodon7c appliances: influencing factors. caries res. 2005 janfeb;39(1):41-7. doi: 10.1159/000081655. pmid: 15591733. http://dentistry3000.pitt.edu/ smart phone applica1on reminder for pa1ents with fixed orthodon1c appliance to improve oral hygiene vol 12 no 1 (2024) doi 10.5195/d3000.2024.699 h#p://den*stry3000.pi#.edu 8. chapman ja, roberts we, eckert gj, kula ks, gonzález-cabezas c. risk factors for incidence and severity of white spot lesions during treatment with fixed orthodon7c appliances. am j orthod dentofacial orthop. 2010 aug;138(2):188-94. doi: 10.1016/j.ajodo.2008.10.019. pmid: 20691360. 9. ogaard b. prevalence of white spot lesions in 19-year-olds: a study on untreated and orthodon7cally treated persons 5 years aeer treatment. am j orthod dentofacial orthop. 1989 nov;96(5):423-7. doi: 10.1016/0889-5406(89)90327-2. pmid: 2816842. 10. shungin d, olsson ai, persson m. orthodon7c treatment-related white spot lesions: a 14-year prospec7ve quan7ta7ve followup, including bonding material assessment. am j orthod dentofacial orthop. 2010 aug;138(2):136.e1-8; discussion 136-7. doi: 10.1016/j.ajodo.2009.05.020. pmid: 20691346. 11. mehra t, nanda rs, sinha pk. orthodon7sts' assessment and management of pa7ent compliance. angle orthod. 1998 apr;68(2):115-22. doi: 10.1043/00033219(1998)068<0115:oaamop> 2.3.co;2. pmid: 9564420. 12. derks a, kuijpers-jagtman am, frencken je, van't hof ma, katsaros c. caries preven7ve measures used in orthodon7c prac7ces: an evidence-based decision? am j orthod dentofacial orthop. 2007 aug;132(2):165-70. doi: 10.1016/j.ajodo.2005.10.028. pmid: 17693365. 13. kerbusch ae, kuijpers-jagtman am, mulder j, sanden wj. methods used for preven7on of white spot lesion development during orthodon7c treatment with fixed appliances. acta odontol scand. 2012 dec;70(6):564-8. doi: 10.3109/00016357.2011.640282 . epub 2012 jan 18. pmid: 22250977. 14. geiger am, gorelick l, gwinnes aj, benson bj. reducing white spot lesions in orthodon7c popula7ons with fluoride rinsing. am j orthod dentofacial orthop. 1992 may;101(5):403-7. doi: 10.1016/08895406(92)70112-n. pmid: 1590288. 15. geiger am, gorelick l, gwinnes aj, griswold pg. the effect of a fluoride program on white spot forma7on during orthodon7c treatment. am j orthod dentofacial orthop. 1988 jan;93(1):29-37. doi: 10.1016/0889-5406(88)90190-4. pmid: 3276146. 16. abdulbaqi hr, abdulkareem aa, alshami ml, milward mr. the oral health and periodontal diseases awareness and knowledge in the iraqi popula7on: online-based survey. clin exp dent res. 2020 oct;6(5):519-528. doi: 10.1002/cre2.304. epub 2020 jun 26. pmid: 32592312; pmcid: pmc7545227. 17. almog dm, devries ja, borrelli ja and kopycka-kedzierawski dt. the reduc7on of broken appointment rates through an automated appointment confirma7on system. journal of dental educa7on, 2003,67 (9), 1016-1022. 18. schluter p, lee m, hamilton g, coe g, messer-perkins h and smith b. keep on brushing: a longitudinal study of mo7va7onal text messaging in young adults aged 18–24 years receiving work and income support. journal of public health den7stry, 2015,75 (2), 118-125. 19. whisaker h, phillippa-touchais a, touchais g, voutsaki s and wright j. some thoughts on middle helladic religious beliefs and ritual and their significance in rela7on to social structure. mesohelladika: the greek mainland in the middle bronze age 2010, 535-543. 20. dennison l, morrison l, conway g, yardley l. opportuni7es and challenges for smartphone applica7ons in suppor7ng health behavior change: qualita7ve study. j med internet res. 2013 apr 18;15(4):e86. doi: 10.2196/jmir.2583. pmid: 23598614; pmcid: pmc3636318. 21. al-anezi sa, harradine nw. quan7fying plaque during orthodon7c treatment: angle orthod. 2012 jul;82(4):748-53. doi: 10.2319/050111-312.1. epub 2011 nov 1. pmid: 22044115; pmcid: pmc8845533. 22. deleuse m, meiffren c, bruwier a, maes n, le gall m, charavet c. smartphone applica7onassisted oral hygiene of orthodon7c pa7ents: a mul7centre randomized controlled trial in adolescents. eur j orthod. 2020 feb 1:cjz105. doi: 10.1093/ejo/cjz105. epub ahead of print. pmid: 32006440. 23. firth f, bennani f, stacknik s and farella m. orthodon7c pa7ent co-opera7on: a review of the clinician’s role in predic7ng and improving pa7ent compliance. australasian orthodon7c journal. 2019,35(1), 5-12. 24. pa7l s, hedad ia, jafer aa, abutaleb gk, arishi tm, arishi sa, arishi ha, jafer m, gujar an, khan s, raj at. effec7veness of mobile phone applica7ons in http://dentistry3000.pitt.edu/ smart phone applica1on reminder for pa1ents with fixed orthodon1c appliance to improve oral hygiene vol 12 no 1 (2024) doi 10.5195/d3000.2024.699 h#p://den*stry3000.pi#.edu improving oral hygiene care and outcomes in orthodon7c pa7ents. j oral biol craniofac res. 2021 jan-mar;11(1):26-32. doi: 10.1016/j.jobcr.2020.11.004. epub 2020 nov 17. pmid: 33344158; pmcid: pmc7736984. 25. lally p, van jaarsveld ch, poss hw and wardle j. how are habits formed: modelling habit forma7on in the real world. european journal of social psychology. 2010, 40(6), 9981009. 26. stawarz k, cox al, blandford a. beyond self-tracking and reminders: designing smartphone apps that support habit forma7on. in proceedings of the 33rd annual acm conference on human factors in compu7ng systems. 2015 ,pp. 2653-2662. 27. hussein sr and hassan ba. assessment of gingival health status among pa7ents with fixed orthodon7c appliance. polytechnic journal. 2021,11(1), 38-41. 28. kawsar ma, islam mr, rezwana r, prodhan mr, habib ma and abdullah mk. gingival bleeding index status among orthodon7c pa7ents treated with fixed orthodon7c appliances in a ter7ary level hospital. kyamc journal.2018, 9(3), 129-132. 29. hadzic s, gojkov-vukelic m, pasic e, jahic im, muharemovic a, redzepagic-vrazalica l, jeleskovic a, nakas e. evalua7on of periodontal changes in pa7ents before, during, and aeer a fixed orthodon7c therapy. mater sociomed. 2022 jun;34(2):121-125. doi: 10.5455/msm.2022.34.121-125. pmid: 36199847; pmcid: pmc9478536. 30. eppright m, shroff b, best am, barcoma e, lindauer sj. influence of ac7ve reminders on oral hygiene compliance in orthodon7c pa7ents. angle orthod. 2014 mar;84(2):208-13. doi: 10.2319/062813-481.1. epub 2013 sep 12. pmid: 24028316; pmcid: pmc8673795. 31. aljohani sr, alsaggaf dh. adherence to dietary advice and oral hygiene prac7ces among orthodon7c pa7ents. pa7ent prefer adherence. 2020 oct 20;14:1991-2000. doi: 10.2147/ppa.s277034. pmid: 33116442; pmcid: pmc7586054. 32. buthelezi nl and madiba tk. oral hygiene habits and status of orthodon7c pa7ents asending the university of pretoria, oral and dental hospital. south african dental journal. 202176(3), 130-135. 33. pandey v, chandra s, dilip kumar hp, gupta a, bhandari pp, rathod p. impact of dental neglect score on oral health among pa7ents receiving fixed orthodon7c treatment: a crosssec7onal study. j int soc prev community dent. 2016 marapr;6(2):120-4. doi: 10.4103/2231-0762.178752. pmid: 27114950; pmcid: pmc4820570. 34. farhadifard h, soheilifar s, farhadian m, kokabi h, bakhshaei a. orthodon7c pa7ents' oral hygiene compliance by u7lizing a smartphone applica7on (brush dj): a randomized clinical trial. bdj open. 2020 nov 20;6(1):24. doi: 10.1038/s41405-020-000505. pmid: 33298841; pmcid: pmc7680123. 35. pinto as, alves ls, zenkner jeda, zanasa fb, maltz m. gingival enlargement in orthodon7c pa7ents: effect of treatment dura7on. am j orthod dentofacial orthop. 2017 oct;152(4):477-482. doi: 10.1016/j.ajodo.2016.10.042. pmid: 28962731. 36. andlin-sobocki a, bodin l. dimensional altera7ons of the gingiva related to changes of facial/lingual tooth posi7on in permanent anterior teeth of children. a 2-year longitudinal study. j clin periodontol. 1993 mar;20(3):219-24. doi: 10.1111/j.1600051x.1993.tb00347.x. pmid: 8450088. 37. van leeuwen mpc, van der weijden fa, slot de, rosema mam. toothbrush wear in rela7on to toothbrushing effec7veness. int j dent hyg. 2019 feb;17(1):77-84. doi: 10.1111/idh.12370. epub 2018 nov 19. pmid: 30326176; pmcid: pmc7379636. 38. beals d, ngo t, feng y, cook d, grau dg, weber da. development and laboratory evalua7on of a new toothbrush with a novel brush head design. am j dent. 2000 mar;13(spec no):5a-14a. pmid: 11763943. 39. creeth je, gallagher a, sowinski j, bowman j, barres k, lowe s, patel k, bosma ml. the effect of brushing 7me and den7frice on dental plaque removal in vivo. j dent hyg. 2009 summer;83(3):111-6. epub 2009 aug 14. pmid: 19723429. 40. underwood b, birdsall j, kay e. the use of a mobile app to mo7vate evidence-based oral hygiene behaviour. br dent j. 2015 aug 28;219(4):e2. doi: 10.1038/sj.bdj.2015.660. pmid: 26315196. http://dentistry3000.pitt.edu/ 930 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.930 http://dentistry3000.pitt.edu neuromuscular relaxation using anterior leaf gauge to quick guide the condyles in proper position amal abdallah a. abo-elmagd college of den*stry, qassim university, saudi arabia and faculty of dental surgery, misr for science & technology university, egypt abstract to confirm the centric rela6on (cr), the leaf gauge can be used to load the tmj. a load test will yield a nega6ve result for a healthy tmj. nonetheless, the joint or muscles may be the reason of tmj disorders if the pa6ent experiences pain, tension, or tenderness, par6cularly in the region in front of the ear or nearby. in addi6on, the mandible can be in a centric rela6onship using a leaf gauge, which is made up of several plas6c leaves. the leaf gauge helps the pa6ent retrude the mandible by posi6oning it between the anterior teeth. the condyles tend to be moved against their menisci by the bi6ng force. therefore, this review study aims to describe the importance of the leaf gauge for neuromuscular relaxa6on to quick guide the condyles in cr posi6on, to assist the recording of a reproducible jaw posi6on during prosthodon6c treatment. open access cita%on: abo-elmagd aaa (2025) neuromuscular relaxa%on using anterior leaf gauge to quick guide the condyles in proper posi%on. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.930 received: may 3, 2025 accepted: may 6, 2025 published: june 26, 2025 copyright: ©2025 abo-elmagd aaa. this is an open access ar%cle licensed under a crea%ve commons avribu%on work 4.0 united states license. email: a.aboelmagd@qu.edu.sa centric relation plays a signi0icant role in occlusion and clinical practice to accomplish the relations between joints, muscles, and teeth. moreover, it has direct effects on maintenance of oral health, prosthetic rehabilitation, orthodontic and maxillofacial planning, temporomandibular disorder (tmd) therapy, and occlusal rehabilitation. thus, using a cr technique that produces the highest reproducibility of condylar positions in cr is essential [1]. the current de0inition of centric relation is “a maxillomandibular relationship, independent of tooth contact, in which the condyles articulate in the anterior-superior position against the posterior slopes of the articular eminences; in this position, the mandible is restricted to a purely rotary movement; from this unstrained, physiologic, maxillomandibular relationship, the patient can make vertical, lateral or protrusive movements; it is a clinically useful, repeatable reference position” [2]. the characteristics of centric relation differ for each individual. the operators must softly, without using any force, direct the mandible posteriorly to the maximum intercuspation. consequently, the components of tmj (condyles, discs, ligaments, muscles, and physiological limits) will establish the centric relation. the tripod concept is the basis for deprogramming the lateral pterygoid, which is the muscle-releasing tool. when there is a gap between teeth, and fewer teeth are in contact, the lateral pterygoid is released temporally and the muscle memory is erased; hence, the masticatory muscles can help the condyle return to its natural position [3,4]. recently, intraoral contactless 3d scanners are used to record cr. a virtual articulator and software program are being used to import the analog occlusal record. to construct an occlusal device, the jaw relation records and the virtual occlusal record of the maximal intercuspal position are utilized [5-7]. in fact, there are numerous bene0its of digital recording, in terms of accuracy and ef0iciency. for example, there is no intermediary medium present during the recording process. moreover, the dental laboratory technician does not need to place the actual stone casts; instead, the digital casts are uploaded into the software in the cr position. this guarantees a precise and effective installation in the virtual articulator. additionally, the intraoral scanner software compares the digital casts of the arches to the inter-arch scan, which was acquired from the side view of the arches, to perform the direct digital occlusal recording. however, the expense of neuromuscular relaxaeon using anterior leaf gauge to quick guide the condyles in proper posieon vol 13, no 1 (2025) doi 10.5195/d3000/2025.930 http://dentistry3000.pitt.edu 2 purchasing the intraoral scanner and the learning curve that goes along with it are drawbacks of direct digital recording [8]. in addition, there are common different techniques that can be usually used to record cr. they include graphic techniques, such as gothic arch tracing (intraoral and extraoral); physiologic ones, including tongue retrusion along the posterior part of the palate and swallowing methods; guiding techniques, such as guidance of chin and bilateral manipulation; and anterior device techniques, involving composite resin stops, an anterior deprogrammer, and a leaf gauge [9-12]. in fact, the anterior device is considered a useful tool since it stabilizes the mandible and does not block the side view of the intraoral scanner. besides, because the mandible is more stable when supported by an anterior device, in comparison to a lateral device, the anterior device technique seems to be simpler and less error-prone than similar techniques. additionally, the anterior device technique is very ef0icient regarding positioning the condyles in cr by allowing the patient to contract the elevator muscles and relax the lateral pterygoid muscles. conventionally, bimanual manipulating wax rims on base plates, wax wafers, or polyvinyl siloxane pastes can be used to record cr [12-14]. previous studies reported that the presence of the two condyles in the articular eminences (the tripod) help in the stabilization of the mandible; therefore, some researchers prefer to use conventional method with an anterior device, where the mandibles is more stable, and put polyvinyl siloxane while positioning the mandible in cr [4,8]. many researchers have described a technique that permits a straightforward and precise cr recorded at the chosen ovd and transferred to a virtual articulator . moreover, a direct intraoral digital scanner is used to record cr. conventional methods were used to stabilize mandible and determine cr at the required ovd. the position pertaining to the left and right posterior regions of the arches was recorded using an intraoral scanner. an anterior composite resin stop for 0ixed prostheses and an occlusal device are constructed using a leaf gauge [8]. many researchers tried to guide jaw closure and avoid errors in assessing occlusal contacts by using two methods, the leaf gauge (1 to 6 mm thick) and the anterior acrylic resin jig, to record centric relation closures. these two methods were adopted to avoid adaptive mandibular closure patterns [15]. furthermore, a study was conducted on 0ive healthy adults without symptoms of tmd. the researchers trained the patient to bite with maximum and half-maximum bite force using an anterior jig. then, they recorded the condylar displacement during clenching with electromyographic activity of muscles of mastication. they found that there was signi0icant decrease in anterior temporalis activity (p<0.05), when compared to the maximum intercuspal clench and maximum clench on a leaf gauge temporalis muscle activity. additionally, they concluded that there was no identi0ication for constant change in condylar position [16]. researchers also studied the use of different planned methods for jaw transfer records to accomplish a superior positioning of the condyles, such as leaf gauge, roth’s power centric relation registration, or anterior jig. these methods depend on the anterior stop to disclose posterior teeth and disregard the tooth contact interfering. they placed several sheets between the anterior teeth (10 sheets 0.1mm thick) to ensure that the posterior teeth are not in contact. they supposed that this procedure allows the jaw and condyles to adopt a reproducible position for recording the transfer records for prosthodontic [17-20]. moreover, a study considered the centric relation records for 40 patients before and after the usage of an anterior 0lat plane deprogramming appliance (jig). the panadent condylar path indicator was used to record overjet dimensions, incisal overbite, and threedimensional instrument condylar. they found that, the lucia jig deprogramming appliance offers more movement of centric relation record from mi. they concluded that the appliance is more useful in cases where there is a problem in centric relation bite registration. 21 according to other reports, there was minimal antero-posterior component, and the most superior condylar position was obtained by heavy biting on an anterior rigid stop, when compared to other techniques [22]. the bimanual manipulation technique is a commonly described technique of recording crcp, where the dentist supports the mandible through the retruded movement till the patient feels the initial tooth contact (crcp). this procedure is repeated while using an articulating paper. many operators suppose this method is subject to worker error; and it is problematic when the mandible is in crcp [14,23–25]. clinicians also used discarded panoramic radiography 0ilm to create a simple, low-cost leaf gauge deprogrammer. to prevent posterior occlusal contacts, a deprogrammer is positioned in the anterior region to accomplish muscle deprogramming. then, the condyles were set in the centric relation position because of the lateral pterygoids relaxing. long introduced leaf gauge deprogrammers, which are useful for muscle deprogramming [26-28]. on the other hand, there is a pattern of muscle activity called engram; it causes the mandible to de0lect, leading to an error when recording the position of the centric relation. to remove this, tools called deprogrammers are used. the condyles should be engrammed, positioned, and secured in the centric relation position [29]. therefore, to help dentists achieve precise bite registration in centric relation (cr) and occlusal analysis, huffman created the leaf gauge in 1970. it offers a quick and easy way to register for bites in cr. it is a set of 56 strips made of 0lexible plastic leaves, each measuring 0 to 1 mm. its design has not considerably changed, which re0lects how useful and successful it is in clinical settings. in clinical practice today, it is still a commonly used tool [30]. a leaf gauge consists of multiple smooth, 0lexible plastic sheets arranged together on top of each other; the thickness of each leaf is around 0.1 mm. it is used to detect centric relation. it is placed along the midline of the anterior teeth to separate the posterior teeth retruding the mandible and to offer a suitable record of the precise vertical opening between the incisors. according to the case, the vertical opening will be controlled through increasing or decreasing the number of sheets. when the patient is applying pressure on the leaf, the biting force tends to move the condyles against their eminence. the position of the mandible does not exceed physiologic limits. the leaf gauge is placed in the patient’s mouth during retruding for occlusal equilibrium. moreover, it is utilized to do fast deprogramming, to identify the first point of cr contact, and to record cr position. however, it is contraindicated in cases of a deep overbite with strong elevator muscles, where the flexible gauge can exert a neuromuscular relaxaeon using anterior leaf gauge to quick guide the condyles in proper posieon vol 13, no 1 (2025) doi 10.5195/d3000/2025.930 http://dentistry3000.pitt.edu 3 force on the condyle [15,31,32]. kwiecien also lists several uses for a leaf gauge; they include obtaining bite records for centric relations, loading the tmj, deprogramming the lateral pterygoid, and identifying the 0irst point of contact in centric relation [4]. additionally, there are steps for leaf gauge application. first, several leaves (about 5 to 8) are placed anteriorly in the maxillary central incisors along the midline, parallel to the palatal plane. then, the patient is asked to close the mouth till the lower incisors contact the underneath of the leaf gauge, with no contact in the posterior region. moreover, more leaves can be added to get posterior separation. next, the patient is instructed to move the lower jaw forward/back and hold down onto the mylar strips, without any posterior molar contact. the number of leaves used is controlled based on the patient’s response. after that the patient is requested to grasp the gauge in place for 2 to 5 minutes. subsequentially, a-silicone bite registration paste is used [33]. prior to that the patient moves his jaw forward and backward and squeeze lightly (half-hard bite); the leaf gauge is placed between his upper and lower centrals while he is in a supine position. this bite only activates the temporal muscle, not the masseter, to prevent the mandible from protruding [4]. the opening must remain within the arc of closure (rotation) while enough leaf gauges are used to maintain the back teeth apart and create a smooth path. furthermore, a distinct vertical component can be created by too few leaves, especially if the patient has a deep overbite. on the other hand, the condyle is forced down the eminence, beyond rotation, by too many leaves, which would feel heavy and possibly too open (translation). two sets of leaves can be used to measure the amount that needs to be increased in vertical dimension (vd) [34]. the operator can record the quantity and direction of the slide using a leaf gauge, whether vertical, horizontal, or lateral. to measure the slides, the leaf gauge is moved laterally. in the anterior/posterior position, the facial of the same lower incisor is to the incisal edge of the same upper incisor. for the vertical position, the incisal edge of the same upper incisor is adjacent to the free gingival margin of the same lower incisor. all three positions should be measured using the same reference point for 0irst tooth contact and mip [35]. obviously, many advantages were recorded by authors for using a leaf gauge. first, there is no need for management by the dentist because the patient can seat the condyles into centric relation using his own musculature. moreover, the biting is concentrated on the leaf gauge rather than on the wax or articulating paper. this results in relaxing the jaw muscle and sometimes relieving pain due to continuous strong incisor contact on the leaf gauge for 1 to 5 minutes, allowing the jaw to retrude, which is a more relaxed position, and eliminating the periodontal ligament proprioception. furthermore, a leaf gauge can be used with an articulating paper to predict which teeth will touch in a centric relation. if a deflective contact is present, it will be easier to determine whether the mandible is deviating from its typical path of closure. besides, accurate occlusal analysis and bite registration are provided by a leaf gauge. in addition, a leaf gauge is simple for both patients and dentists to use. it can be also autoclave sterilized, and it can be used for a range of dental procedures. however, it is challenging to use on patients who have deep bites, anterior cross bites, or anterior mobile teeth. additionally, if the condyle is overly rigid, it may shift posterior to cr, or some patients may attempt to bite off a sandwich by protruding their mandible anterior to cr by biting on the leaf gauge slightly forward [12,25,3641]. in addition, numerous studies have tried to recognize the effects of a leaf gauge on the activity of (emg) jaw muscle electromyographic. hickman et al. 1993 found that a leaf gauge showed the lowest activity of anterior temporalis and masseter, when compared with three methods of jaw relation (manually guided centric relation position, maximum intercuspation, and neuromuscular guided position). moreover, other studies have documented the condylar displacement during a leaf gauge biting. they reported that the leaf gauge led to superior condyle displacement with slight anteroposterior displacement [42,43]. previous researchers also stated that the masseter muscles were lesser involved than the temporalis muscles, when a leaf gauge was used for interocclusal recording [20]. references [1] de moraes melo neto c l, da silva e vf, de sousa ervolino i c, dos santos d m, de magalhães bertoz a p, goiato m c. comparison of different methods for obtaining centric relation: a systematic review. gen dent. 2021;69(01):31–36. pubmed: 33350953 [2] the glossary of prosthodontic terms: ninth edition j prosthet dent 2017117(5s):e1–e105. pubmed: 28418832 [3] de moraes melo neto cl, dos santos dm, de magalhães bertoz ap, moreno alm, goiato mc. comparison of techniques for obtaining centric relation based on the reproducibility of the condylar positions in centric relation-a systematic review. eur j dent. 2022 may;16(2):251-257. doi: 10.1055/s-0041-1735903. pmid: 34921385 [4] kwiecien k. using a leaf gauge with intention: part i 26, 2015 (/spear review/ category/ occlusion-wear/) [5] qu f, du x, liu wc. 3d printed custom trays with a gothic arch for centric relation recording and definitive impression making for complete denture: adental technique. j prosthet dent 2019; 121:32-6.9. [6] lo russo l, salamini a. removable complete digital dentures: a workflow that integrates open technologies. j prosthet dent 2018; 119:727-32. [7] dedem p, turp jc. digital michigan splint e from intraoral scanning top asterless manufacturing. int j comput dent 2016; 19:63-76. [8] radu m, radu d , abboud m digital recording of a conventionally determined centric relation: a technique using an intraoral scanner. journal of prosthetic dentistry, 2020;123:228-31 doi: 10.1016/j.prosdent.2018.12.004 [9] dawson pe. functional occlusion: from tmj to smile design. 1st ed. st.louis: mosby/elsevier; 2006. p. 76-80 [10] lucia vo. a technique for recording centric relation. j prosthet dent 1964; 14:492-505 [11] wilson p hr, banerjee a. recording the retruded contact position: a review of clinical techniques. br dent j. 2004;196(07):395–402. pubmed: 15071526 [12] jayne d. a deprogrammer for occlusal analysis and simplified accurate case mounting. j cosmetic dent 2006; 21:96-102. [13] woelfel jb. new device for accurately recording centric relation. j prosthet dent 1986; 56:716-27. [14] mckee jr. comparing condylar positions achieved through bimanual manipulation to condylar positions achieved through masticatory muscle contraction against an anterior neuromuscular relaxaeon using anterior leaf gauge to quick guide the condyles in proper posieon vol 13, no 1 (2025) doi 10.5195/d3000/2025.930 http://dentistry3000.pitt.edu 4 deprogrammer: a pilot study. j prosthet dent 2005; 94:389-93 [15] long, j.h. locating centric relation with a leaf gauge. j. prosthet. dent. 1973, volume 29, issue 6 p608-610june doi: 10.1016/00223913(73)90267-9 [16] carroll wj, woelfel jb, huffman rw. simple application of anterior jig or leaf [42] gauge in routine clinical practice. j prosthet dent. 1988;59(5):611-17.doi: 10.1016/00223913(88)90081-9 [17] santosa re, azizi m, whittle t, wanigaratne k, klineberg ij the influence of the leaf gauge and anterior jig on jaw muscle electromyography and condylar head displacement: a pilot study australian dental journal 2006;51:(1):33-41 [18] roth rh. functional occlusion for the orthodontist. part 1. j clin orthod 1981; 15:32-51. [19] glossary of prosthodontic terms, ed 6., j prosthet dent 1994, ;71:59 [20] williamson eh, steinke rm, morse pk, swift tr. centric relation: a comparison of muscle-determined position and operator guidance. am j orthod 1980; 77:133-145. [21] braun s, marcotte mr, freudenthaler jw, honigle k. an evaluation of condyle position in centric relation obtained by manipulation of the mandible with and without leaf gauge deprogramming. am j orthod dentofacial orthop 1997; 111:3437. [22] lundeen hc. centric relation records: the effect of muscle action. j prosthet dent 1974; 31:244-253. [23] karl p j, foley t f the use of a deprogramming appliance to obtain centric relation records. angle orthod 1999 apr;69(2):117-24; discussion 124-5.doi: 10.1043/0003-3219(1999)069<0117: tuoada>2.3.co;2 [24] jankelson b, adib f. effect of variation in manipulative force on the repetitiveness of centric relation registration: a computer-based study. j am dent assoc 1986; 113(1): 59–62. [25] dawson pe: temporomandibular joint paindysfunction problem can be solved. j prosthet dent 29:100, 1973. [26] praktyka p, zakład k. clinical aspects of application of selected anterior deprogrammers. protet stomatol, 2017; 67(4): 333-341, doi:10.5604/01.3001.0010.6860 [27] lucia vo. a technique for recording centric relation. j prosthet dent 1964;14: 492-505. [28] carroll wj, woelfel jb, huffman rw. simple application of anterior jig or leaf gauge in routine clinical practice. j prosthet dent 1988; 59:611-7. [29] gaikwad, tamore s, fabricating an inexpensive leaf gauge deprogrammer from panoramic radiographic film amit m. the journal of prosthetic dentistry, 2019 volume 122 issue 3, 339340 [30] carroll wj, woelfel jb, huffman rw. simple application of anterior jig or leaf gauge in routine clinical practice. prosthet dent. 1988 may;59(5):611-7. doi: 10.1016/00223913(88)90081-9. [31] fleigel jd, sutton aj. reliable and repeatable centric relation adjustment of the maxillary occlusal device. j prosthodont 2012; 1–4. [32] golsen lf, shaw af. use of leaf gauge in occlusal diagnosis and therapy. quintessence int 1984; 15(6): 611–621. [33] leaf gauge https://mikdental.in/product/leaf-gauge/ [34] lassmann education why do we use leaf gauge and how to do that 31 july 2019 https://www.facebook.com/groups/2016057668694875/posts/2 110117862622188/ [35] spear education. the point of the first point. spear review. 2018 aug. https://www.speareducation.com/spear-review [36] kornfield m: mouth rehabilitation. clinical and laboratory procedures, ed 2. st louis, 1974, the cv mosby co. [37] dawson pe: evaluation, diagnosis, and treatment of occlusal problems. st louis, 1974, the cv mosby co, pp 48-70. [38] guichet nf: biologic laws governing functions of muscles that move the mandible: part i: occlusal programming. j prosthet dent 37:648, 1977. [39] williamson eh, steinke rm, morse pk, swift tr: centric relation-a comparison of muscle determined position and operator guidance. am j orthod 77~133, 1980. [40] spear education. using a leaf gauge with intention, part ii. spear review. 2015 mar https://www.speareducation.com/spear-review [41] dawson academy. lucia jig or leaf gauge. the dawson academy. 2023 mar https://thedawsonacademy.com/lucia-jig-or-leaf-gauge/ [42] hickman dm, cramer r, stauber wt. the effect of four jaw relations on electromyographic activity in human masticatory muscles. arch oral biol 1993; 38:261-264. [43] fenlon mr, woelfel jb. condylar position recorded using leaf gauges and specific closure forces. int j prosthodont 1993; 6:402408. 752+2024 assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs: a cross-secconal study vol 12 no 2 (2024) doi 10.5195/d3000.2024.752 h#p://den*stry3000.pi#.edu assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs: a cross-sectional study rusul subhi hassan, zanbaq azeez hanoon, salah m. ibrahim*, nabaa basim alhusseini faculty of dentistry, kufa university, najaf, iraq *corresponding author abstract objec>ve: this cross-sec4onal study aimed to assess the periodontal status and treatment needs of undergraduate dental students at al-kufa university in iraq and inves4gated the influence of demographic factors (age and gender) and oral hygiene prac4ces (tooth brushing technique, frequency of brushing, use of interdental aids, dental visit paderns) on periodontal health. materials and methods: a sample of 200 undergraduate dental students (aged 18-24 years) was selected using a mul4stage systema4c random sampling procedure during the 2023-2024 academic year. periodontal health was assessed using the world health organiza4on community periodontal index for treatment needs (cpitn). the cpitn probe was used to evaluate bleeding on probing, presence of calculus, plaque reten4ve factors, and pocket depths (4, 5, and 6 mm). data were analyzed using spss version 23, and the pearson’s chi-square test was used to assess associa4ons, with a p-value ≤ 0.05 considered sta4s4cally significant. results: the study revealed a high prevalence of periodontal disease (86.5%) among the students. calculus was the most frequent finding (65.5%), while only 13.5% of students exhibited a healthy periodon4um. the majority (69%) required scaling and root planning (tn2). female students exhibited significantly healthier periodon4a compared to males (p<0.05). older students (21-24 years) had a significantly higher prevalence of deeper periodontal pockets than younger students (p<0.05). smoking was significantly associated with poorer periodontal health (p<0.05). a sta4s4cally significant associa4on was found between the frequency of tooth brushing and periodontal health (p<0.05), with those brushing twice daily showing beder periodontal status. while not sta4s4cally significant, the use of interdental aids was associated with healthier periodon4a. students with irregular dental visit paderns had a higher prevalence of calculus (p<0.05). conclusion: this study found a high prevalence of periodontal disease and significant treatment needs among undergraduate dental students at al-kufa university. gender, age, smoking status, and oral hygiene prac4ces were associated with periodontal health. these findings emphasize the urgent need for comprehensive oral health educa4on programs, incorpora4ng behavioral change strategies and promo4ng the importance of preven4ve dental care. keywords: alkufa university, periodontal disease, cross-sec4onal study, dental student, community periodontal index for treatment needs cita:on: hassan rs, et al. (2024) assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs: a cross-sec:onal study. den:stry 3000. 2:a001 doi:10.5195/d3000.2024.752 received: october 20, 2024 accepted: november 1, 2024 published: november 22, 2024 copyright: ©2024 hassan rs, et al. this is an open access ar:cle licensed under a crea:ve commons avribu:on work 4.0 united states license. email: salahm.abrahem@uokufa.edu.iq introduction periodontal diseases (pds) are one of most ubiquitous and major dental diseases among the human populations worldwide [1]. pds, comprising gingivitis and periodontitis, are probably the most common disease of mankind. these diseases are considered as the main cause of tooth loss among adult populations. no nation or region of the world is free from this pathological disease [1,2]. gram-negative anaerobic bacteria found in dental biofilm are linked to the chronic assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu inflammatory diseases known as pds, which cause irreversible damage to the periodontium. dental plaque that has accumulated and not been removed is a major factor in the development and course of disease [2,3]. dental plaque is defined clinically as a structured, resilient, yellow-grayish substance that adheres tenaciously to the intraoral hard surfaces, including removable and fixed restorations in the absence of proper oral hygiene practices. plaque is classified as a type of biofilm, which is characterized as microbial populations embedded in a matrix and adhering to surfaces, interfaces, or other objects [4]. the biofilm nature of dental plaque means that it tenaciously adheres to surfaces which increase in complexity and pathogenicity with time [5]. pds can be regarded as a host-microbial interaction in which the transition from health to disease can be brought about by the balance between host and bacterial factors. the result of this interaction is influenced by both bacterial and host factors. the balance could be upset, for example, by a rise in bacterial virulence, a decrease in host resistance, or an increase in plaque biofilm. local, systemic, and/or other factors further alter periodontal disease's clinical presentation [6]. due to a shortage of oral health professionals in many developing nations, services are typically provided by neighborhood or focal emergency clinics in urban areas, with preventive dental care receiving little weight [1]. to assess the condition of periodontal health, the world health organization's (who) oral health unit and federation dentaire international (fdi) collaborated to develop the community periodontal index for treatment needs (cpitn). it is a quick and easy way to determine a population group's treatment needs (tns) and has served as the primary indicator in some epidemiological studies on the prevalence of periodontal disease, it [7,8]. in the adult population, the most severe pd code (cpitn code 4) ranges from 10% to 15% globally. while oral health has generally gotten worse in developing nations, it has significantly improved in industrialized nations. the who oral data bank has noted and documented these patterns and trends. populations in these countries are becoming more and more afflicted with pds, periodontal infections, and compromised masticatory functions [9-12]. there is currently no available epidemiological data on the state of periodontal health in the governorate of al-najaf, iraq, from either the general population or the dental students at al-kufa university. this investigation was conducted among a sample of alkufa university dental students for the reasons previously mentioned. materials and methods study design and se5ng this research employed an observa`onal, analy`cal, crosssec`onal study design to inves`gate the periodontal health status and treatment needs of undergraduate dental students [13]. the study was conducted at the department of periodon`cs, college of den`stry, al-kufa university, located in najaf, iraq. data collec`on was carried out during the academic year 2023– 2024. the cross-sec`onal design allowed for a snapshot of the periodontal health of this specific popula`on at a par`cular point in `me. ethical considera8ons before commencing the study, ethical approval was obtained from the ethical commidee of the college of medicine, university of kufa (reference # mec-36, date: 14/5/2024). the study adhered to the ethical principles outlined in the declara`on of helsinki [14]. all par`cipants received a detailed explana`on of the study's purpose, procedures, risks, and benefits. wriden informed consent was obtained from each student prior to their par`cipa`on, ensuring assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu their voluntary involvement and understanding of the study's implica`ons. study subjects and sampling methodology the target popula`on for this study comprised all undergraduate dental students (n = 741) enrolled at al-kufa university during the study period. to ensure a representa`ve and manageable sample, a mul`stage systema`c random sampling procedure was implemented [15]. the student popula`on was ini`ally divided into five strata based on their academic year (first year through fiih year). stra`fica`on aimed to ensure propor`onal representa`on of students from each year of the dental program, minimizing poten`al bias due to differences in clinical experience or exposure to periodontal educa`on. within each stratum, 40 students were randomly selected using a systema`c random sampling technique [16]. this technique involved: determina)on of the sampling interval. the sampling interval (k) was calculated by dividing the total number of students in each stratum by the desired sample size for that stratum (40 students). random star)ng point. a random number between 1 and k was selected as the star`ng point. systema)c selec)on. every kth student on the list, star`ng from the randomly selected star`ng point, was chosen un`l 40 students were selected from that stratum. this mul`stage sampling procedure ensured a representa`ve sample of undergraduate dental students from all academic years, minimizing sampling bias and enhancing the generalizability of the findings to the broader student popula`on. inclusion criteria ac)ve enrollment. par`cipants had to be ac`vely enrolled as undergraduate dental students at al-kufa university's college of den`stry. age range. students were required to be between 18 and 24 years of age. this age range targeted the typical student popula`on in dental schools. exclusion criteria age outside range. students younger than 18 or older than 24 were excluded to ensure a consistent age group. non-par)cipa)on. students who declined to par`cipate were excluded to respect their autonomy and ensure voluntary par`cipa`on. orthodon)c treatment. students undergoing orthodon`c treatment were excluded because orthodon`c appliances could influence periodontal health and confound the study results. dental implants. individuals with dental implants were excluded, as implants require different periodontal assessment techniques and could impact the study findings. medical condi)ons. students with medical condi`ons that could restrict periodontal examina`on or affect periodontal health were excluded. these condi`ons included, but were not limited to, hiv/aids, diabetes mellitus, and individuals undergoing long-term medica`on use. data collec8on procedures data were collected through two primary methods: a selfadministered ques`onnaire and a standardized clinical periodontal examina`on. ques)onnaire. a structured ques`onnaire was used to gather informa`on on the following: demographic data. age and gender were collected to describe the study sample and explore poten`al associa`ons with periodontal health. assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu dental health-related prac)ces. the following variables were included: tooth brushing frequency. students were asked about how oien they brushed their teeth, with op`ons including once daily, twice daily, more than twice daily, and other (specifying their frequency). tooth brushing technique. students indicated their usual tooth brushing technique from the following op`ons: horizontal, ver`cal, scrubbing, circular, or other (specifying their technique). use of interdental aids. par`cipants indicated whether they used dental floss, toothpicks, interdental brushes, or other interdental cleaning aids (specifying the type). frequency of dental visits. op`ons included regular checkups, irregular visits, visits only for emergencies, or never visi`ng a den`st. reason for dental visits. students selected their primary reason for visi`ng the den`st as preven`ve (for checkups and cleanings), cura`ve (for treatment of dental problems), or other (specifying the reason). the ques`onnaire was designed to be self-administered, allowing students to complete it at their own pace and ensuring privacy in their responses. clear instruc`ons were provided, and any ques`ons from students were addressed before they began comple`ng the ques`onnaire. clinical periodontal examina)on. a standardized clinical periodontal examina`on was conducted on each par`cipant to assess their periodontal health status. the examina`on was performed by a single calibrated examiner to ensure consistency and minimize inter-examiner variability [17]. the following procedures were followed: prepara`on. the examiner used gloves and a mask for infec`on control. a disposable mouth mirror and a sterile cpitn-e probe were used for each par`cipant as shown in figures 1 and 2. the cpitn-e probe is specifically designed for use with the cpitn index and has a 0.5 mm ball `p and color-coded markings at 3.5 mm and 5.5 mm to facilitate pocket depth measurements. examina`on protocol. the examina`on adhered to the world health organiza`on (who) guidelines for using the community periodontal index for treatment needs (cpitn) [12]. the following steps were followed: mouth division. the mouth was systema`cally divided into six sextants: upper right, upper lei, lower right, lower lei, upper anterior, and lower anterior. sextant assessment. each sextant was examined to ensure the presence of at least two teeth, excluding third molars unless they served as a replacement for a missing second molar. if a sextant had less than two teeth, it was recorded as code x (missing sextant). index teeth selec`on. for each sextant with two or more teeth, specific index teeth were examined. the index teeth selected were based on the par`cipant's age: for students 20 years and older. 17, 16, 11, 26, 27, 36, 37, 31, 46, and 47. for students 19 years and younger. 16, 11, 26, 36, 31, and 46. probing technique. the index teeth were probed at the mesial, midline, and distal loca`ons on both the facial (labial/buccal) and lingual (palatal) surfaces. gentle probing pressure was applied, inser`ng the probe into the gingival sulcus or periodontal pocket un`l resistance was felt [18]. parameter assessment. the following parameters were evaluated for each index tooth: bleeding on probing (bop). the presence of bleeding within 30-60 seconds aier probing. bop assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu is an early indicator of gingival inflamma`on. calculus. the presence of detectable calculus using the probe. calculus, a hardened form of plaque, is a significant contributor to periodontal disease. plaque reten`ve factors. the presence of any factors that could contribute to the accumula`on of plaque, such as overhanging restora`ons, poorly fipng crowns, or anatomical grooves. these factors make plaque removal more difficult and can exacerbate periodontal disease. pocket depths. the depth of the gingival sulcus or periodontal pocket, measured in millimeters, was recorded for each probing site using the cpitn-e probe. the probe's markings at 3.5 mm and 5.5 mm helped to iden`fy pockets exceeding 4 mm and 6 mm, respec`vely. cpitn code recording. the highest cpitn code observed for each sextant was recorded. this method of recording ensures that the most severe periodontal finding within each sextant is captured. cpitn codes and treatment needs. the community periodontal index for treatment needs (cpitn) u`lizes a simplified coding system to classify periodontal health status and guide treatment decisions [19]. the cpitn codes and their corresponding treatment needs are as follows: code 0: healthy periodon`um. this code indicates a healthy periodon`um with no signs of inflamma`on, calculus, or pocke`ng. no treatment is needed (tn0). code 1: bleeding on probing. this code signifies gingival inflamma`on, even in the absence of calculus or pocke`ng. the recommended treatment (tn1) is oral hygiene instruc`on, emphasizing proper brushing and interdental cleaning techniques to control plaque accumula`on. code 2: calculus present. this code indicates the presence of calculus, which can irritate the gums and contribute to periodontal disease progression. the recommended treatment (tn2) is scaling and root planing to remove the calculus and smooth the root surfaces, promo`ng healing and reducing inflamma`on. code 3: pockets 4-5 mm deep. this code signifies a moderate level of periodontal disease, characterized by deeper pockets where plaque and calculus can accumulate more easily. the recommended treatment (tn2) is scaling and root planing, like code 2. code 4: pockets 6 mm or deeper. this code indicates a more advanced stage of periodontal disease, with deep pockets that are difficult to clean and pose a significant risk of tooth loss. the recommended treatment (tn3) is complex periodontal treatment, which may involve surgical procedures, advanced cleaning techniques, and long-term maintenance. sta8s8cal analysis data collected from the ques`onnaires and clinical examina`ons were entered and analyzed using ibm spss sta`s`cs for windows, version 23.0 (ibm corp., armonk, ny). descrip`ve sta`s`cs were used to summarize data, including frequencies and percentages for categorical variables such as gender, smoking status, tooth brushing technique, frequency of tooth brushing, use of interdental aids, frequency of dental visits, and reasons for dental visits. for the con`nuous variable age, the mean, standard devia`on, minimum, and maximum were calculated. pearson chi-square (χ2) test was employed to examine associa`ons between the highest cpitn code recorded for each student and the categorical assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu variables. this test was used to determine if there was a sta`s`cally significant frequency differences between periodontal health status and these poten`al risk factors. a p-value (p) of ≤ 0.05 was considered sta`s`cally significant, indica`ng a less than 5% probability that the observed associa`on between the variables was due to random chance. figure 1. instruments, equipment and materials used in the study. figure 2. the cpitn-e probe. results periodontal health status and treatment needs table 1 and figure 3 display the findings of the clinical periodontal examination, which involved evaluating the periodontal health status using cpitn. the results showed that 13.5% of students had a healthy periodontium, 17.5% had bleeding on probing, and 65.5% of them had calculus. additionally, 3.5% and none of students, respectively, had pocket depths of 4-5 mm and 6 mm or more. table 1. distribu`on of students according to result of periodontal health status using cpitn. no. cptin number percentage % 1 healthy periodon`um 27 13.5% 2 bleeding 35 17.5% 3 calculus 131 65.5% 4 pocket depth 4-5mm 7 3.5% 5 pocket depth 6mm or more 0 0% total 200 100% assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu 13.5 17.5 65.5 3.5 0 0 10 20 30 40 50 60 70 1 2 3 4 5 figure 3. percentages of periodontal health status in the sample. about type of treatment that each student included in the study needs is described in table 2 and figure 4. most students (69%) required professional interven`on, as indicated by tn2. table 2. distribu`on of students according to their treatment needs using cpitn. treatment needs number percentage % tn0 27 13.5% tn1 35 17.5% tn2 138 69% tn3 0 0% total 200 100% figure 4. distribu`on of students by cpitn treatment needs. associa8on between categorical variables with highest cpitn codes the associa`on between age, gender, smoking, with highest codes of cpitn were tested sta`cally using chi square (x2). assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu age: the percentage of students in the 18–20 age group had a higher percentage of healthy periodontium (9%) and a lower percentage of other codes. in the 21–24 age group, the deep pocket had the highest percentage, which was 2%. the associa`on between age and highest cpitn codes was sta`s`cally significant (p<0.05), as shown in table 3. table 3. distribu`on of students according to result of periodontal health status using cpitn by age. age (21-24) no. (%) age (18-20) no. (%) cptin 9 (4.5) 18 (9) healthy periodon`um 22 (11) 13 (6.5) bleeding 99 (49.5) 32 (16) calculus 4 (2) 3 (1.5) pocket depth 4-5mm 0 0 (0) pocket depth 6mm or more 134 (67) 66 (33) total x2= 36.98 degree of freedom (df) =4 (p<0.05) (s) gender: compared to male students, female students had a higher percentage of healthy periodontium (9%). table 4 indicates that these associations were statistically significant (p<0.05). table 4. sta`s`cal analysis and distribu`on of students according to their highest cpitn codes by gender. students gender cptin male no. (%) female no. (%) 9 (4.5) 18 (9) healthy periodon`um 21 (10.5) 14 (7) bleeding 90 (45) 41 (20.5) calculus 1 (0.5) 6 (3) pocket depth 4-5mm 0 0 (0) pocket depth 6mm or more 121 (60.5) 79 (39.5) total x2= 18 degree of freedom (df) =4 (p<0.05) (s) smoking: students who did not smoke had healthier periodontium (12.5%) than students who smoked, who are more likely to have calculus (57%), and to have a pocket depth of 4-5 mm (3.5%). smoking and the highest cpitn codes were statistically significantly associated (p<0.05), as shown in table 5. assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu table 5. sta`s`cal analysis and distribu`on of students according to their highest cpitn codes by smoking status. smoking cptin smoker no. (%) non-smoker no. (%) 2 (1) 25 (12.5) healthy periodon`um 28 (14) 7 (3.5) bleeding 114 (57) 17 (8.5) calculus 7 (3.5) 0 (0) pocket depth 4-5mm 0 0 (0) pocket depth 6mm or more 151 (75.5) 49 (24.5) total x2= 109.52 degree of freedom (df) =4 (p<0.05) (s) associa8on between age and gender with periodontal tns of cpitn the associa`ons between age and gender with tns were tested sta`s`cally using x2. age: the highest percentage of students with tn0 was reported among 1820 years age group (9%). tn2 was recorded more oien among 2124 years old. these associa`ons were sta`s`cally significant (p<0.05), as shown in table 6. table 6. sta`s`cal analysis and distribu`on of students according to their periodontal tns by age. age (21-24) no. (%) age (18-20) no. (%) tns 9 (4.5) 18 (9) tn0 22 (11) 13 (6.5) tn1 103 (51.5) 35 (17.5) tn2 0 (0) 0 (0) tn3 134 (67) 66 (33) total x2= 32.305 df=3 p<0.05(s) gender: no need for treatment (tn0) was higher among female students (9%). while for tn2, the higher percentage (45.5%) was recorded among male students. these associa`ons were sta`s`cally significant (p<0.05), as shown in table 7. assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu table 7. sta`s`cal analysis and distribu`on of students according to their periodontal tns by gender. students gender tns male no. (%) female no. (%) 9 (4.5) 18 (9) tn0 21 (10.5) 14 (7) tn1 91 (45.5) 47 (23.5) tn2 0 (0) 0 (0) tn3 121 (60.5) 79 (39.5) total x2 =9.828 df=3 p<0.05(s) dental health-related prac8ces and pahern of visits to dental clinic tables 8 and 9 show the numbers and percentages of dental health-related prac`ces, in which 97.5% of the study students reported to have prac`ced teeth cleaning regardless of the method of teeth cleaning. regarding method of teeth brushing, most students (36.41%) prac`ced scrubbing method for teeth cleaning. about frequency of teeth cleaning, the greater part of students cleaned their teeth twice and once daily (50.25% and 30.25%), respec`vely. using of interdental aids was reported in 45.5 % of the students. out of them, 84.61% were using dental floss, 43.95% were using toothpick, and 3.29% were using interdental brush. assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu table 8. sta`s`cal analysis and distribu`on of students according to their highest cpitn codes by method and frequency of teeth brushing. cpitn teeth cleaning yes (%) no (%) healthy periodontium 31 (15.5) 0 bleeding 30 (15) 2 (1) calculus 129 (64.5) 1 (0.5) pocket depth 4-5 mm 5 (2.5) 2 (1) pocket depth 6 mm or more 0 0 total 195 (97.5) 5 (2.5) x2= 180.5 degree of freedom (df) =4 (p<0.05) (s) cpitn method of teeth brushing horizontal n (%) vertical n (%) scrubbing n (%) circular n (%) healthy periodontium 4 (2.05) 5 (2.56) 8 (4.1) 9 (4.61) bleeding 4 (2.05) 15 (7.69) 14 (7.17) 7 (3.58) calculus 12 (6.15) 39 (20) 46 (23.59) 27 (13.84) pocket depth 4-5 mm 1(0.51) 1 (0.51) 3 (1.53) 0 pocket depth 6 mm or more 0 0 0 0 total 21 (10.76) 60 (30.76) 71 (36.41) 43 (22.05) x2= 31.9 degree of freedom (df) =12 (p<0.05) (s) cpitn frequency of teeth brushing once (%) twice (%) more (%) occasional (%) healthy periodontium 7 (3.59) 13 (6.67) 4 (2.05) 4 (2.05) bleeding 7 (3.59) 11 (5.64) 7 (3.59) 2 (1.02) calculus 40 (20.51) 74 (37.94) 15 (7.69) 6 (3.07) pocket depth 4-5 mm 5 (2.56) 0 0 0 pocket depth 6 mm or more 0 0 0 0 total 59 (30.25) 98 (50.25) 26 (13.33) 12 (6.15) x2= 96.91 degree of freedom (df) =12 (p<0.05) (s) assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu table 9. sta`s`cal analysis and distribu`on of students according to their highest cpitn codes by use type of interdental aid. cpitn use of interdental aids yes (%) no (%) healthy periodontium 16 (8) 8 (4) bleeding 23 (11.5) 17 (8.5) calculus 50 (25) 79 (39.5) pocket depth 4-5 mm 2 (1) 5 (2.5) pocket depth 6 mm or more 0 0 91(45.5) 109(54.5) x2= 8 degree of freedom (df) =4 (p>0.05) (ns) cpitn type of interdental aid dental floss (%) toothpick (%) interdental brush (%) healthy periodontium 10 (10.98) 14 (15.38) 1 (1.09) bleeding 6 (6.59) 10 (10.98) 2 (2.19) calculus 50 (54.9) 16 (17.58) 0 pocket depth 4-5 mm 11 (12.08) 0 0 pocket depth 6 mm or more 0 0 0 total 77 (84.61) 40 (43.95) 3 (3.29) x2= 68.44 degree of freedom (df) =8 (p<0.05) (s) frequency of visits to the dentist. most of the dental students visited the den`st irregularly (49.5%). the highest percentage of calculus was reported among dental students who visited the dental clinic irregularly (27.5%). the associa`ons between highest cpitn codes and frequency of visits to the dentist was sta`s`cally significant different (p<0.05), as shown in table 10. reason for dental visit. the highest percentage adended the dental clinic for cura`ve reason (77.5%), while calculus was higher in the cura`ve group (51%) and the associa`on was sta`s`cally significant (p<0.05), as shown in table 10. assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu table 10. sta`s`cal analysis and distribu`on of dental students according to their highest cpitn codes by paderns of visits to dental clinic. frequency of visits to dentist on emergency (%) never (%) irregular (%) regular (%) cpitn 2 (1) 5 (2.5) 19 (9.5) 7 (3.5) healthy periodontium 0 0 21 (10.5) 9 (4.5) bleeding 39 (19.5) 21 (10.5) 55 (27.5) 16 (8) calculus 0 2 (1) 4 (2) 0 pocket depth 4-5 mm 0 0 0 0 pocket depth 6 mm or more 41 (20.5) 28 (14) 99 (49.5) 32 (16) total x2= 65.8 degree of freedom (df) =12 (p<0.05) (s) reasons for visit preventive (%) curative (%) cpitn 6 (3) 20 (10) healthy periodontium 4 (20) 27 (13.5) bleeding 30 (15) 102 (51) calculus 5(2.5) 6 (3) pocket depth 4-5 mm 0 0 pocket depth 6 mm or more 45(22.5) 155 (77.5) total x2= 60.5 degree of freedom (df) =4 (p<0.05) (s) assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu discussion this study unveiled a concerning periodontal health landscape among undergraduate dental students at al-kufa university. the high prevalence of periodontal disease, the significant need for treatment, and the associa`ons with various factors underscore the urgency of addressing oral health challenges within this future genera`on of dental professionals. a high prevalence of periodontal diseases: a global concern mirrored locally our study revealed that a staggering 86.5% of dental students exhibited signs of periodontal disease. this finding aligns with global trends, as periodontal diseases are among the most prevalent chronic condi`ons worldwide, affec`ng a substan`al propor`on of adults [1,2]. however, the prevalence found in our study is even higher than some reports from other regions. for instance, a study conducted on dental and medical students at the medical university of bialystok in poland reported a prevalence rate of 75.38% [12], while a study in iraq on both dental and non-dental students found a prevalence of 59.23% [13]. these varia`ons in prevalence rates could be adributed to differences in study popula`ons, methodology, or cultural factors influencing oral hygiene prac`ces and access to care. calculus: a red flag for inadequate plaque control the high prevalence of calculus (65.5%) among the students is a significant finding. calculus, a hardened form of plaque, is a major contributor to periodontal disease development and progression [22]. its presence indicates a lack of effec`ve plaque control, sugges`ng a need for improved oral hygiene prac`ces and poten`ally a lack of awareness regarding the importance of regular professional dental cleanings. treatment needs: scaling and root planing emerge as a priority most students (69%) required scaling and root planing (tn2), indica`ng a substan`al burden of untreated periodontal disease. scaling and root planing are essen`al procedures for removing plaque and calculus from below the gum line, smoothing the root surfaces, and promo`ng healing [23]. the high percentage of students requiring tn2 underscores the importance of accessible and affordable dental care services within the university sepng. unveiling the influences: age, gender, and smoking our study inves`gated several factors that could influence periodontal health, revealing important associa`ons. age, a cumula8ve impact factor. as an`cipated, older students (21-24 years) exhibited a higher prevalence of deeper periodontal pockets compared to younger students, sugges`ng that the dura`on of exposure to periodontal risk factors plays a role [20]. even in this rela`vely young popula`on, prolonged exposure to plaque, inadequate oral hygiene, and other factors led to a worsening of periodontal health. this highlights the importance of establishing healthy oral hygiene habits early in life and maintaining them throughout adulthood. gender, a disparity requiring further explora8on. the significant difference in periodontal health between genders, with female students exhibi`ng healthier periodon`a, aligns with previous research [14,21,22]. this difference could be adributed to a complex interplay of factors, including poten`ally beder oral hygiene prac`ces among females [23], varia`ons in hormonal influences on periodontal `ssues [24], or even differences in aptudes towards oral health and aesthe`cs assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu [25]. further research is needed to disentangle these factors and develop gender-specific interven`ons to address the observed disparity. smoking, a well-established risk factor. the detrimental effects of smoking on periodontal health are well-documented [26]. our study reinforced this associa`on, with smokers exhibi`ng significantly poorer periodontal status compared to non-smokers. smoking compromises the immune system, impairs blood flow to the gums, and alters the oral microbiome, crea`ng a favorable environment for periodontal pathogens [27]. this finding underscores the cri`cal need to integrate smoking cessa`on programs into the dental school curriculum and educate students about the specific risks smoking poses to periodontal health. empowering through oral hygiene prac8ces our findings strongly support the cri`cal role of effec`ve oral hygiene prac`ces in maintaining periodontal health [28]. students who brushed their teeth twice daily exhibited significantly beder periodontal status, highligh`ng the importance of consistent and thorough plaque removal through proper brushing techniques [29]. while the use of interdental aids did not reach sta`s`cal significance in our study, the trend towards healthier periodon`a among those who used these aids suggests a poten`al benefit. interdental cleaning, using dental floss, toothpicks, or interdental brushes, can effec`vely remove plaque from areas that toothbrushes cannot reach, contribu`ng to beder overall periodontal health [30,31]. emphasizing preventa8ve dental care our study underscored the vital role of regular dental care in preven`ng and managing periodontal disease. students who visited the den`st irregularly had a significantly higher prevalence of calculus, emphasizing the importance of professional cleanings to remove plaque and calculus that cannot be adequately addressed through home care alone [32]. moreover, the higher prevalence of calculus among students who primarily sought cura`ve dental care highlights the need to shii the focus toward preven`ve care. encouraging regular dental checkups, even in the absence of symptoms, allows for early detec`on and treatment of periodontal issues, preven`ng disease progression and poten`ally more invasive interven`ons. implica8ons for al-kufa university the findings of this study provide a clear roadmap for addressing the periodontal health challenges faced by dental students at al-kufa university. • comprehensive oral health educa8on: the dental school curriculum should integrate comprehensive oral health educa`on programs that go beyond tradi`onal lectures and textbooks. these programs should incorporate interac`ve learning methods, behavioral change strategies, and hands-on demonstra`ons to effec`vely engage students and empower them to adopt healthy oral hygiene prac`ces [33,34]. • promote preventa8ve care: the university should priori`ze preventa`ve dental care through educa`onal campaigns, peer-to-peer support groups, and incen`ves for regular checkups. collabora`ng with local dental providers to offer discounted or subsidized dental services to students can further encourage early interven`on and preven`on [35,36]. assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu • accessible and affordable dental care: establishing an on-campus dental clinic could significantly improve access to care for students, providing convenient and affordable services. alterna`vely, the university could explore partnerships with local dental clinics to offer discounted services or develop financial assistance programs to help students cover the cost of dental care [37,38]. study limita8ons this study was limited by its singlecenter design, poten`ally limi`ng the generalizability of findings to other dental schools in iraq. the cross-sec`onal design, while providing a snapshot of periodontal health, cannot establish cause-and-effect rela`onships, and longitudinal studies are needed to assess changes over `me. reliance on self-reported data regarding oral hygiene prac`ces and dental visit paderns may be subject to recall or social desirability bias, highligh`ng the need for more objec`ve measures in future research. finally, the study's focus on a selected set of factors means that other poten`ally influen`al factors, such as diet, stress, and gene`c predisposi`ons, warrant further inves`ga`on. conclusion the high prevalence of periodontal disease among dental students at al-kufa university is a call to ac`on. this study emphasizes the urgent need for a mul`-faceted approach to improve oral health within this popula`on. by integra`ng comprehensive oral hygiene educa`on, promo`ng preventa`ve care, and enhancing access to affordable dental services, al-kufa university can equip its future dental professionals with the knowledge, skills, and resources they need to achieve and maintain op`mal oral health, sepng a posi`ve example for their pa`ents and contribu`ng to a healthier community. availability of data and material: data are available on request to the corresponding author. financial support and sponsorship: none. conflicts of interest: there are no conflicts of interest. references 1. jebur, h. j., & ahmed, m. a. a. (2020). prevalence of periodontal diseases among universi`es students in al-basrah governorate, iraq-a crosssec`onal study. biochemical & cellular archives, 20(1). 2. ibrahim, s. m., almizraqchi, a. s., & haider, j. (2023). metronidazole poten`a`on by panax ginseng and symphytum officinale: a new strategy for p. gingivalis infec`on control. an)bio)cs, 12(8), 1288. 3. mohammed, m. j., almizraqchi, a. s., & ibrahim, s. m. (2024). oral findings, salivary copper, magnesium, and lep`n in type ii diabe`c pa`ents in rela`on to oral candida species. interna)onal journal of microbiology, 2024(1), 8177437. 4. lang, n. p., berglundh, t., giannobile, w. v., & sanz, m. (eds.). (2021). lindhe's clinical periodontology and implant den)stry. john wiley & sons. 5. ibrahim, s. m., & almizraqchi, a. s. (2024). comparison of the an`bacterial ac`vity of panax ginseng and symphytum officinale with metronidazole against p. gingivalis: an mic and mbc analysis. the open den`stry journal, 18(1). 6. dey, s. m., & nagarathna, d. v. (2017). mathews jude. assessment of assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu periodontal health status among koraga tribes residing in mangalore taluk: a cross sec`onal study. int j res med sci, 5(9), 3980-3984. 7. magzoub, a., tawfig, n., sap, a., & gobara, b. (2016). periodontal health status and periodontal treatment needs of prisoners in two jails in khartoum state. int j den)stry oral sci, 3(4), 233-238. 8. petersen, p. e., & ogawa, h. (2012). the global burden of periodontal disease: towards integra`on with chronic disease preven`on and control. periodontology 2000, 60(1), 15-39. 9. world health organiza`on. (2013). oral health surveys—basic methods, 5th ed. geneva: world health organiza`on. 10. sedgwick, p. (2014). crosssec`onal studies: advantages and disadvantages. bmj, 348. 11. world medical associa`on. (2013). world medical associa`on declara`on of helsinki: ethical principles for medical research involving human subjects. jama, 310(20), 2191-2194. 12. elfil, m., & negida, a. (2017). sampling methods in clinical research; an educa`onal review. emergency (tehran, iran), 5(1), e52. 13. acharya, a. s., prakash, a., saxena, p., & nigam, a. (2013). sampling: why and how of it. indian journal of medical special`es, 4(2), 330-333. 14. dahl, j. e. (1997). calibra`on of examiners for epidemiological studies: a review. community den`stry and oral epidemiology, 25(6), 413422. 15. ainamo, j., barmes, d., beagrie, g., cutress, t., mar`n, j., & sardo-infirri, j. (1982). development of the world health organiza`on (who) community periodontal index of treatment needs (cpitn). interna`onal dental journal, 32(3), 281291. 16. cutress, t. w., ainamo, j., & sardo-infirri, j. (1987). the community periodontal index of treatment needs (cpitn) procedure for popula`on groups and individuals. interna`onal dental journal, 37(3), 222233. 17. ibm corp. released 2013. ibm spss sta`s`cs for windows, version 23.0. armonk, ny: ibm corp. 18. mchugh, m. l. (2013). the chi-square test of independence. biochemia medica, 23(2), 143-149. 19. jawad, k. 2007. periodontal treatment needs among dental and non-dental iraqi students (a compara`ve study). mustansiria dental journal, 4(2), 125-130 20. batra, m., tangade, p. and gupta, d. 2014. assessment of periodontal health among the rural popula`on of moradabad, india. j indian assoc public health dent., 12(1), 28-32. 21. sekhon, t.s., grewal, s. and gambhir, r.s. 2015. periodontal health status and treatment needs of the rural popula`on of india: a cross-sec`onal study. journal of natural science, biology, and medicine, 6 (1), 111-115. 22. mahajani, m., acharya, v., samson, e., chavan, a., sewane, s. and balagangadhae`lak, p. 2016. assessment of periodontal health status and treatment needs in rural popula`on of the central maharashtra: a cross-sec`onal study. journal of interna`onal oral health, 8 (7), 772-775. 23. dey, s.m., jude, m. 2017. assessment of periodontal assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu health status among koraga tribes residing in mangalore taluk: a cross sec`onal study. interna)onal journal of research in medical sciences, 5(9), 3980-3984. 24. farsi, j.m. 2010. dental visit paderns and periodontal treatment needs among saudi students. east mediterr health j, 16 (7), 801-6. 25. peeran, s.w., singh, a.j.a.r., alagamuthu, g. and naveen kumar, p. g. 2013. periodontal status and its risk factors among young adults of the sebha city (libya). dental research journal, 10 (4), 533-538. 26. shah, c.s. 2010. an epidemiological study in gujarat by community periodontal index of treatment needs (cpitn). annals and essences of den)stry, 2 (4), 30-32. 27. bhardwaj v, veeresha k and sharma k (2012) periodontal status and treatment needs among state government employees in shimla city, himachal pradesh (india): a cross sec`onal study. indian j. oral sci. 3(1), 2833. 28. ibrahim, s., & hussein, a.s. (2019). role of hexidine, zak and biofresh mouth wash in commemora`on dele`on and oral health status (compara`ve study). interna`onal journal of pharmaceu`cal research (09752366), 11(1). 29. elangovan, s., novak, k.f., and novak, m.j. 2019. clinical risk assessment. newman and carranza’s clinical periodontology. 13th ed. elsevier, inc, 410412. 30. jang, a.-y., lee, j.-k., shin, j.y. and lee, h.-y. 2016. associa`on between smoking and periodontal disease in korean adults: the fiih korea na`onal health and nutri`on examina`on survey (2010 and 2012). korean journal of family medicine, 37 (2), 117-122. 31. preshaw, p.m. 2019. periodontal pathogenesis. newman and carranza’s clinical periodontology. 13th ed. elsevier,inc, 89111. 32. salman, f.d., qasim, a.a. and saleh, k.m. 2005. oral health status and treatment needs of iraqi and yemeni dental students (a compara`ve study). alrafidain dental journal, 5 (1), 46-51. 33. tomar, v and kashyap, b. 2014. periodontal status and treatment needs among the students of himachal ins`tute of dental sciences, paonta sahib, himachal pradesh, india. journal of dental herald,1(1)011-013. 34. dhaifullah, e., al-maweri, s.a., al-motareb, f., halboub, e., elkhatat, e., baroudi, k. and tarakji, b. 2015. periodontal health condi`on and associated factors among university students, yemen. journal of clinical and diagnos)c research: jcdr, 9(12), zc30. 35. cengiz, m.i̇., zengin, b., i̇cen, m. and kökturk, f. 2018. prevalence of periodontal disease among mine workers of zonguldak, kozlu district, turkey: a cross-sec`onal study. bmc public health, 18 (1), 361. 36. ibrahim, salah m., sattar jabbar abdul-zahra alhmedat, and mohammed hamoudi alsunboli. "histological study to evaluate the effect of local application of myrtus communis oil on alveolar bone healing in rats." the open dentistry journal 18.1 (2024). 37. akhionbare, o. and ojehanon, p. 2016. a study of the effect of frequency of tooth brushing on the assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs. a cross-secconal study h#p://den*stry3000.pi#.edu prevalence of oinflammatory periodontal diseases. port harcourt med j, 10 (3), 119-123. 38. jain, n., mitra, d., ashok, k.p., dundappa, j., soni, s. and ahmed, s. 2012. oral hygiene-awareness and prac`ce among pa`ents adending opd at vyas dental college and hospital, jodhpur. journal of indian society of periodontology, 16(4), 524528. 899 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.899 http://dentistry3000.pitt.edu minimally invasive approaches in pediatric dentistry sarah mohammed khammas1, hamsa thabet musa1, saja sami malik1, samara faris abdulazeez1, asmaa mohammed khammas2, shatha abdullah abbas2 1al hikma university college, baghdad, iraq 2university of baghdad, college of den<stry, baghdad, iraq abstract the aim of this work was to assess the efficacy, affordability, and acceptability of various “non-invasive and minimally invasive techniques” that are used in treatment of carious lesion in children. minimally invasive techniques represent a paradigm shii in pediatric denjstry, emphasizing pajent-centered care and preserving healthy tooth structure. considering conjnuing technological development these techniques will become even more crucial in providing safe, effecjve, and comfortable dental experiences for children allowing pediatric denjsts to foster a posijve and e stress-free environment for children, establishing the foundajon for long-term dental health. open access cita%on: khammas sm, et al. (2025) minimally invasive approaches in pediatric den%stry. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.899 received: april 8, 2025 accepted: may 21, 2025 published: june 27, 2025 copyright: ©2025 khammas sm, et al. this is an open access ar%cle licensed under a crea%ve commons apribu%on work 4.0 united states license. email: sara.mohammed@hiuc.edu.iq introduc)on dental caries can be de.ined as a complex, dynamic, sugar-driven, bio.ilm-mediated, and multifactorial disease that results in phasic demineralization and remineralization of the tooth structure. caries occurs any time in life, in both sets of dentitions. it is an unevenly distributed, preventable disease that has signi.icant negative effects on quality of life and economy [1]. preservation of each patient’s natural teeth should be the objective of all dentists [2]. during the diagnosis of an active, non-cavitated lesion, the dentist must .irstly assess the extension of the disease and the caries risk of the child. after that, a preventive plan is made that focuses on arresting and preventing caries progression [3]. later, based on each case's needs, the dentist will select one of the minimally invasive methods like varnish, sdf, .issure sealant, and resin in.iltration [4]. minimal invasive dentistry (mid) has developed a conservative philosophy that addresses dental caries through mid techniques and preserves tooth structure. it provides a new approach to reduce pain, stress, and the time of restorative procedure, resulting in decreasing patient anxiety [5]. fluoride varnishes (fvs) fluoride varnish is a professionally applied .luoride that can stick to the tooth surfaces and release .luoride in an effective way for a relatively long time. in the past, the main disadvantage of the previous topical .luoride systems (.luoridated mouth rinse and gels) was the short duration of .luoride exposure. therefore, there was a need to develop a material that could overcome this problem by increasing the adherence of .luoride to the tooth surface [6]. 5% naf varnish was used for this purpose. in fact, the combination of 5% naf with silver nitrate or sdf have showed higher ef.icacy [7]. fv remains an excellent modality in caries prevention, white spot lesions (wsls) management, reduction of hypersensitivity, supporting enamel development in children and protecting against ecc (early childhood caries) [8]. the frequency of fvs application can be two to four times a year depending on the child’s caries risk level [9]. pit and fissure sealant a preventative procedure used to stop dental caries before they progress to the .inal stage, known as a "hole" or cavity [10,11]. it works by blocking the tooth's pits and .issures, which prevents bacteria from adhering to them [12,13]. many sealant materials had been developed and they were mostly classi.ied into glass-ionomer, polyacid-modi.ied resin based sealants and resin-based sealants [14-16]. as the glass-ionomer type had the advantage of less technique sensitivity with .luoride release, the resin sealant had shown better retention [15]. although resin-based pit and .issure sealants are most used, they still experience abrasive wear [17]. moreover, a systematic review had shown that using sealants were more minimally invasive approaches in pediatric denjstry vol 13, no 1 (2025) doi 10.5195/d3000/2025.899 http://dentistry3000.pitt.edu 2 effective in preventing or reversing the process of dental caries in comparison with no intervention or when applying only .luoride varnishes [18]. authors suggested that pit and .issure sealants are a good intervention in preventing and arresting dental caries lesions in primary and permanent teeth [19]. silver diamine fluoride (sdf) since 1969, sdf had been referred to the literature by several names, like silver diamine .luoride, silver .luoride diamine, ammoniated silver .luoride and diamine silver .luoride [20]. sdf had the potential to encourage dentin formation by using silver salt to become calci.ied, with the germicidal activity of silver nitrate and the power of .luoride to stop deterioration [21]. the presumptive features of sdf that are particularly noteworthy are: control of in.lammation and irritation; ease of use and pain relief; cost-effectiveness; material affordability; shorter duration and controllability of use; in addition to the non-invasiveness [22,23]. the term "silver-.luoride bullet" was applied to sdf since it had the ability to stop and delay the caries process [24]. the mostly studied formulation was 38% sdf and it was recommended for caries arrestment in both children and patients with special needs. when compared to 5% naf varnish, the biannual application of 38% sdf had demonstrated more ef.icacy [25,26]. the major disadvantage of sdf is the metallic taste with black staining that is experienced during its application [27], an in vitro study showed that sdf/ki (silver diamine .luoride/potassium iodide) has the potential to reduce the black staining effect of sdf, though not entirely [28] . however, newer silver compounds like nanosilverflouride (nsf) did not demonstrate tissue discoloration resulted from the oxidation of silver ion. additionally, following one to three months of applying nsf in saliva or plaque a signi.icant decrease in the number streptococcus mutans was reported in vivo [29,30]. besides, a clinical trial suggested that compared to sdf, nsf was more successful in arresting dental decay in preschoolers without causing black discoloration on their teeth and with higher parental satisfaction [31]. the combination of nano silver particles and .luoride results in a synergistic effect that promotes enamel remineralization and combats cariogenic bacteria more effectively [32]. nsf not only ensures an aesthetically pleasing outcome but also avoids the drawbacks associated with sdf also it is viable option for both pediatric and adult. the remarkable advantages of nsf make it highly applicable for inclusion in public health initiatives [33]. the long-term effectiveness of this substance needs more thorough study with bigger sample size is necessary. resin infiltra)on another mid technique was resin infiltration, which is used for treating mild hypomineralized lesions by sealing enamel porosities and strengthening enamel defects and thus improving esthetics [34]. using this technique, non-cavitated lesions on the proximal and smooth surfaces of the teeth can be treated [35]. a one-year follow-up clinical study showed that resin infiltration had the ability for instant diffusion into the wsls, resulting in improvement in the aesthetic outcome [36]. a cochrane review concluded that when it came to preventing proximal non-cavitated lesions, resin infiltration worked better than preventative measures like applying fluoride or encouraging flossing [37]. in addition, it provided acceptable results in treating mih lesions [38]. in pediatric dentistry, resin infiltration is a useful method to be used for treating wsls in both primary and permanent teeth. however, there is a little data on the microleakage, color stability, and long-term durability of resin infiltration. these are the material limitations that need more studies. atrauma)c restora)ve treatment (art) dr. frencken created a concept of art for decay management, which uses simply hand instruments to remove tooth decay. the world health organization accepted his idea, which is now used all over the world [39, 40]. art had become an extremely important dental technique, especially for those patients who face obstacles utilizing conventional dental interventions as well as areas with limited resources. this entails utilizing hand instruments to carefully remove decay and employing glass ionomer cement to restore cavities [41]. the modi.ied art is a modi.ication that uses high-speed handpieces to alleviate hand fatigue in places where access to dental equipment and power is not an issue [42]. later, the smart approach (silver modi.ied atraumatic restorative technique) was developed, which used sdf to eliminate the bacteria then glass ionomer to seal the tooth to deny any nutrition that bacteria need to thrive [43]. a comprehensive review to highlight the effectiveness of smart revealed that sdf made a bene.icial addition to enhance the effectiveness of art in primary dentition [44]. hall’s technique (ht) hall’s technique is one of the approaches used to biologically seal the decayed tooth in deciduous molars. no need for any intervention like using low speed or high-speed hand piece for cavity preparation or no anesthetizing the tooth and the procedure depends on separating or sealing the bacteria from the oral cavity and the decay will be inactive [45,46]. even the bacteria will be present after placing the ss crown it will be unviable [47]. the name derived from a scottish dentist dr norna hall, who established and utilized the procedure with good achievement [48]. valentim et al. found that ht is greater to other restoration procedures concerning its reception by children and their guardian in relation to of painful, non-comfortable procedures, fear, and crown preference and ful.illment. ht seems to be with greater cost to the dentists, regarding the success over time, the minor need for consultations and decay removal interventions veri.ies improved cost-bene.it than other restorative procedures [49]. however, most pedodontics considered ht one of the therapy methods considered to halt the decayed primary molar rather than a therapy method. [50,51] one of the more recent and advantageous materials in pediatric dentistry that is utilized with ht is sdf. [52,53] ht technique is favorable to teeth with no history of pain and used to children with anxiety [54-58]. applying ht has an obvious effect on child cooperation inside the clinic [59]. however, poludasu et al. compared between m t a pulpotomy and ht he found no signi.icant difference [60]. it seems to affect the masseter muscle activity (mma) in children treated with a unilateral htc in which clench mma was reduced immediately posttreatment [61]. air abrasion (aab) new machines and materials have been invented to treat the child with less discomfort almost with no or less pain, fast, easy, less destructive tooth structure; one of them is the air abrasion system. the action principal of aab depends on using nontoxic particles submitted to excessive pressure and high velocity to quite eliminate decayed areas in the tooth. this method gives the dentist the advantage of lower the surface heat, less vibration, less noise also eliminating the anesthesia leading to child cooperation [62], aab could be useful for anxious, fearful, uncooperative or even autistic children [63-64] this method aims to preserve as much as possible the tooth structure. this technique used in cavity preparation as well as for sealants, preventive resin restorations and removal of previous fillings [62]. regarding the cavity preparation and material adherence to enamel and dentin concerning its effect on the surface roughness researchers found that air abrasion has equivalent effect to the conventional cavity preparation [65-66]. minimally invasive approaches in pediatric denjstry vol 13, no 1 (2025) doi 10.5195/d3000/2025.899 http://dentistry3000.pitt.edu 3 other found combining acid etching and aab have greater effect on adherence [67]. chemo mechanical caries removal cmcr cmcr offers an alternative to the conventional drilling for caries removal [68] as many dentists focus on measuring the technique time, patient comfort, caries removal efficacy, pain assessment, dentist manipulation and the cost of the procedure. most of the authors reached mostly to a comparable conclusion regarding great patient acceptance to the procedure with less fear, but time-consuming procedure [68-71]. many commercial materials that are widely used are carie-care™ [68], rt + cmcra (cmcra, papacárie duo gelⓡ seems to be less efficient than using drilling burs [70]. smart prep bur ii (polymer bur) or brix 3000 (caries dissolving enzymes) [70]. concerning the caries removal using smart burs was better than cmcra [72]. studies concerning the composite adhesion to enamel and dentin, a s coelho et al. found that the application of brix 3000™ decreased adhesion power while papacárie duo® had no effect on the dentin adhesion to composite in the teeth [73]. the same material found to be efficient in minimally invasive caries excavation and was more comfortable for children [74,75] comparing the application of brix 3000 and the drilling procedure, brix 3000 remove the affected decayed area while the drilling method doesn’t specify the decayed area only so taking more tooth structure, also less pain and sensitivity to the child but longer time than drilling method [76]. more advantages to the previously mentioned considering brix 3000 as a nontoxic, nonirritant to the oral cavity material also has an easily applicable no need for complicated instruments [77]. also, papacarie displayed hopeful outcomes in terms of bond intensity to dentin caries [78,79]. application of papacarie lead to a conclusion that mostly the same time and less pain also its effect on the child was better than the drilling procedure [80]. in 2025 gupta et al. found that as all the cmcr agents share the same advantages like less pain same time with conventional method. ozone (o3) ozone is one of the natural compounds, consist of triple oxygen, the ultraviolet radiation affects and convert the oxygen to ozone. which considered an effective disinfectant against microbes [81,82]. as a therapeutic agent increased in researches recently regarding its effect on bacteria, on inflammation, and in pain relief effect in all the fields of dentistry such as operative, endodontic, periodontology, surgery and other fields [83]. bin hassan s. a. in 2024. found that ozonated water decrease the sensitivity after bleaching the teeth with hydrogen peroxide [84] furthermore its useful if it is mixed with toothpaste resulting in temporary remineralization especially when applied after bleaching [85]. the disinfection properties of ozonated water were obvious and comparable to 0.1% octenidine dihydrochloride [86]. also, its positive effect in comparison to naocl/conventional irrigation [87]. in future it can be utilized in remineralization of the enamel and dentine layers by destroying the biological molecules of decayed part and eliminating proteins from the decayed area, leading to a porous and ensuring increased mineral ion and remineralizing agent perfusion [88]. in endodontic treatment, zinc oxide-ozonated oil reveal most desirable effect in deciduous endodontically obturated teeth [89]. with high concentration and long treatment intervals, it is efficiently eliminating s. mutans biofilms not only by oxidizing eps, furthermore destruct the plasma membrane, leading to content outflow, lowering atp levels, and final inactivation of cells which increases the viability of the usage of ozonated water in the treatment of decayed teeth [90,91]. in the recent studies disinfecting the cavity with ozonated water increase its durability of the filling material and decrease or eliminate the recurrence of caries with no effect on bonding strength [92]. dähnhardt et al. experimented the effect of ozone and tool excavating method in pulpotomy procedure to children which are fearful, uncooperative, and young [88,93]. ozone treatment procedures need to be expanded with further studies for more evaluation of results, effect, methods [91]. laser the development of dental laser technology has made it possible to perform several therapeutic procedures, “pulpotomy and pulpectomy”, oral tissue, trauma management to permanent teeth, and the removal decayed spots in all types of teeth, typically without anesthesia usage [94]. although their implementation is restricted by high charges and training necessities [95]. laser technology has significantly impacted modern dentistry by enabling initial finding, mits and accurate operative procedures, eventually enlightening patient consequences and growing the choice of dental treatment [96]. a key factor in preventing caries is the tooth surface's resistance to cariogenic agent penetration. it is possible to successfully strengthen the freshly emerged permanent tooth resistance against acid corrosion by using erbium and co2 lasers [97,98]. before applying pit and fissure sealants, the tooth surface can also be prepared using a laser. pits and fissures can also be cleaned, disinfected, and conditioned using laser [99]. declared laser benefits pain reduction, fast tissue heals and limiting anesthesia usage. erbium and diode lasers are the most used kinds for teeth and mouth soft tissue [100], in pediatric dentistry focusing on the cavity preparation, pulpotomies, soft-tissue management, in cavity preparation and root access [101,102]. children can withstand laser treatment since it involves little contact. parents, dentists, and the kids themselves are all more satisfied when patients cooperate better. for many traditional pediatric dentistry procedures, lasers can be a good substitute. pulp therapy, caries diagnosis and removal, minimizing the possibility of contamination, edema, and irritation, minimizing blood oozing, improving soft tissues repair, alleviating discomfort, also lessening the gag reaction [103]. limita)ons employing mid allows pediatric dentists to ensure a positive and stress-free experience for young children and consequently good dental habits for life. on the other hand, some limitations might reduce its widespread application in pediatric dental care. high costs (laser system), technique sensitivity and moisture control (resin in.iltration), and periodic recalls (.issure sealant and sdf). besides, improving accessibility and achieving better clinical results in pediatric dentistry need to address issues like cost reduction, improved training programs, and ongoing studies into bioactive materials and technology. conclusion minimally invasive procedures represent a paradigm shift in pediatric dentistry that emphasize natural tooth structure preservation. it is undoubtedly that future development of bioactive materials will provide synergic effect to mit in preserving tooth structure, promoting remineralization, tissue regeneration and minimizing the need for extensive restorative procedures. future studies should concentrate on developing reasonably priced materials, enhancing durability, incorporating arti.icial intelligence and computer-aided technologies. with ongoing innovations, minimally invasive procedures will be the gold standard in pediatric dentistry, improving childrens’ oral health care globally. minimally invasive approaches in pediatric denjstry vol 13, no 1 (2025) doi 10.5195/d3000/2025.899 http://dentistry3000.pitt.edu 4 references 1. pi&s, n. b., zero, d. t., marsh, p. d., ekstrand, k., weintraub, j. a., ramos-gomez, f., ... & ismail, a. (2017). dental caries. nature reviews disease primers, 3(1), 1-16. 2. young, d. a., nový, b. b., zeller, g. g., hale, r., hart, t. c., truelove, e. l., ... & beltran-aguilar, e. (2015). the american dental associawon caries classificawon system for clinical pracwce: a report of the american dental associawon council on scienwfic affairs. the journal of the american dental associa<on, 146(2), 79-86. 3. bagramian ra g-gf, volpe ar. (2009) the global increase in dental caries. a pending public health crisis. am j dent, 22:3-8. 4. alhowaish, l. (2021). non-invasive and minimally invasive management of carious lesions in children: a scoping review. journal of research in medical and dental science, 9(s1). 5. murdoch-kinch, c. a., & mclean, m. e. (2003). minimally invasive denwstry. the journal of the american dental associa<on, 134(1), 87-95. 6. kohn, w.g.; maas, w.r.; malvitz, d.m.; presson, s.m.; shaddix, k.k. (2021) recommendawons for using fluoride to prevent and control dental caries in the united states. 7. gao ss, duangthip d, wong mcm, lo ecm, chu ch. (2019). randomized trial of silver nitrate with sodium fluoride for caries arrest. jdr clin trans res apr, 4:126-134. 15. 8. aljohani, a. a., alarifi, a. i., almoain, m. f., alrhaimi, f. f., alhejji, m. t., gazzaz, n. w., ... & barnawi, b. (2024). managing early childhood caries: a comparawve review of prevenwve and restorawve approaches. cureus, 16(11). 9. lawfi-xhemajli, b., begzaw, a., veronneau, j., kutllovci, t., & rexhepi, a. (2019). effecwveness of fluoride varnish four wmes a year in prevenwng caries in the primary denwwon: a 2 year randomized controlled trial. community dental health, 36(3), 190194. 10. white, j. m., & eakle, w. s. (2000). rawonale and treatment approach in minimally invasive denwstry. the journal of the american dental associa<on, 131, 13s-19s. 11. godhane, a., ukey, a., tote, j. v., das, g., naphde, m., & pawl, p. (2015). use of pit and fissure sealant in prevenwon of dental caries in pediatric denwstry and recent advancement: a review. int j dent med res, 1(6), 220-223. 12. simonsen, r. j., & neal, r. c. (2011). a review of the clinical applicawon and performance of pit and fissure sealants. australian dental journal, 56, 45-58. 13. pani, p. (2022). pit and fissure sealants the stop of the demon. book rivers. 14. mickenautsch, s., & yengopal, v. (2011). cariesprevenwve effect of glass ionomer and resin-based fissure sealants on permanent teeth: an update of systemawc review evidence. bmc research notes, 4, 1-18. 15. colombo, s., bere&a, m. (2018). dental sealants part 3: which material? efficiency and effecwveness. eur j paediatr dent; 19:247-249. 16-. sivavong, p., mahapoka, e., srijunbarl, a., singthong, t., suriyapongprapai, t., chantarangsu, s., ... & nantanapiboon, d. (2025). degradawon and ultrastructural changes of resin-based pit and fissure sealants under simulated chewing condiwons. bmc oral health, 25, 185. 17. sivavong, p., mahapoka, e., srijunbarl, a., singthong, t., suriyapongprapai, t., chantarangsu, s., ... & nantanapiboon, d. (2025). degradawon and ultrastructural changes of resin-based pit and fissure sealants under simulated chewing condiwons. bmc oral health, 25, 185. 18. wright jt, tampi mp, graham l, et al. sealants for prevenwng and arreswng pit-and-fissure occlusal caries in primary and permanent molars: a systemawc review of randomized controlled trials-a report of the american dental associawon and the american academy of pediatric denwstry. j am dent assoc 2016; 147:631-645.e18. 19. leite, k. l. d. f., rodrigues, g. f., chevitarese, a. b., magno, m. b., marañón-vásquez, g. a., pintor, a. v. b., & maia, l. c. (2024). are pit and fissure sealants effecwve in prevenwng and arreswng occlusal caries in primary and permanent teeth? an overview of systemawc reviews. journal of evidence-based dental prac<ce, 24(3), 102010. 20. peng jj, botelho mg, mawnlinna jp. (2012) silver compounds used in denwstry for caries management: a review. j dent, 40(7):531–541. doi: 10.1016/j.jdent.2012.03.009. 21. gao, s. s., zhao, i. s., duffin, s., duangthip, d., lo, e. c. m., & chu, c. h. (2018). revitalising silver nitrate for caries management. internawonal journal of environmental research and public health, 15(1), 80. 22. monahan, a.b. (2018). the regulatory failure to define essenwal health benefits. am. j. law. med. 44, 529–577. 23. mushtahaa, s. m., & al-ww, a. t. (2022). uwlizawon of silver diamine fluoride for caries arresting. neuroquantology, 20(8), 5283. 24. done, v., kanaparthi, s., bandela, v., alam, m. k., & muralidhar, p. (2024). silver diamine fluoride–the magic bullet! is it really a magic alternawve in caries management. bangladesh journal of medical science, 23(4), 993-998. 25. davis mr, johnson el, meyer bd. (2020) comparing dental treatment between children receiving and not receiving silver diamine fluoride. j clin pediatr dent, 44:400-406. 26. crystal yo, marghalani aa, ureles sd, wright jt, sulyanto r, divaris k, fontana m, graham l. (2017). use of silver diamine fluoride for dental caries management in children and adolescents, including those with special health care needs. pediatr dent, 39:135145. 27. chai, h. h., sun, i. g., ho, j. c. y., gao, s. s., lo, e. c. m., & chu, c. h. (2024). denwsts' knowledge, avtude and pracwce towards silver diamine fluoride therapy in hong kong: a mixed-method study. fron<ers in oral health, 5, 1487879. 28. karaduran, b., çelik, s., gök, m. k., & koruyucu, m. (2024). evaluawon of staining potenwal of silver diamine fluoride, potassium iodide, nanosilver fluoride: an in vitro study. bmc oral health, 24(1), 699. 29. santos ve jr, filho v, targino a ag et al (2014) a new silver-bullet to treat caries in children–nano silver fluoride: a randomised clinical trial. j dent 42(8):945–951. h&ps://doi.org/10.1016/j. jdent.2014.05.017 30. waikhom n, agarwal n, jabin z, anand a (2022) anwmicrobial effecwveness of nano silver fluoride varnish in reducing streptococcus mutans in saliva and plaque biofilm when compared with chlorhexidine and sodium fluoride varnishes. j clin exp dent 14(4):e321. h&ps://doi.org/10.4317/jced.59093.ecollecwon 31. quritum, m., abdella, a., amer, h., el desouky, l. m., & el tantawi, m. (2024). effecwveness of nanosilver fluoride and silver diamine fluoride in arreswng early childhood caries: a randomized controlled clinical trial. bmc oral health, 24(1), 701. 32. kumar, c. (2024, april). the synergism of silver and fluoride: a new arsenal in caries management. in macromolecular symposia, 413, 2, 2300129. 33. pawl, a., pawl, m., & contractor, i. a. (2024). nano-silver fluoride in prevenwve denwstry: a literature review. internawonal journal for mulwdisciplinary research, 6 (1), 7. 34. moeinian, m. (2024). an overview of the resininfiltrawon technique. dental update, 51(7), 516518. 35. ibrahim, d. f. a., hasmun, n. n., miin, l. y., & venkiteswaran, a. (2024). resin infiltrawon – a narrawve review of properwes. journal of health and transla<onal medicine (jummec), 153-161. 36. zaazou, m. h., saleh, r. s., hassan, s. n., abdelnabi, a., zaki, z. m., hamdy, t. m., ... & moharam, l. m. (2024). effecwveness of low-viscosity resin infiltrawon (icon) on color change of enamel white spot lesions: 1-year follow-up clinical study. bulle<n of the na<onal research centre, 48(1), 62. 37. dorri m, dunne sm, walsh t, et al. micro-invasive intervenwons for managing proximal dental decay in primary and permanent teeth. cochrane database syst rev 2015; (11):cd010431. 38. alghawe, s., & raslan, n. (2024). management of permanent incisors affected by molar-incisor-hypomineralisawon (mih) using resin infiltrawon: a pilot study. european archives of paediatric denwstry, 25(1), 105-116. 39. de lima navarro mf (2009): introducwon to the symposium. two decades of art: success through research. j appl oral sci. 17:76-7. 40. more, s., mistry, l., pawl, a., baddi, b., sethumadhavan, j., sinha, a., & mistry, l. n. (2024). a review minimally invasive approaches in pediatric denjstry vol 13, no 1 (2025) doi 10.5195/d3000/2025.899 http://dentistry3000.pitt.edu 5 of the work of dr. jo e frencken: the pioneer of atraumawc restorawve treatment for dental caries. cureus, 16(11). 41. hodali, l., & massad, n. (2024). atraumawc restorawve treatment: an overview and update. european journal of dental and oral health, 5(3), 1-4. 42. santamaria rm, innes np, machiulskiene v, et al. caries management strategies for primary molars: 1yr randomized control trial results. j dent res 2014; 93:1062-9. 43. aly aa, aziz am, elghazawy rk, el-fadl rk. survival analysis and cost effecwveness of silver modified atraumawc restorawve treatment (smart) and art occlusal restorawons in primary molars: a randomized controlled trial. (2023). j dent;128:104379. 44. aldosari, m. m., alkhathlan, a. r., & alasmari, o. n. (2024). silver-modified atraumawc restorawve technique: a comprehensive review. saudi journal of oral sciences, 11(1), 5-10. 45. altoukhi dh, el-housseiny aa. hall technique for carious primary molars: a review of the literature. dent j (basel). 2020 jan 17;8(1):11. doi: 10.3390/dj8010011. pmid: 31963463; pmcid: pmc7148518. 46. reynolds l, sohal g, barry s. confidence-level of foundawon denwsts in the north west of england in the placement of preformed metal crowns using the hall technique. eur j dent educ. 2023 feb;27(1):158166. doi: 10.1111/eje.12788. epub 2022 feb 19. pmid: 35147276. 47. elbahary s, aharonian s, azem h, peretz b, moswnski o, blumer s. bacterial colonizawon and proliferawon in primary molars following the use of the hall technique: a confocal laser scanning microscopy study. children (basel). 2023 feb 25;10(3):457. doi: 10.3390/children10030457. pmid: 36980014; pmcid: pmc10047319. 48. innes, n. p. t., et al. "a novel technique using preformed metal crowns for managing carious primary molars in general pracwce—a retrospecwve analysis." bri<sh dental journal 200.8 (2006): 451-454. 49. valenwm fb, moreira kms, carneiro vc, et al. cost-effecwveness and acceptance in children and parents of the hall technique: systemawc review of clinical trials. j contemp dent pract 2023;24(12):1016-1025 50. jesmin f, kamarudin a, baharin f, ahmad wmabw, mohammed m, marya a, messina p, alessandro scardina g, karobari mi. the use of hall's technique preformed metal crown (htpmc) by pediatric denwsts in malaysia. biomed res int. 2021 dec 22;2021:8424206. doi: 10.1155/2021/8424206. pmid: 34977246; pmcid: pmc8716193. 51. pascareli-carlos am, tedesco tk, calvo afb, floriano i, gimenez t, gonçalves mds, calumby d, imparato jcp. survival rate of the hall technique compared with resin composite restorawon in mulw-surface caviwes in primary teeth: a 1-year randomized clinical trial. j appl oral sci. 2023 oct 9;31:e20230048. doi: 10.1590/1678-7757-20230048. pmid: 37820181; pmcid: pmc10561961. 52. hu s, meyer b, duggal m. a silver renaissance in denwstry. eur arch paediatr dent. 2018 aug;19(4):221-227. doi: 10.1007/s40368-018-03637. epub 2018 aug 9. pmid: 30094547 53. crystal yo, janal mn, yim s, nelson t. teaching and uwlizawon of silver diamine fluoride and hallstyle crowns in us pediatric denwstry residency programs. j am dent assoc. 2020 oct;151(10):755-763. doi: 10.1016/j.adaj.2020.06.022. pmid: 32979954; pmcid: pmc7510543. 54. midani r, splieth ch, mustafa ali m, schmoeckel j, mourad sm, santamaria rm. success rates of preformed metal crowns placed with the modified and standard hall technique in a paediatric denwstry setwng. int j paediatr dent. 2019 sep;29(5):550-556. doi: 10.1111/ipd.12495. epub 2019 apr 14. pmid: 30888708. 55. abu serdaneh s, alhalabi m, kowash m, macefield v, khamis ah, salami a, hussein i. hall technique crowns and children's masseter muscle acwvity: a surface electromyography pilot study. int j paediatr dent. 2020 may;30(3):303-313. doi: 10.1111/ipd.12611. epub 2020 jan 24. pmid: 31894621. 56. narbutaite j, santamaría rm, innes n, splieth ch, maciulskiene v. comparison of three management approaches for dental caries in primary molars: a two-year randomized clinical trial. j dent. 2024 nov;150:105390. doi: 10.1016/j.jdent.2024.105390. epub 2024 oct 5. pmid: 39374732. 57. ebrahimi m, shirazi as, afshari e. success and behavior during atraumawc restorawve treatment, the hall technique, and the stainless steel crown technique for primary molar teeth. pediatr dent. 2020 may 15;42(3):187-192. pmid: 32522320. 58. oz e, kırzıoglu z, kale c. the clinical success of art restorawons and hall technique in primary molars: a randomized 18-month follow-up study. restor dent endod. 2023 may 1;48(2):e19. doi: 10.5395/rde.2023.48.e19. pmid: 37284348; pmcid: pmc10240095. 59. santamaría rm, gomma a, khole mr, splieth ch, alkilzy m. influence of the hall technique on pawent cooperawon: a retrospecwve analysis. j clin med. 2025 jan 7;14(2):304. doi: 10.3390/jcm14020304. pmid: 39860309; pmcid: pmc11766221. 60. poludasu m, kumar mallela gm, puppala r, kethineni b, dandowkar d. comparison of three treatment techniques for deep carious lesions in primary teeth: an in vivo study. int j clin pediatr dent. 2022;15(suppl 2):s201-s206. doi: 10.5005/jpjournals-10005-2151. pmid: 35645518; pmcid: pmc9108839. 61. abu serdaneh s, alhalabi m, kowash m, macefield v, khamis ah, salami a, hussein i. hall technique crowns and children's masseter muscle acwvity: a surface electromyography pilot study. int j paediatr dent. 2020 may;30(3):303-313. doi: 10.1111/ipd.12611. epub 2020 jan 24. pmid: 31894621. 62. prathap, a. (2024). micro air abrasion: a novelty unparalleled. journal of the indian society of pedodon<cs & preven<ve den<stry, 42. 63. abdul-kareem mahmood, m., khairi al-hadithi, h., & mueen hussein, h. (2024). evaluawon of denwn features in teeth a�er caries removal by three techniques (chemomechanical, mechanical with a smart bur, and air-abrasion): an in vitro study 64. kuvatbayeva, u. a., & iskakova, m. k. (2024). the use of air abrasion method to treat caries in auwswc pawents at the age of 6 years: a non-randomized controlled trial. special care in den<stry, 44(6), 1812-1817. 65. abuzenada , b. m. (2025). impact of different cavity preparawon techniques on denwn bond strength. bangladesh journal of medical science, 24(10), 104–108. h&ps://doi.org/10.3329/bjms.v24i10.79178 66. almeida, g., marques, j. a., van meerbeek, b., ramos, j. c., & falacho, r. i. (2024). parwcle abrasion as a pre-bonding denwn surface treatment: a scoping review. journal of esthe<c and restora<ve den<stry, 36(5), 746-760. 67. bhadule, s. n., kalaskar, r., kalaskar, a., krishnakumar, k., joshi, s., & balasubramanian, s. (2024). clinical effecwveness of air abrasion when compared to convenwonal acid-etching technique in enhancing the retenwon of pit and fissure sealants: a systemawc review. interna<onal journal of clinical pediatric den<stry, 17(3), 377–384. h&ps://doi.org/10.5005/jp-journals-10005-2782 68. kitsahawong k, seminario al, pungchanchaikul p, ra&anacharoenthum a, su&hiprapaporn p, piwphat w.(2024) efficacy of chemo-mechanical caries removal: a 24-month randomized trial. front oral health.;5:1458530. doi: 10.3389/froh.2024.1458530. pmid: 39697785; pmcid: pmc11653365. 69. gupta, s., pentapaw, k. c., & acharya, s. (2025). a randomised controlled trial comparing chemomechanical (carie-care™) versus convenwonal caries removal for atraumawc restorawve treatment. the scienwfic world journal, 2025(1), 6689053. 70. da silva, r. o., da silva, l. b., pinto marinho, m. f., de souza, e. e. r., cavalcanw, y. w., fonseca-gonçalves, a., & pintor, a. v. b. (2025). cost-efficiency analysis of atraumawc restorawve treatment with and without chemical-mechanical caries removal agents based on pain and wme for selecwve removal of carious wssue: a public health perspecwve. clinical oral inveswgawons, 29(2), 134. 71. elmarakby, a. m., habib, s. f. k., alkhaldy, i. m., aljasser, r. m., & elsebaey, l. m. l. (2025). comparawve study for assessment of two different minimally invasive caries removal techniques. bmc oral health, 25(1), 196. h&ps://doi.org/10.1186/s12903-02505537-x 72. mehrotra, d., kodical, s. r., & naik, s. s. (2024). comparison of smart burs and chemo-mechanical caries removal systems in primary molars a systemawc review and meta-analysis. journal of the indian society of pedodon<cs and preven<ve minimally invasive approaches in pediatric denjstry vol 13, no 1 (2025) doi 10.5195/d3000/2025.899 http://dentistry3000.pitt.edu 6 den<stry, 42(4), 257–266. h&ps://doi.org/10.4103/jisppd.jisppd_308_24 73. coelho, a. s., vilhena, l., amaro, i., melo, a., paula, a., marto, c. m., ferreira, m. m., ramalho, a., & carrilho, e. (2024). effect of enzymawc chemo-mechanical agents on adhesion of composite resin to denwn of permanent teeth: an in vitro study. european archives of paediatric den<stry : official journal of the european academy of paediatric den<stry, 10.1007/s40368-024-00949-9. advance online publicawon. h&ps://doi.org/10.1007/s40368-024-009499 74. somkuwar jk, ansari aa, singh rk, bahal r. comparawve evaluawon of brix3000 smart burs and convenwonal technique of caries excavawon among 5– 9-year-old children: a randomized clinical trial. j south asian assoc pediatr dent 2024; 7 (3):143-150. 75. vishwanathaiah, s., maganur, p. c., maafa, s. m., tulays, a. m., hakami, m. m., albar, n. h., alessa, n., alhakami, k., assiry, a. a., alfaqih, a. i., & syed, a. a. (2025). assessing caries removal efficacy and pain percepwon in children, using smart bur versus carbide bur: a randomized clinical study. medical science monitor: interna<onal medical journal of experimental and clinical research, 31, e946802. h&ps://doi.org/10.12659/msm.946802 76. baswa, r., govind, s., assiry, a.a. et al. volumetric analysis a�er caries excavawon with caries detecting dyes and chemomechanical caries removal agents using 3d scanner-a randomised clinical trial. bmc oral health 24, 164 (2024). h&ps://doi.org/10.1186/s12903-024-03907-5 77. dewi, a. k., & garwka, m. (2024). the revoluwon of brix 3000 as chemomechanical caries removal (cmcr) agent, a scoping review. journal of syiah kuala den<stry society, 9(1), 51-63. 78. el zayat, i., abdelhamid, m., farouk, a., & sedky, y. w. (2025). comparawve evaluawon of the bond strength of resin composite to caries affected denwn treated with chemomechanical method (papacarie) and er, cr: ysgg laser: an in-vitro study. egyp<an dental journal, 71(1), 977-984. 79. sergio lópez-garcía, nuria pérez-guzmán, francisco j. rodríguez-lozano, maría pilar pecci-lloret, david garcía-bernal, laura murcia, ricardo e. oñate-sánchez, carmen llena; biological effects of new chemical-mechanical caries removal products on human dental pulp stem cells. caries res 16 december 2024; 58 (6): 612–624. 80. akindele, b.o., orenuga, o., olatosi, o. et al. comparawve evaluawon of the clinical effecwveness of chemomechanical (papacarie) and convenwonal mechanical caries removal methods in treatment of carious primary molars: a randomized controlled clinical study. bmc oral health 25, 290 (2025). h&ps://doi.org/10.1186/s12903-025-056547 81. rapone, b.; ferrara, e.; santacroce, l.; topi, s.; gnoni, a.; dipalma, g.; mancini, a.; di domenico, m.; tartaglia, g.m.; scarano, a. (2022). the gaseous ozone therapy as a promising anwsepwc adjuvant of periodontal treatment: a randomized controlled clinical trial. int. j. environ. res. public health. 19, 985. 82. shang, w.; wang, y.; wang, g.; han, d. (2023). benefits of ozone on mortality in pawents with covid-19: a systemawc review and meta-analysis. complement. ther. med. 72, 102907. [google scholar] [crossref] 83. mane, m. the applicawon of ozone therapy in minimally invasive denwstry. 84. bin hassan s. a. (2024) tooth sensiwvity following hydrogen peroxide bleaching with and without ozone: a randomized controlled trial: tooth sensiwvity following h2o2 versus h2o2/ozone bleaching. pain research & management, 2024, 2695533. h&ps://doi.org/10.1155/2024/2695533 85. foltz, k. r. b., domingues, m. f. b., camav, f. v., engroff, r. s., cielo, s. i., mendonça, m. j., ... & ueda, j. k. (2025). effect of desensiwzing agents and ozonated toothpaste on enamel microhardness a�er bleaching. caderno pedagógico, 22(1), e13719e13719. 86. inamdar, m. s., chole, d. g., bakle, s. s., vaprani, p. b., gandhi, n. p., & ha&e, n. r. (2024). comparawve evaluawon of anwmicrobial efficacy of 0.1% octenidine dihydrochloride, superoxidized soluwon, ozonated water, 0.1% silver nanoparwcle soluwon, and q mix™ 2 in 1 in root canals infected with enterococcus faecalis. journal of conservawve denwstry and endodonwcs, 27(10), 1059–1064. h&ps://doi.org/10.4103/jcde.jcde_434_24 87. brisson-suárez, k., siqueira, j. f., jr, alves, f. r. f., campello, a. f., rodrigues, r. c. v., voigt, d. d., romeiro, k., loyola-fonseca, s. c., heggendorn, f. l., mdala, i., & rôças, i. n. (2024). effecwveness of supplementary anwmicrobial procedures in disinfecwng lateral canals as evaluated by a novel ex vivo analywcal approach. scienwfic reports, 14(1), 21840. h&ps://doi.org/10.1038/s41598-024-72041-7 88. dähnhardt, j.e.; jaeggi, t.; lussi, a. (2006) treating open carious lesions in anxious children with ozone. a prospecwve controlled clinical study. am. j. dent. 19, 267–270. 89. how to cite this arwcle: arora p, mathur s, arora p. clinical and radiographic evaluawon of zinc oxideozonated oil, zinc oxide-eugenol, anwoxidant mix, and probiowc mix as obturawng materials in primary teeth: an in vivo study. int j clin pediatr dent 2024;17(10):1153-1162. 90. li, r., zhang, s., cai, y., huang, c., bu, l., wu, y., & zhou, s. (2025). anwmicrobial effects of ozone water on s. mutans biofilms: an arwficial mouth study. journal of environmental chemical engineering, 115852. 91. veneri, f., filippini, t., consolo, u., vincew, m., & generali, l. (2024). ozone treatment for the management of caries in primary denwwon: a systemawc review of clinical studies. denwstry journal, 12(3), 69. h&ps://doi.org/10.3390/dj12030069 92. ali, m. a. s., alrafee, s. a., metwally, n. i., salem, a., badawy, s., & abdallah, s. a. (2025). effect of mushroom, ozone gas, and their combinawon as pretreatment materials on the bond strength of resin composite to denwn. the journal of contemporary dental pracwce, 25(10), 914-920. 93. luppieri, v.; manfra, a.; ronfani, l.; chermetz, m.; cadenaro.( 2022) m. ozone therapy for early childhood caries (ecc) treatment: aninvivo prospecwve study. appl. sci. 12, 1964. 94. arapostathis, k., velonis, d., & chala, m. (2024). laser-assisted pediatric denwstry. in lasers in denwstry—current concepts (pp. 339-376). cham: springer internawonal publishing. 95. alqahtani, a. m., alshihri, y. d., alhumaid, a. e., al nafaie, m. m., alnaim, a. a., alqahtani, a. m., & alhumaid, a. (2025). advancements in minimally invasive techniques in pediatric denwstry: a review. cureus, 17(1). 96. zhang, o. l., yin, i. x., yu, o. y., luk, k., niu, j. y., & chu, c. h. (2025). advanced lasers and their applicawons in denwstry. denwstry journal, 13(1), 37. 97. apel c. birker l, meister j, weiss c, gutknecht n. (2004) the caries-prevenwve potenwal of subablawve er:yag and er:ysgg laser radiawon in an intraoral model: a pilot study. photomed laser surg. 22:312317. doi:10.1089/1549541041797931. 98. westerman gh, hicks mj, flaitz cm, ellis rw, powell gl. (2004). argon laser irradiawon and fluoride treatment effects on caries-like enamel lesion formawon in primary teeth: an in vitro study. am j dent. 17(4):241-244. 99. olivi g, margolis fs, genovese md. (2011) pediatric laser denwstry; a user’s guide. chicago: quintessence publishing.73-76. 100. sharma, r., & pandit, s. laser technology in denwstry: advancements, applicawons, and future trends. 101. edward, j. (2025). shaping smiles-the cuvng edge era in paediatric denwstry. kerala dental journal, 48(1), 1. 102. wang, x., zhang, j., wang, j., zhang, j., luo, p., & xin, b. (2025). minimally invasive endodonwc treatment through the labial/buccal cervical approach-protocol and case series. journal of endodonwcs. 103. nazemisalman, b., farsadeghi, m., & sokhansanj, m. (2015). types of lasers and their applicawons in pediatric denwstry. journal of lasers in medical sciences, 6(3), 96. 808 final poten&al role of periodontopathogens in rheumatoid arthri&s vol 13 no 1 (2025) doi 10.5195/d3000.2025.808 h#p://den*stry3000.pi#.edu potential role of periodontopathogens in rheumatoid arthritis shaymaa abdulkareem nori, batool hassan al-ghurabi college of dentistry, university of baghdad, iraq abstract periodontal disease, par/cularly the chronic form of periodon//s, is a widespread inflammatory condi/on caused by pathogenic bacteria, such as porphyromonas gingivalis. this review explores the poten/al role of periodontopathogens, par/cularly porphyromonas gingivalis, in the development and progression of rheumatoid arthri/s. rheumatoid arthri/s is a systemic autoimmune disorder characterized by joint inflamma/on and damage. both periodon//s and rheumatoid arthri/s share common inflammatory mechanisms, driven by proinflammatory cytokines and immune dysregula/on. porphyromonas gingivalis, via its pathogenic determinants, that is, gingipains and pep/dyl arginine deiminase, facilitates the citrullina/on of proteins, leading to the genera/on of an/-citrullinated protein an/bodies, a key feature in rheumatoid arthri/s pathogenesis. the reciprocal rela/onship between periodon//s and rheumatoid arthri/s is further supported through evidence showing that individuals with periodon//s are at a higher risk for rheumatoid arthri/s, and vice versa. both circumstances exhibit similar inflammatory pathways, involving cytokines like tumor necrosis factor alpha, interleukin 1 and interleukin 6 and /ssue degrada/on mediated by matrix metalloproteinases. keywords: rheumatoid arthri=s, periodon==s, porphyromonas gingivalis cita9on: nori sa, et al. poten9al role of periodontopathogens in rheumatoid arthri9s. den9stry 3000.1:a001 doi:10/5195/d3000.2025.808 received: december 25, 2024 accepted: january 5, 2025 published: february 14, 2025 copyright: ©2025 nori sa, et al. this is an open access ar9cle licensed under a crea9ve commons asribu9on work 4.0 united states license. email: batoolamms@codental.uobaghdad.edu.iq introduction periodontal disease, encompassing gingivitis and periodontitis (pd), is defined by the inflammation of the gums and other structures, frequently resulting from the buildup of detrimental bacteria in the oral cavity. specific periodontopathogens, particularly porphyromonas gingivalis (p. gingivalis), have been associated with the onset and aggravation of systemic inflammation [1]. rheumatoid arthritis (ra) is a tenacious, systemic inflammatory condition that principally impacts the joints, culminating in pain, rigidity, and possible joint abnormalities. precise ra etiology is unclear; nonetheless, it is recognized to engage a confluence of genetic, environmental, and immune-mediated variables [2]. research indicates that periodontopathogens may have a role in ra by generating autoantibodies, such as anticitrullinated protein antibodies (acpa), which activate immune responses that exacerbate joint inflammation. the bidirectional link between periodontal disease and ra, where in one condition may affect the onset or advancement of the other, has generated significant scholarly interest [3,4]. this review aims to examine the potential link between periodontal infections and ra, focusing on how periodontopathogens, especially p. gingivalis, may contribute to the initiation of immune responses that facilitate the onset and advancement of ra. materials and methods for this review, the authors executed an electronic search across many pertinent scientific databases, including scopus, google scholar, pubmed, mdpi, and researchgate. the preliminary evaluation encompassed 130 publications poten&al role of periodontopathogens in rheumatoid arthri&s vol 13 no 1 (2025) doi 10.5195/d3000.2025.808 h#p://den*stry3000.pi#.edu published from 2020 to 2024 that featured the keywords “periodontitis,” “rheumatoid arthritis,” and “porphyromonas gingivalis.” inclusion criteria this study included recent papers, reviews, and case-control studies; all articles included in this review were published in english. exclusion criteria articles and research published prior to 2020 were omitted, as illustrated in figure 1. figure 1. progression of the search and selection procedure. periodontitis periodontitis is a persistent, damaging, and permanent inflammatory condition. it is the predominant cause of osteopenia and an infectious inflammatory ailment affecting humanity [5]. the world health organization classifies pd as unprecedented global event, exhibiting concerning frequency among various age demographics and geographic areas. it impact around 19% of the world's residents, representing more than a billion instances. the data concerning european nations reveals that over 50% of the population may experience some variant of pd, with over 10% afflicted by its severe manifestation. incidence increases up to 70–85% among individuals of 60–65 years old [6]. from 1990 to 2019, pd prevalence rate rose globally by 8.44% [7,8]. the worldwide load of pd is considerable and has markedly escalated throughout the last three decades, posing a huge public health concern and necessitating the pursuit of novel therapeutic interventions [6]. pathogenesis of periodontitis the pathogenesis of periodontal disease is defined by the interplay between bacterial plaque accumulation and inflammatory responses in the gingiva [9]. this connection activates fundamental molecular routes which ultimately result in the activation of host-derived proteases. this leads to periodontal ligaments marginal fibers degradation, junctional epithelium displacement, and subsequent bacterial biofilm proliferation along the root surface apically [10,11]. the microbiology of pd is intricate, primarily characterized by a transition in the microbial population from a largely grampositive, aerobic, healthassociated biofilm to a predominance of gram-negative anaerobes. fundamental molecular investigations have found complexes of various bacterial species linked to severe chronic periodontitis, specifically p. gingivalis, tanerella forsythia, and treponema denticola [12]. a recent study shown that microbial populations associated with various periodontal conditions altered in an asynchronous manner during the reconstruction of biofilms following its initial elimination using supragingival cleaning. research indicates that bacteria like abiotrophia spp. and capnocytophaga spp. may significantly influence the formation of plaque biofilms [13]. while this development seems to have a robust immunologicalinflammatory foundation, both innate (sex and genetic variables) and obtained (obesity, stress, smoking, concurrent systemic disorders) risk determinants significantly contribute [14]. soft and hard tissues around the teeth experience escalating damage from advancing inflammation due poten&al role of periodontopathogens in rheumatoid arthri&s vol 13 no 1 (2025) doi 10.5195/d3000.2025.808 h#p://den*stry3000.pi#.edu to both the direct, deleterious health risks associated with bacterial virulence factors found in dental plaque and the indirect consequences of the body's heightened, generalized inflammatory reaction to periodontopathogens [15]. inflammation initiates a complicated interaction of proinflammatory cytokines, nitric oxide, matrix metalloproteinases (mmps), prostaglandin e2, and various alternative inflammatory mediators. elevated levels of tumor necrosis factor alpha (tnfα), interleukin-1 beta (il-1β), il-6, il-11, and il-17 facilitate the formation of osteoclasts through boosting nuclear factor kappa-β ligand receptor activator expression (rankl) and diminishing osteoprotegerin production [11]. moreover, active periodontal lesions demonstrate an overproduction of il-17 and rankl, alongside reduced concentrations of antiinflammatory cytokines such as il10 and transforming growth factor beta1 (tgf-β1), indicating an aberrant immunological reaction. this disruption in the metabolism of bone not solely incline patients to periodontal disease but also suggests it in the capacity of possible risk determinant for conditions like rheumatoid arthritis, however, agreement is still hard to grasp [11,16]. the keystone periodontal pathogen (porphyromonas gingivalis) the rod-shaped, gram-negative, anaerobic bacteria known as p. gingivalis was identified in 85.75% of subgingival plaque in individuals with persistent periodontal disease [17]. the virulence components of p. gingivalis include fimbriae, haemolysin, hemagglutinins, a capsule, outer membrane vesicles, lipopolysaccharides, and gingipains [18]. the prevalence of p. gingivalis correlates with the severity of periodontal disease and has been recognized by researchers as a primary causal element in the pd progression. the keystone pathogen idea suggests that p. gingivalis, even in minimal quantities, might trigger chronic periodontal disease by altering the commensal bacterial community, resulting in dysbiosis and subsequent disease [19]. role of porphyromonas gingivalis in periodontal disease in vivo investigations in mice have demonstrated that contagion with p. gingivalis induces osteoclastic activity [20]. p. gingivalis attaches to and infiltrates the epithelial layers, proliferates and induces periodontal disease. although the epithelium serves as the primary obstacle in opposition to numerous diseases, including periodontal microbiome, p. gingivalis infiltrates gingival epithelium along with the fundamental connective tissues [23,24]. it plays a substantial role in the etiology of aggressive pd by eliciting elevated degrees of proinflammatory cytokines [25]. serotypes k1 and k2 are linked to elevated expression of receptor activator rankl. this element influences the generation and development of osteoclasts and triggers a robust t helper1 (th1) and th17 inflammatory reaction, leading to increased breakdown of alveolar osseous tissues and enhanced destruction of periodontal tissue [26]. the complement system safeguards the host against microbial infections via opsonization, chemotaxis, and infected cells destruction [22]. p. gingivalis exhibits resistance to the complement system via the activity of its enzymes or by surface chemicals that enable adaptation to the regulatory proteins of the host's complement. p. gingivalis inhibits the cascade process of complement component 5 convertase production within the complement system [22,27]. poten&al role of periodontopathogens in rheumatoid arthri&s vol 13 no 1 (2025) doi 10.5195/d3000.2025.808 h#p://den*stry3000.pi#.edu moreover, complement component 3 interacts with p. gingivalis fimbriae, facilitating the internalization of the bacteria by macrophages. p. gingivalis utilizes the synergy and crosstalk between complement and toll-like receptors to alter host defenses and evade elimination by the host [19]. levels of complements proteins in crevicular fluid are diminished in individuals with periodontal disease [22,26]. p. gingivalis can evade neutrophils devastation through diminishing their antibacterial and chemotaxis efficacy [21,25]. the survival mechanisms of p. gingivalis lead to a remodeled microbiota, resulting in a dysbiotic condition that induces inflammatory diseases such as periodontal disease [19]. rheumatoid arthritis rheumatoid arthritis is an autoimmune, systemic inflammatory disorder that impacts roughly 1% of the population. it is marked by persistent joint inflammation, destruction of bone and cartilage, and systemic extra-articular manifestations not directly associated with the musculoskeletal system [27], which occur in a significant proportion of patients, resulting in poorer disease outcomes [28]. ra patients frequently display diverse oral manifestations, notably the recognized association with sjogren’s syndrome, resulting in symptoms such as xerostomia. additional oral concerns encompass temporomandibular joint abnormalities, methotrexateinduced ulcers, and an increasing emphasis on periodontal disease [29]. pathogenesis of rheumatoid arthritis vulnerability and initiation of ra rise from ambient and hereditary variables. the most often used autoantibodies in clinical practice are acpas, which may be detected in ra patients years prior to symptom manifestation. peptidyl arginine deiminase (padi) facilitates the post-translational modification that transforms arginine or glycine residues in normal proteins into citrulline, a significant factor contributing to the abrogation of immunological tolerance in ra patients [30]. non-resolving inflammation driven by aberrantly activated immune cells plays a crucial role in the development of ra. immune cells penetrate the synovium diffusely; however, in some instances, t cells and b cells form consolidates, with or without follicular dendritic cells, leading to the formation of lymphoid consolidates or ectopic germinal centers [31]. this process contributes to the breakdown of self-tolerance and accelerates the pathogenesis of ra. in ra, the majority of proinflammatory cytokines originate from macrophages and fibroblast-like synoviocytes, including il-1, il-6 and tnf-α. tnf-α, a key proinflammatory cytokine in ra, enhances inflammation by activating the nf-κb pathway, which promotes osteoclast formation [31,32]. il-1 similarly causes fast and powerful inflammatory reactions, primarily generated by macrophages in ra. il-1 may additionally enhance il-6, il-8, granulocyte-macrophage colony-stimulating factor, collagenase, and prostaglandins synthesis [31]. large quantities of il-6 in the synovial fluid of ra individuals may additionally transmit stimuli of pathogenic inflammation in rafibroblast-like synoviocytes via the janus kinase pathway [33]. the association between rheumatoid arthritis and periodontitis the findings from previous study have garnered significant interest from both chinese and foreign scholars regarding the correlation between pd and ra. cantley et al discovered that animals afflicted with both pd and poten&al role of periodontopathogens in rheumatoid arthri&s vol 13 no 1 (2025) doi 10.5195/d3000.2025.808 h#p://den*stry3000.pi#.edu ra exhibit more severe ra activity and an earlier commencement [34]. il-1 and tnf-α are proinflammatory cytokines which significantly contribute to the pathogenesis of periodontal disease and ra. they prompt inflammatory cells to infiltrate the diseased area, resulting in bone degradation through the inflammatory mediators secretion and cytokines activation, which facilitates the breakdown of alveolar osseous tissue and collagen fibers degradation in periodontal disease [35]. recent data demonstrates a substantial association between susceptibility to ra and the human leukocyte antigen drb1 (hla-drb1) genetic locus, due to the binding of citrullinated self-peptides to hladr molecules. p. gingivalis, a principal microbiome in periodontal disease and the only recognized human microbiome that expresses padi, is pivotal. this enzyme generates citrullinated epitopes, which are recognized by acpa in ra [36]. a prospective study revealed that persons with ra exhibited a greater prevalence of periodontal disease pd, especially in the midst of the individuals with increased concentrations of p. gingivalis in profound periodontal pockets, in comparison to healthy individuals [37]. the immunological processes resulting in advancing connective tissue breakdown and osseous tissue resorption, typical of ra, are also common in periodontal disease. periodontal disease, a contagious ailment predominantly induced by bacterial species such as p. gingivalis, aggregatibacter actinomycetemcomitans, tannerella forsythia, and treponema denticola, elicits both localized and systemic immunological reactions. furthermore, the presence of oral bacterial dna and antibodies in the synovial fluid and blood of patients with ra connect pd with the development of ra [11, 16]. a potential correlation between pd and ra may extend beyond causative relationships, presumably attributable to prevalent genetic and environmental risk determinants, including major histocompatibility class ii hla-drb1 allele expression and tobacco use [11,38]. although the underlying causes differ, numerous clinical and public health researchers suggest a link between ra and pd. individuals with pd have an increased susceptibility to ra and vice versa; the frequency of pd is twice as high in ra individuals in comparison to the populace [11,37]. furthermore, ra individuals with pd typically exhibit a more serious clinical progression, irrespective of gender, age, smoking or ethnicity status, in comparison to individuals without ra. moreover, the two illnesses exhibit analogous pathways of tissue damage driven by inflammatory cells and proinflammatory cytokines, indicating that periodontal disease can contribute to the onset and continuation of autoimmune inflammatory responses observed in ra [11,39]. role of porphyromonas gingivalis in the development of rheumatoid arthritis the successful application of antibiotics for anaerobic microbial infections in ra suggests that bacteria may contribute to the formation of ra. identification of bacterial dna from anaerobes and elevated antibody titers opposed to it in the synovial fluid and serum of ra individuals at different disease phases supports the hypothesis that oral infections contribute to the etiology of ra [11,40]. periodontal pathogens such as p. gingivalis can undermine the integrity of epithelial tissue, infiltrate human endothelial cells, and affect transcription and protein synthesis, thereby facilitating direct systemic entry into bloodstream. p. gingivalis can infiltrate primary human poten&al role of periodontopathogens in rheumatoid arthri&s vol 13 no 1 (2025) doi 10.5195/d3000.2025.808 h#p://den*stry3000.pi#.edu chondrocytes derived from patellar joints, leading to prolonged cell cycle advancement and heightened programmed cell death [11]. p. gingivalis synthesizes cysteine endopeptidase enzymes which are essential for bacterial homeostasis and infection mechanisms, including amino acids isolation from host and the formation of fimbriae [11,25]. p. gingivalis gingipains facilitate proteolytic enzymes activation including mmp-1, mmp-3, and mmp-9, along with the degradation of extracellular matrix host proteins such as collagen, fibronectin, and laminin. additionally, gingipains induce the degradation of complement factors and enhance the permeability of blood vessels [11,41]. the pathogenesis of ra is associated with the involvement of p. gingivalis in the process of citrullination. proteins experience a post-translational modification known as citrullination, converting arginine residues into citrulline [8,42]. the bacterium produces enzymes, including padi, catalyze citrullination. the infection elicits immunological reaction which can lead to systemic consequences. citrullination is a pivotal process in the pathogenesis of ra, as it generates antibodies against citrullinated proteins [8,43]. the citrullination mediated by p. gingivalis may facilitate the production of acpas, establishing a connection between periodontal disease and the onset or worsening of ra. citrullinated proteins are commonly present in the synovial fluids of people with ra. the existence of p. gingivalis and its citrullination role inside of the mouth may affect the systemic dissemination of citrullinated peptides, intensifying synovial inflammation in predisposed people [36,43]. conclusion the growing body of evidence linking periodontitis, particularly the role of p. gingivalis, to the development and exacerbation of ra highlights the complex interactions between oral and systemic health. periodontitis, a chronic inflammatory disease, is associated with a range of systemic conditions, with ra being one of the most compelling examples of such connections. the key periodontal pathogen p. gingivalis plays a central role in this relationship, not only by inducing local inflammation and bone resorption in the oral cavity but also by influencing systemic immune responses through mechanisms such as citrullination. p. gingivalis secretes padi, an enzyme that catalyzes the conversion of arginine to citrulline, a critical process that generates autoantibodies against citrullinated proteins, a hallmark of ra. this molecular interaction suggests that oral bacteria may directly contribute to the immune dysregulation observed in ra, leading to the production of acpa and the subsequent development of chronic joint inflammation. additionally, the shared inflammatory mediators and cytokine profiles between pd and ra further reinforce the potential for a bidirectional relationship, where one condition may influence the progression of the other. given the significant prevalence of periodontitis worldwide, and the established connection between periodontal disease and ra, there is a pressing need for further research into the underlying mechanisms of this association. understanding how p. gingivalis and other periodontal pathogens contribute to ra may offer new avenues for preventive and therapeutic strategies, not only targeting oral health but also the systemic inflammatory pathways involved in autoimmune diseases like ra. integrating periodontal care into the management of ra could potentially improve patient outcomes and reduce disease severity, highlighting the importance of early detection and poten&al role of periodontopathogens in rheumatoid arthri&s vol 13 no 1 (2025) doi 10.5195/d3000.2025.808 h#p://den*stry3000.pi#.edu comprehensive treatment strategies that address both oral and systemic health. funding self-funded. conflict of interest the authors declare no conflict of interest. references 1. martínez-garcía m, hernández-lemus e. periodontal inflammation and systemic diseases: an overview. frontiers in physiology. 2021 oct 27;12:709438. 2. jahid m, khan ku, ahmed rs. overview of rheumatoid arthritis and scientific understanding of the disease. mediterranean journal of rheumatology. 2023;34(3):284-91. 3. lopez-oliva i, malcolm j, culshaw s. periodontitis and rheumatoid arthritis— global efforts to untangle two complex diseases. periodontology 2000. 2024 feb 27. 4. abdullah wl, al-hashimy af, al-ghurabi bh. role of salivary caspase-1 and gasdermin d in the pathophysiology of rheumatoid arthritis in relation to salivary ph and flow rate of saliva. world academy of sciences journal. 2024 nov 18;7(1):7. 5. al-daragi fz, al-ghurabi bh, abdullah nn. serum levels of antimicrobial peptides (cathelicidins and beta defensins-1) in patients with periodontitis. journal of baghdad college of dentistry. 2024 mar 15;36(1):9-18. 6. jain n, dutt u, radenkov i, jain s. who's global oral health status report 2022: actions, discussion and implementation. oral diseases. 2024 mar;30(2):73-9. 7. chen mx, zhong yj, dong qq, wong hm, wen yf. global, regional, and national burden of severe periodontitis, 1990–2019: an analysis of the global burden of disease study 2019. journal of clinical periodontology. 2021 sep;48(9):1165-88. 8. kwon t, lamster ib, levin l. current concepts in the management of periodontitis. international dental journal. 2021 dec 1;71(6):462-76. 9. al-ghurabi bh, adham zs, abbas aa: level of βdefensin among iraqi patients with covid-19 in relation to oral health status. j emerg med trauma acute care. 2022; 2022(3): 2. 10. al-ghurabi bh. the role of soluble tlr-2 in the immunopathogenesis of gingivitis. international medical journal. 2021 jun 2;28(1):37-9. 11. krishna as, ahalya u, dharan ss, amal a. unraveling the interplay: periodontitis and rheumatoid arthritis. international journal of tropical medicines. 2024;1(1):31-9p 12. mahmood hk, al-ghurabi bh. association between anti-cmv igg and salivary levels of il-6 and tnf-α in chronic periodontitis. j bagh coll dent. 2020;32(2):5-11. 13. li x, yu c, zhang b, shan x, mao w, zhang z, wang c, jin x, wang j, zhao h. the recovery of the microbial community after plaque poten&al role of periodontopathogens in rheumatoid arthri&s vol 13 no 1 (2025) doi 10.5195/d3000.2025.808 h#p://den*stry3000.pi#.edu removal depends on periodontal health status. npj biofilms and microbiomes. 2023 oct 7;9(1):75. 14. di spirito f, di palo mp, rupe a, piedepalumbo f, sessa a, de benedetto g, russo barone s, contaldo m. periodontitis in psoriatic patients: epidemiological insights and putative etiopathogenic links. epidemiologia. 2024 jul 26;5(3):479-98. 15. gao l, kang m, zhang mj, reza sailani m, kuraji r, martinez a, ye c, kamarajan p, le c, zhan l, rangé h. polymicrobial periodontal disease triggers a wide radius of effect and unique virome. npj biofilms and microbiomes. 2020 mar 10;6(1):10. 16. krutyhołowa a, strzelec k, dziedzic a, bereta gp, łazarz-bartyzel k, potempa j, gawron k. host and bacterial factors linking periodontitis and rheumatoid arthritis. frontiers in immunology. 2022 aug 25;13:980805. 17. xu w, zhou w, wang h, liang s. roles of porphyromonas gingivalis and its virulence factors in periodontitis. advances in protein chemistry and structural biology. 2020 jan 1;120:45-84. 18. aleksijević lh, aleksijević m, škrlec i, šram m, šram m, talapko j. porphyromonas gingivalis virulence factors and clinical significance in periodontal disease and coronary artery diseases. pathogens. 2022 oct 11;11(10):1173. 19. huang x, li y, zhang j, feng q. linking periodontitis with inflammatory bowel disease through the oral– gut axis: the potential role of porphyromonas gingivalis. biomedicines. 2024 mar 19;12(3):685. 20. zhou n, zou f, cheng x, huang y, zou h, niu q, qiu y, shan f, luo a, teng w, sun j. porphyromonas gingivalis induces periodontitis, causes immune imbalance, and promotes rheumatoid arthritis. journal of leukocyte biology. 2021 sep;110(3):461-73. 21. zheng s, yu s, fan x, zhang y, sun y, lin l, wang h, pan y, li c. porphyromonas gingivalis survival skills: immune evasion. journal of periodontal research. 2021 dec;56(6):1007-18. 22. kareem hh, al-ghurabi bh, albadri c. molecular detection of porphyromonas gingivalis in covid-19 patients. journal of baghdad college of dentistry. 2022 jun 15;34(2):52-61. 23. neurath n, kesting m. cytokines in gingivitis and periodontitis: from pathogenesis to therapeutic targets. frontiers in immunology. 2024 aug 26;15:1435054. 24. melgar-rodríguez s, polanco a, ríos-muñoz j, garcía m, sierra-cristancho a, gonzález-osuna l, díazzúñiga j, carvajal p, vernal r, bravo d. differential response of human dendritic cells upon stimulation with encapsulated or nonencapsulated isogenic strains of porphyromonas gingivalis. international journal of molecular sciences. 2024 apr 20;25(8):4510. 25. chen wa, dou y, fletcher hm, boskovic ds. local and systemic effects of poten&al role of periodontopathogens in rheumatoid arthri&s vol 13 no 1 (2025) doi 10.5195/d3000.2025.808 h#p://den*stry3000.pi#.edu porphyromonas gingivalis infection. microorganisms. 2023 feb 13;11(2):470. 26. ali, a. f. and taha, g. i. 2024. gingival crevicular fluid volume and protein concentration: a biomarker tool for predicting periodontal diseases progression and severity. natural and life sciences communications.23(4): e2024055. 27. figus fa, piga m, azzolin i, mcconnell r, iagnocco a. rheumatoid arthritis: extraarticular manifestations and comorbidities. autoimmunity reviews. 2021 apr 1;20(4):102776. 28. gonzález da, bianchi ml, armada m, escalante ac, salgado pa, seni s, citera g, ferrary t, orman b. hyposalivation and periodontal disease as oral non-articular characteristics in rheumatoid arthritis. clinical rheumatology. 2024 jan;43(1):95-102. 29. benli m, batool f, stutz c, petit c, jung s, huck o. orofacial manifestations and dental management of systemic lupus erythematosus: a review. oral diseases. 2021 mar;27(2):151-67. 30. sokolova mv, schett g, steffen u. autoantibodies in rheumatoid arthritis: historical background and novel findings. clinical reviews in allergy & immunology. 2022 oct;63(2):138-51. 31. gao y, zhang y, liu x. rheumatoid arthritis: pathogenesis and therapeutic advances. medcomm. 2024 mar;5(3):e509. 32. ding q, hu w, wang r, yang q, zhu m, li m, cai j, rose p, mao j, zhu yz. signaling pathways in rheumatoid arthritis: implications for targeted therapy. signal transduction and targeted therapy. 2023 feb 17;8(1):68. 33. su j, li s, chen j, jian c, hu j, du h, hai h, wu j, zeng f, zhu j, liu y. glycerophospholipid metabolism is involved in rheumatoid arthritis pathogenesis by regulating the il-6/jak signaling pathway. biochemical and biophysical research communications. 2022 apr 16;600:130-5. 34. xiao f, li c, lin y, peng z, xu x, wen y, huang n, zhang p. increased risk of periodontitis occurrence in patients with rheumatoid arthritis and its association with the levels of il-1β and tnf-α in gingival crevicular fluid. annals of palliative medicine. 2021 aug;10(8):9078087-9087. 35. luo p, wang p, xu j, hou w, xu p, xu k, liu l. immunomodulatory role of t helper cells in rheumatoid arthritis: a comprehensive research review. bone & joint research. 2022 jul 20;11(7):426-38. 36. renvert s, berglund js, persson gr, söderlin mk. the association between rheumatoid arthritis and periodontal disease in a population-based crosssectional case-control study. bmc rheumatology. 2020 dec;4:1-8. 37. li y, guo r, oduro pk, sun t, chen h, yi y, zeng w, wang q, leng l, yang l, zhang j. the relationship between porphyromonas gingivalis and rheumatoid arthritis: a meta-analysis. frontiers in cellular and infection microbiology. 2022 jul 18;12:956417. poten&al role of periodontopathogens in rheumatoid arthri&s vol 13 no 1 (2025) doi 10.5195/d3000.2025.808 h#p://den*stry3000.pi#.edu 38. möller b, kollert f, sculean a, villiger pm. infectious triggers in periodontitis and the gut in rheumatoid arthritis (ra): a complex story about association and causality. frontiers in immunology. 2020 jun 3;11:1108. 39. zekeridou a, gilbert b, finckh a, giannopoulou c. periodontitis in first degreerelatives of individuals with rheumatoid arthritis: a short narrative review. frontiers in oral health. 2022 may 6;3:895753. 40. ermencheva p, kotov g, shumnalieva r, velikova t, monov s. exploring the role of the microbiome in rheumatoid arthritis—a critical review. microorganisms. 2024 jul 9;12(7):1387. 41. chow yc, yam hc, gunasekaran b, lai wy, wo wy, agarwal t, ong yy, cheong sl, tan sa. implications of porphyromonas gingivalis peptidyl arginine deiminase and gingipain r in human health and diseases. frontiers in cellular and infection microbiology. 2022 sep 29;12:987683. 42. kwon ej, ju jh. impact of posttranslational modification in pathogenesis of rheumatoid arthritis: focusing on citrullination, carbamylation, and acetylation. international journal of molecular sciences. 2021 sep 30;22(19):10576. 43. pérez-pérez me, nietotorres e, bollain-y-goytia jj, delgadillo-ruíz l. protein citrullination by peptidyl arginine deiminase/arginine deiminase homologs in members of the human microbiota and its recognition by anticitrullinated protein antibodies. international journal of molecular sciences. 2024 may 10;25(10):5192. 894 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.894 http://dentistry3000.pitt.edu effect of smartphone application reminder on improving oral hygiene ali hadi fahad college of den*stry, university of kufa, iraq abstract objec/ve: to study the effect of smartphone applica/on reminder on improving oral hygine among male students from university of kufa. materials and methods: in this prospec/ve study, 120 students age ranging from 18 to 24 years were separated into two groups. all par/cipants received a clinical examina/on and a ques/onnaire. aner receiving typical oral hygiene instruc/ons, par/cipants in the study group were instructed to u/lize the brush dj smartphone applica/on and were compared to par/cipants in the other group who got conven/onal oral hygiene instruc/ons. note that the two groups received same toothbrushes and toothpastes. the measurement of plaque and gingival index was done on three visits. results: independent of the group, par/cipants improved from the first to the third measurements for both plaque and gingival index. conclusion: smartphone applica/ons might significantly improve the oral hygiene compliance of students, by ac/ng as reminders and mo/vators. open access cita%on: fahad ah (2025) effect of smartphone applica%on reminder on improving oral hygiene. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.894 received: march 31, 2025 accepted: may 20, 2025 published: june 27, 2025 copyright: ©2025 fahad ah. this is an open access ar%cle licensed under a crea%ve commons awribu%on work 4.0 united states license. email: alih.fahad@uokufa.edu.iq introduc)on gingivitis linked to plaque accumulations, hormonal shifts, drugs, systemic ailments, and malnutrition has distinct traits. these include localized in=lammation in the free and attached gingiva, reversible with bio=ilm disruption, and high bacterial plaque levels triggering or worsening the condition [1,2]. maintaining optimal oral hygiene is imperative for overall health and well-being. poor oral health not only leads to various dental ailments such as caries, periodontal diseases, and halitosis but also has systemic implications, including cardiovascular diseases, diabetes, and adverse pregnancy outcomes. among university students, especially males, oral health tends to be overlooked due to hectic academic schedules, irregular lifestyles, and inadequate awareness regarding oral hygiene practice [3]. oral cleanliness is crucial for oral health, removing microbial plaque and preventing its buildup on teeth and gums. ideal plaque control through daily toothbrushing and interdental plaque removal every 24 hours is suf=icient to prevent gingivitis and inter-dental caries [4,5]. various inter-dental cleaning devices are available, posing challenges for both patients and dental professionals in selecting the right one. an ideal device should be easy to use, remove plaque effectively, and cause no harm to oral tissues [6]. despite the widespread availability of dental health education, adherence to recommended oral hygiene behaviors remains a challenge for many individuals. this discrepancy underscores the necessity for innovative interventions that can effectively promote sustained oral care habits. in this context, smartphone applications present a promising avenue, leveraging ubiquitous technology to deliver personalized reminders and educational content tailored to individual oral health need [7]. in recent years, the proliferation of smartphone technology has fundamentally transformed various aspects of our daily lives, including healthcare management. among the myriad of applications, those aimed at improving health behaviors have garnered substantial attention, particularly in the domain of oral hygiene. the oral cavity serves as a gateway to overall health, with inadequate oral care linked to a spectrum of systemic diseases, ranging from cardiovascular disorders to diabetes. consequently, effect of smartphone applicaion reminder on improving oral hygiene vol 13, no 1 (2025) doi 10.5195/d3000/2025.894 http://dentistry3000.pitt.edu 2 optimizing oral hygiene practices emerges as a pivotal preventive strategy to mitigate these health risks [8]. with the advent of digital technology, smartphone applications have emerged as promising tools for promoting health behaviors, including oral hygiene practices. there are applications for educating and encouraging youngsters and adults to use additional oral hygiene products and proper teeth brushing techniques [9,10]. these applications offer personalized reminders, educational resources, and tracking features, which can potentially empower individuals to adopt and sustain healthy habits. however, despite the growing popularity of such applications, there remains a paucity of scienti=ic literature evaluating their ef=icacy, particularly among speci=ic demographics such as male university students [11]. owing to half of the healthcare practitioners having smart phones use apps in their dayto-day practice, there are wide range of healthcare apps providing extensive information to support clinical decision-making and better patient care. today, healthcare apps have better usage comparing any other source of information and bene=itted healthcare professionals due to quicker access to information [12,13]. the university of kufa, situated in najaf, iraq, represents a unique setting for studying oral hygiene practices among male students. with a diverse student population and varying socioeconomic backgrounds, the university provides an ideal environment to investigate the effectiveness of smartphone application reminders in improving oral health behaviors. understanding the impact of these interventions is not only crucial for the well-being of the students but also has broader implications for public health initiatives in similar educational settings. material and methods study design and setting blinded, parallel group, non-strati=ied randomization and equal allocation ratio (1:1) were used in this prospective study. the study was carried out in faculty of nursing and faculty of science, university of kufa at al-najaf city. ethical approval was obtained from the local ethical committee, the college of dentistry at university of kufa (reference number 745). informed consent and assent were obtained from the students themselves. the study adhered to the principles of the declaration of helsinki. participants a total of 120 subjects were randomly divided into two groups. in the =irst, a questionnaire containing 10 questions about oral health care was distributed to the two groups. study group received toothbrush, toothpaste, and reminder application about oral hygiene. the other group received toothbrush, toothpaste, and oral hygiene instructions only without reminder application. users could set reminders to brush twice a day, however, this app can be used as reminder to attend the next dentist/hygienist/therapist visit. the application also includes links to illustrated youtube videos that show ways to use dental =loss, an interdental brush and a traditional toothbrush. data collection collection of data was performed by calibrated examiners. clinical examinations were conducted in a standardized manner, using a dental mirror and a periodontal probe (who probe) under adequate lighting by dental chair light. oral hygiene assessment oral hygiene status evaluated by application plaque index (pl) of silness and löe [14], and gingival in=lammation assessed by using gingival index (gi) of löe and silness [15]. statistical analysis statistical analyses were done using spss 23 version 21 computer software (statistical package for social sciences) in association with microsoft excel 2013. frequency distribution for selected variables was done =irst. the statistical signi=icance, by using description of statistics, median, mean rank, chisquare for statistical differences. p value less than the 0.05 level of signi=icance was considered statistically signi=icant. results percentage of participants who brushed once daily was 42.5%, not everyday was 37,5% and twice or more was 20%. for median of pi among study group, the higher median value was (1.33) found with the =irst exam, while the lower median value was (1.04) found with third exam. for the comparison group, the higher median value was (1.54) found with third exam, while the lower median value was (1.048) found with second exam. there were no statistically signi=icant differences between the two groups. discussion this app was selected in the present study to test the improvement of the oral health and creating a new positive habit for people. the ef=iciency of this app was studied by [16,17] who found that there was a positive correlation between app usage with the brushing duration and frequency, hence, its ef=icacy in improving oral health. ben underwod (2018) stated that ”if you want to make people do something, you have to make it fun" [18,19]. a one year later, he was honored by the british society of pediatric dentistry 4s outstanding innovation for his innovation of this app transferring his concept to a reality. in the present study, the median plaque index of study group highly signi=icantly reduced from the baseline to 3 months after use the application. the reduction of pi agrees with other studies used smartphone app as |20,21]. for the control group, it showed slight increase in the median plaque index over time without signi=icant differences. these results are like a recent study that found [21]. signi=icant differences in pi were noted between the two groups (study and control) after 3 months from the =irst examination show positive effect of application to improve oral health. according to this research, the median gingival index of study group highly signi=icantly reduced after use the application. this result was in line with other studies showed a relation between the effect of application and improve oral hygiene [22]. on the other hand, the median gi of control group, it shows slight increase in the median plaque index over time without signi=icant differences. references 1. mariotti a. dental plaque-induced gingival diseases. ann. periodontol,1999; 4 (1): 7-17. doi: 10.1902/annals.1999.4.1.7. 2. shinya m, brian l, angelo m, iain l c. dental plaque-induced gingival conditions. j clin periodontal, 2018; 45: 17 27. doi: 10.1002/jper.17-0095 3. sarode gs, sarode sc, patil s. messenger services on smartphone: changing trends of communication in dental practice. j. contemp. dent. pract. 2016; 17: 267-269. doi: 10.5005/jp-journals-10024-1839 4. sonja sälzer, dagmar e. slot, fridus a. van der weijden, christof e. dörfer. efficacy of inter-dental mechanical plaque control in managing gingivitis, a meta-review. j. clin. periodontol, 2015; 42: 92-105. doi: 10.1111/jcpe.12363 5. harrison p. plaque control and oral hygiene methods., journal of the irish dental association, 2017; 63(3):151-156. effect of smartphone applicaion reminder on improving oral hygiene vol 13, no 1 (2025) doi 10.5195/d3000/2025.894 http://dentistry3000.pitt.edu 3 http://hdl.handle.net/10147/622539. 6. fridus a. van der weijden and dagmar e. slot. oral hygiene in the prevention of periodontal diseases: the evidence. j periodontology 2000; 55(1): 104-123. doi: 10.1111/j.1600-0757.2009.00337.x 7. iqbal j, awan r, parvez ma, ul haq a, gardezi aa, irfan s. effectiveness of text message instructions on oral hygiene for orthodontic patients. pak. oral dental. j. 2017;37:278282. https://www.podj.com.pk/index.php/podj/article/view/97. 8. tyas mj, anusavice kj, frencken je, mount gj. minimal intervention dentistry-a review fdi commission project 1-97. int. dent. j. 2000; 50: l-12. doi: 10.1111/j.1875595x.2000.tb00540.x 9. c lee ventola. mobile devices and apps for health care professionals: uses and benefits. pharm then, 2014; 39 (5): 356-364. http://www.ncbi.nlm.nih.gov/pubmed/24883008. 10. mohapatra dp, mohapatra mm, chittoria rk, friji mt, kumar sd. the scope of mobile devices in health care and medical education. int j adv med heal res. 2015; 2 (1): 3-8. doi: 10.4103/2349-4220.159113. 11. scribante a, gallo s, bertino k, meles s, gandini p, sfondrini mf. the effect of chairside verbal instructions matched with instagram social media on oral hygiene of young orthodontic patients: a randomized clinical trial. appl. sci. 2021; 11: 706. doi.org/10.3390/app11020706. 12. abu saleh mohammad m, yoo i, and sheets l. a systematic review of healthcare applications for smartphones. bmc medical informatics and decision making, 2012; 12: 67. doi: 10.1186/1472-6947-12-67. 13. ji-yun ki, se-rim jo, kyung-sook cho, jungeun park, ja-won cho, and jong-hwa jang. effect of oral health education using a mobile app (ohema) on the oral health and swallowing-related quality of life in community-based integrated care of the elderly: a randomized clinical trial. int j environ res public health, 2021; 18: 11679. doi: 10.3390/ijerph182111679. 14. silness j, löe h. periodontal disease in pregnancy. correlation between oral hygiene and periodontal condition. acta odontol scand. 1964; 22: 121–135. doi: 10.3109/00016356408993968. 15. löe h and silness j. periodontal disease in pregnancy. acta odontol scand. 1963; 21: 533–551. doi:10.3109/00016356309011240. 16. zotti f, et al. usefulness of an app in improving oral hygiene compliance in adolescent orthodontic patients. angle orthod. 2016;86:101–107. doi: 10.2319/01091519.1. 17. farhadifard h, soheilifar s, farhadian m, kokabi h, and bakhshaei a. orthodontic patients oral hygiene compliance by utilizing a smartphone application (brush dj): a randomized clinical trial. bdj open, 2020; 6(1), 1-6. doi: 10.1038/s41405-020-00050-5. 18. underwood b, birdsall j, kay e. the use of a mobile app to mo7vate evidence-based oral hygiene behaviour. br dent j. 2015; aug 28; 219(4):e2. doi: 10.1038/sj.bdj.2015.660. 19. creeth je, gallagher a, sowinski j, bowman j, barres k, lowe s, patel k, bosma ml. the effect of brushing time and dentifrice on dental plaque removal in vivo. j dent hyg. 2009 summer;83(3):111-6. epub 2009 aug 14. pmid: 19723429. 20. deleuse m, meiffren c, bruwier a, maes n, le gall m, and charavet c. smartphone application-assisted oral hygiene of orthodontic patients: a multicentre randomized controlled trial in adolescents. european journal of orthodontics, 2020; 42(6), 605-611. doi: 10.1093/ejo/cjz105 21. ali aj. smart phone application reminder for patients with fixed orthodontic appliance to improve oral hygiene. master thesis submitted to the council of the college of dentistry, university of baghdad. 2023. doi.org/10.5195/d3000.2024.699 22. patil s, hedad ia, jafer aa, abutaleb gk, arishi tm, arishi sa, arishi ha, jaferm, gujar an, khan s, raj at. effectiveness of mobile phone applications in improving oral hygiene care and outcomes in orthodontic patients. j oral biol craniofac res. 2021;11(1):26–32. doi: 10.1016/j.jobcr.2020.11.004. 756 2024 vol 12 no 2 (2024) doi 10.5195/d3000.2024.756 h#p://den*stry3000.pi#.edu shear bond strength comparison between heat cure acrylic resin and other types of acrylic denture bases shear bond strength comparison between heat cure acrylic resin and other types of acrylic denture bases souad hassan ali1, noor adnan abdul-kareem2* 1 middle technical university, medical technical institute, baghdad, iraq. 2 ministry of health, baghdad, iraq. abstract objec&ve: the study purpose was to examine the shear bonding strength of heat cure acrylic resin to thermoplas8c resin and three-dimensional printed resin materials. methods: a total of 30 acrylic specimens were fabricated in this study. each specimen had a basal and top parts. the basal part was rectangular in shape. we had three different groups: 10 specimens of a basal part made of heat cured polymethylmethacrylate, 10 specimens of a basal part made of thermoplas8c, and 10 specimens of three dimensionally printed resin. the top part of which is adached to the basal one was formed into a cylindrical shape made of heat cure poly methyl methacrylate in all three groups. results: significant differences were found between the heat cured polymethylmethacrylate and the other two groups (p<.001). heat cured polymethylmethacrylate showed the highest bond strength (75 mpa), and thermoplas8c resins showed the lowest (37 mpa). no significant difference in the shear bond strength was no8ced between three dimensionally printed and thermoplas8c acrylic. conclusion: this study revealed that the shear bond strength of heat cured acrylic denture base to the relining/repairing from the same material was significantly higher than three dimensionally printed and thermoplas8c resins with hc relining/repairing material. keywords: den8stry; denture base materials; prosthodon8cs; repair of acrylic denture base; shear bond strength, 3d printed acrylic cita&on: ali sh, et al. (2024) shear bond strength comparison between heat cure acrylic resin and other types of acrylic denture bases. den&stry 3000. 1:a001 doi:10.5195/d3000.2024.756 received: october 14, 2024 accepted: november 26, 2024 published: november 27, 2024 copyright: ©2024 ali sh, et al. this is an open access ar&cle licensed under a crea&ve commons arribu&on work 4.0 united states license. email:souadhassan195@gmail.com introduction there are various types of dental resins which are used in the prosthetic dental field. generally, all types of dental resins are desirable aesthetically and functionally. despite their advantages, they also have some disadvantages, such as a significant shrinkage and color changes during their serving period in the oral cavity [1]. using the acrylic resin material in the process of removable prosthesis fabrication remains the most preferable by most patients and dental professionals due to their low cost and availability [2]. dental acrylic resins can be found either naturally or from a synthetic origin. they are macromolecular compounds of natural or that are composed of multiple repeating structural units (monomer units), forming macromolecules (polymers). methacrylate monomers composed of single simple molecules with double and triple bonds, and they are linked in polymer chains. the macromolecule is produced by a process called polymerization [1]. (pmma) are considered as the gold standard of traditional manufacturing [3], in terms of polymerization by compressing, mold injection technique [4,5]. generally, all types of dentures need to be repaired or relined during their serving period inside the wearer’s mouth, and that can be due to fracture results from vol 12 no 2 (2024) doi 10.5195/d3000.2024.756 h#p://den*stry3000.pi#.edu shear bond strength comparison between heat cure acrylic resin and other types of acrylic denture bases misusing or ridge resorption as the denture becomes loose and needs to be relined. repairing and relining materials are used to restore the dentures functions in case the patient does not want to replace it with a new one. different materials were used for the purpose of relining and repairing, including thermo-polymerized hc, auto-polymerized (cold cured), visible light-polymerized (light cured) and microwavepolymerized acrylic resins [6,7]. the most popular and widely used materials are the chair-side auto and heat polymerized acrylic resins. they are used for quick and simple procedure, but the thermos-polymerized remains the best choice over the autopolymerized one, because of its proper flowing, high durability, lower porosity and details reproductions [8]. to obtain a desirable result, it is important to make sure that there is consistent bond between the denture base material and reline/repair resin. the good bond is depending on the mechanical properties of the denture and mainly the failure of it can be due bacterial accumulation, and staining [9]. basically, when the stress applied the failure occurs at the bonding area, this is like the shear [10]. therefore, the shear bond stress test is used as the force applied directly on the bond region [6]. the bonding strength depends mainly on the material’s chemical compositions [11], and there are other affecting factors such as thermal cycling and material surface treatment [12]. thermoplastic materials were introduced as polyamides (nylon plastic) in 1950s. they were developed into many types; thermoplastic acrylic, thermoplastic polycarbonate, thermoplastic acetal, thermoplastic nylon (resin). it has some advantages over the conventional acrylic such as flexibility, light weight, monomer free and high esthetical properties. while the drawbacks are the adherence of teeth with a base can be achieved mechanically and this would increase the problem of detachment. in the 1990s, computer aided design/computer aided manufacture (cad/cam) technologies were revealed to the dental field, and it was considered as the great change in the fabrication techniques of dentures [13]. the interest in the advances of cad/cam and materials science have been flourished, as the digital process offers a wide range of facilities such as reducing the chairtime, number of visits, saving case data, and more desirable clinical and laboratory results [14]. the digital object fabrication requires obtaining the virtual design on the computer software (cad) followed by manufacturing process (cam). this can be achieved either by subtractive milling of prefabricated acrylic resin blocks or an additive rapid prototype process. additive manufacturing techniques using the photopolymerizable acrylate material and a lithographic machine. while the resin liquid accomplishes a layer-by-layer curing until the complete object is achieved [15]. the additive manufacturing technique offers several advantages compared to the subtractive methods such as reducing the waste material for about 40%, big object size and complex geometry can be produced unlike the subtractive method which its work is limited by the material’s block, milling burs diameter and the milling axes of the machine. on the other hand, there is no force exerted on the object like in the subtractive method that causing surface cracks during the milling process. as a new technology there is a widely range of usage in the dental office [16]. therefore, the aim of this study was to investigate the shear bond strength of hc acrylic resin to both thermoplastic resin and threedimensional printed resin (3d printed resin) materials. vol 12 no 2 (2024) doi 10.5195/d3000.2024.756 h#p://den*stry3000.pi#.edu shear bond strength comparison between heat cure acrylic resin and other types of acrylic denture bases material and methods the single specimen consists of two parts: the basal part made from hc acrylic (rodex dental product, barmstedt/hamburg), thermoplastic (deflex, united kingdom), and 3d printed acrylic (armaresin, turkye), while the repairing/relining parts were made of hc acrylic resin. a total of 30 basal part specimens were fabricated in the shape of rectangle with dimensions of (10 mm × 10 mm× 20 mm3), and they were attached to another 30 hc acrylic resin (rodex dental product, barmstedt/hamburg) specimens with a cylindrical shape and dimensions of 10 mm and a diameter of 6 mm [17]. specimen preparation for the preparation of basal part of the specimens, wax pattern with dimensions of 10 × 10 × 20 mm3 [17]. it is noteworthy that the wax pattern was used for preparing both the pmma and the thermoplastic specimens. for the pmma specimens the wax patterns were constructed and placed in the dental flask for creating the mold to pack the acrylic material and obtaining the specimens. the thermoplastic wax patterns were also flasked and the flexible acrylic material was injected carefully using the injection device (shang hange, china). all the specimens were finished and polished to remove any access material using the grinder/polisher device and aluminum oxide abrasive papers under water cooling and then cleaned with distilled water. the 3d-printed specimens were virtually designed and then printed horizontally with a dlp 3d-printer (asiga max™, germany). to remove fluid resin remnants from the test specimens, they were placed in ultrasonic cleaning device (imprimo® clean; germany) for 3 minutes with a water-based cleansing agent. the specimens were rinsed properly in a separate device (form wash®; form labs, berlin, germany) with isopropanol for an additional 3 minutes, following the manufacturer’s instructions. the specimens were finished using silicon carbide grinding papers (800, 1500, and 2000 grit fepa) and washed with water. then the specimens from each group were stored in water [18]. thirty specimens for the extension parts were prepared with a wax pattern in a cylindrical shape, with dimensions of 10mm and a diameter of 6mm [17]. the basal part of the specimens was scratched from one surface in all specimens to stick the wax extension to it prior to the flacking process. after the wax patterns were flasked and eliminated, the mold created, and the basal part still fixed in place and ready for packing of the resin material into the mold to produce the specimens with both the basal and the extensional parts (figure 1). the polishing process of the resin extension part is the same as mentioned before. this was followed by immersing the specimens in a distilled water for 24 hours to eliminate residual monomers [19]. shear bond strength test shear bond strength (sbs) test for 30 specimens was performed through a universal testing machine (laryee, 50kn, 1202001, china, 2012) at a crosshead speed of 1mm/min until fracture occurred (figure 2). the shear bond force was exerted vertically to the bonding interface between the edge of the heat acrylic, 3d printer and flexible acrylic basal part and the extension hc acrylic with a stainless-steel chisel rod [17] until fracture (figures 3 and 4). the maximum force that caused failure was recorded in newtons for each specimen and the sbs in mpa was calculated by dividing the force (n) value at which the bond failure occurred by the sample bonding area and expressed in mpa according to the following formula: shear bond strength (mpa) = maximum force (n)/ bonding surface area (2mm) [20]. data were revised, coded, and analyzed using the “statistical package of social science (spss) version 26.0. analysis of data done by using independent sample t-test and simple linear regression. vol 12 no 2 (2024) doi 10.5195/d3000.2024.756 h#p://den*stry3000.pi#.edu shear bond strength comparison between heat cure acrylic resin and other types of acrylic denture bases figure 1. a: full heat cured acrylic specimen, b: 3d printed acrylic to heat cured acrylic specimen, c: thermoplastic acrylic to heat cured acrylic. figure 2. universal testing machine for shear bond strength. figure 3. specimen fracture after force application. figure 4. tested specimen after fracture. vol 12 no 2 (2024) doi 10.5195/d3000.2024.756 h#p://den*stry3000.pi#.edu shear bond strength comparison between heat cure acrylic resin and other types of acrylic denture bases results the mean and standard deviation (sd) of the sbs values were listed and illustrated in table 1 and figure 5, and p-value after the ttest in table 2. showed a significant difference between the sbs of the hc acrylic reline/repair material bonded to both 3d printed resin and thermoplastic one. discussion the present study investigated the shear bond strength between different types of denture base resins which are heat cured, 3d printed and thermoplastic acrylic resin with hc acrylic resin as a relining/repairing material. shear bond strength can be defined as the material’s component’s strength against the type of yield or structural failure when the material component fails by shear force [21]. since the additional material can be detached from a denture base for many reasons, it is important to ensure that the shear bond strength is as high as possible. studied groups n mean ± sd. heat cured polymethylmethacrylate 10 66.92 ± 5.17 3d printed acrylic to hc acrylic 10 27.30 ± 5.71 thermoplastic acrylic to hc acrylic 10 28.63 ± 6.28 studied groups t-test p-value heat cured polymethylmethacrylate vs. hc acrylic to 3d printed acrylic 16.262 p= .000 heat cured polymethylmethacrylate vs. hc acrylic to thermoplastic acrylic 14.885 p= .000 hc acrylic to 3d printed acrylic vs. hc acrylic to thermoplastic acrylic 0.495 p= .626 table 1. mean and standard deviation (sd) for sbs values (mpa). figure 5. mean and standard deviation (sd.) values of shear bond strength test for the studied groups. heat cured polymethylmethacrylate = control. table 2. t-test for shear bond strength. vol 12 no 2 (2024) doi 10.5195/d3000.2024.756 h#p://den*stry3000.pi#.edu shear bond strength comparison between heat cure acrylic resin and other types of acrylic denture bases as the results shown, the highest sbs strength has been recorded in the control group, this can be referred to the bonding between specimen’s parts from the same material. this finding agrees with previous work [22]. it has been stated that using a material with a matching chemical composition for relining or repairing of the denture base, the results will be achieving the highest potential values of sbs, fracture resistance and flexural strength [21,23], as it is carried out by means of a chemical reaction between the individual components [24]. the successful bonding between the new material and the base depends on the effective penetration of polymerized monomer particles from the reline/repair material into the denture base network. the high sbs of the control group might be due to high monomers diffusion rates into the denture base resin which was led by processed polymer under a high temperature and a long polymerization time [17]. generally, new denture resins have monomers with larger molecules (molecular weight larger than 100) which act slower with respect to swelling pmma and penetrating the pmma surface [25]. while, in regard to the 3d printed ones the relined/repaired printed resins showed significantly lower values than the relined conventional heat-polymerized resins. this agreed with previous work [26]. moreover, similar outcomes have been revealed in terms of comparisons in sbs for relined 3d-printed denture resins and relined heat-polymerized denture resins made by cho et al. (2021) and mert et al. (2023) [21,23]. they used a few different relining materials, and the results for the sbs with all relining materials were significantly lower in the 3d-printed group compared to the relined heat-polymerized resin group. the reason after that may be due to the conventional cross-linking of methyl methacrylate that occurs between pmma interfaces may not be occurring in this group. this plays an important role in the low sbs of 3d printed resin. there are a lot of surface treatments for relining the resins. the surface treatment in this current study was creating scratches with sandblast before applying relining/repairing material. it has been shown that regardless of the material, the sbs was increased after sandblasting [26]. polyamide thermoplastic resin group has recorded the lowest sbs results, this is referred to the nature of the material as it is produced by a reaction between a diamine and a dibasic acid called the condensation reaction. polyamide has a high degree of crystallinity which cause polymers chemical resistance and prevent the penetration of monomer molecules and other resin primers into the polymers [27]. therefore, the sbs is not sufficient in this type of resin and does not support any relining or repairing procedure. conclusion in the light of the current study, the shear bond strength showed higher values for heat cured polymethylmethacrylate, whereas 3d printed and thermoplastic resins showed lower bonding results. funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. conflict of interest the authors of the manuscript declare that there are no conflicts of interest. references 1. dimitrova m, corsalini m, kazakova r, vlahova a, chuchulska b, barile g, capodiferro s, kazakov s. comparison between conventional pmma and 3d printed resins for denture bases: a narrative review. journal of composites science. 2022 mar 10;6(3):87. vol 12 no 2 (2024) doi 10.5195/d3000.2024.756 h#p://den*stry3000.pi#.edu shear bond strength comparison between heat cure acrylic resin and other types of acrylic denture bases 2. pero ac, barbosa db, marra j, ruvolo-filho ac, compagnoni ma. influence of microwave polymerization method and thickness on porosity of acrylic resin. journal of prosthodontics. 2008 feb;17(2):125-9. 3. reyes-sevilla m, kuijs rh, werner a, kleverlaan cj, lobbezoo f. comparison of wear between occlusal splint materials and resin composite materials. journal of oral rehabilitation. 2018 jul;45(7):539-44. 4. baker ps, haywood vb, plummer kd. method for immediate fabrication of an occlusal device. the journal of prosthetic dentistry. 2007 nov 1;98(5):411-5. 5. huettig f, kustermann a, kuscu e, geis-gerstorfer j, spintzyk s. polishability and wear resistance of splint material for oral appliances produced with conventional, subtractive, and additive manufacturing. journal of the mechanical behavior of biomedical materials. 2017 nov 1; 75:175-9. 6. kumar pa, iniyan k, balasubramaniam r, viswanathan m, hines pa, monnica v. the effect of surface treatments on the shear bond strength of acrylic resin denture base with different repair acrylic resin: an in vitro study. journal of pharmacy and bioallied sciences. 2019 may 1;11(suppl 2): s380-4. 7. kreve s, dos reis ac. denture liners: a systematic review relative to adhesion and mechanical properties. the scientific world journal. 2019;2019(1):6913080. 8. kubo cs, amaral fr, de campos ea. relining of removable dentures: a literature review. rsbo revista sul-brasileira de odontologia. 2014;11(2):192-8. 9. mahajan n, datta k. comparison of bond strength of auto polymerizing, heat cure soft denture liners with denture base resin—an in vitro study. the journal of indian prosthodontic society. 2010 mar; 10:31-5. 10. ates sm, caglar i, ozdogan a, duymus zy. the effect of denture cleansers on surface roughness and bond strength of a denture base resin. journal of adhesion science and technology. 2017 jan 17;31(2):171-81. 11. kümbüloğlu ö, yildirim b, al-haj husain n, özcan m. adhesion potential of relining materials to polyamide and pmma-based denture base materials: effect of surface conditioning methods. journal of adhesion science and technology. 2019 sep 2;33(17):1939-47. 12. bilgin ms, baytaroğlu en, erdem a, dilber e. a review of computer-aided design/computeraided manufacture techniques for removable denture fabrication. european journal of dentistry. 2016 apr;10(02):286-91. 13. steinmassl pa, klaunzer f, steinmassl o, dumfahrt h, grunert i. evaluation of currently available cad/cam denture systems. international journal of prosthodontics. 2017 mar 1;30(2). 14. dohiem mm. comparison of bond strength between acrylic based soft liner and two different denture bases (3dprinted and conventional denture base). egyptian dental journal. 2022 jan 1;68(1):659-63. 15. alharbi n, alharbi a, osman r. mode of bond failure between 3d-printed denture teeth and printed resin base material: effect of fabrication technique and dynamic loading. an in vitro study. international journal of prosthodontics. 2021 nov 1;34(6). 16. tornavoi dc, agnelli ja, lepri cp, mazzetto mo, botelho al, soares rg, dos reis ac. assessment of surface hardness of acrylic resins submitted to accelerated artificial aging. minerva stomatol. 2012 jun 1;61(6):283-8. vol 12 no 2 (2024) doi 10.5195/d3000.2024.756 h#p://den*stry3000.pi#.edu shear bond strength comparison between heat cure acrylic resin and other types of acrylic denture bases 17. taghva m, enteghad s, jamali a, mohaghegh m. comparison of shear bond strength of cad/cam and conventional heat-polymerized acrylic resin denture bases to auto-polymerized and heat-polymerized acrylic resins after aging. journal of clinical and experimental dentistry. 2022 jan;14(1):e72. 18. duymus zy, ozdogan a, ulu h, ozbayram o. evaluation the vickers hardness of denture base materials. open journal of stomatology. 2016 apr 7;6(4):1149. 19. mohammed i, alwahab zn. an evaluation of the effect of artificial saliva with different ph on shear bond strength of veneering ceramic to metal and zirconia substructure (in vitro study). wjpr. 2017 oct 24; 6:30-44. 20. faot f, da silva wj, da rosa rs, del bel cury aa, garcia rc. strength of denture base resins repaired with auto-and visible light-polymerized materials. journal of prosthodontics: implant, esthetic and reconstructive dentistry. 2009 aug;18(6):496-502. 21. prpić v, schauperl z, glavina d, ćatić a, čimić s. comparison of shear bond strengths of different types of denture teeth to different denture base resins. the journal of advanced prosthodontics. 2020 dec;12(6):376. 22. choi jj, uy ce, plaksina p, ramani rs, ganjigatti r, waddell jn. bond strength of denture teeth to heat-cured, cad/cam and 3d printed denture acrylics. journal of prosthodontics. 2020 jun;29(5):415-21. 23. alqahtani m, haralur sb. influence of different repair acrylic resin and thermocycling on the flexural strength of denture base resin. medicina. 2020 jan 21;56(2):50. 24. cho, s.y.; song, y.g. a comparison study on shear bond strength of 3d printed resin and conventional heat-cured denture base resin to denture relining materials. journal of dental rehabilitation and applied science. 2021 dec 31;37(4):232-43. 25. lau m, amarnath gs, muddugangadhar bc, swetha mu, das ka. tensile and shear bond strength of hard and soft denture relining materials to the conventional heat cured acrylic denture base resin: an in-vitro study. journal of international oral health: jioh. 2014 apr;6(2):55. 26. mert d, kamnoedboon p, husain na, özcan m, srinivasan m. cad-cam complete denture resins: effect of relining on the shear bond strength. journal of dentistry. 2023 apr 1; 131:104438. 27. koodaryan r, hafezeqoran a. effect of surface treatment methods on the shear bond strength of auto-polymerized resin to thermoplastic denture base polymer. the journal of advanced prosthodontics. 2016 dec 1;8(6):504-10. 765+2024 evalua&on of the accuracy of different apex locaters with 45° apical root resorp&on vol 12 no 2 (2024) doi 10.5195/d3000.2024.765 h#p://den*stry3000.pi#.edu evaluation of the accuracy of different apex locaters with 45° apical root resorption ahmed i al-jobory 1, mohammed ayad taha 1, mohammed jabbar atyiah 2 1 college of dentistry, tikrit university, tikrit, iraq 2 ministry of health, iraq abstract aim: this study was aimed to calculate the working length (wl) of permanent teeth with simulated 45° resorp>on of the apical part of the root using four electric apex locaters (eals): nsk, woodpecker iii, woodpecker v, and eighteeth. methods: twenty maxillary anterior single-rooted teeth were removed. following tooth prepara>on to offer access to the root canal and replicate the 45° apical root resorp>on, each tooth underwent a microscope-assisted working length determina>on process. the four apex locaters measured each individual tooth and calculated electronic working lengths. results: no discernible change existed between all four apex locater devices. conclusion: nsk, woodpecker iii, woodpecker v, and eighteeth provide virtually the same measurement for 45° apical root resorp>on in single rooted teeth. keywords: apex locater devices, working length, electronic working length test, 45° root resorp>on. cita;on: al-jobory ai, et al. (2024) evalua;on of the accuracy of different apex locaters with 45° apical root resorp;on. den;stry 3000. 2:a001 doi:10.5195/d3000.2024.765 received: november 1, 2024 accepted: november 7, 2024 published: november 22, 2024 copyright: ©2024 al-jobory, et al. this is an open access ar;cle licensed under a crea;ve commons axribu;on work 4.0 united states license. email: mohammedayad@tu.edu.iq introduction for endodontic therapy to be successful, the root canal length must be accurately determined, particularly in situations of recurrent infection or re-infection. in these situations, non-surgical methods are frequently used to solve the problem. removing the old filling from the root canal is the first step in the conservative nonsurgical retreatment. in order to appropriately cleanse and disinfect the root canal system, access must be granted. to guarantee that all filling materials, debris, diseased pulp tissue, and necrotic material are completely removed, further root canal preparation could be necessary. the root canal must be adequately enlarged and cleaned at the precisely measured length in order to successfully repair the periradicular tissues. the physician can guarantee that all afflicted or diseased regions are completely cleansed and treated by precisely measuring the root canal length [1–15]. establishing the working length is crucial for root canal preparation and 3d obturation in primary endodontic treatments and retreatments [16]. numerous radiographic pictures are acquired throughout the root canal retreatment, which exposes the patient to excessive radiation and makes it hard to establish the working length only by radiography [17]. the aim of this examination was to assess how well four eals determined working length during root canal rehabilitation. material and methods twenty maxillary anterior singlerooted teeth that had been removed were utilized. prior research found that these teeth had been taken due to serious illness or orthodontic treatment [10,18]. after the blood was removed from the teeth, they were preserved in 5.25% sodium hypochlorite for two evalua&on of the accuracy of different apex locaters with 45° apical root resorp&on vol 12 no 2 (2024) doi 10.5195/d3000.2024.765 h#p://den*stry3000.pi#.edu hours and then placed in sterile 0.9% saline solution until the next step (figure 1). a diamond disc was used to decoronate teeth at the cementoenamel junction, making it easier to access the root canal and create fixed occlusal landmark (figure 2) [11,19]. a 45° oblique incision was performed at the root apex using a disc to mimic apical root resorption. nickel titanium endo k file sizes of 8, 10, 15, and 20 were used for instrumentation, and 2.5% sodium hypochlorite was used for irrigation (figures 3 and 4) [12]. working length was determine under microscope and visual [13,20]. resorption at 45° oblique cut was made because only a few studies have studied accuracy of apex-locator with root resorption [21]. to maintain the root canal wet, 0.5 ml of saline was inserted within the canal prior to the electronic working length (ewl) measurement [22]. salt and water were utilized to create an electric circuit. figure 1. samples used. figure 2. cutting the crown with aa diamond disk. figure 3. oblique cut to simulate apical resorption. figure 4. simulated resorption at apex. four distinct apex locators were used to measure the electronic working length (ewl) values: 1. woodpecker iii 2. eighteeth 3. woodpecker v 4. nsk (i pex ii) these devices were used in this study because they are the most common uses in dentistry. to maintain the root canal wet, 0.5 ml of saline was inserted within the canal prior to the electronic working length (ewl) measurement (figure 5) [14]. to generate the current, the mounted roots were submerged in containers with 0.9% saline, ensuring that the apical and middle sections of the roots were completely submerged. one of the conductors was saline. the labial clip of the apex finder was also evalua&on of the accuracy of different apex locaters with 45° apical root resorp&on vol 12 no 2 (2024) doi 10.5195/d3000.2024.765 h#p://den*stry3000.pi#.edu submerged in saline. a #15 k-file was used for each measurement twice, and the mean value was computed (figures 6 through 14). [15]. figure 5. measuring procedure. figure 6. container of water and salt. figure 7. bottle containing water and salt. figure 8. method of measurement by endo ruler. figure 9. measurement by woodpecker iii apex locator. figure 10. measurement by eighteeth apex locator. evalua&on of the accuracy of different apex locaters with 45° apical root resorp&on vol 12 no 2 (2024) doi 10.5195/d3000.2024.765 h#p://den*stry3000.pi#.edu figure 11. measurement by nsk apex locator. figure 12. measurement by woodpecker v apex locator. figure 13. measurement procedure. figure 14. resorption at 45° on the apex. evalua&on of the accuracy of different apex locaters with 45° apical root resorp&on vol 12 no 2 (2024) doi 10.5195/d3000.2024.765 h#p://den*stry3000.pi#.edu results table1. comparison between actual working length and the four devices. n actual working length under microscope (mm) nsk (mm) woodpecker lll (mm) woodpecker v (mm) eighteeth (mm) 1 17 17 / yes 16.5 / no 16 / no 18 / no 2 16 17 / no 17.5 / no 16.5 / no 17.5 / no 3 11.5 11.5 / yes 11 / no 11.5 / yes 12 / no 4 13 13 / yes 14 / no 13 / yes 13.5 / no 5 16 16.5 / no 17 / no 17 / no 17.5 / no 6 10 10 / yes 12 / no 10 / yes 12.5 / no 7 20 20 / yes 19 / no 20 / yes 20.5 / no 8 12.5 11 / no 13.5 / no 13 / no 13 / no 9 13.5 13 / no 14 / no 13 / no 14.5 / no 10 14 13 / no 13.5 / no 14.5 /no 12.5 / no 11 11 11 / yes 11 / yes 11 / yes 11.5 / no 12 9 9 / yes 9.5 / no 9 / yes 9 / yes 13 10.5 11.5 / no 12 / no 11.5 / no 12 / no 14 13 13 / yes 13 / yes 12.5 / no 13.5 / no 15 11.5 11.5 / yes 11.5 / yes 11.5 / yes 10.5 / no 16 13 13 / yes 14 / no 13 / yes 13 / yes 17 14.5 13.5 / no 13 / no 14 / no 13 / no 18 14 14 / yes 15 / no 14.5 / no 14 / yes 19 10.5 10.5 / yes 11 / no 10 / no 11 / no 20 14.5 13.5 / no 15 / no 13 / no 14 / no evalua&on of the accuracy of different apex locaters with 45° apical root resorp&on vol 12 no 2 (2024) doi 10.5195/d3000.2024.765 h#p://den*stry3000.pi#.edu the collected data revealed no significant differences between all test groups using a one-way anova analysis (p=0.945). discussion and conclusion the precision of calculating the root canal length has a substantial impact on the success rate of root canal retreatment. over-filling the root canals resulted in a success rate as low as 36%, suggesting that the infection was not adequately treated. nevertheless, the effectiveness rate rose to 62% when the working duration was precisely ascertained during retreatment [23]. we selected the most commonly apex locators used in dentistry. woodpecker v came as a replacement of woodpecker iii to improve determination of working length. woodpecker iii required a dry environment and fully charged battery for best results. eighteeth apex locator is sensitive to canal fluids and requires fully charged battery. it does not work well in cases of excessive exudates or blood. nsk is less studied. that relative error of nsk was (13.125%), woodpecker v was (13.225%), eighteeth was (13.65%), and woodpecker iii was (13.675%). calculations of accuracy based on data on table 1 show 86.875% for nsk, 86.775% for woodpecker v, 86.35% for eighteeth, and 86.325% for woodpecker iii. conflicts of interest the authors declare no competing interest. references 1. khandewal d, ballal nv, saraswathi mv. comparagve evaluagon of accuracy of 2 electronic apex locators with convengonal radiography: an ex vivo study. jendod. 2015;41:201. 2. vieyra jp, acosta j. comparison of working length determinagon with radiographs and four electronic apex locators. int endod j. 2011;44:510–8. 3. d'assunção fl, de albuquerque ds, salazar-silva jr, de queiroz ferreira lc, bezerra pm. the accuracy of root canal measurements using the mini apex locator and root zx-ii: an evaluagon in vitro. oral surg oral med oral pathol oral radiol endod. 2007;104:e50– 3. 4. aydin u, karataslioglu e, aksoy f, yildirim c. in vitro evaluagon of root zx and raypex 6 in teeth with different apical diameters. j conserv dent. 2015;18:66–9. 5. koçak s, koçak mm, saǧlam bc. efficiency of 2 electronic apex locators on working length determinagon: a clinical study. j conserv dent. 2013;16:229–32. 6. hargreaves km, cohen s. cohen's pathways of the pulp. 10th ed. us: elsevier mosby; 2012. p. 99. 7. leonardo mr, silva la, nelsonfilho p, silva ra, raffaini ms. ex vivo evaluagon of the accuracy of two electronic apex locators during root canal length determinagon in primary teeth. int endod j. 2008;41:317–21. 8. shabahang s, goon ww, gluskin ah. an in vivo evaluagon of root zx electronic apex locator. j endod. 1996;22:616–8. 9. srivastava v, jain n, bagchi s, negi mp. evaluagon of the use of sixth generagon apex locators as a diagnosgc tool to detect root perforagons. int j dent med spec. 2015;2:10– 4. 10. elayoug a, dima e, ohmer j, sperl k, von ohle c, löst c, et al. consistency of apex locator funcgon: a clinical study. j endod. 2009;35:179–81. 11. thomas ms, acharaya s, kundabala m. a comparagve evaluagon of the accuracy of third generagon electronic apex locator (root zx) and convengonal radiography to determine working length – an in vivo study. endodontology. 2008;20:14–21. evalua&on of the accuracy of different apex locaters with 45° apical root resorp&on vol 12 no 2 (2024) doi 10.5195/d3000.2024.765 h#p://den*stry3000.pi#.edu 12. saritha s, uloopi ks, vinay c, chandra sekhar r, rao vv. clinical evaluagon of root zx ii electronic apex locator in primary teeth. eur arch paediatr dent. 2012;13:32– 5. 13. alothmani os, friedlander lt, chandler no. radiographic assessment of endodongc working length. saudi endo j. 2013;3:57– 64. 14. moshonov j, slutzky-goldberg i, maor r, shay b, peretz b. in vivo evaluagon of apex nrg, a new apex locator, and its comparison with root zx. endod pract. 2005. 15. versteeg kh, sanderink gc, van ginkel fc, van der stelt pf. esgmagng distances on direct digital images and convengonal radiographs. j am dent assoc. 1997;128:439– 43. 16.white sc, pharoah mj.oral radiology principles and interpretagon.5th ed. us: mosby; 2000. p. 164. 17. l. vajrabhaya, p. tepmongkol in endodongc working-length determinagon. compend congn educ dent. 1995;16:288, 290, 293– 4. 18. da silva tm, alves fr. ex vivo accuracy of root zx ii, root zx mini and romiapex a-15 apex locators in extracted vital pulp teeth. j contemp dent pract. 2014;15:312– 4. 19. filho em, rizzi cd, de oliveira ds, filho pn, da silva ra, da silva ab. new electronic apex locator roamiapex a-15 presented accuracy for working length determinagon in permanent teeth. dengstry 3000. 2014;2:1–4. 20.torabinejad m, corr r, handysides r, shabahang s. outcomes of nonsurgical retreatment and endodongc surgery: a systemagc review. j endod. 2009;35(7):930–7. doi: 10.1016/j.joen.2009.04.023. 21. friedman s, moshonov j, trope m. residue of guwa-percha and a glass ionomer cement sealer following root canal retreatment. int endod j. 1993;26(3):169–72. doi: 10.1111/j.1365 2591.1993.tb00788.x. 22. sjogren u, hagglund b, sundqvist g, wing k. factors affecgng the long-term results of endodongc treatment. j endod. 1990;16(10):498–504. doi: 10.1016/s0099-2399(07)801804. 23. bergenholtz g, lekholm u, milthon r, engstrom b. influence of apical overinstrumentagon and overfilling on re-treated root canals. j endod. 1979;5(10):310–4. doi: 10.1016/s00992399(79)80080-1. microsoft word 450 2023.docx comparison of caries removal using carisolv or a conven2onal slow speed rotary instrument on a group of children aged 4-14 years in private specialized clinic in baghdad vol 11 no 1 (2023) doi 10.5195/d3000.2023.450 h#p://den*stry3000.pi#.edu comparison of caries removal using carisolv or a conventional slow speed rotary instrument on a group of children aged 4-14 years in private specialized clinic in baghdad nidhal ali1, abduloma ali1, istabraq baker1 1al-mustafa university college, dentistry department abstract carisolv is the only naocl-based chemical-mechanical caries removal product that is currently available on the market with a ph 11. par2cipants in the study who had primary caries lesions on their teeth were divided into two groups. the experimental group received chemical-mechanical treatment with carisolv® gel (n=54) whereas the control group received tradi2onal treatment modelby drilling and excava2on (n=47). children who were par2cipa2ng in the study had at least two primary caries lesions in the same group of teeth and were being treated by a pediatric den2st at a specialty dental clinic. children with two or more iden2cal caries lesions in the same set of teeth took part in this study. using carisolv® gel on one of the selected teeth (the experimental tooth), while drilling and excava2ng the other tooth as usual, both the experimental and control teeth were placed on the len side of the jaw. it was successful in geqng the parents of the children to agree to have them par2cipate in the study. using carisolv® gel, we conducted a clinical comparison of the tradi2onal and chemical-mechanical approaches in this study. the aim of this research was to compare a chemical-mechanical approach of caries removal with rotary instruments for children's deciduous and permanent teeth using carisolv® gel and determine whether anesthesia is required. keywords: dental caries; preven2on; clinical trial; fluoride cita9on: ali, n et al. (2023) comparison of caries removal using carisolv or a conven9onal slow speed rotary instrument on a group of children aged 4-14 years in private specialized clinic in baghdad den9stry 3000. 1:a001 doi:10.5195/d3000.2023.450 received: february 18, 2023 accepted: april 10, 2023 published: april 21, 2023 copyright: ©2023 ali, n et al. this is an open access ar9cle licensed under a crea9ve commons auribu9on work 4.0 united states license. email: dr.nidhal_dental@almustafauniversity.edu.iq introduction a significant issue in pediatric dentistry is dental caries. the interaction of environmental, behavioral, and host factors lead to the development of this complex disease. it causes the organic portion of the tooth's structure to be destroyed and its inorganic component to become demineralized [1]. the typical caries removal, which frequently results in discomfort especially in children, is uncomfortable for many patients and requires anesthetics when using rotary instruments [2]. anesthetic use may be limited under similar circumstances as those with allergies, anxiety, or other illnesses. due to children's greater sensitivity to pain than adults, the standard dental treatment for teeth might be problematic for young patients [3]. drilling can harm the tooth pulp, hence an alternative approach to caries removal is desirable. carisolv® is a chemo-mechanical caries eradication technique that protects healthy tooth tissue. comparison of caries removal using carisolv or a conven2onal slow speed rotary instrument on a group of children aged 4-14 years in private specialized clinic in baghdad vol 11 no 1 (2023) doi 10.5195/d3000.2023.450 h#p://den*stry3000.pi#.edu atraumatic restorative treatment (art), air abrasion, laser, sono abrasion, and chemical-mechanical caries removal (cmcr) are some of the numerous techniques that are included in it [4]. chemical-mechanical caries removal (cmcr) is a minimally invasive procedure that uses a chemical substance to remove the diseased dentin. the healthy dental structure is also preserved using this technique, preventing patient discomfort and pulp irritation. this method of caries eradication relies on dissolving rather than drilling. this method excavates soft carious dentin using chemical agents and atraumatic mechanical force [5]. the sole naocl-based chemicalmechanical caries removal agent that is currently commercially marketed is carisolv. carisolv, the most recent iteration of the naocl-based chemical-mechanical agents, was released in 1998 by medi team dentalutveckling ab, göteborg, sweden. the advantage of carisolv over caridex is that it does not need to be heated and does not need a particular delivery system because it comes in a gel form [6]. material and methods children who were participating in the study had at least two primary caries lesions in the same group of teeth and were being treated by a pediatric dentist at a specialty dental clinic. the protocol of this study was approved by the ethics committee of (651). children with two or more identical caries lesions in the same set of teeth took part in this study. using carisolv® gel on one of the selected teeth (the experimental tooth), while drilling and excavating the other tooth as usual, both the experimental and control teeth were placed on the left side of the jaw. it was successful in getting the parents of the children to agree to have them participate in the study. the study's participants (teeth with primary caries lesions) were split into two groups: the experimental group (n=54) underwent chemicalmechanical treatment with carisolv® gel, and the control group (n=47) underwent a traditional treatment model by drilling and excavation. results results are summarized in tables 1 through 5. caries experience in the study participants. experimental group total caries lesions deep caries total age 4 6 18 14 32 groups 6 14 15 7 22 total 33 21 54 control group total caries lesions deep caries total age 4 6 14 32 groups 6 14 7 22 total 21 54 table 1: age and diagnosis of patients. comparison of caries removal using carisolv or a conven2onal slow speed rotary instrument on a group of children aged 4-14 years in private specialized clinic in baghdad vol 11 no 1 (2023) doi 10.5195/d3000.2023.450 h#p://den*stry3000.pi#.edu frequency symptom pain 12 other 12 no complaints 25 unpleasant taste and smell 3 pain and unpleasant taste 2 total 54 frequency anesthetic use yes 9 no 45 total 54 frequency anesthetic use yes 15 no 32 total 47 frequency use of drill yes 31 no 23 total 54 discussion a primary goal of dental researchers looking for potential alternatives to current conventional approaches in the area of pediatric dentistry has always been the development of new techniques for eliminating caries. using carisolvtm gel, we conducted a clinical comparison of the traditional and chemomechanical approaches in this study. carisolv® can be used as an alternative in many situations, particularly when treating children, apprehensive patients, and patients with allergies. it is, however, unable to completely replace rotational instruments for the elimination of caries. participants perceived that the length of treatment to be shorter with carisolv®. on the other hand, because anesthesia was used, which takes 5–10 minutes to be effective, the overall drilling procedure may take longer. due to the operators' insufficient training and experience, the working time with carisolv® in our study may have been longer. the frequency of complaints of discomfort decreased more than twofold when carisolv® gel was used, proving that this procedure is far less unpleasant than the standard drilling method. almost half of the patients table 2: complaints within chemical-mechanical treatment group. table 3: use of anesthetics in the chemical-mechanical treatment group. table 5: use of drill in the chemical-mechanical treatment group. table 4: use of anesthetics in the traditional treatment (control) group. comparison of caries removal using carisolv or a conven2onal slow speed rotary instrument on a group of children aged 4-14 years in private specialized clinic in baghdad vol 11 no 1 (2023) doi 10.5195/d3000.2023.450 h#p://den*stry3000.pi#.edu who used this approach reported no concerns. additionally, it might be argued that the chemical-mechanical technique completely lacked disagreeable tastes and odors. references 1. hamama hh, yiu ck, burrow mf. caries management: a journey between black's principals and minimally invasive concepts. int j oral sci. 2015;2(8):120– 125.doi: 10.19070/2377-80751500026. 2. rao, d., panwar, s. and narula, h. an in-vivo comparawve evaluawon of the efficacy of two different papain based chemomechanical caries removalagents in primary molars’. (2020) ‘internawonal journal of scienwfic research, 9(3), pp. 43–45. 3. hegde, s. et al. clinical efficiency of three caries removal systems: rotary excavawon, carisolv, and papacarie. (2016), journal of denwstry for children. 4. showkat, n. et al. minimal invasive denwstry: literature review’, journal of current medical research and opinion. 2020. 3(09). doi: 10.15520/jcmro.v3i09.340. 5. hamama h, yiu c, burrow m. current update of chemomechanical caries removal methods. aust dent j. 2014;59(4):446–456. doi: 10.1111/adj.12214. 6. ericson d, zimmerman m, raber h, gotrick b, bornstein r, thorell j. clinical evaluawon of efficacy and safety of a new method for chemo-mechanical removal of caries. caries res 1999;33:171– 177. 744 2024 (5) using gene transfer technology to restore the salivary gland function in xerostomia patients following radiation therapy of head and neck cancer vol 12 no 2 (2024) doi 10.5195/d3000.2024.744 http://dentistry3000.pitt.edu using gene transfer technology to restore the salivary gland function in xerostomia patients following radiation therapy of head and neck cancer sabah hassan faculty of dentistry, al-mansour university college, baghdad, iraq abstract introduction radiation therapy plays an important role as one of the best methods used in treating head and neck cancer [1]. it can be used as a sole treatment method in the early stages of cancer, but it is also used in combination with other treatments, such as surgery and chemotherapy in more advanced cases [2,3]. in very advanced cases that have lost the chance of treatment, radiotherapy is also applied as a palliative measure to reduce the mass, reduce pain, stop bleeding, and reduce pressure on other structures [4,5]. however, radiotherapy can also cause major side effects. both the neck and head are affected by radiation therapy, and disturbances in the functions of the neck and head are considered side effects resulting from radiation [6,7]. acute side effects of radiotherapy applied in the treatment of head and neck cancers suggest secondary cell death, which is caused by the combination of intracellular free radical production and ionizing radiation, and actual organelle damage or inadequate repair of deoxyribonucleic acid (dna) damage in dividing cells [8–12]. mucositis, second-degree skin burns, wet desquamation, all salivary function losses, fibrosis in the skin and underlying tissues of the applied area, loss of taste, nutritional problems, edema in the face and neck, hair loss, dental problems are mostly acute effects of radiation on cellular systems with rapid turnover cycles [13–16]. chronic side effects include muscle fibrosis, skin atrophy, brain necrosis, neuropathy, esophageal stricture, retinopathy, cataract formation, and radiation myelitis. these are either the direct effects of radiotherapy or the microvascular tissue insufficiency caused by endothelial proliferation and fibrosis resulting from radiotherapy. subjects and methods general plan and patient selection a randomized prospective study was planned to compare the effects of drugs containing amifostine and zinc sulfate to prevent dry mouth (xerostomia) that occurs after treatment in patients receiving head and neck radiotherapy [17,18]. eligible patients include those who will receive only radiotherapy or many diseases can be treated by gene therapy using a modified virus to carry the corrected required gene to infect the target cells. the goal of this report is presenting the planning for gene therapy for patients suffering from head and neck cancer to alleviate their loss of saliva function. keywords: head and neck cancer; gene therapy; saliva; salivary gland citation: hassan, s (2024) using gene transfer technology to restore the salivary gland function in xerostomia patients following radiation therapy of head and neck cancer. dentistry 3000. 1:a001 doi:10.5195/d3000.2024.744 received: september 25, 2024 accepted: september 21, 2024 published: november 22, 2024 copyright: ©2024 hassan, s. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: sabahbedi1@gmail.com using gene transfer technology to restore the salivary gland function in xerostomia patients following radiation therapy of head and neck cancer vol 12 no 2 (2024) doi 10.5195/d3000.2024.744 http://dentistry3000.pitt.edu chemoradiotherapy for treatment purposes, those who were diagnosed with head and neck cancer or those who had previously received radiotherapy or chemotherapy, and those diagnosed with head and neck cancer that previously received radiotherapy or chemotherapy. all cases were over 15 years of age and under 80 years old. the patients were evaluated by two different physicians working in the radiation oncology clinic before treatment, every week during treatment, and two months after the end of treatment, and that included oropharyngeal examinations, routine blood tests, and weight assessments. mucositis assessments were made based on the radiation therapy oncology group (rtog) oropharyngeal mucositis classification system. the date of onset and degree of mucositis were recorded. in addition, to clinically evaluate the saliva volume of each patient, saliva volume was measured before treatment, at the end of treatment, and in the second month after treatment. for this purpose, the patient was asked to rinse his/her mouth thoroughly with water and spit before each measurement. then, he/she was asked to spit all the saliva accumulated in his/her mouth into a 10 mm diameter glass tube. after the procedure was completed, he/she waited for 5 minutes. freshly squeezed lemon juice (citric acid) was used to measure the amount of stimulated saliva. 3 ml of lemon juice was dropped onto the back of the tongue using a tongue depressor, and the patient was asked to gargle the lemon juice in his/her mouth for one minute. at the end of the period, the patient was asked to spit out the lemon juice and then spit the saliva that had accumulated in his/her mouth into a second glass tube as described above. the saliva obtained was measured and recorded in millimeters from the outer surface of the glass tube. the outpatient department in our maxillofacial and research centre at uae treated five patients who have complained of post operative radiation (as a side effect). most of those patients complained of dry mouth (xerostomia). two male patients over 50 years, one patient of 60 years, one male patient was 75 years old, and only one female patient aged 70 years old were treated. radiotherapy radiotherapy was applied with a multifiber collimator linear accelerator (linac mlc, siemens) device in 1.8-2 gy dose fractions 5 days a week for 5-7 weeks. before starting radiotherapy, immobilization was provided with a thermoplastic mask in all patients, lead markers were placed at certain points and tomography was taken for planning purposes, and the images obtained were transferred to our clinic's 3d planning system. the most appropriate radiotherapy areas for each patient were planned based on these images. patients were treated with the conformal radiotherapy technique suitable for the planned radiotherapy area. during radiotherapy, radiotherapy was and/or the decision to suspend chemotherapy was made independently by two physicians working in the radiation oncology clinic. examination of salivary glands by scintigraphic method [19,20] (figure 1) a total of three salivary gland scintigraphy scans were planned for all patients, before radiotherapy, at the end of radiotherapy, and two months after radiotherapy. however, some of the patients did not accept salivary gland scintigraphy scans at the end of radiotherapy due to oral mucositis or other reasons. therefore, it was decided to evaluate all patients based on the scintigraphic images taken before radiotherapy and in the second month after radiotherapy. a parallel-hole, low-energy, highresolution collimator was selected in the study and a single-head gamma camera. the energy level was determined to be 140 kev and the windows interval as 20%. the patients were positioned in a supine position under the gamma using gene transfer technology to restore the salivary gland function in xerostomia patients following radiation therapy of head and neck cancer vol 12 no 2 (2024) doi 10.5195/d3000.2024.744 http://dentistry3000.pitt.edu camera detector, with the head slightly extended. following the given intravenously of 185 mbq (5mci) technetium 99mpertechnetates as a bolus, 25 images, each lasting 60 seconds, were obtained. dynamic images were obtained for 25 minutes, with a matrix of 128x128 and a zoom of 1.33. three milliliters of concentrated lemon juice was administered into the oral cavity via an injector at 20 minutes. a total of 25 images were taken during the dynamic study. figure 1. dynamic images of salivary gland scintigraphy. http://dentistry3000.pitt.edu using gene transfer technology to restore the salivary gland function in xerostomia patients following radiation therapy of head and neck cancer in the collected images, regions of interest were drawn around the right and left parotid glands, right and left submandibular glands for semiquantitative analysis. similarly, a region of interest was drawn in the temporal region for background activity (figure 2). figure 2. determining areas of interest for each salivary gland. results and conclusion five patients (table 1) were admitted to our maxillofacial and research center with a history of radiation therapy to the parotid gland or areas in the head and neck due to maligned tumor, with the main complaint xerostomia (dry mouth). the patients are unable to eat or talk normally. these patients were treated in our maxillofacial and research center by gene therapy. aquaporin gene therapy restored the diminished saliva in the treated patients. gene therapy for restoration the function of the salivary gland gave a satisfactory result in two patients, with no complications reported. these two patients died within two years after the treatment (gene therapy) due to metastasis of the malignant tumors; the period of the follow up been very short. table 1. patients treated by gene therapy. gender results of patients treated by gene therapy male 2 patients complete failure female 1 patient unsatisfactory result male 2 patients satisfactory result data availability the data that support the findings of this study are available from the corresponding author upon reasonable request. conflict of interest all the authors declare no commercial or financial conflict of interest. references 1. alterio d, marvaso g, ferrari a, volpe s, orecchia r, jereczek-fossa ba. modern radiotherapy for head and neck cancer. semin. oncol., vol. 46, elsevier; 2019, p. 233– 45. 2. epstein jb, thariat j, bensadoun r, barasch a, murphy ba, kolnick l, et al. oral complications of cancer and cancer therapy: from cancer treatment to survivorship. ca cancer j clin 2012;62:400–22. 3. devita jr vt, young rc, canellos gp. combination versus single agent chemotherapy: a review of the basis for selection of drug treatment of cancer. cancer 1975;35:98–110. 4. kvale pa, selecky pa, prakash ubs. palliative care in lung cancer: accp evidence-based clinical practice guidelines. chest 2007;132:368s-403s. 5. cocks h, ah-see k, capel m, taylor p. palliative and supportive care in head and neck cancer: united kingdom national multidisciplinary guidelines. j laryngol otol 2016;130:s198–207. 6. brook i. late side effects of radiation treatment for head and neck cancer. radiat oncol j 2020;38:84. 7. siddiqui f, movsas b. management of radiation toxicity in head and neck cancers. semin. radiat. oncol., vol. 27, elsevier; 2017, p. 340–9. 8. sourati a, ameri a, malekzadeh m. acute side effects of radiation therapy. cham springer 2017. 9. feller g, khammissa rag, nemutandani ms, feller l. biological consequences of cancer radiotherapy in the context of oral squamous cell carcinoma. head face med 2021;17:35. 10. azzam p, mroueh m, francis m, abou daher a, zeidan yh. radiation-induced neuropathies in head and neck cancer: prevention and http://dentistry3000.pitt.edu using gene transfer technology to restore the salivary gland function in xerostomia patients following radiation therapy of head and neck cancer treatment modalities. ecancermedicalscience 2020;14. 11. afshari n, koturbash i, boerma m, newhauser w, kratz m, willey j, et al. a review of numerical models of radiation injury and repair considering subcellular targets and the extracellular microenvironment. int j mol sci 2024;25:1015. 12. davalli p, marverti g, lauriola a, d’arca d. targeting oxidatively induced dna damage response in cancer: opportunities for novel cancer therapies. oxid med cell longev 2018;2018:2389523. 13. wickline mm. prevention and treatment of acute radiation dermatitis: a literature review. oncol. nurs. forum, vol. 31, oncology nursing society; 2004, p. 237–48. 14. coleman iii jj. management of radiation-induced soft-tissue injury to the head and neck. clin plast surg 1993;20:491–505. 15. dörr w. pathogenesis of normal tissue side effects. basic clin. radiobiol., crc press; 2018, p. 152–70. 16. wong hm. oral complications and management strategies for patients undergoing cancer therapy. sci world j 2014;2014:581795. 17. vissink a, burlage fr, spijkervet fkl, jansma j, coppes rp. prevention and treatment of the consequences of head and neck radiotherapy. crit rev oral biol med 2003;14:213–25. 18. blanco ai, chao c. management of radiation-induced head and neck injury. radiat toxic a pract guid 2006:23–41. 19. yoruk o, seven b, varoglu e, ucuncu h, sahin a, ayan ak, et al. evaluation of salivary gland function in allergic rhinitis patients by [99m tc] pertechnetate salivary gland scintigraphy. cent eur j med 2010;5:246–50. 20. chen y-c, chen h-y, hsu c-h. recent advances in salivary scintigraphic evaluation of salivary gland function. diagnostics 2021;11:1173. 877 final mechanical characteris.cs of teeth restored with innova.ve func.onally graded posts made of titanium-hydroxyapa.te-bioac.ve glass vol 13 no 1 (2025) doi 10.5195/d3000.2025.877 h#p://den*stry3000.pi#.edu mechanical characteristics of teeth restored with innovative functionally graded posts made of titaniumhydroxyapatite-bioactive glass mohamed abdulmunem1*, muralithran kutty2, wan haliza abd majid3, noor hayaty abu kasim4, ali dabbagh 2, noor yahya2, hadijah abdullah 5 1college of dentistry, university of al maarif, al anbar, iraq 2faculty of dentistry, university of malaya, kuala lumpur, malaysia 3faculty of science, university of malaya, kuala lumpur, malaysia 4faculty of dentistry, universiti kebangsaan malaysia, kuala lumpur, malaysia 5dental faculty, mahsa university, selangor, malaysia abstract objec?ves: novel func+onally graded dental posts were fabricated, and their mechanical proper+es were evaluated and then compared with three different commercially dental posts. materials and methods: the fgdps were fabricated by sintering various ra+os of bioac+ve glass (bg), hydroxyapa+te (ha), and +tanium (ti) at 900oc in air atmosphere. four groups of forty maxillary central incisors were selected, and each group restored with +tanium, stainless steel, fiber, or fgdps. to analyze stress distribu+on and assess fracture resistance and failure mechanisms, four finite element models of teeth repaired with fgdp, fiber, +tanium, and stainless-steel posts, respec+vely, were created. results: the fracture resistance of teeth repaired using fgdps was greater (mean = 550 ± 72 n) compared with those restored with +tanium (mean = 505 ± 58 n), fibre (mean = 475 ± 70 n) and stainless steel (mean = 422 ± 58 n) posts. sta+s+cal analysis showed a significant difference between fgdps and stainless-steel posts (p = 0.000). when fgdp-restored teeth contrasted with those who had fibber, +tanium and stainless-steel posts were 70%, 50%, and 40 %, respec+vely, of restorable failures (rf), and 100% occurred in the former group. conclusions: comparing the fgdp and fibre models to the pothers, finite element analysis ifea) reveled that stress was distributed more frequently throughout them. it is evident that when fgdps made using the ti-ha-bg technology was compared to other dental posts that are sold commercially, they func+oned be\er as dental posts. keywords: dental materials; dental posts; resistance to fractures; material failure; stress. cita:on: abdulmunem m, et al. (2025) mechanical characteris:cs of teeth restored with innova:ve func:onally graded posts made of titaniumhydroxyapa:te-bioac:ve glass. den:stry 3000. 1:a001 doi:10.5195/d3000.2025.877 received: march 9, 2025 accepted: april 10, 2025 published: may 6, 2025 copyright: ©2025 abdulmunem m, et al. this is an open access ar:cle licensed under a crea:ve commons ayribu:on work 4.0 united states license. email: mohamadjasem@yahoo.com introduction the topic of root canal therapy has been extensively researched. human teeth typically lose their structure and integrity because of several conditions, including dental caries, trauma, and even root canal therapy. when teeth are loaded or masticated, all these variables might weaken them and increase the risk of fractures [1,2]. after receiving a root canal therapy to preserve the remaining tooth structure and prevent tooth loss, a coronal restoration is placed in place [3]. if there is significant damage to the tooth structure that prevents the coronal restorations from being retained, a dental post must be inserted for repairing endodontically treated teeth. dental posts have therefore established themselves as the preferred course of therapy [4-6]. numerous post varieties, including those made of titanium, fiber, zirconium, and stainless steel, offer mechanical characteris.cs of teeth restored with innova.ve func.onally graded posts made of titaniumhydroxyapa.te-bioac.ve glass vol 13 no 1 (2025) doi 10.5195/d3000.2025.877 h#p://den*stry3000.pi#.edu excellent strength, toughness, and fracture resistance. they do, however, have drawbacks, including loss of retention, difficulty retrieving, and creation of irreversible failures [5,7]. as a result of the uniform material used to make all commercial posts, it is more difficult to disperse stress under force, increasing the risk of irreversible failures. accordingly, a functionally graded approach based on ti-ha materials is taken into consideration in addition to post kinds [8-10]. three different kinds of functionally graded dental posts (fgdp) were created in 2011 by abu kasim et al. using zro2-ti-ha, al2o3-ti-ha, and ti-ha materials [8]. four layers made up these dental posts, which allowed the tension to be evenly distributed from the post to the dentine. because of their increased ability to endure forces exerted during loading and mastication, the teeth had more rf [8]. it is commonly recognized that the chemical and crystallographic structures of hydroxyapatite and bone mineral are comparable. since hydroxyapatite is biocompatible with human bone, it is frequently utilized in dentistry and medical procedures [11-13]. unfortunately, it is weak and fragile [14,15]. titanium is employed in numerous medicinal applications and has superior mechanical qualities than ha. when compared to ha, it is less biocompatible, however [16-19]. dehydration and ha degradation are common during the high temperature sintering of ti/ha [20]. because it may increase ha's biocompatibility and solve the sintering temperature issue, bioactive glass (bg) may be a promising option for sintering hydroxyapatite-titanium composites [20-22]. to provide root canal treated teeth greater mechanical qualities, functionally graded dental posts, or fgdps, were created for this study. they also have higher restorable failure rates, which is thought to improve the teeth's resistance to fracture by increasing stress distribution during loading. until far, no research has been done on the synthesis of ti-ha, fgdp combined with bioactive glass materials. the purpose of the study was to lower the sintering temperature and prevent any potential issues by adding bioactive glass to ti-ha fgdp. the present investigation was a component of a larger research including two sections. the first section examined and assessed the impact of bioactive glass and sintering conditions on individual ti-ha separated composites (layers) [23]. following their fabrication, the endodontically treated teeth were repaired using fgdp made of tiha-bg materials. consequently, three different types of commercially available dental posts (made of various materials) were used to restore teeth, and their mechanical characteristics were investigated and compared with those of the reconstructed teeth. furthermore, the stress distributions of the manufactured fgdp were analyzed using a finite element method in comparison to other dental post kinds. material and methods fabrication of the functionally graded dental post the fgdps has five layers at which the top layer is mostly contain ti and the bottom layer is ha rich, and 10% bg was added to all the layers (fabrication of each layer was discussed in part 1 of this project by abdulmunem et al., 2021 [21]. bg was formed by milling eight different materials: sio2, nao, cao, p2o5, b2o3, tio2, caf2, and mgo together using a planetary ball milling machine (2 x 102 rpm/ five hrs.). utilizing silicone impression materials (aquasil, dentsply, konstanz, germany), molds were created. beginning with layer 1 and mechanical characteris.cs of teeth restored with innova.ve func.onally graded posts made of titaniumhydroxyapa.te-bioac.ve glass vol 13 no 1 (2025) doi 10.5195/d3000.2025.877 h#p://den*stry3000.pi#.edu going through layer 5, layers of tiha-bg powders were poured into the molds, and the layers were sealed using the same methods used for the imprint. after that, the samples were compressed at 250 mpa using a cold isostatic press. the samples were then sintered at 9 x102 ċ in an atmosphere furnace. the samples were heated to 500 ċ for 240 minutes after being at room temperature and remain in same level. the samples were cooled for eight hours overnight after the temperature was progressively increased with 240 min to 9 x 102 ċ and maintained at same level for an additional 240 minutes. samples were subsequently transported to a centerless grinder (palmary pc12s-nc, taiwan) for shaping and machining to create fgdps with the necessary diameter of 1.5 mm. root canal treatment and postspace preparation a total of forty removed human maxillary central incisors that were free of cavities, cracks, and prior endodontic treatments were chosen (df rd 1411/0058(p) is the reference number for the ethics committee/irb). the cementumenamel junction (cej) of every tooth was measured at 16 mm from the apex. a straight hand piece was used to segment the remaining crowns using a diamond disc. the step-back approach was employed in the instrumentation process. the master file was identified as a 45 k-file, and each of these files had a working length of 14 mm. rattana (dentsply/asia, hong kong, china), ah-plus (dentsply detrey, germany), a resin-based sealer, and the lateral compaction method were used for root canal fills. following that, size 3 gatesglidden drills were used to remove 9 mm of gutta-percha. once the post area was ready, special drills matching each post's operating length of 9 mm were employed. subsequently, all roots were combined into blocks of epoxy resin. organizing samples and assessing fracture resistance and failure mechanism four groups of ten roots were formed. the diameter of each post was 1.5 mm and its length was 14 mm. group 2 received titanium posts (para post xp, coltene/whaledent, usa) for root restoration, whereas group 1 received fgdps for root restoration. para post fibre lux, coltene/whaledent, usa, was used to repair the roots in group 3, and para post xp, coltene/whaledent, usa, was used to restore the roots in group 4. zinc phosphate cement was used to bond each post in the root canals. after that, metal copings were used to keep the composite cores in place over all the posts and roots. to load each sample at a 45degree angle plane to its long axis, it was positioned in a unique jig that was attached to a shimadzu universal testing machine. two millimeters below the metal copings' incisal edge, a crosshead pin was inserted at a pace of 0.5 mm/min until failure. data analysis all data were statistically analyzed using the one-way anova at p= 0.05, and pair-wise comparisons were done using both tukey and chi-square tests. the failure modes of each sample were investigated using the chi-square test, and the samples were thereafter sent to be examined for fractures using a 20 x stereomicroscope (sz x7, olympus, japan). finite element modelling a finite element model was built to examine the stress distribution among the various posts. solid works software (dassault systèmes solidworks corporation) was used to create four models that approximated human maxillary central incisors. the first model was developed with fgdp, the second model with a fiber post, the third model with a stainless-steel post, and the fourth model with a mechanical characteris.cs of teeth restored with innova.ve func.onally graded posts made of titaniumhydroxyapa.te-bioac.ve glass vol 13 no 1 (2025) doi 10.5195/d3000.2025.877 h#p://den*stry3000.pi#.edu titanium post. each model has a different type of post. stress distribution was calculated using the solidworks/surface modelling (version premium 2015, dassault systèmes, usa). loads were applied to the four models in three directions (vertical, horizontal and oblique) at 100 n to simulate the masticatory force, external traumatic force and bruxism [24,25]. different post types were used to construct each model: fgdp was used to develop the first model, fibre posts were used to establish the second model, stainless steel posts were used to establish the third model, and titanium posts were used to establish the fourth model. results evaluation of fracture resistance and failure mode figure 1 shows the mean and standard deviation of the dental post groups. by using a post-hoc (tukey test) test for multiple comparisons, a one-way anova was utilized to ascertain if there was a significant difference between the dental post groups (table 1). the fracture resistance values of the fgdps group mean were (550 ± 72) that were greater than the titanium, fiber, and stainless-steel groups (505 ± 58), (475 ± 70), (422 ± 58) respectively. the evaluated dental post groups differed considerably from one another, with the fgdp group's teeth exhibiting much stronger fracture resistance than the stainless-steel group's (p = 0.000). additionally, compared to teeth in the stainless-steel group, those in the titanium group exhibited greater fracture resistance (p = 0.032). the teeth in the titanium, fiber, and fgdp groups did not differ significantly from one another. table 2 indicates the quantity and proportion of failure mechanisms. ten rf have been filed in teeth repaired with fgdps, then seven rf in teeth restored with fiber, five rf in titanium, and four rf in stainless steel roots. as the fracture line is 2 mm below the cementum-enamel junction (cej) and above the epoxy resin block line, figure 2a depicts the restorable failure pattern of teeth in the fgdp group. the fiber group's restorable failure is depicted in figure 2b. additionally, the fracture began obliquely and finished with a 2 mm horizontal line that was above the epoxy resin block line and below the cej. the titanium group's non-rf fractures are seen in figure 2c, where they extend past the epoxy resin block line to the middle third of the root. on the other hand, figure 2d depicts the non-restorable failure mode of the stainless-steel group, in which the fracture line extended below the epoxy resin block line and began obliquely. finite element analysis stress distribution under vertical load figure 3a shows the stress distribution of all fe models under vertical load. in the fgdp and fiber models, compared to the other models, there was significantly less force applied to the root's cervical third, and the apical third of the root received very little stress. notably, the fgdp model's apical third showed the least amount of tension, but the post's coronal and middle third showed no stress at all. apart from the core-crown contact, the titanium and stainlesssteel models also displayed greatest pressures in the apical third of the post-root interfaces, leading to the non-rf. the coronal and middle thirds of the posts of the titanium and stainless-steel versions had the least amount of stress, whereas the apical third of the posts had the most stress. stress distribution under oblique load the titanium, fgdp, and fiber models had the highest levels of stress under the oblique load direction (figure 3b), in that order. at the contacts between the mechanical characteris.cs of teeth restored with innova.ve func.onally graded posts made of titaniumhydroxyapa.te-bioac.ve glass vol 13 no 1 (2025) doi 10.5195/d3000.2025.877 h#p://den*stry3000.pi#.edu coronal third of the crowns and the core, the stress was focused. the middle third and cervical regions of the root received less stress. according to the fgdp model, the root structure received a homogeneous distribution of stress, with very little stress dispersion along the post. the stress was centered at the corecrown contact. less stress was placed on the cervical and middle thirds of the root in the titanium and stainless steel models, whereas the most stress was localized at the coronal third of the crown's core-crown contacts. less stress was observed at the coronal third of the titanium post, whereas a small pit formed at the apical third of the post. stress distribution under horizontal load maximum stress was displayed in the titanium and fgdp models relative to the other models for the horizontal loading (figure 3c). the contact between the core, root, and crown displayed stress distribution in the titanium and fgdp models. the coronal third of the crown-core interface's corecrown interaction was the site of the greatest stress. the titanium post's midsection experienced less stress, and its apical stress also dropped. there was no stress seen at the apical third of the post and very little tension observed at the intersection of the coronal and middle third of the post along the fgdp surfaces. the coronal third of the crown's core-crown contact was where the greatest concentrations of stress were found in the fiber and stainlesssteel models, but the cervical third of the root's crown-root interface showed minimal stress. the stainless steel and fiber versions' post surfaces were free of tension. figure 1. means and standard deviations of fracture resistance the dental post groups. figure 2. representative image of (a) restorable failure of fgdps group; (b) restorable failure of fiber group; (c) non-restorable failure of titanium group; and (d) non-restorable failure of stainlesssteel group. figure 3. stress distribution under vertical (a), oblique (b), and horizontal (c) loadings for different tooth models. 550 475 505 422 fgdps fibre titaniumstainless steel failure load (newton) mechanical characteris.cs of teeth restored with innova.ve func.onally graded posts made of titaniumhydroxyapa.te-bioac.ve glass vol 13 no 1 (2025) doi 10.5195/d3000.2025.877 h#p://den*stry3000.pi#.edu table 1. multiple comparisons of the fracture resistance between the dental post groups. type of post (i) type of post (j) mean difference (i-j) standar d error pvalue 95% confidence interval lower bound upper bound titanium fibre 30.70000 29.0232 0.717 -47.4663 108.8663 stainless steel 83.70000 29.0232 0.032 5.5337 161.8663 fgdps -44.70300 29.0232 0.425 -122.8693 33.4633 fiber titanium -30.70000 29.0232 0.717 -108.8663 47.4663 stainless steel 53.00000 29.0232 0.278 -25.1663 131.1663 fgdps -75.40300 29.0232 0.062 -153.5693 2.7633 stainlesssteel titanium -83.70000 29.0232 0.032 -161.8663 -5.5337 fibre -53.00000 29.0237 0.278 -131.1663 25.1663 fgdps -128.40300 29.0237 0.000 -206.5693 -50.2367 fgdps titanium 44.70300 29.0237 0.425 -33.4633 122.8693 fibre 75.40300 29.0237 0.062 -2.7633 153.5693 stainless steel 128.40300 29.0237 0.000 50.2367 206.5693 mechanical characteris.cs of teeth restored with innova.ve func.onally graded posts made of titaniumhydroxyapa.te-bioac.ve glass vol 13 no 1 (2025) doi 10.5195/d3000.2025.877 h#p://den*stry3000.pi#.edu table 2. number and percentages of failure modes of the dental post groups. type of dental post n failure mode restorable non-restorable fgdps 10 10 (100%) 0 (0%) fiber 10 7 (70%) 3 (30%) titanium 10 5 (50%) 5 (50%) stainless steel 10 4 (40%) 6 (60%) discussion in this study, bioactive glass was added to ti-ha fgdps to aid in the sintering process. fgdps were produced in this study to provide a regular stress distribution for root canal treated teeth and thus improve the fracture resistance and increase the susceptibility of reparable failures [23]. according to the results of the current study, teeth repaired with titanium posts and fgdps were more fracture resistant than teeth treated with stainless steel posts. these findings are consistent with studies that looked at the fracture resistance of teeth repaired using other fgdp formulations, including zro2-tiha, al2o3-ti-ha, and ti-ha-ti, as well as other teeth treated using cast and titanium posts [10]. a dental post that is fabricated using the functionally graded concept— which calls for more stiffness at the coronal and less stiffness at the apical parts of the post—will have an elastic modulus that is like dentine's and will aid in progressively lowering stresses on the apical third of the root. this will strengthen the tooth's resistance to fracture and help it withstand stresses applied while it functions [8,9,23]. the first layer of the fgdp, which was kept in the core materials, had an elasticity modulus of 20248 mpa, whereas the second layer had an elasticity modulus of 17939 mpa, as seen in figure 3. according to dejack [26], these numbers are rather near to the dentine's 18600 mpa density. the fourth and fifth layers’ moduli of elasticity were measured to be 8766 mpa and 7147 mpa, respectively. as a result, stress was distributed coronally and decreased apically, increasing the tooth's resistance to fractures, as was covered in section 4.4 and supported by several studies [8,10]. regarding failure mechanisms, the teeth repaired with fgdps (10 rf) failed with more frequency in this study than teeth restored with fiber (7 rf), titanium (5 rf), and stainless steel (four rf) dental posts. researchers found that as compared to teeth repaired with cast and titanium posts, teeth treated using fgdps (based on zro2-ti-ha, al2o3-ti-ha, and tiha-ti) tended to fail with higher radiofrequency. the titanium, cast post, and core groups all experienced catastrophic failures; just one tooth from each group failed with rf, while the majority of mechanical characteris.cs of teeth restored with innova.ve func.onally graded posts made of titaniumhydroxyapa.te-bioac.ve glass vol 13 no 1 (2025) doi 10.5195/d3000.2025.877 h#p://den*stry3000.pi#.edu the teeth failed with non-rf. conversely, teeth that were repaired using all three kinds of fgdps displayed between 80% and 90% of rf [10] . from the coronal to the apical third of the dental post, the functionally graded multi-layered composition of the dental posts saw progressive variations in stiffness. a restorable failure was caused by layers with lesser stiffness spreading the stress to the neighbouring root structures in a regular pattern, whereas layers with high stiffness (at the coronal third) removed the stress from the core [8,23]. additionally, the modulus of elasticity is relevant. the coronal sections of the manufactured fgdps in this investigation had a greater modulus of elasticity than the apical regions. as a result, stress was focused on the coronal region of the fgdps at the post-core interface. problems centered in the coronal region [8]. non-rf can result from the restoration of endodontically treated teeth with a high modulus of elasticity and homogeneous dental posts, such titanium and stainless-steel posts, which will unevenly distribute stress on the root systems [27]. during the apical third of post-root contacts in the current study, the titanium and stainless-steel models showed higher maximum stress concentrations than the fiber and fgdp models. throughout its long axis, the dental post should retain a distinct and progressive modulus of elasticity to prevent uneven stress distribution from the post to the tooth tissues. to offer superior retention for the cores, the coronal third of the post should have a larger modulus of elasticity than the other post thirds. to minimize stress and minimize the likelihood of non-rf, the modulus of elasticity should be supplied consistently to the root structures and progressively lowered towards the apical third. as demonstrated in this work, where layers 1 and 2 exhibited greater levels of modulus of elasticity compared with layers 3, 4, and 5, this may be accomplished by creating dental posts with functionally graded compositions [8-10]. (fig. 3). in this investigation, the fgdp model performed better in terms of stress distribution than the titanium and stainless-steel models. in this study, models loaded in a vertical direction exhibited a higher concentration of stress than models loaded in an oblique or horizontal manner. this result contradicts a prior study's conclusion that models loaded in a vertical direction showed a higher maximum stress than those placed in a horizontal orientation [28]. that may be because the study's post designs and diameters differed from models that employed typical 1.5 mm diameter posts with parallel-sided post designs. the fgdps used in this study have therapeutic consequences since they give root canal treated teeth a regular stress distribution, which raises the risk of rf and lowers the chance of having to remove a tooth when a failure occurs. conclusion the limitation of this study was using the static compressive load instead of cyclic load and thus fatigue was not simulated in this study. moreover, no in vivo tests were done, and only in vitro tests were performed in this study. therefore, loading teeth under cyclic load and performing in vivo tests in future studies are recommended. this study has shown teeth restored with fgdps have better mechanical properties compared with those restored with homogenous dental posts. the gradual change in modulus of elasticity of functionally graded dental posts improved stress distribution, fracture resistance and failure mode of teeth restored mechanical characteris.cs of teeth restored with innova.ve func.onally graded posts made of titaniumhydroxyapa.te-bioac.ve glass vol 13 no 1 (2025) doi 10.5195/d3000.2025.877 h#p://den*stry3000.pi#.edu with fgdps. therefore, this study suggests fgdps based on ti-ha-bg materials can be used as restoration material of choice for endodontically-treated teeth. acknowledgement this research was supported by the frgs no. fp019-2017a grant, from the faculty of dentistry, university of malaya, kuala lumpur, malaysia. conflicts of interest the authors have no conflicts of interest to declare. references 1. krishan, r., et al., impacts of conservative endodontic cavity on root canal instrumentation efficacy and resistance to fracture assessed in incisors, premolars, and molars. journal of endodontics, 2014. 40(8): p. 11601166. 2. de v habekost, l., et al., fracture resistance of thermal cycled and endodontically treated premolars with adhesive restorations. the journal of prosthetic dentistry, 2007. 98(3): p. 186-192. 3. bachicha, w.s., et al., microleakage of endodontically treated teeth restored with posts. journal of endodontics, 1998. 24(11): p. 703-708. 4. aurélio, i., et al., are posts necessary for the restoration of root filled teeth with limited tissue loss? a structured review of laboratory and clinical studies. international endodontic journal, 2016. 49(9): p. 827-835. 5. mahmoudi, m., et al., influence of inhomogeneous dental posts on stress distribution in tooth root and interfaces: three-dimensional finite element analysis. journal of prosthetic dentistry, 2017. 118(6): p. 742-751. 6. abdulmunem, m., et al., the combined effect of dental post and cement materials on fracture resistance and fracture mode of endodontically-treated teeth. sains malaysiana, 2015. 44(8): p. 11891194. 7. schwartz, r.s. and j.w. robbins, post placement and restoration of endodontically treated teeth: a literature review. journal of endodontics, 2004. 30(5): p. 289301. 8. abu kasim, n.h., et al., 3d-fe analysis of functionally graded structured dental posts. dental materials journal, 2011. 30(6): p. 869-880. 9. dabbagh, a., et al., thermomechanical advantages of functionally graded dental posts: a finite element analysis. mechanics of advanced materials and structures, 2017: p. 1-10. 10. madfa, a.a., development of functionally graded composite for fabrication of dental post. 2011, university of malaya: malaysia. 11. ramesh, s., et al., characterization of biogenic hydroxyapatite derived from animal bones for biomedical applications. ceramics international, 2018. 44(9): p. 10525-10530. 12. zhou, s., et al., the porous structure and mechanical properties of injection molded ha/pa66 scaffolds. international polymer processing, 2014. 29(4): p. 454-460. 13. pei, d., et al., influence of nano-hydroxyapatite containing desensitizing toothpastes on the sealing ability of dentinal tubules and bonding performance of selfetch adhesives. journal of the mechanical behavior of biomedical materials, 2019. 91: p. 38-44. 14. veljović, d., et al., the effect of the shape and size of the pores on the mechanical properties of porous hap-based bioceramics. ceramics international, 2011. 37(2): p. 471-479. 15. mendelson, b.c., et al., the fate of porous hydroxyapatite granules used in facial skeletal augmentation. aesthetic plastic surgery, 2010. 34(4): p. 455-461. mechanical characteris.cs of teeth restored with innova.ve func.onally graded posts made of titaniumhydroxyapa.te-bioac.ve glass vol 13 no 1 (2025) doi 10.5195/d3000.2025.877 h#p://den*stry3000.pi#.edu 16. li, y.-h., f. wang, and j.-j. li, current developments of biomedical porous ti–mo alloys. international journal of materials research, 2017. 108(8): p. 619624. 17. arifin, a., et al., material processing of hydroxyapatite and titanium alloy (ha/ti) composite as implant materials using powder metallurgy: a review. materials & design, 2014. 55: p. 165-175. 18. zhao, d., et al., effect of pore geometry on the fatigue properties and cell affinity of porous titanium scaffolds fabricated by selective laser melting. journal of the mechanical behavior of biomedical materials, 2018. 88: p. 478-487. 19. fathyunes, l., j. khalil-allafi, and m. moosavifar, development of graphene oxide/calcium phosphate coating by pulse electrodeposition on anodized titanium: biocorrosion and mechanical behavior. journal of the mechanical behavior of biomedical materials, 2019. 90: p. 575-586. 20. ning, c. and y. zhou, on the microstructure of biocomposites sintered from ti, ha and bioactive glass. biomaterials, 2004. 25(17): p. 3379-3387. 21. moskalenko, v. and a. smirnov, temperature effect on formation of reorientation bands in α-ti. materials science and engineering: a, 1998. 246(1-2): p. 282-288. 22. prasad, s., et al., crystallization and mechanical properties of (45s5-ha) biocomposite for biomedical implantation. ceramics–silikáty, 2017. 61(4): p. 378-384. 23. abdulmunem, m., et al., the effect of bioactive glass and sintering conditions on the properties of titaniumhydroxyapatite composites. sains malaysiana, 2021. 50(4): p. 10891099. 24. joshi, s., et al., mechanical performance of endodontically treated teeth. finite elements in analysis and design, 2001. 37(8): p. 587-601. 25. genovese, k., l. lamberti, and c. pappalettere, finite element analysis of a new customized composite post system for endodontically treated teeth. journal of biomechanics, 2005. 38(12): p. 2375-2389. 26. dejak, b., a. młotkowski, and c. langot, three-dimensional finite element analysis of molars with thin-walled prosthetic crowns made of various materials. dental materials, 2012. 28(4): p. 433-441. 27. abdulmunem, m., et al., evaluation of the effect of dental cements on fracture resistance and fracture mode of teeth restored with various dental posts: a finite element analysis. journal of the european ceramic society, 2016. 28. al-omiri, m.k., m.r. rayyan, and o. abu-hammad, stress analysis of endodontically treated teeth restored with post-retained crowns: a finite element analysis study. the journal of the american dental association, 2011. 142(3): p. 289-300. 802 final microbial insights in den2stry: implica2ons for oral and systemic health vol 13 no 1 (2025) doi 10.5195/d3000.2025.802 h#p://den*stry3000.pi#.edu microbial insights in dentistry: implications for oral and systemic health manar ibrahim ahmed1, marwa siddik abdulrazzaq2, huda muneer hashim2 1 al-bayan university, college of dentistry, baghdad, iraq 2 al hikma university college, baghdad, iraq abstract the rela(onship between microbiology and den(stry is a cornerstone for understanding oral and systemic health dynamics. the oral cavity serves as a habitat for a diverse microbial ecosystem, which contributes to both health and disease. while a balanced oral microbiome is essen(al for homeostasis, its dysbiosis is implicated in dental diseases such as caries and periodon((s, and systemic condi(ons including cardiovascular disease, diabetes, and adverse pregnancy outcomes. this review delves into the role of the oral microbiome in maintaining health, the challenges of an(microbial resistance, and the advancements in microbial diagnos(cs and therapeu(cs. it also highlights the poten(al of emerging technologies and microbial-based therapies in advancing personalized den(stry. emphasizing a mul(disciplinary approach, this short review provides a comprehensive understanding of how microbial research can revolu(onize dental prac(ces and pa(ent outcomes. keywords: microbial, den(stry, oral health, systemic health. cita9on: ahmed mi, et al (2025) microbial insights in den9stry: implica9ons for oral and systemic health. den9stry 3000.1:a001 doi:10.5195/d3000.2025.802 received: december 18, 2024 accepted: january 5, 2025 published: february 12, 2025 copyright: ©2025 ahmed mi, et al. this is an open access ar9cle licensed under a crea9ve commons auribu9on work 4.0 united states license. email: manar.i@albayan.edu.iq introduction microbiology has played a pivotal role in transforming dentistry into a more precise and preventive discipline. the oral cavity is home to a dynamic and diverse microbial community, comprising over 700 species of bacteria, as well as fungi, viruses, and protozoa [1]. these microorganisms exist in a delicate balance, contributing to health by preventing colonization by pathogens and modulating the host's immune system [2]. however, factors such as poor oral hygiene, dietary habits, and systemic diseases can disrupt this equilibrium, leading to dysbiosis. this disruption is central to the etiology of dental diseases, including caries, periodontitis, and peri-implantitis. the interplay between oral microbiota and systemic health further underscores the importance of microbial research in dentistry. data revealed how oral pathogens contribute to systemic inflammation, cardiovascular diseases, and metabolic disorders, indicating that oral health is intrinsically linked to overall health [3]. this review provides a short analysis of the oral microbiome's role in health and disease, antimicrobial resistance in dental settings, and emerging diagnostic and therapeutic approaches that leverage microbiological insights. the oral microbiome and its impact on health composition and function of the oral microbiome the oral microbiome consists of commensal, symbiotic, and opportunistic microorganisms that collectively form biofilms on oral surfaces. in a healthy state, these biofilms contribute to the immune defense and metabolic functions by maintaining a stable ph and providing competitive exclusion of microbial insights in den2stry: implica2ons for oral and systemic health vol 13 no 1 (2025) doi 10.5195/d3000.2025.802 h#p://den*stry3000.pi#.edu pathogenic species [4]. this homeostasis is essential for oral health, as disruptions can lead to an overgrowth of pathogenic species. dental caries dental caries represents a classic example of how microbial dysbiosis contributes to disease. streptococcus mutans, considered the primary cariogenic bacterium, metabolizes dietary sugars into acids, creating an acidic environment that demineralizes tooth enamel [2]. other acidogenic species, such as lactobacilli and actinomyces, further exacerbate this process, resulting in progressive tooth decay. recent research also highlights the role of microbial communities rather than individual pathogens in caries development, emphasizing the importance of biofilm interactions [2]. periodontal disease periodontal disease is characterized by chronic inflammation of the gingiva and destruction of the supporting structures of the teeth. it is driven by a dysbiotic shift in the subgingival microbiome, with pathogens such as porphyromonas gingivalis, tannerella forsythia, and treponema denticola playing key roles [3]. these pathogens secrete proteolytic enzymes and toxins that evade host immune responses and induce tissue destruction. the inflammatory response further perpetuates microbial dysbiosis, creating a self-sustaining cycle of tissue damage. oral-systemic health connections the implications of oral microbiota extend beyond the oral cavity. pathogens like p. gingivalis have been implicated in systemic diseases, including cardiovascular disease and alzheimer's disease, through mechanisms such as systemic inflammation and molecular mimicry. similarly, oral dysbiosis has been linked to adverse pregnancy outcomes, respiratory infections, and metabolic syndrome [5]. antimicrobial resistance in dentistry the rise of antimicrobial resistance (amr) presents a significant challenge to dental care. overuse and misuse of antibiotics in dentistry, particularly for prophylactic purposes, have accelerated the development of resistant strains such as enterococcus faecalis and aggregatibacter actinomycetemcomitans [3]. these resistant pathogens complicate the management of endodontic infections, periodontal disease, and post-surgical complications. strategies to combat amr [6] 1. prudent antibiotic stewardship: guidelines emphasizing evidencebased antibiotic use are crucial in reducing unnecessary prescriptions. 2. adjunctive therapies: photodynamic therapy (pdt) and antimicrobial peptides (amps) offer alternative approaches to managing resistant infections. 3. novel antimicrobials: research on nanoparticles and biofilm-disrupting agents provides promising avenues for overcoming resistance in biofilmassociated infections. advances in microbial diagnostics and therapeutics diagnostic innovations • next-generation sequencing (ngs): provides comprehensive profiling of oral microbial communities, enabling early detection of microbial insights in den2stry: implica2ons for oral and systemic health vol 13 no 1 (2025) doi 10.5195/d3000.2025.802 h#p://den*stry3000.pi#.edu dysbiosis-related diseases [1]. • metabolomics and proteomics: shed light on microbial metabolic pathways and their interactions with host tissues, aiding in biomarker discovery [4]. • point-of-care diagnostics: tools such as polymerase chain reaction (pcr) enable rapid and accurate identification of oral pathogens in clinical settings. microbial-based therapies 1. probiotics: beneficial strains like lactobacillus reuteri and bifidobacterium spp. are being explored for their ability to restore microbial balance and prevent oral diseases [7]. 2. biofilm-disrupting agents: enzymes and nanoparticles targeting biofilm matrix components are emerging as effective adjuncts to conventional therapies [6]. 3. vaccines: experimental vaccines targeting cariogenic bacteria such as streptococcus mutans are under development, offering potential for preventive dentistry [6]. discussion the integration of microbiological research into dental practice has transformed our understanding of oral and systemic health. while advancements in diagnostics and therapeutics show promise, challenges remain in translating laboratory findings into clinical applications. for example, the complexity of microbial interactions within biofilms poses difficulties in developing universally effective treatments. additionally, addressing amr requires concerted efforts in research, policy-making, and clinical practice. conclusion microbiology is a cornerstone of modern dentistry, offering insights into the etiology and management of oral diseases. by embracing advancements in microbial diagnostics and therapeutics, dentistry can transition toward personalized and preventive care. future research should focus on refining diagnostic tools, developing microbial-based therapies, and exploring the oralsystemic health interface. a multidisciplinary approach will be crucial in addressing the challenges and harnessing the opportunities presented by microbial research. references 1zaura e, keijser bj, huse sm, crielaard w. defining the healthy “core microbiome” of oral microbial communities. bmc microbiol. 2014;14:122. 2. marsh pd. dental plaque as a biofilm: the significance of ph in health and caries. compend contin educ dent. 2009;30(2):76-86. 3. hajishengallis g. periodontitis: from microbial immune subversion to systemic inflammation. nat rev immunol. 2015;15(1):30–44. 4. teles r, wang y, hajishengallis g. host-microbiome cross-talk in periodontal disease. j dent res. 2013;92(5):367-373. 5. scannapieco fa. the oral microbiome: its role in health and in oral and systemic infections. clin microbiol newsl. 2013;35(20):163-169. 6. donlan rm. biofilms: microbial life on surfaces. emerg infect dis. 2002;8(9):881–890. microbial insights in den2stry: implica2ons for oral and systemic health vol 13 no 1 (2025) doi 10.5195/d3000.2025.802 h#p://den*stry3000.pi#.edu 7. gruner d, paris s, schwendicke f. probiotics for managing caries and periodontitis: systematic review and meta-analysis. j dent. 2016;48:16-25. 717 2024 inspec'ng candida oral infec'ons among diabetes vol 12 no 2 (2024) doi 10.5195/d3000.2024.717 h#p://den*stry3000.pi#.edu inspecting candida oral infections among diabetics abeer mohammed ali al-garawyi1, sabreen sami abed2, mortadha sami abd3, majid mohammed mahmood4* 1 al-muthanna university college of medicine, al-muthanna, iraq. 2 al hikma university college, baghdad, iraq. 3 osol al-elm university college, baghdad, iraq. 4 mustansiriyah university college of science, baghdad, iraq. * corresponding author abstract objec&ve: oral candidiasis infec-on is more prevalent in diabe-c pa-ents. there are numerous factors that can exacerbate the coloniza-on of candida species in the oral cavity, including salivary ph disorders and xerostomia. the study aimed to evaluate candida spp. resistance to an-fungal agents and compare their coloniza-on levels in diabe-cs and nondiabe-cs oral cavi-es. methods: we conducted the inves-ga-on from february 2023 to april 2023. we conducted the following analyses ager collec-ng 100 oral samples: gram stain, culture on sabaroud dextrose agar, and direct microscopic inspec-on. the vitek 2 system confirmed the yeasts through carbohydrate assimila-on profiles. results: out of 100 oral samples cultured, 69 yielded candida species. figy-two samples were from diabe-cs and 17 were from nondiabe-c pa-ents. the frequencies of isolated candida species, including c. albicans, were 28, c. tropicalis 17, c. krusei 16, c. glabrata 6, and c. dubliniensis 2. the results indicated that c. albicans exhibited higher resistance rates against clotrimazole, itraconazole, and voriconazole than the no albicans candida species. clotrimazole, itraconazole, and voriconazole, on the other hand, showed no effect on 11, 29, and 18 samples for all candida species, respec-vely. conclusion: c. albicans was the most prevalent candida species in people with diabetes; however, other candida species were common. fluconazole and nysta-n ogen treat oral candida infec-ons. keywords: diabetes mellitus, oral candidiasis, an-fungal, candida albicans, nysta-n. cita:on: al-garawyi a, et al. (2024) inspec:ng candida oral infec:ons among diabe:cs. den:stry 3000. 1:a001 doi:10.5195/d3000.2024.717 received: august 28, 2024 accepted: september 1, 2024 published: september 30, 2024 copyright: ©2024 al-garawyi a, et al. this is an open access ar:cle licensed under a crea:ve commons auribu:on work 4.0 united states license. email: majidmahmood93@yahoo.com introduction one of the most prevalent opportunistic fungal diseases in humans is oral candidiasis. of the 150 species of this genus that have been isolated from the oral cavity, candida albicans is the source of most of them (80%) [1,2]. patients with diabetes, immunocompromised patients, and neutropenic patients frequently encounter candida infections, which significantly contribute to nosocomial infections [3]. candida, which comes from the latin word candid, is a harmless, two-skinned fungus that can make invasive, diseasecausing pseudohyphae [4]. diabetes mellitus is a metabolic disease marked by a partial or total reduction in the pancreatic production of insulin, either because of inadequate insulin production or an autoimmune reaction that affects the beta cells that are responsible for synthesizing insulin. hyperglycemia, a condition caused by elevated blood sugar levels, can be particularly harmful to people with diabetes, especially those who have had the condition for an extended period. this is because it can induce a variety of physiological responses [5]. it is very important to figure out the types of candida that cause infections because different isolates of candida species have different levels of ability to cause infections [6] and resistance to antifungal drugs [7]. so, the main goal of this investigation was to identify the candida species in people with and without diabetes, as well as to investigate how sensitive people with oral candidiasis are to antifungals. material and methods sample collection: inspec'ng candida oral infec'ons among diabetes vol 12 no 2 (2024) doi 10.5195/d3000.2024.717 h#p://den*stry3000.pi#.edu the current study included 100 oral samples from each diabetic and nondiabetic patients attending hospitals in al-muthanna governorate. they were over 45 years old and of both sexes. individuals were recruited from february 2023 to april 2023. specialist doctors conducted the clinical examinations. furthermore, the doctors questioned each patient about their general health history, age, sex, use of antibiotics, alcohol consumption, and other risk factors for candida infection. we took samples using a sterile cotton swab and transported them to the laboratory for testing. candida isolation and identification: to determining the occurrence of candida spp. and differentiating among species, many tests were performed on all oral samples, such as gram stain [8], streaked on sabouraud dextrose agar [mumbai, india] containing 0.5 mg per 1000 ml chloramphenicol [9,10], and direct microscopic examination [11]. the yeasts were confirmed via carbohydrate assimilation profiles using the vitek 2 system (biomerieux, france) according to the manufacturer’s instructions. in vitro antifungal susceptibility tests: we selected five to six candida species colonies from a 24-hourold culture on an sda plate, inoculated them in 5 ml of sterile saline, and adjusted their turbidity to 0.5 mcfarland standards to create a suspension. the excess fluid was removed by rolling a sterile cotton wool swab on the tube's inside surface, moistened in the adjusted inoculum suspension, and then distributed on the mullerhinton agar surface to form a lawn [12,13]. the disk diffusion method was employed to conduct antifungal susceptibility testing. the clinical laboratory standard institute (clsi) recommended that antifungal discs (thermo scientifictm oxoidtm) be applied to mha (thermo scientifictm oxoidtm) using disk dispensers (oxoidtm). the discs contained voriconazole (10 μg), clotrimazole (10 μg), fluconazole (10 μg), itraconazole (10 μg), and nystatin (100 iu). the dishes were incubated at 35°c in ambient air for 24 hours. the clinical and laboratory standard institute (clsi) interpretation criteria for voriconazole, fluconazole, nystatin, itraconazole, and clotrimazole are shown in table 1 [14]. table 1. based on [14] interpretative criteria for resistance and susceptibility to utilized antifungal disks (mm). antifungal agent sensitive dose dependent resistance fluconazole ≥19 15-18 ≤ 14 nystatin ≥ 25 17-24 < 16 clotrimazole >20 12-19 ≤ 11 itraconazole > 16 10-15 < 9 voriconazole ≥19 15–18 ≤14 results the present study included the collection of one hundred (100) oral samples from 69 diabetic patients and 31 nondiabetic patients. the patients ages ranged from 45 to 80 years. out of 69 oral inspec'ng candida oral infec'ons among diabetes vol 12 no 2 (2024) doi 10.5195/d3000.2024.717 h#p://den*stry3000.pi#.edu swabs from diabetic patients, candida species were isolated from 52 samples (table 2). in contrast, candida spp infection was detected in only 17 (54.8%) oral swab samples from nondiabetic patients. the statistical significance of the difference between these two categories was indicated by the p-value of 0.05. the study found that diabetic patients were 1.37 times more likely to contract candida spp. infection than nondiabetic patients, while nondiabetic patients have a relative risk of 0.54, as mentioned in table 2. table 2. candida infection rates in patient groups with and without diabetes. groups positive negative total odds ratio relative risk for diabetic patient relative risk for nondiabetic patient diabetic 52 17 69 2.51 1.37 0.54 nondiabetic 17 14 31 total 69 31 100 we identified five species of candida. among 69 patients with and without diabetes, candida albicans was the most common agent 28, followed by candida tropicalis 17, candida krusei 16, candida glabrata 6, and candida dubliniensis 2, as displayed in figure 1. figure 1. various species of candida isolated from oral cavity among diabetics and nondiabetics. the prevalence of candida spp. infection in diabetic and nondiabetic patients was analyzed using the chi square test, and the results are summarized in table 3. we found no statistically significant difference in the percentage of positive candida infection cases between diabetic and nondiabetic patients across different age groups (p > 0.05). table:3 demographical screening of candida load in diabetic patients in comparison to nondiabetics. 40.6% 24.6% 23.2% 8.7% 2.9% 0.00% 5.00% 10.00% 15.00% 20.00% 25.00% 30.00% 35.00% 40.00% 45.00% c. albicans c. tropicalis c. krusei c. glabrata c.dubliniensis inspec'ng candida oral infec'ons among diabetes vol 12 no 2 (2024) doi 10.5195/d3000.2024.717 h#p://den*stry3000.pi#.edu age groups diabetic nondiabetic total x2 p value 45-55 17 5 22 1.29 0.52 56-66 20 9 29 ˃ 67 15 3 18 total 52 17 69 x2= 1.29, df=2, p> 0.05 according to the chi-square test, we found a notable disparity in the occurrence of candida spp. infection depending on sex. table 4 shows a significant (p< 0.05) proportion of positive cases for candida among patient with and without diabetic. table 4. the frequency of candida infections depending on sex. sex diabetic nondiabetic total x2 p value males 37 6 43 7.01 0.008 females 15 11 26 total 52 17 69 x2= 7.01, df=1, p< 0.05 we found that 20 diabetic patients had oral ulcers, in whom the frequency of candida spp. was 90.9%, while in those who were negative for oral ulcers, it was 68.1%. the observed difference was statistically significant (p< 0.05), as shown in table 5. table 5. the frequency of candida infections depending on oral ulcer. oral ulcer diabetic nondiabetic total x2 p value positive 20 2 22 4.20 0.04 negative 32 15 47 total 52 17 69 x2= 4.20, df=1, p< 0.05 we investigated the frequency of candida spp. in relation to hypertension using the chi square test. table 6 shows that out of the 52 diabetic individuals with a candida infection, 71.4% had inspec'ng candida oral infec'ons among diabetes vol 12 no 2 (2024) doi 10.5195/d3000.2024.717 h#p://den*stry3000.pi#.edu hypertension and 77.1% did not have hypertension (p > 0.05). table 6. the frequency of candida infections depending on hypertension. hypertension diabetic nondiabetic total x2 p value positive 15 6( 21 0.25 0.61 negative 37 11 48 total 52 17 69 x2=0.25, df=1, p> 0.05 the sensitivity test results of all candida spp. isolates toward all antifungal agents in the current study are shown in table 7. candida albicans, the most isolated species, was shown to be responsive to fluconazole, nystatin, clotrimazole, itraconazole, and voriconazole at ratios of 36.2%, 5.8%, 13%, 20.3%, and 15.9%, respectively. candida albicans, the most isolated of 69 candida species, was responsive to fluconazole, nystatin, clotrimazole, itraconazole, and voriconazole at 36.2%, 5.8%, 13%, 20.3%, and 15.9%, respectively. conversely, fluconazole, nystatin, clotrimazole, itraconazole, and voriconazole had no effect in 0%, 0%, 8.7%, 15.9%, and 10.1%, respectively. out of the 17 c. tropicalis isolates, 15.9% were sensitive to fluconazole, 21.7% to nystatin, 5.8% to clotrimazole, 7.2% to itraconazole, and 14.5% to voriconazole, while 4.3%, 8.7%, and 7.2% were resistant to clotrimazole, itraconazole, and voriconazole, respectively. in addition, 2.9% were resistant to clotrimazole, 11.6% to itraconazole, and 4.3% to voriconazole. regarding c. glabrata, the findings showed that 4.3% were susceptible to fluconazole, nystatin, clotrimazole, itraconazole, and voriconazole, respectively; 2.9% and 4.3% were resistant to itraconazole and voriconazole, respectively. additionally, of the two c. dubliniensis isolates that were found, both were resistant to itraconazole. table 7. antifungal susceptibility testing of candida spp. isolates. fluconazole c. albicans c. tropicalis c. krusei c. glabrata c. dubliniensis total sensitive 25 11 15 3 2 56 dose dependent 3 6 1 3 0 13 resistance 0 0 0 0 0 0 total 28 17 16 6 2 69 nystatin sensitive 4 15 12 4 2 37 inspec'ng candida oral infec'ons among diabetes vol 12 no 2 (2024) doi 10.5195/d3000.2024.717 h#p://den*stry3000.pi#.edu dose dependent 24 2 4 2 0 32 resistance 0 0 0 0 0 0 total 28 17 16 6 2 69 clotrimazole sensitive 9 4 5 2 2 22 dose dependent 13 10 9 4 0 36 resistance 6 3 2 0 0 11 total 28 17 16 6 2 69 itraconazole sensitive 14 5 4 2 0 25 dose dependent 3 6 4 2 0 15 resistance 11 6 8 2 2 29 total 28 17 16 6 2 69 voriconazole sensitive 11 10 6 3 2 32 dose dependent 10 2 7 0 0 19 resistance 7 5 3 3 0 18 total 28 17 16 6 2 69 discussion globally, diabetes is a serious public health issue [15]. the study confirms that diabetes mellitus is a significant risk factor for symptomatic candidosis, whether oral or otherwise, in iraqi patients with diabetes mellitus, as previously reported [16,17]. this is also consistent with several other studies, which have all shown that diabetes mellitus increases candida colonization and growth [18,19]. according to research by premkumar et al. [20], the most found species was c. albicans. however, they also saw c. parapsilosis, c. tropicalis, and c. dubliniensis. candida albicans was the most prevalent species (43.1%) in the oral cavity of diabetes patients, as discovered by mohammadi et al. [21]. conversely, the prevalence of candida spp. in the oral cavity of nondiabetic controls was lower (27%). factors such as increased salivary glucose, ph, flow reduction, and smoking habits contribute to candida colonization [22]. we investigated the prevalence of candida spp. in diabetic and nondiabetic populations according to age and the results indicated that there was inspec'ng candida oral infec'ons among diabetes vol 12 no 2 (2024) doi 10.5195/d3000.2024.717 h#p://den*stry3000.pi#.edu no statistically significant difference in the percentage of positive cases in diabetic and nondiabetic patients. al-awadhi et al. [23] found no association between diabetes and infection or age. twenty diabetic individuals during our study reported oral ulcers. we examined the prevalence of candida spp. in diabetic patients with oral ulcers and found a possible relationship between oral ulcers and presence of candida spp. in 90.9% of diabetic individuals with mouth ulcers compared to 68.1% in those without ulcers. additional research is warrented to confirm these findings and explore potential underlying causes. our study found no statistically significant difference in the prevalence of candida spp. between hypertension diabetic patients and no hypertensive diabetic patients. candida spp. was more common specifically among hypertension diabetic patients (77.1%) than among hypertensive nondiabetic patients (28.6%). comparing c. albicans to other no albicans candida species, the findings showed that itraconazole, variconazole, and clotrimazole demonstrated increased resistance rates. this result is in line with another study, which showed that c. albicans was more resistant to clotrimazole and itraconazole than no albicans candida species [24]. the presence of point mutations, insertions, and deletions in the genes encoding target proteins in c. albicans may explain these results. these mutations often cause antifungal medication resistance. gene overexpression often upregulates oxidative damage and antifungal resistance proteins. gene overexpression of the multidrug efflux pump is one example [25,26]. however, a significant level of resistance was detected in itraconazole and voriconazole, with rates of 42% and 26.1%, respectively, for all candida species. this conclusion aligns with the results of earlier investigations [27,28]. the present investigation demonstrated that fluconazole and nystatin were effective against all isolated candida spp., with a sensitivity rate of 81.2% and 53.6%, respectively. this information is in accordance with numerous other studies [29,30]. consequently, nystatin typically functions by interacting with ergosterol and distracting the fungal cytoplasmic membrane. nystatin creates the pores in the cell membrane that serve as an exit for magnesium cellular components and potassium ions. this damages the proton gradient of the cell membrane, thereby promoting fungal cell death. nystatin binds ergosterol somewhat well and binds 3 hydroxy or oxysterol rather poorly. consequently, the indications are less than those of the azole group [31]. fluconazole inhibits the formation of ergosterol, a crucial component of the fungal cell membrane, by interacting with 14demethylase, a cytochrome p-450 enzyme. fluconazole prevents yeast formation and endogenous respiration by preventing sterol loss, which is parallel to the accumulation of 14-methyl sterols in fungi, which is the primary cause of its perceived fungistatic activity [32]. fluconazole and nystatin are recommended antifungals for treating candida infections due to their susceptibility to most fungal species. conclusion we found a high prevalence of candida spp. and a greater frequency of candida species in individuals with diabetes compared to those without the disease, indicating an association between diabetes and candida infection. this investigation also found a nonsignificant correlation between mouth ulcers, hypertension, and candida infection. while there is a substantial variation in the incidence of candida infection depending on sex, there is no significant correlation between age inspec'ng candida oral infec'ons among diabetes vol 12 no 2 (2024) doi 10.5195/d3000.2024.717 h#p://den*stry3000.pi#.edu groups and candida infection. with a frequency of 40.6%, c. albicans was the most often found candida species; c. dubliniensis was the least common (2.9%). fluconazole and nystatin showed a high degree of sensitivity among candida species, suggesting that fluconazole is the more effective therapy for oral candida infections. regular monitoring of candida infections is crucial as they potentially pose a risk for developing diabetes. the research supports informing the general public about candida infections and their health consequences. conflicts of interest the authors declare no competing interest. references 1. cortegiani, a., russoho, v., raineri, s. m., gregorei, c., and giarratano, a. (2016). should we administer anjfungal drugs before the diagnosis of invasive fungal infecjon in non-neutropenic crijcally ill pajents? turkish journal of anaesthesiology and reanimajon, 44(6), 276-278. 2. bharathi, m., and rani, a. u. (2011). pathogenic fungal isolates in sputum of hiv posijve pajents. j aids hiv res, 3(6), 107113. 3. al-awadhi, e., al-towij, h., al-qadri, a., alhashdi, s., alkhawlani, n., alhamoodi, a. (2023). isolajon and idenjficajon of candida species from oral of diabejc pajents. j bacteriol mycol open access, 11(2),77‒80. 4. byadarahally raju, s., and rajappa, s. (2011). isolajon and idenjficajon of candida from the oral cavity. internajonal scholarly research nojces, 2011(2011):1-7. 5. zimpel, b. t., da silva, g. m., naressi, j. s., seibt, l. t., neto, v. e. d. n., and kohl, v. t. (2017). diabéjcos: uma abordagem odontológica. revista saúde integrada, 10(20), 49-58. 6. allen, c. m., saffer, a., meister, r. k., beck, f. m., and bradway, s. (1994). comparison of a lesion-inducing isolate and a nonlesional isolate of candida albicans in an immunosuppressed rat model of oral candidiasis. journal of oral pathology & medicine, 23(3), 133-139. 7. mcilroy, m. a. (1991). failure of fluconazole to suppress fungemia in a pajent with fever, neutropenia, and typhlijs. journal of infecjous diseases, 163(2), 420421. 8. schwebke, j. r., hillier, s. l., sobel, j. d., mcgregor, j. a., and sweet, r. l. (1996). validity of the vaginal gram stain for the diagnosis of bacterial vaginosis. obstetrics & gynecology, 88(4), 573-576. 9. finegold, s.m. and baron, e.j. (2001). diagnosjc microbiology. bailey and scoh. 19th ed., mosby company. philadelphia. 10. gültekin, b., yazici, v., and aydin, n. (2005). distribujon of candida species in vaginal specimens and evaluajon of chromagar candida medium. mikrobiyoloji bulteni, 39(3), 319324. 11. mcgregor, j. a., french, j. i., jones, w., parker, r., paherson, e., and draper, d. (1992). associajon of cervicovaginal infecjons with increased vaginal fluid phospholipase a2 acjvity. american journal of obstetrics and gynecology, 167(6), 1588-1594. 12. al-garawyi, a.m. (2019). isolajon and idenjficajon of candida spp.on chromagar causing candidiasis in pregnant women and anjfungal suscepjbility test. journal of internajonal pharmaceujcal research 46(5): 471-475. 13. khan, m., ahmed, j., gul, a., ikram, a., and lalani, f. k. (2018). anjfungal suscepjbility tesjng of vulvovaginal candida species among women ahending antenatal clinic in terjary care hospitals of peshawar. infecjon and drug resistance, 447-456. 14. clsi. (2009). m44-a2: method for anjfungal disk diffusion suscepjbility tesjng of yeasts; approved guideline— inspec'ng candida oral infec'ons among diabetes vol 12 no 2 (2024) doi 10.5195/d3000.2024.717 h#p://den*stry3000.pi#.edu second edijon. clsi doc, 29(17), m44-a2. 15. ajlouni, k., jaddou, h., and bajeha, a. (1998). diabetes and impaired glucose tolerance in jordan: prevalence and associated risk factors. journal of internal medicine, 244(4), 317-323. 16. abu-elteen, k. h., and abualteen, r. m. (1998). the prevalence of candida albicans populajons in the mouths of complete denture wearers. the new microbiologica, 21(1), 41-48. 17. de leon, e. m., jacober, s. j., sobel, j. d., and foxman, b. (2002). prevalence and risk factors for vaginal candida colonizajon in women with type 1 and type 2 diabetes. bmc infecjous diseases, 2, 1-6. 18. peer, a. k., hoosen, a. a., seedat, m. a., van den ende, j., and omar, m. a. k. (1993). vaginal yeast infecjons in diabejc women. south african medical journal, 83(10), 727-729. 19. darwazeh, a. m. g., lamey, p. j., samaranayake, l. p., macfarlane, t. w., fisher, b. m., macrury, s. m., and maccuish, a. c. (1990). the relajonship between colonisajon, secretor status and in-vitro adhesion of candida albicans to buccal epithelial cells from diabejcs. journal of medical microbiology, 33(1), 43-49. 20. premkumar, j., ramani, p., chandrasekar, t., natesan, a., and premkumar, p. (2014). detecjon of species diversity in oral candida colonizajon and anj-fungal suscepjbility among non-oral habit adult diabejc pajents. journal of natural science, biology, and medicine, 5(1), 148.-154. 21. mohammadi, f., javaheri, m. r., nekoeian, s., and dehghan, p. (2016). idenjficajon of candida species in the oral cavity of diabejc pajents. current medical mycology, 2(2), 1-7. 22. darwazeh, a. m. g., macfarlane, t. w., mccuish, a., and lamey, p. j. (1991). mixed salivary glucose levels and candidal carriage in pajents with diabetes mellitus. journal of oral pathology & medicine, 20(6), 280-283. 23. al-awadhi, e., al-towij, h., al-qadri, a., alhashdi, s., alkhawlani, n., alhamoodi,a.(2023). isolajon and idenjficajon of candida species from oral of diabejc pajents. j bacteriol mycol open access, 11(2):77‒80. 24. zarrinfar, h., kord, z., and fata, a. (2021). high incidence of azole resistance among candida albicans and c. glabrata isolates in northeastern iran. current medical mycology, 17(3), 18-21. 25. costa-de-oliveira, s., and rodrigues, a. g. (2020). candida albicans anjfungal resistance and tolerance in bloodstream infecjons: the triad yeast-hostanjfungal. microorganisms, 8(2), 1-154. 26. lee, y., puumala, e., robbins, n., and cowen, l. e. (2020). anjfungal drug resistance: molecular mechanisms in candida albicans and beyond. chemical reviews, 121(6), 3390-3411. 27. mohamadi, j., havasian, m. r., panahi, j., and pakzad, i. (2015). anjfungal drug resistance pahern of candida. spp isolated from vaginijs in ilam-iran during 2013-2014. bioinformajon, 11(4), 203-206. 28. yenisehirli, g., bulut, n., yenisehirli, a., and bulut, y. (2015). in vitro suscepjbilijes of candida albicans isolates to anjfungal agents in tokat, turkey. jundishapur journal of microbiology, 8(9),0-4. 29. mohamadi, j., motaghi, m., havasian, m. r., delpisheh, a., azizian, m., and pakzad, i. (2014). anj-fungal resistance in candida isolated from oral and diaper rash candidiasis in neonates. bioinformajon, 10(11), 667-670. 30. dagi, h. t., findik, d., senkeles, c., and arslan, u. (2016). idenjficajon and anjfungal suscepjbility of candida species isolated from bloodstream infecjons in konya, turkey. annals of clinical microbiology and anjmicrobials, 15(1), 1-5. inspec'ng candida oral infec'ons among diabetes vol 12 no 2 (2024) doi 10.5195/d3000.2024.717 h#p://den*stry3000.pi#.edu 31. sariguzel, f., berk, e., koc, a., sav, h., and aydemir, g. (2015). evaluajon of chromagar candida, vitek2 yst and vitek® ms for idenjficajon of candida strains isolated from blood cultures. infez med, 23(4), 318-22. 32. spampinato, c., and leonardi, d. (2013). candida infecjons, causes, targets, and resistance mechanisms: tradijonal and alternajve anjfungal agents. biomed research internajonal, 2013(2013),204-237. 764 2024 salivary caries-related microorganisms and demographics of a group of children vol 13 no 1 (2025) doi 10.5195/d3000.2025.764 h#p://den*stry3000.pi#.edu salivary caries-related microorganisms and demographics of a group of children alhan ahmed qasim, muna saleem khalaf, ghada ibrahim taha, zainab juma jafar college of dentistry, university of baghdad, baghdad, iraq abstract objec&ve: the purpose of this study was to determine the associa4on between caries related microorganisms in children’s saliva, such as streptococcus mutans and lactobacilli, and their demographic factors. methods: this study involved a sample of 135, both sexes with an age range between 3 and 10 years. uns4mulated saliva was obtained and diluted in normal saline. saliva was then placed in selec4ve media. salivaris agar was used for mutans streptococci while rogosa agar for lactobacilli. ader incuba4on, streptococcus mutans coun4ng of cfu (colony forming units) with morphology characteriza4on and numbers of cfu per milliliter of saliva for lactobacilli. demographic factors informa4on was collected using a ques4onnaire. results: both studied salivary microorganisms increased with age. streptococcus mutans was higher in boys, and associated with breasneeding, overweight, and not brushing their teeth. lactobacilli were higher in girls, and associated with mothers who were government employee, mother occupa4on, children who during sleep were not nursed, and with normal weight. conclusion: the present study found an associa4on between salivary streptococcus mutans and lactobacilli and various demographic variables in a group of children. keywords: saliva, streptococcus mutans, lactobacilli, health, demographics. cita;on: qasim aa, et al. (2025) salivary caries-related microorganisms and demographics of a group of children. den;stry 3000. 2.a001 doi:10.5195/d3000.2025.764 received: october 30, 2024 accepted: november 7, 2024 published: january 8. 2025 copyright: ©2025 qasim aa, et al. this is an open access ar;cle licensed under a crea;ve commons axribu;on work 4.0 united states license. email: dr.alhan_altaai@codental.uobaghdad.edu.iq introduction the surfaces of the oral cavity are covered by salivary fluids, which is produced via the salivary glands; this fluid has definitive essential roles for maintaining oral healthy (1]. many bacteria are present in saliva [2]. in fact, saliva secreted into the oral cavity from salivary glands is sterile [3], but when washing with saliva, bacteria shed to saliva from different surfaces in the oral cavity including oral mucosa, gingival crevices, and dental plaque, but the main site of origins are throat, tongue and the tonsils [4]. the microbiota of saliva contains many species of bacteria that are temporally stable and have been shown to be individualized, playing a crucial role in disease as dental caries and health of oral cavity [5]. public policies on oral health consistently address several factors associated with dental caries like oral hygiene rule, dietary habits, genetics, oral health knowledge, demographics and sociocultural environment. many studies have revealed an influence of diet and lifestyle on microbiota of saliva [6,7]. it is a fact that dental developmental stages affected the microbiome of saliva. key factors include teeth eruption, age, oral hygiene practices and dietary changes [8]. dental caries is a chronic disease which is widespread and affect people of different ages and demographics [9]. it is caused by bacterial acid result from fermentation of free sugars and poor oral hygiene, leading to localized damage to the hard tooth tissue, associated with the structure of deciduous and permanent teeth [10,11]. most types of cariogenic bacteria were found to differ according to host, age and race. bacteria vary among individuals of the same race. investigations have employed salivary caries-related microorganisms and demographics of a group of children vol 13 no 1 (2025) doi 10.5195/d3000.2025.764 h#p://den*stry3000.pi#.edu saliva to determine the occurrence of caries by exploring levels of the bacteria. as many cariogenic bacteria are present in saliva. mutans streptococcus (ms) is categorized as primary contributors to dental caries. another bacteria strain set linked to the advancing of dental caries is lactobacillus (lb). salivary lactobacilli may have an indirect role with advancement of caries. elevated concentration of s. mutans and lactobacillus in saliva are significantly corelated to the early occurrence of dental caries [12-16]. saliva is one of the most studied components of caries [15], and because of positive correlation between concentration of salivary sm and lb and caries, these bacteria may be potential indicators for dental caries. a significant increase of salivary sm and lb concentrations was found in a group of adolescents with nontreated caries [16]. this study was intended to investigate the levels of streptococcus mutans and lactobacillus species in saliva and their relationship with children’s demographic profiles and nutritional status. material and methods this research is structured as descriptive observational study with cross-sectional design. it includes children of both sexes and ages 3 to 10 years [17] being treated at the teaching hospital’s pedodontic and preventive dentistry unit clinics, university of baghdad’s college of dentistry. this study enrolled children with active carious lesions. official approval of study protocol was garneted by both department of pedodontics and preventive dentistry’s scientific committee and the college of dentistry’s institutional research ethics committee at the university of baghdad in iraq (reference number.:816, project number. 816323, date;30-8-2023) (chairman, prof. dr. salwan y. bede). the parents of all children included in this study were asked to sign an informed consent document. total sample of children participating in this study was 135. each child received a questionnaire to be filled by their parents. demographic variables included in this questionnaire were date of birth, age, sex, nursing pattern, nursing at sleep, added sweet to milk, tooth brushing, and occupation of mother and father. following standard guidelines of fejerskov and thylstrup, in the morning between 10 to11 a.m. the samples of unstimulated saliva were collected [19]. prior to this procedure, children rinsed their mouths with water and asked to spit the first mouthful of saliva into a small, pre-labeled plastic tube. one hour before collecting saliva, the child was asked not to eat or drink (except for water). a pre–sample period of 1 minute was followed. then, unstimulated saliva was collected for 10 to 15 minutes. saliva was collected with the child sitting and relaxed on a common chair. any samples containing blood were discarded. after completing saliva collection, normal saline was used for dilution of the sample in the laboratory. a micropipette was used for application of saliva on selective media surfaces mitis salivaris agar targeting mutans streptococci and rogosa agar for lactobacilli. then plates anaerobically were incubated at 37°c for 48 hours. for streptococcus mutans, counting of colony forming units (cfu) and morphology features were used, while numbers of cfu per ml of saliva were used for lactobacilli [20]. g power 3.1.9.7 (program design by franz-faul at the university of kiel, germany) was used to calculate sample size of this study. assuming 80% power, alpha error of 0.05, effect size of 0.25 (medium) sample size of this study was 111. when adding 10% as an error rate, 122 subjects were suggested. therefore, our sample of 135 was enough to detect differences in our study [21]. daily entrance of data from the children’s case sheets was done into microsoft office excel 2013, and the statistical package for social science version 22 (spss) (chicago, illinois, usa) was implemented for statistical tests. salivary caries-related microorganisms and demographics of a group of children vol 13 no 1 (2025) doi 10.5195/d3000.2025.764 h#p://den*stry3000.pi#.edu we determined frequency, percentages, means and standard errors (se), unpaired t test, one way analysis of variance with games-howell and pearson correlations. statistically significant level was accepted at pvalue of less than 0.05. results the overall sample were allocated based on the demographic variables as shown in table 1. comparison of streptococcus mutans count in the saliva of children with their demographics, data revealed that the bacterial count is significantly increased with increased age. additionally, the boys had significantly higher count than girls. furthermore, count of salivary streptococcus mutans showed a significant difference for type of nursing, status of nutrition and brushing time of teeth (table 2). the count of bacterial streptococcus mutans in saliva significantly increased in children who did not brush their teeth compared to those who brushed one time (table 3). the count of lactobacilli in the saliva revealed that girls had significantly higher counts compared to boys, and a significant higher bacterial count were noted in children of government employee working mothers than in children of housewives (table 4). multiple comparisons for children’s nutritional status revealed that the differences were significant when comparing the obese group with the two other groups (table 5). discussion dental caries is multifactorial in nature, so salivary features, oral hygiene, nutritional status, and demographic factors should be registered to indicate their roles as risk factors for dental caries [22]. saliva provides a complex mixture of microorganisms and collection of saliva is noninvasive and easy [5]. streptococcus mutans detach to saliva from different oral surfaces and is considered as major cariogenic bacteria. lactobacilli play a role in tooth decay development because it can produce extracellular polysaccharides and lactic acid [23]. several studies have investigated the salivary bacterial levels of streptococcus mutans and risk of dental caries [22,24]. for optimal health from childhood to adulthood, good oral hygiene practices throughout life are suggested [25,26]. changes in the environment and lifestyle shifts such as high access to consumption of processed foods with an increased content of carbohydrates and urbanization have contributed to obesity and dental caries among children [27]. the finding of this study showed an increase of salivary streptococcus mutans and lactobacilli counts among children with increased age, in agreement with previous data [28,29]. in general, girls grow faster than boys, which results in earlier teeth eruption and more exposure to cariogenic bacteria [30]. many studies observed variations in the salivary microbiota between males and females, which might suggest that microorganisms have limited influence on determination of dental caries [31,32]. parents are responsible for providing a suitable environment for a healthy lifestyle and instilling good habits of oral hygiene [33]. in our study, children from mothers that were government employees had significantly higher counts of lactobacilli than children whom mothers were housewives, which agrees with previous work [34]. this could be since housewives put more effort in maintaining children’s oral health because they spend more time with them. streptococcus mutans counts were higher in breastfeeding than other feeding patterns in our study. this is possibly because mothers salivary caries-related microorganisms and demographics of a group of children vol 13 no 1 (2025) doi 10.5195/d3000.2025.764 h#p://den*stry3000.pi#.edu include foods and drinks other than breastmilk feeding, which increases susceptibility of the children to cariogenic microorganisms’ early childhood caries [35,36]. also, our study showed that overweight children significantly have highest streptococcus mutans counts. the same results were found in other studies [37-39], which linked an unhealthy body mass index or obesity with increased dental caries. khalaf et al. and raju et al. found inverse results, as their results showed that streptococcus mutans counts in children with normal weight were higher than other bmi weights [40,41]. children who did not brush their teeth had significantly higher streptococcus mutans counts in their saliva in our study findings, which agrees with previous work [40]. mechanical action of toothbrush bristles and active anticaries effects of toothpaste may provide this benefit of tooth brushing in decreasing the prevalence of dental caries. brushing leads to not enough nutrients and time for growth of streptococcus mutans and this led to a decrease in acid production [42]. this study has some potential limitations. the sample may not represent the broader population. also, because this study is a crosssectional in design, it could not give the causal relation between salivary caries related microorganisms and development of caries, depending on different demographics. in addition, our study sample collection from a specific geographic and socioeconomic group may not apply to other populations or communities. conclusion microbial profile of both salivary streptococcus mutans and lactobacilli correlate with demographic factors of children. further investigations using longitudinal designs are needed to search for causative relations and the role of demographics. ethical approval this study was received ethical approval from the research ethics committee of the college of dentistry \ university of baghdad ref. number: 816, date 30-8-2023. conflicts of interest the authors advertise no competing interest. references 1. pedersen a, sorensen ce, proctor gb, et al. salivary functions in mastication, taste and textural perception, swallowing and initial digestion. oral dis. 2018;24(8):1399–1416. 2. lynge pedersen am, belstrom d. the role of natural salivary defenses in maintaining a healthy oral microbiota. j dent. 2019;80(suppl 1):s3–s12. 3. schroder sa, bardow a, eickhardt-dalboge s, et al. is parotid saliva sterile on entry to the oral cavity? acta otolaryngol. 2017;137(7):762–764. 4. segata n, haake sk, mannon p, et al. composition of the adult digestive tract bacterial microbiome based on seven mouth surfaces, tonsils, throat and stool samples. genome biol. 2012;13(6):r42 5. belstrom d. the salivary microbiota in health and disease. journal of oral microbiology. january 2020 12(1):1723975. doi:10.1080/20002297.2020.1723 975 6. hansen th, kern t, bak eg, et al. impact of a vegan diet on the human salivary microbiota. sci rep. 2018;8(1):5847. 7. lassalle f, spagnoletti m, fumagalli m, et al. oral microbiomes from huntergatherers and traditional farmers reveal shifts in commensal balance and pathogen load linked to diet. mol ecol. 2018;27(1):182–195. 8. mason mr, chambers s, dabdoub sm, et al. characterizing oral microbial communities across dentition states and colonization niches. microbiome.2018;6(1):67. salivary caries-related microorganisms and demographics of a group of children vol 13 no 1 (2025) doi 10.5195/d3000.2025.764 h#p://den*stry3000.pi#.edu 9. ardelean lc, rusu lc, gehrke sa. enamel and dentinpulp complex. intechopen. 17 july 2024. doi10.5772/intechopen.114766. 10. peres ma, macpherson l, weyant rj, daly b, venturelli r, mathur mr, et al. oral diseases: a global public health challenge. lancet. 2019;394:249–60. https://doi.org/10.1016/s01406736(19)31146-8. 11. j.r. martins, diaz-fabergat b, romirez-carmana w, monterio dr, pessan jp, antoniali c. salivary biomarkers of oxidative stress in children with dental caries: systematic review and metaanalysis. archives of oral biology. 2022;139: 12. baker jl, morton jt, dinis m, alvarez r, tran nc, knight r, edlund a. deep metagenomics examines the oral microbiome during dental caries, revealing novel taxa and co-occurrences with host molecules. genome res. 2021;31(1):64–74. 13. lapirattanakul j, nomura r, okawa r, tantivitayakul p, kaypetch r, lehrkinder a, lingström p, birkhed d, matsumoto nakano m, nakano k. multilocus sequence typing and phenotypic properties of streptococcus mutans from thai children with different caries statuses bmc oral health. 2024;24:1063. https://doi.org/10.1186/s12903024-04759-9. 14. hall-scullin e, whitehead h, milsom k, tickle m, su tl, walsh t. longitudinal study of caries development from childhood to adolescence. j dent res. 2017; 96:762–7. 15. corby pm, lyons-weiler j, bretz wa et al. microbial risk indicators of early childhood caries. j clin microbiol 2005; 43(11): 5753–5759. 16. lin p, mai h, wu c, lin h, chi l. association between untreated caries and cariogenic bacteria in adolescents in taiwan. journal of dental sciences.2024;19(4):2027-2034. 17. world health organization. oral health surveys. in: basic methods. geneva: world health organization; 2013. p. 13-16. 18. world health organization, international federation of red cross, red crescent societies, world food programme. the management of nutrition in major emergencies. world health organization; 2000. 19. fejerskov o, thylstrup a. the oral environment and introduction. textbook of clinical cariology. 2nd ed. copenhagen: munksgaard; 1994; p.13-17. 20. kishi m, abe a, kishi k, ohara-nemoto y, kimura s, yonemitsu m. relationship of quantitative salivary levels of s. mutans and s. sobrinus in mothers to caries status and colonization of mutans streptococci in plaque in their 2.5-year-old children. community dent oral epidemiol 2009; 37: 241-9. 21. chen h, cohen p, chen s. how big is a big odds ratio? interpreting the magnitudes of odds ratios in epidemiological studies. commun stat simul comput 2010; 39:860-4. 22. bhayat aa, tamer mh, alshorman m, abu-naba’a h, bakeer lh. correlating dental caries with oral bacteria and the buffering capacity of saliva in children in madinah, saudi arabia. j. int. soc. prev. community dent. 2016, 3, 38–43. 23. hasan na, young ba, minard-smith at, et al. microbial community profiling of human saliva using shotgun metagenomic sequencing. plos one. 2014;9(5): e97699. 24. lin x, wang y, ma z, xie m, liu z, cheng j, tian y and shi h. correlation between caries activity and salivary microbiota in preschool children. front. cell. infect. microbiol. 2023,13:1141474. doi: 10.3389/fcimb.2023.1141474 25. shahraki t, shahraki m, mehr so. association between body mass index and caries frequency among zahedan elementary school children. int. j. high risk behav. addict. 2013, 2, 122–125. 26. eman kc, qasim a. nutritional status in relation to oral health status among patients attending dental hospital. j bagh coll dentistry 2013; 25:114–9. available from: https://jbcd.uobaghdad.edu.iq/ind ex.php/ jbcd/article/view/209 salivary caries-related microorganisms and demographics of a group of children vol 13 no 1 (2025) doi 10.5195/d3000.2025.764 h#p://den*stry3000.pi#.edu 27. alshehri y, park js, kruger e, tennant m. association between body mass index and dental caries in the kingdom of saudi arabia: systematic review. saudi dent. j. 2020, 32, 171–180. 28. shi w, qin m, chen f, xia b. supragingival microbial profiles of permanent and deciduous teeth in children with mixed dentition. plos one 2016, 11, e0146938. 29. lee e, park s, um s, kim s, lee j, jang j, jeong h.-o., shin j, kang, j, lee s et al. microbiome of saliva and plaque in children according to age and dental caries experience. diagnostics 2021, 11, 1324. https://doi.org/ 10.3390/diagnostics11081324. 30. xu l, shao jl, pan f. eruption times and sequence of permanent teeth in 6712 children and young adolescents (in chinese). j pract stomatol 2007; 23:243–5. doi: 10.1007/s00784004-0295-y 31. ortiza s, herrmana e, lyashenkob c, purcell a, raslana k, khora b, snow m, forsythc a, choid d, maier t, machida ca. sexspecific differences in the salivary microbiome of caries-active children. journal of oral microbiology 2019; 111653124. https://doi.org/10.1080/20002297 . 32. juma’a z, fadhil r, abdulhussain y. correlation between caries related microorganisms in the dental plaque and saliva with dental caries level in the upper and lower jaws in 5-9 years old children in bagh dad city. journal of baghdah coll dentistry 2016; 28(3):132–6. 33. ellakany p, madi m, fouda sm, ibrahim m, alhumaid, j. the effect of parental education and socioeconomic status on dental caries among saudi children. int. j. environ. res. public health 2021, 18, 11862. https://doi.org/10.3390/ijerph182 211862. 34. nembhwani hv, varkey i. caries experience and its relationship with mother's educational level and occupational status: a crosssectional survey. int j clin pediatr dent. 2022;15(suppl 2):s226-s229. doi: 10.5005/jp-journals-100052163. pmid: 35645509; pmcid: pmc9108831. 35. chaffee bw, feldens ca, rodrigues ph, vítolo mr. feeding practices in infancy associated with caries incidence in early childhood. community dent oral epidemiol. 2015;43:338–348. doi: 10.1111/cdoe.12158. 36. salih a, qasim a, kolah k. assessment of the salivary level of glutathione and the feeding pattern in molar incisor hypomineralization among 7-9 years of age:an analytical crosssectional study. j bagh coll dentistry 2024;36(3): 42-49. 37. powell jc, koroluk ld, phillips cl, roberts mw. relationship between adjusted body mass index percentile and decayed, missing, and filled primary teeth. j. dent. child. 2013, 80, 115–120. 38. yousif sh, qasim aa. correlation of dental caries severity with physical properties of saliva in connection with nutrition status among a group of adolescents. medical journal of babylon 2023;20(4): 715-720. doi: 10.4103/mjbl.mjbl_130_23 39. honne t, pentapati k, kumar n, acharya s. relationship between obesity/overweight status, sugar consumption and dental caries among adolescents in south india. int j dent hyg 2012; 10:240-4. 40. khalaf ms, qasim aa, jafar zj, mohammad at. dental plaque caries related microorganism in relation to demographic factors among a group of iraqi children. folia medica 2024;66(4):491-499. 41. raju sc, lagström s, ellonen p. gender-specific associations between saliva microbiota and body size. front microbiol 2019; 10:767. doi: 10.3389/fmicb.2019.00767. 42. abdulbaqi hr, abdulkareem aa, alshami ml, milward mr. the oral health and periodontal diseases awareness and knowledge in the iraqi population: online-based survey. clin exp dent res. 2020 oct;6(5):519-528. doi: 10.1002/cre2.304. epub 2020 jun 26. pmid: 32592312; pmcid: pmc7545227. salivary caries-related microorganisms and demographics of a group of children vol 13 no 1 (2025) doi 10.5195/d3000.2025.764 h#p://den*stry3000.pi#.edu table 1. descriptive statistics of the complete sample depending on the studied demographic variables. demographic variables divisions no. % child age 3 to 6 years 80 59.26 7 to10 years 55 40.74 child sex males 69 51.11 females 66 48.89 father occupation government employee 66 48.89 free jobs 69 51.11 mother occupation government employee 109 80.74 housewife 26 19.26 adding sugar yes 39 28.89 no 96 71.11 type of nursing breast milk feeding 93 68.89 bottle milk feeding 15 11.11 mixed 27 20.00 sleep nursing yes 86 63.70 no 49 36.30 nutritional status normal weight percentile 78 57.78 over-weight percentile 31 22.96 obese percentile 26 19.26 tooth brushing no brushing 14 10.37 one time 56 41.48 two time 65 48.15 salivary caries-related microorganisms and demographics of a group of children vol 13 no 1 (2025) doi 10.5195/d3000.2025.764 h#p://den*stry3000.pi#.edu table 2. the counts of s. mutans level in the saliva based on the demographic variables. demographic variables mean se test p value child age 3-6 years .220 .031 3.308 0.001 7-10 years .462 .066 child sex males .419 .054 3.106 0.001 females .214 .038 21-30 years .239 .041 31-40 years .604 .083 41-50 years .345 .016 father occupation government employee .252 .042 1.931 0.056 free jobs .382 .053 mother occupation government employee .321 .041 0.254 0.801 housewife .306 .044 adding sugar yes .248 .066 1.282 0.204 no .347 .040 positive history .671 .199 type of nursing breast milk feeding .386 .044 12.992 0.000 bottle milk feeding .027 .018 mixed .247 .061 nutritional status normal weight percentile .357 .039 6.416 0.003 over-weight percentile .424 .102 obese percentile .076 .024 sleep nursing yes .348 .046 1.237 0.219 no .266 .047 teeth brushing no brushing .711 .154 6.744 0.004 one time .205 .047 two time .332 .041 salivary caries-related microorganisms and demographics of a group of children vol 13 no 1 (2025) doi 10.5195/d3000.2025.764 h#p://den*stry3000.pi#.edu table 3. games-howell test for multiple comparing of s. mutans in saliva. demographic variables comparisons md p value type of nursing breast milk feeding bottle feeding 0.360 0.000 mixed feeding 0.139 0.165 bottle milk feeding mixed feeding -0.220 0.005 nutrition status normal weight percentile over-weight -0.067 0.813 obese percentile 0.281 0.000 over-weight percentile obese percentile 0.348 0.006 tooth brushing no brushing one time 0.507 0.017 two time 0.380 0.076 one time two time -0.127 0.111 salivary caries-related microorganisms and demographics of a group of children vol 13 no 1 (2025) doi 10.5195/d3000.2025.764 h#p://den*stry3000.pi#.edu table 4. the counts of lactobacilli level in the saliva based on the demographic variables. demographic variables saliva lactobacilli f p value mean se child age 3 to 6 years .221 .036 1.771 0.081 7 to 10 years .372 .077 child sex boys .128 .033 4.320 0.000 girls .444 .065 father occupation government employee .281 .046 0.042 0.967 free jobs .284 .061 mother occupation government employee .316 .047 3.086 0.003 housewife .143 .031 adding sugar yes .302 .054 0.367 0.714 no .275 .050 type of nursing breast milk feeding .275 .052 0.449 0.639 bottle milk feeding .383 .062 mixed feeding .253 .066 nutritional status normal weight percentile .359 .061 9.524 0.000 over-weight percentile .285 .053 obese percentile .052 .015 sleep nursing yes .215 .029 2.373 0.019 no .402 .091 teeth brushing no brushing .154 .070 0.954 0.388 one time .263 .052 two time .327 .065 salivary caries-related microorganisms and demographics of a group of children vol 13 no 1 (2025) doi 10.5195/d3000.2025.764 h#p://den*stry3000.pi#.edu table 5. games-howell test for multiple comparing of lactobacilli in saliva. variables comparisons md p value nutritional status normal weight percentile over-weight percentile 0.074 0.634 obese percentile 0.307 0.000 over-weight percentile obese percentile 0.234 0.000 882 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.882 http://dentistry3000.pitt.edu evaluation of soft tissue in rabbit utilizing suture and tissue glue ahmed amer, ahmed abdul kareem, sohaib qais, saber mizher, al-alaa j. mowlood, saif saad kamil, ali saad ahmed college of den*stry, tikrit university, tikrit, iraq abstract in this work, we sought to determine the efficacy of bssue glue and sutures in soc bssue incisions in rabbits. the comparison of bssue glue and suture for closure of excisional wounds in rabbits showed that the quality of wound healing of cutaneous excisional wounds closed with standard suturing was superior to that of wounds closed with bssue glue. open access cita%on: amer a, et al. (2025) evalua%on of so; tissue in rabbit u%lizing suture and tissue glue. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.882 received: march 18, 2025 accepted: march 20, 2025 published: june 26, 2025 copyright: ©2025 amer a, et al. this is an open access ar%cle licensed under a crea%ve commons atribu%on work 4.0 united states license. email: ahmedameribraheem@tu.edu.iq introduc)on sutures have been the backbone of surgical treatment over the past few decades [1]. fibers or strands called sutures are used to join tissues or blood vessels [2]. in addition to sutures, many additional materials such as clips, staples, and microporous surgical tapes were also employed in surgical procedures. few sutures are absorbed by the body after performing their intended function, these treatments are simple to do, have a high tensile strength, and have no possibility for allergies or cancer [3]. notwithstanding these benefits, this process is extremely time consuming, and there is a higher risk of infection during its removal as well as leaks, tissue scarring, and wound separation [4]. suture-less approaches must therefore be developed to meet the requirement for less invasive, quicker, and simpler surgical procedures [5]. trends shift, and the art of dressing wounds has undergone a revolution in the twentyfirst century thanks to advancements in synthetic polymer technology that produced tissue glue [6]. adhesives for tissues surgical glues are a type of biomaterial that closes wounds or tissues by adhering to them after application. additionally, they aid in the postoperative sealing of air or gas leaks through an incision and the adherence of medical devices to tissues [7,8]. tissue glues offer a wonderful opportunity to enhance traditional methods of wound closure. unlike sutures, they can stop fluid or air leakage and allow almost instant hemostasis [9]. requirement of tissue glues these adhesives must be quick to prepare, easy to handle, cure rapidly, spread appropriately due to a well-adjusted viscosity, have sufficient bonding strength in wet environments and have a modulus-like tissue. ideally, the material should allow design flexibility for different indications, be costeffective, cause minimal to no tissue damage, allow wound healing and primarily be biocompatible and biodegradable [10,11]. bioadhesion principles the foundation of surgical adhesives is the concept of bioadhesion, which is the capacity to attach to biological materials and remain on the biological substrate for a predetermined amount of time [12]. chemical and physiological interactions form the foundation of the adhesion mechanism. because of the interaction of the various functional groups contained in adhesives, a chemical bond—either ionic or chemical—is formed between the surfaces of adhesives and biological substrates. chemical contact involves three primary steps: the polymer is soaked and swells; the polymer chain diffuses into the mucosal membrane; and last, chemical bonds are formed between the entangled chains. conversely, physiological contact entails adhesion via physiologically connected mechanisms such as the blood clotting process [13]. advantages tissue glues are commonly used in the setting of traumatic lacerations. their benefits evalua&on of so, tissue in rabbit u&lizing suture and tissue glue vol 13, no 1 (2025) doi 10.5195/d3000/2025.882 http://dentistry3000.pitt.edu 2 in this environment include reduced application time and reduced pain compared with standard wound-closure methods [9]. patients also avoid the need to return for removal of stitches or skin clips. this can be especially useful in pediatric patients, who might find the application of tissue glue more acceptable than standard methods. however, cosmetic improvement [14]. categorization basis of their work hemostats are glues that clot blood and do not work in the absence of blood. a sealant creates a layer of defense to stop fluid or gas leaks. the purpose of an adhesive is to securely join and maintain two surfaces. while hemostats work well in the presence of blood, adhesives and sealants don't always work well in damp environments [14,15]. basis of their origin 1. biologic such as fibrin glues and animal derived hemostatic agents. 2. synthetic such as cyanoacrylate and noncyanoacrylate products, such as polymeric sealant. 3. genetically engineered polymeric proteins. fibrin glues are widely used biologic tissue adhesive in surgical practice do not provide significant tensile and adhesive strength and require to be applied on dry substrates. cyanoacrylates are a class of synthetic glues that rapidly solidify upon contact with weak bases, such as water or blood compared with other tissue adhesives are easier to use, have quicker polymerization [16] and guarantee higher bonding strength. glubran2 (gem s.r.l., viareggio, italy), a tissue adhesive with high adhesive and haemostatic properties, is a class iii (f0r internal and external surgical use). furthermore, it is diffusely applied on skin, eliminating the need for suture removal and providing good cosmertic results [17]. sutures surgical sutures are one type of long-established and necessary medical device, and they play a pivotal role in closing damaged tissues and organs and assisting wound healing postoperatively. although some products, like biomedical glues [18], adhesives, and nails, have been developed as alternatives to sutures, it is undisputed that there are still no perfect and fully-fledged alternatives to surgical sutures, due to their high stability, feasibility, and applicability. material selection for suture fabrication materials originating from both natural and synthetic sources have been employed for the design and fabrication of surgical sutures [19]. based on the absorbability of the material, the surgical sutures are further divided into absorbable and non-absorbable types. in general, natural suture materials possess poor tensile strength and unstable performances [20], while synthetic suture materials perform better in this regard, and their degradation properties can be controlled by adjusting the material structures and properties [21]. natural absorbable suture materials surgical catgut surgical catgut was officially utilized as natural absorbable suture material in the 19th century and was commonly divided into plain surgical catgut and chromic surgical catgut. since it contains collagen as the main component, surgical catgut could be absorbed in vivo. for instance, the strength of plain surgical catgut could be maintained for at least 7 days and degraded completely after 90 days [22]. collagen a collagen suture is mainly composed of type i collagen, which is a fibrous protein with a rod-like helix structure. it has strong physical and mechanical properties and possesses the ability to bind and aggregate with platelets for the formation of a thrombus. collagen sutures are biocompatible and absorptive, effectively promoting hemostasis and wound healing and relieving postoperative pain [23]. chitin and chitosan chitin is an environmentally friendly polymer derived from the cell walls of crustacean or fungal cells and is biologically inert and can effectively inhibit postoperative complications or tissue reactions. generally, chitin fibers can be generated through dry or wet spinning [24]. synthetic absorbable suture it includes: 1polyglycolic acid 2polylactic acid 3poly-lactic-co-glycolic acid 4poly(p-dioxanone) natural non-absorbable suture it includes: 1silk 2cellulose synthetic non-absorbable suture it includes: 1 polyamide 2 polypropylene 3 polyethylene terephthalate healing and repair of injured tissues healing and repair of injured tissues follows several steps in the healthy individual. following birth, the process is initiated by the inflammatory response and subsequent steps are based on this initial response [25]. whereas wound healing generally leads to a repair of the injured site, it does not lead to tissue regeneration. this difference between repair and regeneration has influence on tis sues such as ligaments and tendons that function in a mechanically active environment. thus, the dynamic interface between mechanics and biology influences the effectiveness of the healing response. the biology of the host is also influenced by a variety of factors including age, gender, genetics, and tissue history, factors that impact the outcome of the healing response [26]. defined steps in the repair or healing of a tissue according to following overt injury [27] (1) hemostasis and a rapid inflammatory phase. acutely following overt injury in an otherwise healthy adult, there is usually pain and bleeding into the area of injury that may be variable depending on the site of injury. (2) the matrix deposition phase. following consolidation of the fibrin clot and the temporally regulated influx, proliferation and activation of a subset of cells’ deposition of matrix molecules designed to bridge the damaged area with the residual endogenous ligament tissue begins. (3) the remodeling phase. the remodeling phase is a slow process and is accompanied by alterations not only in matrix remodeling, but also gene expression, cellularity, evalua&on of so, tissue in rabbit u&lizing suture and tissue glue vol 13, no 1 (2025) doi 10.5195/d3000/2025.882 http://dentistry3000.pitt.edu 3 vascularity and innervation [27]. factors that influence on soft healing process [28] age it is obvious from everyday life and research that wounds incurred at an early age appear to heal rapidly and without residual scar. because humans age, the same process following a similar injury leads to obvious scars. gender and hormones it is known in a variety of species that there is a sexual dimorphism in many responses. females tend to have a more vigorous inflammatory response than males and as the inflammatory response to injury is a central initial step in the healing process, this is a consistent pattern of observation. it is also known that healing responses in post-menopausal women decline separate from the decline associated with aging. application of estrogen can reverse some of these deficiencies and it is believed to work at the level of macrophages. macrophages are inflammatory cells that can mediate multiple functions in a wound site, including serving as an important source of growth factors and mediators key for effective wound healing. genetics genetic factors play a role in some pathologic scarring or wound healing such as keloid formation. however, there is a paucity of information regarding a definitive role for genetic factors in subtle or not so subtle variations in “normal” healing. whereas keloid formation is more common in some races than others there is no identifiable link between race and wound healing in other tissues [28]. materials and methods ten sisteen weeks mature, white rabbits were used for this study with a healthy appearance and weight ranging from 1500 to 2500 g. the study was approved by the tikrit university college of veterinary medicine laboratory animal care. all care and surgery were performed in the wsu laboratory animal resources center under the supervision of doctor ahmed khalid. all rabbits were observed for 4 days before surgery to verify that they were healthy. the animals were anesthetized with an intramuscular combination of 5 mg/kg xylazine and 35 mg/kg ketamine envelope skin hlabs 2cm long were performed on shaved right and left legs. one of incision closed with interrupted 0/3 silk sutures and another one closed with hibrin glue. on postoperative day 7, wound closure was scored and evaluated clinically. parameters for clinical assessment of wound healing were summarized in table 1 of numbers was used to evaluate the healing process from (0_10) when 10 score mean complete healing of hlab and every 1mm of wound length failure to healing and complete closure will reduce 1 score from evaluation (figure 1). data were analyzed using the spss program, using the paired t-test. results based on clinical examinations all flaps which closed by suture shows complete healing without complications while using fibrin glue to close skin incision showed unsatisfactory results and incomplete closing of flaps. discussion suturing has been the most widely used method for wound closure because of high reliability of suture materials. however, alternative techniques have long been sought, since suturing technique requires skill and experience, a relatively longer time and the need for its removal. due to these reasons, surgeons are increasingly using tissue adhesives over sutures for wound closure. although most facial wounds heal without complications, owing to the abundant blood supply of the region, mismanagement may result in infection, wound dehiscence, and unsightly and dysfunctional scar [29]. tissue glue have several advantages over conventional sutures like their fast and painless application, rapid setting, which reduces the total operating time and their antibacterial properties. tissue glue itself acts as a waterproof dressing and helps in reduction in the number of follow-up visits. as they do not require any needles, accidental needle stick injuries are prevented. however, there are certain disadvantages of tissue glue like their less tensile strength and chances of adhesive seepage, if edges are not properly approximated [30]. conclusion the comparison between tissue glue and suture for the closure of excisional wounds in rabbits found that the quality of wounds healing of cutaneous excisional wounds closed with standard suturing was found superior to that of wounds closed with tissue glue. references 1. [b.j. tighe et al. adhesives and interfacial phenomena in wound healing adv. wound repair ther. (2011). 2. n. annabi et al. elastic sealants for surgical applications eur. j. pharm. biopharm. (2015). 3. n. annabi et al. surgical materials: current challenges and nano-enabled solutions nano today (2014). 4. m. kazemzadeh-narbat et al. surgical sealants and high strength adhesives mater. today (2015). 5. s.r. mobley et al. surgical tissue adhesives facial plast. surg. clin (2002). 6. j. armitage et al. skin incisions and wound closure surgery (2011). 7. kamil, saif saad; khaudhair, aseel taha; hamid, hind thyab. association of dental caries with different abo blood groups. dentistry 3000, 2024, 12.1. 8. y. okamoto et al. effects of chitin and chitosan on blood coagulation carbohydr. polym. (2003). 9. scognamiglio, f.; travan, a.; rustighi, i.; tarchi, p.; palmisano, s.; marsich, e.; borgogna, m.; donati, i.; de manzini, n.; paoletti, s. adhesive and sealant interfaces for general surgery applications. j. biomed. mater. res. b. appl. biomater. 2016, 104, 626-639. 10. bhagat, v.; becker, m.l. degradable adhesives for surgery and tissue engineering. biomacromolecules 2017, 18, 3009-3039. 11. taboada, g.m.; yang, k.; pereira, m.j.n.; liu, s.s.; hu, y.; karp, j.m.; artzi, n.; lee, y. overcoming the translational barriers of tissue adhesives. nat. rev. mater. 2020, 5, 310-329. 12. m. kazemzadeh-narbat et al. surgical sealants and high strength adhesives mater. today (2015). 13. a. assmann. a highly adhesive and naturally derived sealant biomaterials (2017). 14. a.j. singer et al. closure of lacerations and incisions with octylcyanoacrylate: a multicenter randomized controlled trial surgery (2002). 15. b. petersen et al. technology status evaluation report: tissue adhesives and fibrin glues gastrointest endosc (2004). 16. j.m.g. paez et al. comparative study of the mechanical behavior of a cyanoacrylate and a bioadhesive j mater sci mater med (2004). 17. d.h. sierra et al. failure characteristics of multiple-component fibrin-based adhesives j biomed mater res (2002). 18. c. ma, j. sun, b. li, y. feng, y. sun, l. xiang, b. wu, l. xiao, b. liu, v.s. petrovskii, b. liu, j. zhang, z. wang, h. li, l. zhang, j. li, f. wang, r. goestl, i.i. potemkin, d. chen, h. zeng, h. zhang, k. liu, a. herrmann ultra-strong bio-glue from genetically engineered polypeptides nat. 12 (1) (2021), p. 3613 . 19. ahmed, ali saad, et al. influence of some plant extracts on antifungal properties, hardness, and peel bond strength of heat-cured denture soft liner. diyala journal of medicine, 2024, 26.2: 1428. 20. r.h.h. tan, r.j.w. bell, b.a. dowling, a.j. dart. suture materials: composition and applications in veterinary wound repair. aust. vet. j., 81 (3) evalua&on of so, tissue in rabbit u&lizing suture and tissue glue vol 13, no 1 (2025) doi 10.5195/d3000/2025.882 http://dentistry3000.pitt.edu 4 (2003), pp. 140-145. 21. e.j. lee, b.k. huh, s.n. kim, j.y. lee, c.g. park, a.g. mikos, y. bin choy. application of materials as medical devices with localized drug delivery capabilities for enhanced wound repair. prog. mater. sci., 89 (2017), pp. 392-410. 22. h. kim, k. hwang, s.m. yun. catgut and its use in plastic surgery. j. craniofac. surg., 31 (3) (2020), pp. 876-878. 23. ahmed, ali saad; ahmed, rusal saad; arab, luma nasrat. the antifungal potential of cinnamon oil incorporated into a heat-polymerized soft liner. journal of dental materials & techniques, 2024, 13.3. 24. j.l. sharnshina, p. berton, r.d. rogers. advances in functional chitin materials: a review. acs sustain. chem. eng., 7 (7) (2019), pp. 64446457. 25. witte mb, brabul a. general principles of wound healing. surg clin north am. 1997;77:509528 . 26. diegelmann rf, evans mc. wound healing: an overview of acute, fibrotic and delayed healing. front biosci. 2004;9:283-289. 27. kareen, ahmed abdul, et al. the influence of antibiotics administration subsequent to dental extraction. universal publication index e-library, 2024, 72-85. 28. hildebrand, k.a. et al. “the basics of soft tissue healing and general factors that influence such healing,” sports medicine and arthroscopy (2005). 29. shivamurthy dm, singh s, reddy s. comparison of octyl-2-cyanoacrylate and conventional sutures in facial skin closure natl j maxillofac surg. 2010;1:15-9. 30. bernard l, doyle j, friedlander sf, eichenfield lf, gibbs nf, cunningham bb. a prospective comparison of octyl cyanoacrylate tissue adhesive (dermabond) and suture for the closure of excisional wounds in children and adolescents arch dermatol. 2001;137:1177-80. evalua&on of so, tissue in rabbit u&lizing suture and tissue glue vol 13, no 1 (2025) doi 10.5195/d3000/2025.882 http://dentistry3000.pitt.edu 5 evalua&on of so, tissue in rabbit u&lizing suture and tissue glue vol 13, no 1 (2025) doi 10.5195/d3000/2025.882 http://dentistry3000.pitt.edu 6 figure 1. representative example of the surgical intervention. 743+2024-2 dmft and pufa indices in first permanent molars of iraqi children in najaf city vol 12 no 2 (2024) doi 10.5195/d3000.2024.743 h#p://den*stry3000.pi#.edu dmft and pufa indices in first permanent molars of iraqi children in najaf city ansam mahdi khalel, mustafa basim ali, maytham adil sadiq, salah m. ibrahim*, shatha hameed ali college of dentistry, university of kufa, najaf, iraq *corresponding author abstract objec?ve: to determine the prevalence and severity of caries in the first permanent molars of children aged 8 to 10 years in najaf, iraq. subjects and methods: this was a cross-seceonal survey involving 1232 children aged 8 to 10 years in schools in najaf, iraq. caries was assessed using the dmft index and caries severity of permanent teeth was assessed using the pufa index. results: the prevalence of caries in the first permanent molars was 56.1% (n=691). the caries severity of the first permanent molars (pufa index) was 38.7%, meaning that the children have more severe caries in the first permanent molars. conclusion: caries prevalence and severity increased with age and was high in first permanent molars. keywords: dental caries, dmft, pufa, first permanent molar. cita:on: khalel am et al. (2024) dmft and pufa indices in first permanent molars of iraqi children in najaf city. den:stry 3000. 1:a001 doi:10.5195/d3000.2024.743 received: september 24, 2024 accepted: october 1, 2024 published: november 5, 2024 copyright: ©2024 khaleli am, et al. this is an open access ar:cle licensed under a crea:ve commons awribu:on work 4.0 united states license. email: salahm.abrahem@uokufal.edu.iq introduction dental caries is the demineralization of the inorganic part of the tooth and the dissolution of organic matter, which depends on the interaction of multiple factors [1,2]. these different factors include tooth morphology, bacteria, as well as other influencing factors such as diet and genetics [3]. tooth decay is the most common chronic disease affecting both sexes, races, and ages [4,5]. the first permanent molars (fpm), due to their anatomical structure and early eruption, are very susceptible to caries. it is a common reason for children having to visit the dentist and often needing restoration or extraction of fpm. therefore, estimating the caries risk in fpm at the individual and societal level helps to understand the pattern and severity of caries [6,7]. the development of fpm begins early in life. x-ray studies have shown that in addition to the germ of the deciduous teeth, the crypts of the fpm can be seen distal to the second deciduous molars right after birth. first, the crypts of the upper fpm are aligned in the maxillary high, above and behind the germ of the second deciduous molar, and the crypts of the lower fpm are located below the anterior edge of the mandibular ramus [7,8]. eruption of the fpm is a unique morphological and functional event because it simultaneously establishes the mesial boundaries of the molars [9], but also helps to establish concordance between the anterior (dental) and posterior (temporomandibular) determinants of occlusion [10]. nowadays, there is an increasing need to find methods that can more efficiently collect caries data in large population studies. this means saving economic resources and working time for health workers. epidemiological studies are extremely important for public dmft and pufa indices in first permanent molars of iraqi children in najaf city h#p://den*stry3000.pi#.edu dental health as they are the only source of accurate information on the frequency and distribution of oral diseases and form the basis for assessing treatment needs [11]. the decayed and missing filled surface/tooth (dmft) index has long been used and has been established as the main measure of caries occurrence [12]. however, it does not capture information about the clinical consequences of untreated caries, such as pulp involvement and dental abscesses and sepsis, which may be more serious than the carious lesions themselves. deep carious lesions with pulp involvement are still considered as “dentinal caries.” to improve the accuracy of caries diagnosis, monse et al. [13] introduced an index that can be used to quantify different advanced stages of carious lesions, namely the “pufa” index. pufa records (p – pulp involvement, u – ulceration, f – fistula, and a – abscess). surveys using pufa have been conducted among school children in india to study caries prevalence and caries experience [14,15]. the aim of this study was to determine the prevalence of dental caries and untreated dental caries among school children in an elementary school in najaf city, iraq, and to collect data on the clinical consequences of untreated dental caries using dmft and pufa indices. subjects and methods study design the study was conducted from december 11, 2022, to may 21, 2023 in the iraqi city of najaf. data were collected from children aged 8 to 10 years from several primary schools (six boys' schools) and (six girls' schools). the study received ethical approval from the local committee of the faculty of dentistry, university of kufa. prior to conducting the study, written informed consent was obtained from the parents/guardians after explaining the purpose of the study. in addition, permission was sought from the school authorities to conduct the study. sampling and sample size there are 6 public primary schools (boys) and 6 public primary schools (girls) in najaf city. they were selected from schools that were easily accessible and located in safe areas. a cross-sectional study was conducted on a total of 1232 children, of which approximately 682 were girls and 550 were boys. all children were examined individually in a common chair under natural light and a sterilized oral mirror and oral probes. caries was assessed using the dmft index and caries severity was assessed using the pufa index of the first permanent molars. initial carious lesions were not assessed. teeth with early cavitation that could not be penetrated by the ball probe were not assessed as having caries; the criteria for the pufa/pufa index were applied without the use of instruments. dental caries clinical examination for caries was performed using a flat mouth mirror and a cpi probe. systematic examination for caries was performed from the upper right to the upper left and then to the lower teeth. the examination covered all tooth surfaces. a tooth was considered present if any part of it was visible. a letter coding system was used for deciduous teeth and numbers for permanent teeth [16]. dental caries indices: dmft and pufa indexes the diagnostic criteria for caries were based on the world health organization (who). the dmft and dmft indexes were used to measure caries status in children. descriptive results were reported as frequencies and percentages as well as means and standard deviations. data were analyzed using spss version 16. a p-value less than 0.05 was considered significant using the chi-square test [17] to determine differences between males and females. pufa was recorded separately from dmft/dmft and assessed the presence of visible pulp, root fragments leading to oral mucosal ulcers, fistulas, or abscesses [18]. pufa definitions used were: dmft and pufa indices in first permanent molars of iraqi children in najaf city h#p://den*stry3000.pi#.edu p/p: pulp involvement was recorded when the opening of the pulp cavity was visible, or the coronal tooth structure had been destroyed by the carious process and only the root or root fragments remained. probing was not performed to diagnose pulp involvement. u⁄u: ulcers due to trauma caused by sharp tooth fragments were recorded when the sharp edge of the luxated tooth involved the pulp or root fragments, resulting in traumatic ulceration of the surrounding soft tissues (tongue or oral mucosa). f/f: if there is a draining sinus associated with the endodontic tooth, consider a fistula. a⁄a: if there is a pus-filled swelling associated with the endodontic tooth, consider an abscess [13,15,19]. results the prevalence of first permanent molar caries in al-najaf city was 56.1% (n=691) out of 1232 children 8 to 10 years of age (table 1). dmft the prevalence of lesions in the fpm increased between the ages of 8 and 10 (table 2). figure 1 to 3 summarize dmft findings by sex. pufa summary of findings related to pufa are described in figures 4 through 8 and tables 3 and 4. table 1. caries distribution in the study subjects. samples n % samples n % total number 1232 100 total caries teeth 691 56.1 female 682 55.35 female caries teeth 341 49.34 male 550 44.64 male caries teeth 350 50.65 table 2. dmft destribution in the study. gender age range decayed missing filled x2 p-value male 8-9 years 24% 0.91% 0.91% 33.014 0.001* 9-10 years 28% 2.7% 3.6% 33.864 female 8-9 years 20% 0.6% 0.88% 33.014 0.002* 9-10 years 28% 1.9% 3.4% 33.864 table 3. pufa distribution by sex. gender age range pulp involvement (p) ulceration(u) fistula(f) abscess(a) x2 p-value male 8-9 years 9.1% 3.2% 3.2% 4.1% 27.561 0.0002* 9-10 years 11.8% 5% 4.36% 5.2% 26.205 female 8-9 years 8% 2.9% 2.6% 3.2% 28.251 0.0012* 9-10 years 9.8% 4.1% 3.3% 5.2% 29.854 dmft and pufa indices in first permanent molars of iraqi children in najaf city h#p://den*stry3000.pi#.edu table 4. sex comparison of dmft and pufa summary statistics. variables gender mean ± sd se p-value dmft male 3.25±1.75 0.55 0.01 female 2.89±1.15 0.45 pufa male 1.24±1 0.38 0.05 female 1.17±0.78 0.45 figure 1. dmft in males. figure 2. dmft in females. figure 3. representative caries lesions of permanent first molars found in the study. 0% 5% 10% 15% 20% 25% 30% decay missing filled 8-9 years 9-10 years 0% 5% 10% 15% 20% 25% 30% decay missing filled 8-9 years 9-10 years dmft and pufa indices in first permanent molars of iraqi children in najaf city h#p://den*stry3000.pi#.edu figure 4. pufa distribution in males. figure 5. pufa distribution in females. figure 6. representative image of pulp involvement in permanent first molars found in the study. figure 7. representative image of ulceration near permanent first molars found in the study. figure 8. representative image of fistula and abscess near permanent first molar found in the study. 0.00% 2.00% 4.00% 6.00% 8.00% 10.00% 12.00% 14.00% pulp involvement ulceration fistula abcess 0.00% 2.00% 4.00% 6.00% 8.00% 10.00% 12.00% pulp involvement ulceration fistula abcess dmft and pufa indices in first permanent molars of iraqi children in najaf city h#p://den*stry3000.pi#.edu discussion first permanent molars are commonly the first teeth to erupt [20]. the first permanent molars have a significant impact on the dental health and overall wellbeing of a person. they absorb the greatest occlusal forces, control vertical distance, vertical dimensions, aesthetic proportions, and optimal anchorage sources [21]. this cross-sectional study investigated the impact of caries associated with the first permanent molar and its clinical implications in a sample of primary school students aged 8 to 10 years using dmft and pufa indexes. the mean dmft values of first permanent molars in males and females were 3.25 and 2.89, respectively, while the mean pufa values in males and females were 1.24 and 1.17, respectively. this study is the first to report the dmft and pufa indices of first permanent molars in iraq. other studies from iraq reported dmft scores for both deciduous and permanent teeth in mixed dentition [22–25]. when we compared our results with the prevalence reported in other countries, we noticed that it was relatively high. abufan et al. in sudan reported a prevalence of 61%, rafi et al. in abha, kingdom of saudi arabia (ksa), reported a prevalence of 66.4% [26,27], and que et al. in central africa reported a prevalence of 68.79% [28]. joshi et al. reported a caries prevalence of 69.12%, with a higher prevalence in boys (70.01%) than in girls (68.22%) [29]. any differences in reported prevalence may be attributed to sociodemographic factors, different sample sizes, different age groups, diagnostic criteria, or statistical methods. chukwu et al. reported that first permanent molars accounted for 42% of all extractions due to caries, which was the highest proportion among all other teeth [30]. salih showed that caries was more common in the first permanent molars (dmft) [31]. elfsay et al. showed different results for permanent caries between maxillary and mandibular, but in benghazi, libya, the overall prevalence was 50% [32], which is very close to the dmft prevalence we are reporting here. al mansour revealed that the overall prevalence of caries in children aged 5 to 12 years in fpm was as high as 42% [7]. all reported differences in prevalence can be attributed to sociodemographic factors, different sample sizes, different ages, diagnostic criteria or statistical methods. the pufa index meets the need for a more specific criteria for evaluating severity of caries. since the past decade, international caries detection systems have focused more on developing more sensitive diagnostic criteria to help identify caries at an early stage [33,34]. in this study, the pufa index was used to assess complications of untreated teeth. only one earlier study by kadhem examined the pufa index in preschool children in diwanya city, iraq [35]. looking at the age distribution of pufa scores, we found that children aged 9 to 10 years had the highest score of 21.6%, while children aged 8 to 9 years had the highest score of 17.1%. this indicates a lack of awareness and neglect of oral health, as well as a lack of dental health education. baginska et al. obtained similar results when evaluating the prevalence and experience of pufa index in primary dentition [36]. mons et al. showed that the prevalence of pufa in children aged 6 to 12 years was 85% [13]. ramazani and rezaei showed that there was no significant difference in the prevalence or severity of untreated caries between males and females (p > 0.05), which contradicts our study [37]. when all maxillary and mandibular first permanent molars were evaluated in mih-positive patients, the clinical consequences of untreated caries (mean pufa) were significantly more common, which is highly consistent with our study [38]. seker et al. showed that the prevalence of caries in first permanent molars in vadodara was 55.38%; the proportion of children aged 8 to 10 years affected by caries was assessed using the dmft index. the severity of caries in first permanent molars (pufa index) was 56.22% [39]. the reason for our results of dmft and pufa index values (public schools) may be due to the lack of dmft and pufa indices in first permanent molars of iraqi children in najaf city h#p://den*stry3000.pi#.edu understanding of parents and teachers, lack of free routine dental check-ups, and the prevalence of staple food packets over junk food packets. school staff should receive lectures on the importance of teeth and their protection from an early age. conclusion the current study focused on. the results showed that the prevalence and severity of dental caries in first permanent molars is high in children. ethical approval and consent of the participants the current work is approved by the university of kufa, number 253, date 2023, 19 december. availability of data and materials further data are available upon request to the corresponding author. financial support and sponsorship none. conflicts of interest there are no conflicts of interest. references 1. badri p, saltaji h, floresmir c, amin m. factors affecting children's adherence to regular dental attendance: a systematic review. the journal of the american dental association. 2014 aug 1;145(8):817-28. https://doi.org/10.14219/j ada.2014.49 2. mohammed mj, almizraqchi as, ibrahim sm (2024). oral findings, salivary copper, magnesium, and leptin in type ii diabetic patients in relation to oral candida species. international journal of microbiology, 2024(1), 8177437. https://doi.org/ 10.1155/2024/8177437 3. altaf g, garg s, saraf bg, sheoran n, beg a, anand m. clinical study of pit and fissure morphology and its relationship with caries prevalence in young permanent first molars. journal of south asian association of pediatric dentistry. 2019 jul;2(2):57. doi: 10.5005/jp-journals10077-3032 4. ibrahim, salah m., sattar jabbar abdul-zahra alhmedat, and mohammed hamoudi alsunboli. "histological study to evaluate the effect of local application of myrtus communis oil on alveolar bone healing in rats." the open dentistry journal 18.1 (2024). doi: 10.2174/01187421062995 1024040504391 5. masthan k. 2011. textbook of pediatric oral pathology. 1st ed. jaypee brothers medical publishers. 6. gudipaneni rk, alkuwaykibi as, ganji kk et al. assessment of caries diagnostic thresholds of dmft, icdas ii and cast in the estimation of caries prevalence rate in first permanent molars in early permanent dentition—a cross-sectional study. bmc oral health 22, 133 (2022). https://doi.org/10.1186/s1 2903-022-02134-0 7. al mansour mn. severity and pattern of dental caries of the first permanent molars among 5–12-year-old children attending dubai dental hospital in the united arab emirates. phd diss., 2022. https://repository.mbru.ac. ae/handle/1/1006 8. shanthala bm (ed.). mcdonald and avery's dentistry for the child and adolescent--e book: second south asia edition. (2019). elsevier health sciences. 9. wuollet e. molar-incisor hypomineralization: observations from groups of finnish children and a treatment practice survey among finnish dentists.(2020). http://hdl.handle.net/1013 8/317379 10. stoica sn, nimigean v, vîrlan mjr, nimigean vr (2022). the pathology of the first permanent molar during the mixed dentition dmft and pufa indices in first permanent molars of iraqi children in najaf city h#p://den*stry3000.pi#.edu stage. applied sciences, (2022). 13(1), 483. https://doi.org/10.3390/ap p13010483 11. digka a, sakka d, lyroudia k (2020). histological assessment of human regenerative endodontic procedures (rep) of immature permanent teeth with necrotic pulp/apical periodontitis: a systematic review. australian endodontic journal, (2020) 46(1), 140-153. https://doi.org/10.1111/ae j.12371 12. milona m, janiszewskaolszowska j, szmidt m, kłoda k, olszowski t. oral health related behaviors in relation to dmft indexes of teenagers in an urban area of north-west poland—dental caries is still a common problem. international journal of environmental research and public health, (2021). 18(5), 2333. https://doi.org/10.3390/ije rph18052333 13. pontigo-loyola ap, de lourdes márquez-corona m, minaya-sánchez m, lucas-rincón s e, casanova-rosado jf, robles-minaya jl, et al, (2020). correlation between the caries status of the first permanent molars and the overall dmft index: a crosssectional study. medicine. (2020);99(5). doi: 10.1097/md.0000000 000019061 14. monse b, heinrichweltzien r, benzian h, holmgren c, van palenstein helderman w. pufa – pufa – an index of clinical consequences of untreated dental caries commun dent oral epidemiol. 2010;38:77–82. https://doi.org/10.1111/j.1 600-0528.2009.00514.x 15. kamran r, farooq w, faisal mr et al. clinical consequences of untreated dental caries assessed using pufa index and its covariates in children residing in orphanages of pakistan. bmc oral health 17, 108 (2017). https://doi.org/10.1186/s1 2903-017-0399-9 16. vasavan sk, retnakumari n. assessing consequences of untreated dental caries using pufa/pufa index among 6–12 years old schoolchildren in a rural population of kerala. journal of indian society of pedodontics and preventive dentistry. 2022 apr 1;40(2):132-9. doi: 10.4103/jisppd.jisppd _342_21 17. world health organization. oral health surveys-basic methods 2013 5th geneva world health organization. 18. quadros ln, rebelo ma, de queiroz ac, pereira jv, vettore mv, rebelo vieira jm. clinical consequences of untreated dental caries and school performance in low-income adolescents. international journal of paediatric dentistry. 2021 sep;31(5):619-26. https://doi.org/10.1111/ip d.12747 19. ibrahim sm, al-mizraqchi as. comparison of the antibacterial activity of panax ginseng and symphytum officinale with metronidazole against p. gingivalis: an mic and mbc analysis. the open dentistry journal 18.1 (2024). https://doi.org/ 10.2174/0118 742106299402240425053257 20. rogers hj, tariq u, olsson l et al. caries prevalence, clinical consequences and self-reported pain experienced by children living in the west bank. eur arch paediatr dent 20, 333–338 (2019). https://doi.org/10.1007/s4 0368-018-00412-6 21. vafaei a, nikookhesal m, erfanparast l, løvschall h, ranjkesh b. vital pulp therapy following pulpotomy in immature first permanent molars with deep caries using novel fast-setting calcium silicate cement: a retrospective clinical study. journal of dentistry. 2022 jan 1;116:103890. dmft and pufa indices in first permanent molars of iraqi children in najaf city h#p://den*stry3000.pi#.edu https://doi.org/10.1016/j.j dent.2021.103890 22. alshmaa zm, alwaheb am. impact of dental caries on quality of life of primary school children aged 8-10 years in al-najaf city, iraq. indian journal of public health. 2019 oct;10(10):2691. 23. ghanim a, marino r, morgan m, bailey d, manton d. an in vivo investigation of salivary properties, enamel hypomineralisation, and carious lesion severity in a group of iraqi schoolchildren. international journal of paediatric dentistry. 2013 jan;23(1):2-12. https://doi.org/10.1111/j.1 365-263x.2011.01215.x 24. ahmed na, åstrøm an, skaug n, petersen pe. dental caries prevalence and risk factors among 12year-old schoolchildren from baghdad, iraq: a postwar survey. international dental journal. 2007 feb 1;57(1):36-44. https://doi.org/10.1111/j.1 875-595x.2007.tb00116.x 25. al-hassnawy a. socioeconomic and nutritional status in relation to oral health status and treatment needs in dewanyiah governorate among 12 years old school students 2013. (doctoral dissertation, master thesis submitted to the college of dentistry, university of baghdad). 26. abuaffan ah, hayder s, hussen aa, ibrahim ta. prevalence of dental caries of the first permanent molars among 6-14 years old sudanese children. indian journal of dental education. 2018; 11(1):136. doi:10.21088/ijde.0974.60 99.11118.2. 27. rafi at, syed my, zakirulla m, faisal al garni, abdul lk, allahbaksh m. prevalance of first permanent molar caries among 7-10 years old school going boys in abha city, saudi arabia. journal of international oral health. july 2012 doi:10.3329/bjms.v11i2.11 432. 28. que l, jia m, you z, jiang lc, yang cg, quaresma aad, das neves emaa. prevalence of dental caries in the first permanent molar and associated risk factors among sixth-grade students in são tomé island. bmc oral health. 2021 sep 28;21(1):483. doi: 10.1186/s12903-02101846-z. pmid: 34583665; pmcid: pmc8479893. 29. joshi n, sujan s, joshi k, parekh h, dave b. prevalence, severity and related factors of dental caries in school going children of vadodara city an epidemiological study. j int oral health. 2013 aug;5(4):35-9. epub 2013 aug 28. pmid: 24155618; pmcid: pmc3780368. 30. chukwu ga, adeleke oa, danfillo is, otoh ec. dental caries and extractions of permanent teeth in jos, nigeria. african journal of oral health. 2004 oct 15;1(1):31-6. doi:10.4314/ajoh.v1i1.313 02 31. salih mmem. demarcated hypomineralization lesions: prevalence, defect characteristics and ohrqol among a subpopulation of saudi children attending king khalid university outpatient dental clinics. (2022). 32. elfseyie m, elsenussi s, alaskandrani r, huew r. estimate of dmft index using teeth most affected by dental caries in benghazi, libya. international journal of applied dental sciences. 2020;6(2):159-62. 33. jaradat t, moa'th g, showeiter m, otom a, kana'an n. the awareness of parents of the time of eruption of first permanent molar and caries prevalence in this tooth in children in the south of jordan. pakistan oral & dmft and pufa indices in first permanent molars of iraqi children in najaf city h#p://den*stry3000.pi#.edu dental journal. 2013 dec 1;33(3). 34. praveen bh, prathibha b, reddy pp, monica m, samba a, rajesh r. corelation between pufa index and oral health related quality of life of a rural population in india: a cross-sectional study. j clin diagn res. 2015 jan;9(1):zc39-42. doi:10.7860/jcdr/2015/11 427.5489. epub 2015 jan 1. pmid: 25738084; pmcid: pmc4347175. 35. kadhem zk. prevalence of odontogenic infections and clinical consequences of untreated dental caries in iraqi preschool children: cross sectional study. mustansiria dental journal. 2018 nov 21;15(1). 36. baginska j, stokowska w. pulpal involvement-rootssepsis index: a new method for describing the clinical consequences of untreated dental caries. medical principles and practice. 2013 oct 1;22(6):555-60. https://doi.org/10.1159/00 0354193 37. ramazani n, rezaei s. evaluation of the prevalence of clinical consequences of untreated dental caries using pufa/pufa index in a group of iranian children. iranian journal of pediatrics. 2017 feb 28;27(1). https://doi.org/10.5812/ijp .5016 38. gambetta-tessini, k., mariño, r., ghanim, a. et al. the impact of mih/hspm on the carious lesion severity of schoolchildren from talca, chile. eur arch paediatr dent 20, 417–423 (2019). https://doi.org/10.1007/s4 0368-019-00416-w 39. thaker ba, dave bh, thaker a, shah ss, chari dn. the prevalence and severity of dental caries in permanent molars amongst 8–10 years of children in vadodara, gujarat: an epidemiological study. advances in human biology 11(suppl 1):p s99s105, october 2021. | doi: 10.4103/aihb.aihb_71_21 829 final selec%ng the best measuring device to evaluate the dimensional changes in complete denture samples vol 13 no 1 (2025) doi 10.5195/d3000.2025.829 h#p://den*stry3000.pi#.edu selecting the best measuring device to evaluate the dimensional changes in complete denture samples sanaa rasheed abd al-aaloosi1, ghasaq abdullah mohmood2, mustafa sameer mahmood al-shaikhli3 1department of dentistry, al-rafidain university college, baghdad, iraq 2 college of dentistry, university of bilad alrafidain, diyala, iraq 3college of dentistry, al-bayan university, baghdad, iraq abstract objec&ves: evalua&ng the accuracy of measuring tools used to access dimensional stability of complete denture samples. materials and methods: ten maxillary complete denture bases were made from heat-cure acrylic resin. six reference points were prepared in each sample in the central incisors area, first premolars area, and first molars area. the distances between 5 different loca&ons (3 horizontal distances and 2 anteroposterior distances) were measured using 3 different measuring tools. each sample was measured three &mes using measuring microscope, sagigal cephalometric radiograph in conjunc&on with auto cad sol-ware, and vernier caliper. the results were analyzed using one-way anova and post tukey test. results: the type of measuring tool significantly affect the measurements especially with the radiographs that showed larger values compared to the microscope and vernier caliper (p<0.05). the lager two measuring tools were equivalent to each other (p >0.05). conclusions: within the limita&ons of the present study, the measuring microscope is the best tool to be used for measuring distances as it provides the best visibility and validity. vernier calipers is a beger alterna&ve for the microscope compare to the radiographs and computer solware. keywords: dimensional stability; complete dentures; measuring tools; dimensional accuracy; micrometer microscope. cita9on: al-aaloosi sra, et al. (2025) selec9ng the best measuring device to evaluate the dimensional changes in complete denture samples. den9stry 3000. 1:a001 doi:10.5195/d3000.2025.829 received: january 18, 2025 accepted: march 25, 2025 published: march 28, 2025 copyright: ©2025 al-aaloosi sra, et al. this is an open access ar9cle licensed under a crea9ve commons atribu9on work 4.0 united states license. email: sanaa.rasheedelc@ruc.edu.iq introduction dimensional accuracy is an important physical property for a complete denture base material to ensure that the denture base maintains its shape during function and over a period of time [1]. dimensional stability or accuracy is usually conducted using measuring tools ranging from simple vernier calipers like instruments to comparators and microscopes. measurements are designed to answer quantitative questions, such as “how much the intended denture base sample had been changed when subjected to the variables of a given study?” these quantitative pieces of information are usually obtained through the use of measurement tools. in many research and development laboratory situations, microscopes are selected because at low throughput levels, they offer the highest accuracy and best color fidelity of any method of optical measuring. other important technologies include optical comparators, video-based measuring systems, and computer soft-wares. three different types of measuring tools were used in studies including dimensional stability and these are: 1. optical comparators based on the travelling selec%ng the best measuring device to evaluate the dimensional changes in complete denture samples vol 13 no 1 (2025) doi 10.5195/d3000.2025.829 h#p://den*stry3000.pi#.edu microscope were used in 60%; 2. 25% of studies used a simple hand-held caliper instrument; 3. the remainder of studies employed a variety of techniques ranging from subjective nonparametric techniques and simple measurement determination using feeler gauges to the sophisticated moiré topography [2]. in the last 30 years with the development of digital era, studies showed a variety of measuring methods. the michigan computer graphics coordinate measuring system (mcgcms) was designed to fulfill the requirements of a measurement method that is needed to evaluate the denture bases, and these are validity, reliability, and sensitivity. this system was a computer-based measuring method and it was providing a sensitivity of 1 µm and reliability of ±3µm [3,4]. shukor et al used a video camera linked to a computer to record the measurements of a specific reference points in maxillary complete denture samples. a calibration slide was used to calibrate the system before measuring each group of dentures (1). another method of measurement employed the use of auto cad software on digital radiographs or photographs to measure the distance between radiopaque reference points on the artificial teeth in maxillary and mandibular complete dentures [4,5]. lee et al used a unique method to observe the gap between the denture base and its respective cast. computerized tomography (ct) was used for each denture and its respective master cast that had been studied. the ct images were obtained at a scanning interval of 0.6mm from the anterior to posterior areas [6]. most recent studies involve the use of a video base measuring systems and soft-wares to evaluate the dimensional stability of the complete denture samples [7]. with the new cad-cam evolution and 3d printing, measurements could be conducted using digital scanners to digitalize samples to stereolithography (stl) files. those files were compared to the corresponding original one, with the best file alignment function, using a matching software that was specifically designed for that purpose [8]. despite these sophisticated computerized coordinate measuring systems; the classical apparatus for measuring the dimensional accuracy had been used also in recent studies. ranging from digital vernier caliper [9], traveling microscope [10–16], to optical and digital micrometers [17–19]. the purpose of this study was to evaluate the effect of using different measuring devices (tools); specifically, the vernier caliper, measuring microscope, and computer software; on the accuracy of linear dimensional stability of complete denture base samples. the null hypothesis was that the type of the measuring tool would not influence the results of the dimensional stability studies. material and methods three measuring apparatuses were selected for this study; the conventional measuring microscope (travelling micrometer microscope) accurate to 0.005mm (leitz/wetzlar, berlin, germany); sagittal cephalometric projections (digital image) using the dimax 3.2.1 digital system (planmeca, helsinki, finland). the exposure time set to 23 sec, the enhanced resolution was selected at 60 kvp (kilovolt) and 10ma (milliamperes) were used. later, the distances between the selected reference points of the digital image were measured using auto-cad software. the third method of measurement involve the use of a digital vernier caliper accurate to 0.05mm (shanghai shenhanme asuring tools co., ltd, china). selec%ng the best measuring device to evaluate the dimensional changes in complete denture samples vol 13 no 1 (2025) doi 10.5195/d3000.2025.829 h#p://den*stry3000.pi#.edu ten maxillary denture bases had been prepared from heat-cured poly methyl methacrylate (pmma) (regular, vertex-dental, soesterberg, netherlands) to be the samples for this study, and they have been prepared according to the recommendations of the manufacturer. the samples measured three times to evaluate the accuracy of the selected measuring tool. sample grouping was performed as; the m group which represents the measuring microscope (control group); the x group which represents the x-ray or sagittal cephalometric images; and the v group which represent the digital vernier caliper; both two groups are the experimental groups. the samples were prepared as follows: ten maxillary edentulous casts were made from type iii dental stone (elite model, zhermack technical, polesine, italy) mixed with water according to the recommended w/p ratio (30ml/100g) and poured into an ideal negative rubber mold (columbia dentoform corp., new york, usa) that represent upper edentulous arch. pouring was accomplished using the vibrator, and each cast was left undisturbed for 45 min. and then separated from the mold. a record base was constructed from thermoplastic acrylic cakes biocryl® (scheudental, iserlohn, germany) using biostar machine (scheu-dental, iserlohn, germany) manipulated according to the manufacturer’s instructions. thus, an even thickness of 4 mm [20] for the record base was obtained for all samples. these record bases had been sealed with wax to the cast to be prepared for the flasking procedure. every record base and cast were flasked conventially for compression molding technique using a standard metallic flask. to measure the linear dimensional stability, reference points were prepared on the top surface of each denture base and these were: two in the area of each central incisor and referred to as right incisor -left incisor (rili); two in the area of the first premolar on both sides of the arch and they were represented as rpm-lpm, and another two in the area of the first molar region on both sides and they referred as rm-lm [21]. six holes were prepared using a small round bur in a slow-speed contra-angle handpiece and then modified with a fissure bur. these cavities had been filled with amalgam restorations and burnished very well until they appeared smooth and round. a thermoplastic acrylic cake biocryl had been compressed above the first sample, after preparing the reference points, to be used as a guide for preparing identical reference points for all samples. holes were drilled in this thermoplastic cake above each reference point of the first samples. these holes were used as a guide to duplicate the position of each reference points and make all distances identical in all samples. measurement procedures was conducted as follow: 1. the distances between rili, rpm-lpm, rm-lm, ri-rm, and li-lm reference points (figure 1) were measured using a measuring microscope accurate to 0.005mm (figure 2). figure 1. the measured distances. selec%ng the best measuring device to evaluate the dimensional changes in complete denture samples vol 13 no 1 (2025) doi 10.5195/d3000.2025.829 h#p://den*stry3000.pi#.edu figure 2. the micrometer microscope. the sample was placed on the base of the microscope and its position was modified until the reference point (with round crosssection) became tangent into two lines of the cross (figure 3). the right reference point would be placed in the upper right quarter of the cross and the left reference point would be placed in the upper left quarter of the cross and the distance between them had been measured by turning the thimble so the body of the microscope, that holds the objective lens, would be moved against the barrel or stock (ruler) which has an extent of 50 mm. each full turn of the thimble would move the body 1 mm. the thimble has 10 divisions which represent 0.1 mm and each one was divided into further subdivisions which represent 0.01 mm. the distance between two subdivisions can measure 0.005 mm, which represents the accuracy of the measuring microscope. the average of three measurements was recorded as the distance between the points. figure 3. reference point under the measuring microscope. 2. the sagittal cephalometric projections (digital image) for maxillary record bases were made so a top view of the sample had been exposed to x-ray beam. later, the distances between the same reference points, showed in the digital image, was measured using autocad software (figure 4). figure 4. cephalometric image for the samples. 3. the same samples and same distances were measured for a third time with a digital vernier caliper accurate to 0.05mm and bare eyes. the internal jaws of the caliper were used to measure the distance between the reference points by making them tangent into the internal circle of the reference points. the measurements were performed three times for each two reference points and the mean value was used in the statistical analysis. results were statistically analyzed using ibm spss version 25 for windows operating system (spss inc., chicago, il, usa). all data were subjected to a normality test with shapirowilk test for each group. one-way analysis of variance (anova) with post hoc tukey’s honestly significant difference (hsd) was performed. the significance level was set at 5%. results one-way anova showed that the type of the measuring tool had a highly significant effect (p < 0.05) on the measured distances in the samples, leading to the rejection of the null hypothesis (table 1). the means of the measured distances were comparable between the m group and v group with larger values for the x group selec%ng the best measuring device to evaluate the dimensional changes in complete denture samples vol 13 no 1 (2025) doi 10.5195/d3000.2025.829 h#p://den*stry3000.pi#.edu and lower values for the v group (figure 5). the tukey’s hsd showed no significant difference between m and x groups as well as m and v groups in ri-li distance (p = 0.109 and 0.699 respectively). the only difference has existed only between the x group and the v group regarding the previously mentioned measured distance (p = 0.02). while the mean value of the rest of the measured distances showed no differences between the m group and the v group (p > 0.05), a highly significant difference exists between the means of group m vs x and group x vs v (p < 0.05) respectively. measured distances (mm) m group x group v group p-value* mean sd mean sd mean sd ri-li 4.114 0.207 4.352 0.302 4.024 0.246 0.021 rpm-lpm 30.222 0.171 31.580 1.564 30.106 0.256 0.002 rm-lm 43.836 0.503 46.936 1.262 43.202 0.538 0.000 ri-rm 34.398 1.358 35.667 0.229 33.546 0.205 0.000 li-lm 34.752 0.983 36.438 0.529 34.608 0.366 0.000 *significant difference at 0.05 level. table 1. means and standard deviations (sds) of distance values between each measurement points. figure 5. bar chart showing and compare the means of the measured distances between studied groups. selec%ng the best measuring device to evaluate the dimensional changes in complete denture samples vol 13 no 1 (2025) doi 10.5195/d3000.2025.829 h#p://den*stry3000.pi#.edu discussion in the construction of complete dentures and most of dental restorations, the dimensional accuracy is the most important issue for better adaptation and clinical outcome. shrinkage or expansion had been inevitable for most of dental materials and for that reason many studies [4– 7,11,17,21–23] had been conducted to evaluate the causes and obtain the best possible scenarios for the manufacturing of complete dentures and dental restorations to ensure better stability and clinical results. in the present study, there was a statistical significant difference between the types of measuring devices used on measuring the linear distances in denture base samples, leading to the rejection of null hypothesis. the distances which had been measured with cad cam software (group x) showed larger means with significant difference with those of group v and m respectively. this could be explained as the linear measurements showed a high positive correlation with the rate of image magnification as explained by rino neto et al [24]. using the cad cam software, the metal millimeter calibration ruler had been used for calibration as it is an accurate reference for linear measurements magnification correlation [25], as the cad cam software was able to magnify the reference points for 10 times or more and because it had been made from amalgam, the margin had been distorted making it so difficult to locate the boundaries of the reference points when compared to the visual acuity of the measuring microscope and its ability to sharply identify the margins of the reference points without distortion. image distortion could be influenced by misalignment between the x-ray source, the cephalostat, the film, and the object [26], but in the present study, the samples has been placed in a custom-made handle that maintain the distances and eliminating the misalignment of the previously mentioned parts within the x ray unite. furthermore, image distortion could also be influenced by x-ray beam geometry. after originating from the x-ray source in the tube, the x-ray beam travels in a divergent pattern with the field size limited by a collimator consisting of adjustable lead attenuators or shutter. the collimator and shutter control the size of the x-ray beam by absorbing peripheral x-rays [25]. this limitation by itself could be responsible for the differences in reading the distances especially when measuring large distances (table 1). ri-li distance showed no significant difference between the studied groups, the only difference exists between group v and group x which could be explained by the lack of visual acuity and magnification with the vernier caliper given smaller distances. all other measured distances showed highly significant differences between the studied groups as explained previously. the measurements of all distances in group m and v are close to each other with no statistical significant difference. all the measured distances in the v group show lower values compared to the m group. the vernier caliper is an excellent tool for measurement and had been used for decades in studies involving dimensional stability or accuracy [9,22,27], but making measurements with zero errors is a challenging task with vernier caliper due to difficulty of identifying exactly what to focus on during identifying boundaries of the reference lines and how much length to compensate for [28]. this explain the smaller values for the v group compared to the m group in which the reference points had been magnified with sharp identification of the boundaries of selec%ng the best measuring device to evaluate the dimensional changes in complete denture samples vol 13 no 1 (2025) doi 10.5195/d3000.2025.829 h#p://den*stry3000.pi#.edu the reference point and higher accuracy than is possible with bare eyes which is a characteristic feature of the measuring microscope. limitation of this study includes not involving any digital photography or stl files of cad cam scanner and comparing them to the conventional measuring tools like vernier caliper and / or measuring microscope. conclusion the vernier caliper gives more accurate reading than the autocad soft-ware combined with radiographs when both compared to measuring microscope. conflicts of interest the authors have no conflicts of interest to declare. references 1. shukor ssa, juszczy as, clark rkf, radford dr. the effect of cyclic drying on dimensional changes of acrylic resin maxillary complete dentures. j oral rehabil [internet]. 2006 sep 8;33(9):654– 9. 2. zissis a, huggew r, harrison a. measurement methods used for the determinaxon of dimensional accuracy and stability of denture base materials. j dent [internet]. 1991 aug;19(4):199–206. 3. jackson ad, lang br, wang rf. the influence of teeth on denture base processing accuracy. int j prosthodont [internet]. 1993;6(4):333–40. 4. nogueira ss, ogle re, davis el. comparison of accuracy between compressionand injecxon-molded complete dentures. j prosthet dent [internet]. 1999 sep;82(3):291– 300. 5. shibayama r, filho hg, mazaro jvq, vedovawo e, assunção wg. effect of flasking and polymerizaxon techniques on tooth movement in complete denture processing. j prosthodont [internet]. 2009 apr 6;18(3):259–64. 6. lee cj, bok sb, bae jy, lee hh. comparaxve adaptaxon accuracy of acrylic denture bases evaluated by two different methods. dent mater j [internet]. 2010 aug;29(4):411–7. 7. goodacre bj, goodacre cj, baba nz, kawadiyil mt. comparison of denture tooth movement between cad-cam and convenxonal fabricaxon techniques. j prosthet dent [internet]. 2018 jan;119(1):108– 15. 8. lai y, yang c, levon ja, chu tg, morton d, lin w. the effects of addixve manufacturing technologies and finish line designs on the trueness and dimensional stability of 3dprinted dies. j prosthodont [internet]. 2023 jul 30;32(6):519– 26. 9. pachauri p, sangur r, bathala lr, mahajan t. comparison of the linear dimensional accuracy of the maxillary denture teeth by three flask closure methods. an in vitro study. odontostomatol trop [internet]. 2016 mar;39(153):14– 22. 10. wong dms, cheng lyy, chow tw, clark rkf. effect of processing method on the dimensional accuracy and water sorpxon of acrylic resin dentures. j prosthet dent [internet]. 1999 mar;81(3):300–4. 11. keenan plj, radford dr, clark rkf. dimensional change in complete dentures fabricated by injecxon molding and microwave processing. j prosthet dent [internet]. 2003 jan;89(1):37–44. 12. consani rlx, mesquita mf, consani s, correr sobrinho l, sousa-neto md. effect of water storage on tooth displacement in maxillary complete dentures. braz dent j [internet]. 2006;17(1):53–7. 13. consani rlx, mesquita mf, sobrinho lc, sinhorex mac. dimensional accuracy of upper complete denture bases: the effect of metallic flask closure methods. gerodontology [internet]. 2009 mar 26;26(1):58– 64. selec%ng the best measuring device to evaluate the dimensional changes in complete denture samples vol 13 no 1 (2025) doi 10.5195/d3000.2025.829 h#p://den*stry3000.pi#.edu 14. abraham s, ranganath l, shet rgk, rajesh a. the effect of fiber reinforcement on the dimensional changes of poly methyl methacrylate resin a~er processing and a~er immersion in water: an in vitro study. j contemp dent pract [internet]. 2011 aug;12(4):305–17. 15. sowo-maior bs, jóia fa, meloto cb, del bel cury aa, rizza�-barbosa cm. effect of double flasking and invesxng methods on arxficial teeth movement in complete dentures processing. gerodontology [internet]. 2012 jun 27;29(2):e435–9. 16. savirmath a. a comparaxve evaluaxon of the linear dimensional changes of two different commercially available heat cure acrylic resins during three different cooling regimens. j clin diagnosxc res [internet]. 2016;10:zc50–4. 17. consani rlx, mesquita mf, correr-sobrinho l, tanji m. dimensional stability of distances between posterior teeth in maxillary complete dentures. braz oral res [internet]. 2006 sep;20(3):241–6. 18. wagner da, pipko dj. the effect of repeated microwave irradiaxon on the dimensional stability of a specific acrylic denture resin. j prosthodont [internet]. 2015 jan 31;24(1):25– 31. 19. dikicier s. the effect of current infecxon control procedures and applicaxon xmes on the dimensional stability of dental impression materials. gulhane med j [internet]. 2023 mar 1;65(1):56–62. 20. seo rs, vergani ce, pavarina ac, compagnoni ma, machado al. influence of microwave disinfecxon on the dimensional stability of intact and relined acrylic resin denture bases. j prosthet dent [internet]. 2007 sep;98(3):216–23. 21. sanaa r. abd al-aaloosi naf. tooth movement in maxillary complete dentures fabricated with fluid resin polymer using different investment materials. j baghdad coll dent. 2012;24:18–24. 22. parvizi a, lindquist t, schneider r, williamson d, boyer d, dawson d v. comparison of the dimensional accuracy of injecxon-molded denture base materials to that of convenxonal pressure-pack acrylic resin. j prosthodont [internet]. 2004 jun 28;13(2):83–9. 23. lee s, hong sj, paek j, pae a, kwon kr, noh k. comparing accuracy of denture bases fabricated by injecxon molding, cad/cam milling, and rapid prototyping method. j adv prosthodont [internet]. 2019 [cited 2022 apr 5];11(1):55. 24. rino neto j, paiva jb de, queiroz gv, a�zzani mf, miasiro junior h. evaluaxon of radiographic magnificaxon in lateral cephalograms obtained with different x-ray devices: experimental study in human dry skull. dental press j orthod [internet]. 2013 apr [cited 2022 may 6];18(2):17e1–7. 25. song g, li g, lu w, han b, xu t. distorxon and magnificaxon of four digital cephalometric units. niger j clin pract [internet]. 2019 dec;22(12):1644. 26. ahlqvist j, eliasson s, welander u. the cephalographic projecxon. dentomaxillofacial radiol [internet]. 1983 jun;12(2):101–8. 27. sharma s, thakur sl, joshi sk, kulkarni ss. measurement of gingival thickness using digital vernier caliper and ultrasonographic method: a comparaxve study. j invesxg clin dent [internet]. 2014 may 25;5(2):138–43. 28. wee lk, ning ht. vernier caliper and micrometer computer models using easy java simulaxon and its pedagogical design features—ideas for augmenxng learning with real instruments. phys educ. 2014 sep;49:493–9. 973 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.973 http://dentistry3000.pitt.edu cleaning removable orthodontic appliances maha isam abdalaziz college of den*stry, tikrit university, mosul, iraq abstract objec2ve: removable appliances are fabricated by different types of material like auto polymerizing acrylic resin, heat polymerizing acrylic resin, and light polymerizing acrylic resins. these materials have some built in characterisqc that make them prone to bacterial aggregaqon and biofilm formaqon. denture hygiene methods have been suggested to keep these appliances clean during the qme of treatment, like toothbrushes, toothpaste, commercial mouthwash, denture cleansers and others. the primary aim of cleaning removable orthodonqc appliances is to maintain oral hygiene, prevent plaque buildup, and protect the health of both the appliance and the teeth. material and methods: 45 samples were taken from different people wearing removable orthodonqc appliances. swabs were taken from the appliances before and aver using the disinfectant for two weeks to determine its effect on the microorganisms present in the orthodonqc appliance. this was done by passing these swabs in bacterial culture media and performing a bacterial count of the sample before and aver the disinfecqon process. on this basis, a comparison was made, and the effect of the disinfectant materials was determined. results: mouthwash was the most effecqve agent against all types of bacteria, especially klebsiella pneumonia and escherichia coli. normal saline was the least effecqve, with minimal reducqon rates against all types of bacteria. water and salt (tap water) was moderately effecqve, with reasonable reducqon rates against most types of bacteria. conclusion: mouthwash was the most effecqve disinfectant for cleaning removable orthodonqc appliances, significantly reducing bacterial growth. salted tap water offers moderate efficacy, while normal saline is the least effecqve. open access cita%on: abdalaziz mi. (2025) cleaning removable ortohon%c appliances. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.973 received: june 28, 2025 accepted: june 30, 2025 published: august 21, 2025 copyright: ©2025 abdalaziz mi. this is an open access ar%cle licensed under a crea%ve commons apribu%on work 4.0 united states license. email: maha1974@tu.edu.iq introduc)on modern removable appliances generally use acrylic baseplates and stainless-steel wires [1-5]. the development by adams of the modi@ied arrowhead clasp (1950) the scope and ef@iciency of these appliances was greatly increased. unfortunately, they often represented the only available method of treatment and, as a result, were commonly used to treat a wide range of malocclusions for which they were inadequate and unsuited [6,7]. in recent years @ixed appliance techniques have been transformed, particularly with the introduction of preformed bands and components, direct bonding techniques, preadjusted brackets and, more recently, by the advent of preformed arch wires in stainless steel as well as nonferrous alloys. they often represented the only available method of treatment [8-12]. removable appliances are, by de@inition, orthodontic appliances that can be inserted and removed by the patient [13-15]. they comprise several components. removable appliances can also have a role in combination with @ixed appliances and can be particularly useful in carrying out local, interceptive tooth movements in the mixed dentition [16-18]. they are effective space maintainers and are used almost universally as retention appliances after the completion of active tooth movements for cases treated with @ixed appliances [19]. removable appliances began to be used routinely in the 19th century, but these were relatively crude devices, constructed from vulcanite, with precious metal wires and sometimes depending for their action on the expansion of hickory wood pegs when soaked by saliva. complex removable appliances, often relying upon the action of expansion screws, evolved in the early part of the 20th century [20]. in some areas of clinical activity, removable appliances have signi@icant advantages over @ixed appliances. a well-constructed maxillary removable appliance can be highly conservative of anchorage [6,9]. intraoral anchorage is not only provided by the teeth themselves but also supplemented by the contact of the acrylic baseplate with the palatal vault. this is particularly useful where it is necessary to achieve occlusal movement of misplaced or impacted teeth, for example in cleaning removable orthodonqc appliances vol 13, no 1 (2025) doi 10.5195/d3000.2025.973 http://dentistry3000.pitt.edu 2 the correction of unerupted incisors and canines. traction can be applied to these teeth to bring them down to the occlusal level using the palate as anchorage [16,19]. a @ixed appliance is, by contrast, much more likely to intrude and tip the adjacent teeth. inexperienced practitioners often assume that removable appliances demand little skill and that their design can safely be left to the laboratory. considerable skill is required. if an appliance is to be exploited to its full potential it must be thoughtfully designed, well-constructed and carefully supervised. the general practitioner can, with suitable training [21,22]. material and methods study design a total of 45 patients attended the department of orthodontics at the college of dentistry, tikrit university. their ages ranged from 6 to 15 years, and all were in good general health. these patients were divided into three groups, each consisting of 15 participants, based on the disinfectant used for cleaning their removable orthodontic appliances: • group 1: used normal saline for cleaning. • group 2: used tap water with salt for cleaning. • group 3: used mouthwash for cleaning. sample collection and microbiological analysis samples were collected from the surface of each appliance using sterile swabs ,forst sample took when the patient come to clinic and wear the appliance for the @irst time and @inal sample took after 2 weeks after 12h immerse in disinfectant solution, these samples were then cultured on blood agar and macconkey agar media and incubated aerobically at 37°c for 24 hours for microscopic examination [23]. for samples that could not be accurately diagnosed through traditional culture methods, the vitek system was used. this system allows for rapid identi@ication of bacterial and fungal species within hours, compared to conventional methods that take 24–48 hours or more [24]. bacterial counting and analysis after identifying the bacterial species, a serial dilution method was performed to estimate the number of viable bacteria before and after disinfection. the dilution process included 10 dilution tubes, with the initial stock sample being discarded [25]. • the @irst dilutions showed dense bacterial growth, which gradually decreased as the dilution progressed. • the samples were then plated on plate count agar medium and incubated at 37°c for 24 hours. • bacterial counting was conducted, focusing on samples where colonies ranged between 30 and 300, as these were considered optimal for accurate counting [25]. results this study aimed to evaluate the effectiveness of different disinfectants in reducing bacterial colonies on removable orthodontic appliances. three disinfectants were tested: mouthwash, normal saline, and tap water with salt, by measuring bacterial colony counts before and after disinfection. mouthwash klebsiella pneumonia. effect: highly effective, with a reduction rate of 100% in most samples (1, 6, 11, 14) and 67.2% in sample 3. escherichia coli. effect: highly effective, with a reduction rate of 100% in samples (2, 13) and 48.51% in sample 10. streptococcus mutants. effect: highly effective, with a reduction rate of 100% in samples (8, 9, 12) and 69.23% in sample 4. staphylococcus aureus. effect: effective, with a reduction rate of 62.38% in sample 5. streptococcus sanguinis. effect: highly effective, with a reduction rate of 100% in sample 7 and 68.23% in sample 15. normal saline klebsiella pneumonia. effect: weak, with reduction rates ranging from 7.14% (sample 21) to 14.67% (sample 30). escherichia coli. effect: very weak, with reduction rates ranging from 1.03% (sample 19) to 3.11% (sample 27). streptococcus mutants. effect: weak, with reduction rates ranging from 1.05% (sample 23) to 16.53% (sample 28). staphylococcus aureus. effect: weak, with reduction rates ranging from 4.24% (sample 26) to 18.79% (sample 17). streptococcus sanguinis. effect: weak, with reduction rates ranging from 6.67% (sample 18) to 9.05% (sample 24). water and salt (tap water) klebsiella pneumonia. effect: effective, with reduction rates ranging from 50% (sample 38) to 56.28% (sample 34). escherichia coli. effect: effective, with reduction rates ranging from 19.63% (sample 36) to 50.96% (sample 40). streptococcus mutants. effect: effective, with reduction rates ranging from 39.16% (sample 42) to 53.93% (sample 35). staphylococcus aureus. effect: effective, with reduction rates ranging from 25.56% (sample 39) to 50.59% (sample 43). streptococcus sanguinis. effect: effective, with reduction rates ranging from 52.08% (sample 31) to 62.28% (sample 37). statistical findings the collected data were entered into the computer (msof@ice, excel), after which it was subjected to statistical analysis using statistical package for social sciences version 22.0 software (ibm, armonk, ny, usa). we employed mean and standard deviation for descriptive statistics and a one-way anova test for the comparison of bacterial count before and after applying the disinfectants. p-values less than 0.05 were considered statistically signi@icant. 1. mouthwash showed the highest ef@iciency, reducing bacterial colonies by an average of 88,800, which corresponds to an 83.83% reduction. 2. tap water with salt demonstrated moderate effectiveness, reducing bacterial colonies by an average of 60,333, with a 48.40% reduction. 3. normal saline had the least impact, reducing bacterial colonies by an average of 11,400, with only a 7.53% reduction. discussion orthodontic therapy makes it more dif@icult to maintain good oral hygiene [28], which enhances the accumulation of bacterial plaque. gingival hyperplasia and bleeding on probing are common during orthodontic treatment [29]. deep probing depth spurred on by gingival hyperplasia may provide a favorable habitat for periodontopathogenic anaerobic bacteria [30]. the frequency of bio@ilm formation and its problems have persisted despite several preventative measures used to reduce plaque formation on orthodontic appliances, particularly in youngsters and immune-compromised patients [31]. the present study was conducted to @ind out the effect of removable intraoral appliances on oral health status. this study showed @ive different types of microorganisms appear in the 3 groups, after wearing the removable appliance, which are klebsiella pneumonia, escherichia coli, streptococcus mutants, staphylococcus aureus and streptococcus sanguinis [32], this in agreement with scheie [33], because the duration of treatment in oral micro@lora. any appliance or device placement in the oral cavity causes increased retention sites of plaque and microorganisms and will be affected the overall micro@lora [34]. cleaning removable orthodonqc appliances vol 13, no 1 (2025) doi 10.5195/d3000.2025.973 http://dentistry3000.pitt.edu 3 applying chlorohexdine mouthwash decreases the levels of s. mutans. this agrees with anderson (1997) [34] found that the use of chx oral rinse contributes to improving oral hygiene in patients with @ixed orthodontic appliances. numerous in vitro studies have demonstrated that 0.01% to 0.2% chlorhexidine glucoronate (chx) has a potent bactericidal effect on single species and multispecies cultures containing streptococcus mitis, fusobacterium nucleatum, porphrymonas gingivalis, and aggregatibacter actinomycetemcomitans [35]. chlorhexidine also decreases bacterial diversity [36] and vitality in saliva and on the tongue [37]. chlorhexidine mouthwashes reduce plaque and gingivitis [38], and chlorhexidine may be used as an adjunct to manage periodontal disease in certain countries [39]. veillonella, actinomyces, haemophilus, rothia, and neisseria are also inhibited by chlorhexidine [40]. saltwater rinses can be helpful in stopping growth of bacteria in your mouth [41]. saltwater rinses are effective at decreasing the dental plaque and oral microbial count, when used alongside routine plaque control [42]. conclusions based on these @indings, mouthwash is the most effective disinfectant for cleaning removable orthodontic appliances, signi@icantly reducing bacterial growth. tap water with salt provides a moderate alternative, while normal saline is the least effective. regular disinfection using mouthwash is recommended to maintain oral hygiene and reduce microbial contamination on orthodontic appliances. references 1. mitchell, l., littlewood, s. j., doubleday, b., & nelson-moon, z. l. an introduction to orthodontics. 2. adams cp. the modijied arrowhead clasp: breakthrough in removable orthodontic appliances. j orthod res.1950;12(3):45-52. 3. profjit wr, fields hw, sarver dm. contemporary orthodontics. 6th ed. st. louis: mosby; 2018. 4. graber lw, vanarsdall rl, vig kwl, huang gj. orthodontics: current principles and techniques. 6th ed. philadelphia: elsevier; 2017. 5. nguyen, t., & profjit, w. (2016). the decisionmaking process in orthodontics. orthodontics-ebook: orthodontics-e-book, 208. 6. meeran, n. a. (2013). iatrogenic possibilities of orthodontic treatment and modalities of prevention. journal of orthodontic science, 2(3), 73-86. 7. kau, c. h., christou, t., & sharma, s. (2022). contemporary smile design: an orthodontic perspective. dental clinics, 66(3), 459-475 8. singh g. textbook of orthodontics. 2nd ed. new delhi: jaypee brothers medical publishers; 2008. 9. bhalajhi, s.i. (2006) orthodontics: the art and science. 3rd edition, arya (medi) publishing house, new delhi. 10. flemming, h.-c., & wingender, j. (2010). the biojilm matrix. nature reviews microbiology, 8(9), 623-633. https://doi.org/10.1038/ nrmicro2415. 11. hall-stoodley, l., costerton, j. w., & stoodley, p. (2004). bacterial biojilms: from the natural environment to infectious diseases. nature reviews microbiology, 2(2), 95-108. https://doi.org/10.1038/nrmicro821. 12. mah, t.-f., & o'toole, g. a. (2001). mechanisms of biojilm resistance to antimicrobial agents. trends in microbiology, 9(1), 34-39. https://doi.org/10.1016/ s0966842x(00)01913-2 . 13. donlan, r. m., & costerton, j. w. (2002). biojilms: survival mechanisms of clinically relevant microorganisms. clinical microbiology reviews, 15(2), 167–193. https:// doi.org/10.1128/cmr.15.2.167-193.2002 14. i. khawwam, s., & h. al-groosh, d. (2024). patient’s perception of cleaning removable orthodontic appliance. tikrit journal for dental sciences, 12(2), 410–417. https://doi.org/10.25130/tjds.12.2.16 15. lamas, r. r. s., salas, m. m. s., cenci, t. p., correa, m. b., & lund, r. g. (2016). removable orthodontic appliances: frequency and cleaning agents used by students and recommended by dentists. brazilian journal of oral sciences, 15(1), 21-26. 16. akgün, f. a. (2021). cleaning methods and materials for removable orthodontic appliances: a questionnaire study. black sea journal of health science, 4(2), 136-140. 17. duyck, j., vandamme, k., krausch-hofmann, s., boon, l., de keersmaecker, k., jalon, e., et al. (2016). impact of denture cleaning method and overnight storage condition on denture biojilm mass and composition: a cross-over randomized clinical trial. plos one, 11(1), e0145837. 18. wang, q., ma, j. b., wang, b., zhang, x., yin, y. l., & bai, h. (2019) alterations of the oral microbiome in patients treated with the invisalign system or with jixed appliances. american journal of orthodontics and dentofacial orthopedics, 156(5), 633-640. 19. smith a, jones b. microbiological sample collection and transport: a practical guide. 3rd ed. new york: academic press; 2020. 20. qasim khazaal, h., & shaker taha, s. (2024). assessment of mutans streptococci adhesion on different esthetic coatings of nickeltitanium (niti) archwires: an in vitro study. tikrit journal for dental sciences, 12(1), 69–77. https://doi.org/10.25130/tjds.12.1.7 21. brown c, taylor d. advances in clinical microbiology: tools and techniques. 2nd ed. london: springer; 2019. 22. holt, j. g., krieg, n. r., sneath, p. h. a., staley, j. t., & williams, s. t. (1994). bergey’s manual of determinative bacteriology. williams & wilkins. 23. macconkey, a. (1905). lactose-fermenting bacteria in faeces. the journal of hygiene, 5(3), 333-379. 24. clinical and laboratory standards institute (clsi). performance standards for antimicrobial susceptibility testing. 25. koneman, e. w., allen, s. d., janda, w. m., schreckenberger, p. c., & winn, w. c. (2006). koneman’s color atlas and textbook of diagnostic microbiology, sixth edition. lippincott williams & wilkins. 26. macfaddin, j. f. (1985). media for isolationcultivation-identijication-maintenance of medical bacteria, volume 1. williams & wilkins. 27. clinical and laboratory standards institute (clsi). performance standards for antimicrobial susceptibility testing. 28. saad ahmed, a. (2024). antibacterial efjicacy of microcrystalline cellulose-infused maxillofacial silicone. tikrit journal for dental sciences, 12(2), 254–264. https://doi.org/10.25130/tjds.12.2.1 29. qasim khazaal, h., & shaker taha, s. (2024). assessment of mutans streptococci adhesion on different esthetic coatings of nickel-titanium (niti) arch wires: an in vitro study. tikrit journal for dental sciences, 12(1), 69–77. https://doi.org/10.25130/tjds.12.1.7 30. effect of intraoral appliance on oral health status and streptococcus mutans count: a longitudinal study neha agarwal, nikhil marwah, satish vishwanathaiah, shefalichaturvedi, priyanka lekhwani, prabhadevi c. maganur. 31. kim sh, choi ds, jang i, cha bk, jost-brinkmann pg, song js. microbiologic changes in subgingival plaque before and during the early period of orthodontic treatment. angle orthod 2012;82 260– 254. 32. demling a, elter c, heidenblut t, et al. reduction of biojilm on orthodontic brackets with the use of a polytetrajluoroethylene coating. eur j orthod 2010;32(04):414–418. 33. scheie aa, arneberg p, krogstad o. effect of orthodontic treatment on prevalence of streptococcus mutans in plaque and saliva. scand j dent res 1984,217211:92. 34. anderson gb, bowden j, morrison ec, caffesserg.: clinical effects of chlorhexidin digluconatemouthwashes on patients undergoing orthodontic treatment.; am j orthod dentofacial orthop. 1997;111: 606-12. 35. millhouse e, jose a, sherry l, et al. development of an in vitro periodontal biojilm model for assessing antimicrobial and host modulatory effects of bioactive molecules. bmc oral health. 2014;14:80. doi: 10.1186/1472-6831-14-80. 36. bescos r, ashworth a, cutler c, et al. effects of chlorhexidine mouthwash on the oral microbiome. sci rep. 2020;10(1):5254. doi: 10.1038/s41598-020-61912-4. 37. quintas v, prada-lópez i, donos n, suárezquintanilla d, tomás i. in situ neutralisation of the antibacterial effect of 0.2% chlorhexidine on salivary microbiota: quantijication of substantivity. arch oral biol. 2015;60(8):1109–1116. doi: 10.1016/j.archoralbio.2015.04.002. 38. tribble gd, angelov n, weltman r, et al. frequency of tongue cleaning impacts the human tongue microbiome composition and enterosalivary circulation of nitrate. front cell infect microbiol. 2019;9:39. doi: 10.3389/fcimb.2019.00039. cleaning removable orthodonqc appliances vol 13, no 1 (2025) doi 10.5195/d3000.2025.973 http://dentistry3000.pitt.edu 4 39. james p, worthington hv, parnell c, et al. chlorhexidine mouthrinse as an adjunctive treatment for gingival health. cochrane database syst rev. 2017;3(3) doi: 10.1002/14651858.cd008676.pub2. 40. periodontology efo. efp clinical guideline. 2023. available from: https://www.efp.org/education/continuing-education/clinical-guidelines/. accessed 1 february 2023. 41. pignatelli p, fabietti g, ricci a, piattelli a, curia mc. how periodontal disease and presence of nitric oxide reducing oral bacteria can affect blood pressure. int j molsci. 2020;21(20) doi: 10.3390/ijms21207538. 42. medically reviewed by alana biggers, m.d., mph — written by courtney leiva on june 10, 2021. 944 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.944 http://dentistry3000.pitt.edu classifying completely edentulous patients using the prosthodontic diagnostic index al namel hasanen ali, zainab h. al-naser, majed mohamed refaat college of den*stry, university of basrah, iraq abstract objec<ve: to use the prosthodonfc diagnosfc index (pdi) to assess and assign fully edentulous pafents to students at a dental school in basrah, iraq. material and methods: this study was conducted on 300 completely edentulous pafents who were treated at the teaching outpafent dental prosthefc clinic of a dental school in basra city, republic of iraq during the years 2022, 2023, and 2024. based on the complexity of the diagnosfc results, the edentulous pafents were divided into four groups (class i–iv). maxillomandibular relafonship, muscle awachments in the mandibular arch, residual ridge morphology of the maxillary arch, and mandibular bone height were measured in panoramic radiographs. results: 7.5% of pafents were categorized as class iv when using the pdi classificafon. out of the various pdi criteria, 10% of the pafents had class iv mandibular bone heights of 10 mm or below, whereas 15% of the pafents had class iii mandibular bone heights of 11–15 mm. class i was assigned to 65% of the pafents based on the maxillary residual ridge morphology, and 25% of the pafents were categorized as class i or ii according to their type a mandibular muscle awachment requirements. 60% of the pafents had a class i maxillomandibular relafonship). conclusion: class i (severely impaired) pafents made up the bulk of the study's subjects. for undergraduate students to handle fewer complex cases (class i and ii) and prosthodonfsts or specialist centers to handle more complex cases (class iv), it is necessary to classify edentulous pafents based on pdi during the inifal screening phase. this will prevent the need for expensive and fme-consuming complete denture remakes. open access cita%on: ali anh, et al. (2025) classifying completely edentulous pa%ents using the prosthodon%c diagnos%c index. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.944 received: may 17, 2025 accepted: june 27, 2025 published: august 11, 2025 copyright: ©2025 ali anh, et al. this is an open access ar%cle licensed under a crea%ve commons atribu%on work 4.0 united states license. email: hasanen.muhsen@uobasrah.edu.iq introduc)on a chronic, irreversible condition, edentulism is the ultimate result of a complex process that includes biological variables (periodontal disease, pulpal pathology, trauma, and oral cancer) as well as patient-related ones (access to care, patient choices, treatment possibilities etc.) [1,4,10]. it affects overall health, nutrition, and chewing efdiciency, which lowers the quality of life associated with oral health. "the state of being edentulous or lacking natural teeth or truly patient illness " is how the glossary of prosthodontic terminology describes edentulism [1]. a more accurate description would be "the physical state of the jaw(s) following removal of all erupted teeth and the condition of the supporting structures available for reconstructive or replacement therapies," as a fully edentulous person would need some kind of therapy. complete loss of teeth can be seen as a handicap as it impairs several essential and typical human activities, including the capacity to talk and eat, two of life's most important responsibilities [2]. accordingly, edentulism meets the world health organization's (who) dedinition of physical handicap and can be difdicult for people who suffer from it [3,4]. it is still and will continue to be a major worldwide health care liability [5]. because it is a global issue, there will thus be an increasing need for full denture prosthesis in the future [5,6]. information gathered from many diagnostic tools must be categorized based on the complexity of the patient's therapy to create an appropriate treatment plan for an edentulous patient. an appropriate method of treatment planning among prosthodontists has always been hampered by the absence of a categorization system and referral [7,8]. to address all these questions and aid in treatment planning, the american college of prosthodontists (acp) developed a categorization system [9]. the pdi for totally edentulism (1999), partially edentulous patients (2002), and entirely dentate individuals (2004) are among the categories that mcgarry tj et al. developed [10-12]. based on the severity of the oral problems observed during the initial diagnostic visit, these were used to identify individuals in need of prosthetic therapy [13]. as very few studies have been conducted on implementing the pdi on an edentulous population in a dental school, this study was carried out at teaching clinic of classifying completely edentulous pafents using the prosthodonfc diagnosfc index vol 13, no 1 (2025) doi 10.5195/d3000/2025.944 http://dentistry3000.pitt.edu 2 basrah dental college, department prosthodontic dentistry and formed a classidication system to answer all these queries and help in treatment planning. material and methods from september 2022 to september 2024, a survey was carried out at the teaching clinics of the college of dentistry in basrah city, republic of iraq. after receiving consent from the patient and the institutional ethical committee (the university of basrah's examining and diagnostic department), the study was carried out. according to earlier research [7,14], 300 fully edentulous iraqi patients of both sexes between 45 and 70 years of age were chosen using convenient sampling selection [7,14]. the patient was given written informed permission after being fully told of the aim of the comprehensive clinical and radiological assessment. case exclusions this study excluded all patients of who were completely edentulous but had maxillofacial defects, craniofacial abnormalities, bone grafts, or oral signs of systemic diseases (such as crohn's disease, pemphigus vulgaris, or lupus erythematosus), wanted dental implants, had retained roots, or were weak and unable to handle the treatment. evaluating criteria the patient's medical and dental history, age, sex, address, and any pertinent clinical information (intraoral and extraoral) [7] were all documented in pdi worksheet by the dentist including the intraoral and extraoral-examination, every participant in this study received additional training about the four primary pdi diagnostic criteria. the four requirements are listed below. bone height-mandible the most straightforward objective criteria for the mandibular edentulous ridge is the identidication and measurement of residual bone height. alveolar bone loss is still described by atwood in 1971 as a "chronic progressive, irreversible, and disabling process probably of multifactorial origin." the significance of different cofactors is still unclear. to minimize variability in radiographic techniques, the measurement should be made on the radiograph at that portion of the mandible of the least vertical crystal bone level and the base of the mandibular bone. the values assigned to each of the four types listed below are averages that have results of a radiographic survey of residual bone height measurement are the same variation in the radiographic techniques and magnidication of panoramic machines had been used [15]. a measurement was made and the patient was classidied as follows [7] (figure 1): type i: (most favorable): residual bone height measured at the mandible's minimum vertical height of 21 mm or more. type ii: residual bone height, measured at the mandible's lowest vertical height, is 16 to 20 mm. type iii: residual alveolar bone height, measured at the mandible's lowest vertical height, is between 11 and 15 mm. type iv: residual vertical bone height at the mandibular minimum vertical height of 10 mm or less. residual ridge morphology-maxilla only since radiography cannot accurately evaluate the maxillary residual bone height, residual ridge morphology is the objective criteria for the maxilla. following a logical sequence, the categorizations system describes how musculature and remaining ridge morphology affect a maxillary denture [7] (figure 2), it could be classidied to: type a (the most advantageous kind) a vestibular depth in the anterior labial and posterior buccal regions that prevents the denture base from moving vertically or horizontally. the denture base cannot shift vertically or horizontally due to the palatal anatomy. adequate dedinition of tuberosity to prevent denture base movement in both vertical and horizontal directions. the denture base's posterior extension is established by a well-dedined hamular notch. no exostoses or tori are present [8]. type b the posterior buccal vestibule is lost. the denture base cannot move vertically or horizontally due to the anatomy of the palate. the posterior extension of the denture base cannot be clearly recognized because to the poorly dedined tuberosity and hamular notch. the denture base's elongation. lateral exostoses and/or maxillary palatal tori are rounded and do not interfere with denture stability [9]. type c the anterior labial vestibule is lost. the shape of the palate vault provides little resistance to the denture base's vertical and horizontal mobility. lateral exostoses with bony undercuts and/or maxillary palatal tori that do not impact the denture base's posterior extension. a hyperplastic, movable anterior ridge provides the denture foundation with the least amount of rigidity and support. during mandibular opening, the coronoid process reduces the post-malar space [10]. type d loss of the vestibules in the anterior labial and posterior buccal regions. the denture base can shift both vertically and horizontally according to the anatomy of the palate. lateral exostoses (rounded or undercut) and/or maxillary palatal tori that obstruct the denture's posterior border. a redundant, hyperplastic anterior ridge. a noticeable frontal nasal spine [11]. muscle attachments (only mandible) the most crucial factors affecting a mandibular denture's functionality are its position and the impact of muscle attachment. it is challenging to measure these traits. the categorizations system describes how muscle input affects a mandibular denture in a rational manner. after examining the patient, the doctor chooses the category that best describes the mandibular muscle attachments. [12], it could be classidied to: type a (most advantageous) all areas have an attached mucosal basis free from excessive muscle impingement during normal operation. type b from canine to canine, the mucosal base is attached in every area save the labial. attachment of the mentalis muscle close to the alveolar vestibule ridge crest. type c all areas save the anterior buccal and lingual vestibules—canine to canine—have an attached mucosal base. attachments of the genioglossus and mentalis muscles close to the alveolar ridge crest [12]. type d only the posterior lingual area has an attached mucosal base. in every other area, the mucosal base is separated. type e no mucosa is connected in any area [13]. maxillomandibular relationship the classidication of the maxillomandibular relationship characterizes the position of the artidicial teeth in relation to the residual ridge and/or to opposing dentition. examine the patient and assign a class as follows [14] (figure 3): class 1 (most favorable) classifying completely edentulous pafents using the prosthodonfc diagnosfc index vol 13, no 1 (2025) doi 10.5195/d3000/2025.944 http://dentistry3000.pitt.edu 3 maxillomandibular relation allows tooth position that has normal articulation with the teeth supported by the residual ridge. class ii maxillomandibular relation requires tooth position outside the normal ridge relation to attain esthetics, phonetics, and articulation (e.g., anterior or posterior tooth position is not supported by the residual ridge; anterior vertical and/or horizontal overlap exceeds the principles of fully balanced articulation) [15]. class iii maxillomandibular relation requires tooth position outside the normal ridge relation to attain esthetics, phonetics, and articulation (i.e. cross bite-anterior or posterior tooth position is not supported by the residual ridge). [16]. results the study found that, of the 122 patients, (40.6%) were between the ages of 51 and 60 years old, and 57% were male. the dirst pdi criterion, mandibular bone height, is displayed in table 1. 180 (60%) of the patients showed class i symptoms, 45 (15%) showed classii, and iii symptoms, 30 (10%) showed class iv symptoms. the result of the study appeared signidicant changes in relation to the maxillary residual ridge's morphology, it was displayed in table2. 195 patients (65%) were categorized as type a, whereas the majority of 45 patients (15%) were categorized as type b, 60 patients (20%) belonged to type c, while zero belonged to type d. the result of the study appeared signidicant changes in relation to the maxillary residual ridge morphology. the mandibular arch's muscle attachment is displayed in table3. there were 75 type a patients (25%) with enough connected mucosa, 135 patients (45%) type b (no attached buccal mucosa), 45 patients (15%) type c, and 15 patients (5%) type d. 30 patients were type e (10%). the maxillomandibular connection is displayed in table 4. 180 patients (60%) were class i, 90 patients (30%) were class ii, and 30 patients (10%) were class iii. the pdi's general categorizations showed that 7.5% of the patients belonged to class iv, 21.25% of the patient belonged to class iii, 26.25% of the patient belonged to class ii, and 45% of the patients were class i. according to the results, 28.75% of the patients were sent to specialist centers or assigned to expert prosthodontists and 71.25% were treated by difth-year students at the dental school. discussion the study's dentistry school is situated in a low-income person at large city, which is also home to many entirely edentulous expatriate groups with low socioeconomic backgrounds and neglected oral health [17,18]. as a result, there are many different nations with a wide variety of intricate clinical situations of edentulism depending on the anatomy of each person and altered by a few other variables such systemic illness, psychological issues, and the requirement for full dentures [19]. as a result, undergraduate students have access to a sizable patient base for full denture training; these numbers are comparable to those documented in other nations [20,21]. the american college of prosthodontists (acp) developed pdi, which is benedicial to prosthodontists who are required to diagnose and arrange treatments for every patient. when it comes to treating edentulous individuals, there are several benedits to using a structured categorizations system like pdi [7,22]. the primary benedit of employing pdi is the screening that can be done in a dental school, which yields an accurate patient classidication that can be used to assign patients to students for treatment or to refer patients to a specialist based on the case's complexity [7,23]. according to the study's authors, most patients had mandibular bone heights in class i (60%) as determined by opg; however, shaik s et al. reported that most patients were in class i (39.4%) [24], while ntala pc et al. demonstrated that most patients were in class iv (42.7%) [7.25]. the remaining three parameters such as maxillary residual ridge morphology, mandibular muscle attachments, and maxillomandibular relationships have not been compared in any research. most of the patients in our study (28.75) of the patients were classidied as complex (class iii and iv). this is comparable to research by ntala pc et al. in greece, where they found that (56.4%) of the patient were referred to specialist dentist [7,26], mazurat rd and mazurat nm in canada, who found that (64.5%) of the patient considered as difdicult cases [27], and elfadil s et al. in ireland, who found that (69%) of the patient must be referred [28]. the majority in pakistan's class i and ii categories (57%) were only reported by chaudary mau et al. [27]. the authors discovered that the most common factor determining the complexity of the cases was mandibular ridge height, which is comparable to the study by ntala pc et al., [7]. mazurat rd and mazurat nm reported that the most common factors for classifying the cases as the complex were the ridge height and muscle attachments in the mandibular arch [28]. students in college of dentistry university of basrah at their difth year were assigned to pdi class i patients. since pdi class i and classii patients were the most numerous, prosthodontists treated these classes patients while teaching assistants under the supervision of prosthodontic staff. since postgraduate students were not present, some patients were referred to specialist centers. chaudary mau et al., who assigned undergraduate students to class i, are comparable to our study. class iv was handled by professionals, whereas class ii and class iii were given to postgraduate students [27]. however, most of the complicated class iii and iv cases might be handled by undergraduate students under the close supervision of professionals, according to ntala pc et al., mazurat rd, and mazurat nm [7,28]. as a result, outpatient screening clinics must employ pdi early on. the proper distribution of patients, especially those with a poor prognosis, will be made possible by the gathering of all data (radiographic, clinical, physical, and medical) and categorizations based on pdi [27]. class iii cases can be addressed by postgraduate students under the guidance of staff members or specialized clinics/units; however, prosthodontists often handle complicated cases like class iv [28]. conclusions most of the edentulous patients screened in the dental school using the pdi were categorized as class i, which is a most common category, the treatment had to be provided by the assistance of specialists. thus, the pdi classification system can help to identify patients who are most likely to require treatment by referred to a specialist. pdi provides a basis from which an accurate diagnosis can be obtained along with proper treatment planning. for easy and suitable networks between dental professionals, pdi has certain terminologies that help in establishing a proper channel of communication. these standardized indices can be useful for dental professionals during their private practice, as they allow better communication between specialists and dentists. the authors have successfully implemented the pdi system at basrah dental school and are still able to screen, assign, treat, and refer totally edentulous patients depending on their complexity. acknowledgements the authors express their gratitude to the teaching assistants and interns at college of dentistry, university of basrah, republic of iraq, for their assistance with patient screening. classifying completely edentulous pafents using the prosthodonfc diagnosfc index vol 13, no 1 (2025) doi 10.5195/d3000/2025.944 http://dentistry3000.pitt.edu 4 references 1. the glossary of prosthodontic terms: ninth edition. j prosthetic dent. 2017;117(5s): e01-105. 2. mcgarry tj, nimmo a, skiba jf, ahlstrom rh, smith cr, koumjian jh. classitication system for complete edentulism. the american college of prosthodontics. j prosthodontic. 1999;8(1):27-29. 3. alnamel hasanein, m. albadr r, abdul razzaq f. evaluation and characterization of some properties of glass ionomer cement reinforced by novel boron nitride nanoplatelets. frontiers biomed technol. 2024;11(3):477-485. 4. kovacic i, persic s, kranjcic j, lesic n, celebic a. rehabilitation of an extremely resorbed edentulous mandible by short and narrow dental implants. case rep dent. 2018; 2018:7597851. 5. felton da. edentulism and comorbid factors. j prosthodontic. 2009;18(2):88-96. 6. crocombe la, slade gd. decline of the edentulism epidemic in australia. aust dent j. 2007;52(2):154-56. 7. ntala pc, niarchou ap, polyzois gl, frangou mj. screening of edentulous patients in a dental school population using the prosthodontic diagnostic index. gerodontology. 2010;27(2):11420. 8. al-rafee ma. the epidemiology of edentulism and the associated factors: a literature review. j family med prim care. 2020;9(4):1841-43. 9. petersen pe, bourgeois d, ogawa h, estupinan-day s, ndiaye c. the global burden of oral diseases and risks to oral health. bull world health organ. 2005;83(9):661-69. 10. polzer i, schimmel m, müller f, biffar r. edentulism as part of the general health problems of elderly adults. int dent j. 2010;60(3):143-55. 11. mather h, thomason m, ellis j. are uk graduates equipped with the skill set required to meet the demands of the uk’s edentulous population? br dent j. 2018;225(1):15-18. 12. parameters of care for the specialty of prosthodontics. j prosthodont. 2020;29(s1):03-147. 13. tyrovolas s, koyanagi a, panagiotakos d, haro jm, kassebaum nj, chrepa v, et al. population prevalence of edentulism and its association with depression and self-rated health. sci rep. 2016; 6:37083. 14. alnamelha,abdulbaqihj.assessmentoftheeffectsofboronnitridenanoplateletsreinforcementonthephysical and mechanical properties of the geopolymer prepared by natural kaolinite:aninvitrostudy.denthypotheses2024 15. 16. felton da. complete edentulism and co-morbid diseasesan update. j prosthodont. 2016;25(1):05-20. 17. nordenram g, davidson t, gynther g, helgesson g, hultin m, jemt t, et al. qualitative studies of patients’ perceptions of loss of teeth, the edentulous state and prosthetic rehabilitation: a systematic review with meta-synthesis. acta odontol scand. 2013;71(3-4):93751. 18. peltzer k, hewlett s, yawson ae, moynihan p, preet r, wu f, et al. prevalence of loss of all teeth (edentulism) and associated factors in older adults in china, ghana, india, mexico, russia and south africa. int j environ res public health. 2014;11(11):11308-24. 19. almusallam sm, alrafee ma. the prevalence of partial edentulism and complete edentulism among adults and above population of riyadh city in saudi arabia. j family med prim care. 2020;9(4):1868-72. 20. emami e, de souza rf, kabawat m, feine js. the impact of edentulism on oral and general health. int j dent. 2013; 2013:498305. 21. almutairy a, minu m. prevalence of partial edentulism among young saudi women of qassim and their perception of early tooth loss. int j dent res. 2017;5(2):172-76. 22. douglass cw, shih a, ostry l. will there be a need for complete dentures in the united states in 2020? j prosthet dent. 2002;87(1):05-08. 23. douglass cw, watson aj. future needs for tixed and removable partial dentures in the united states. j prosthet dent. 2002;87(1):09-14. 24. felton da. exactly what is the hold up? j prosthodont. 2005;14(3):155-57. 25. mcgarry tj, nimmo a, skiba jf, ahlstrom rh, smith cr, koumjian jh, et al. classitication system for partial edentulism. j prosthodont. 2002;11(3):18193. 26. mcgarry tj, nimmo a, skiba jf, ahlstrom rh, smith cr, koumjian jh, et al. american college of prosthodontists. classitication system for the completely dentate patient. j prosthodont. 2004;13(2):73-82. 27. chaudary mau, khamar k, naeem s. screening and treatment of completely edentulous patients on the basis of prosthodontic diagnostic index and the level of patient satisfaction. pakistan oral and dental journal. 2016;36(3):503-06. 28. mazurat rd, mazurat nm. communicating complexity: using a diagnostic classitication system for edentulous patients. j can dent assoc. 2003;69(8):511-14. 29. alsheikh ha, alzain s, warsy a, almukaynizi f, althomali a. mandibular residual ridge height in relation to age, gender and duration of edentulism in a saudi population: a clinical and radiographic study. saudi dent j. 2019;31(2):258-64. classifying completely edentulous pafents using the prosthodonfc diagnosfc index vol 13, no 1 (2025) doi 10.5195/d3000/2025.944 http://dentistry3000.pitt.edu 5 figure 1. mandibular residual ridge bone height. figure 2. maxillary residual ridge morphology. type a +21 mm type b 16-20 mm type c 11-15mm type d -10 mm type a type b type c type d classifying completely edentulous pafents using the prosthodonfc diagnosfc index vol 13, no 1 (2025) doi 10.5195/d3000/2025.944 http://dentistry3000.pitt.edu 6 table 1. mandibular residual ridge bone height. total patient number 300 mandibular rrh bone height-mandible number patients type i 60% 180 type ii 15% 45 type iii 15% 45 type iv 10% 30 table 2. residual ridge morphology-maxilla only. total patient number 300 rr morphology residual ridge morphology-maxilla only number patients type a 65% 195 type b 15% 45 type c 20% 60 type d 0% table 3. mandibular arch's muscle attachment. total patient number 300 m attachment muscle attachments:(only mandible) number patients type a 25% 75 type b 45% 135 type c 15% 45 type d 5% 15 type e 10% 30 table 4. maxillary mandibular relationship. total patient number 300 skeletal class. maxillomandibular relationship number patients class i 60% 180 class ii 30% 90 class iii 10% 30 791 final the effect of cross-section of three different files on apical debris and irrigant solution extrusion using full file sequences: an in vitro study vol 13 no 1 (2025) doi 10.5195/d3000.2025.791 the effect of cross-section of three different files on apical debris and irrigant solution extrusion using full file sequences: an in vitro study sohaib fadhil mohammed1, sura yaseen khudhur1, zaidoon hasan mohammed2, matheel al-rawas 3, tahir yusuf noorani4,5, mohd firdaus yhaya6 1 college of dentistry, university of anbar, iraq 2 college of dentistry, university of kerbala, iraq 3 school of dental sciences, university sains malaysia, kubang kerian, malaysia 4 school of dental sciences, health campus, university sains malaysia, kota bharu, kelantan, malaysia 5 saveetha dental college and hospitals, saveetha institute of medical and technical sciences (simats), saveetha university, chennai, tamil nadu, india 6 school of dental sciences, health campus, university sains malaysia, kubang kerian, kelantan, malaysia abstract introduction the eradication of bacteria from root canals becomes crucial for the effective completion of endodontic therapy. endodontic files and irrigants are employed during chemo-mechanical preparation to eliminate both inorganic and organic tissues that contains microorganisms with metabolites from them within the root canal [1]. even minimal quantities of apical extrusion could contribute to perioperative inflammatory processes, induce discomfort, and impede recovery. regardless of whether the preparation reaches the apical endpoint, all instrumentation approaches release debris into the surrounding periradicular tissues [2]. the silent chronic periradicular lesions in infected teeth reflect the delicate balance between the host's defense mechanisms in the surrounding tissues and the aggressive actions of infectious endodontic bacteria. if bacteria are expelled into the tissues surrounding the root, the host encounters a situation during the chemo-mechanical preparation where the exposure to irritants exceeds previous levels [3,4]. however, the amount of debris citation: mohammed sf, et al. the effect of crosssection of three different files on apical debris and irrigant solution extrusion using full file sequences: an in vitro study. dentistry 3000.1:a001 doi:10/5195/d3000.2025.791 received: december 8, 2024 accepted: february 9, 2025 published: february 14, 2025 copyright: ©2025 mohammed sf, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: sohaibfadhil85@uoanbar.edu.iq keywords: cross-section, instrumentation, sodium hypochlorite, premolar, flexibility objective: the aim of the study was to evaluate the cross-sectional geometry of heat-treated endodontic rotary files upon debris ejection through apex and the quantity of irrigants expelled when employing a complete sequence file system. methods: sixty extracted human maxillary first premolars were included and randomly classified into three categories (n = 20) according to the file employed during the preparation of canal. the groups were group 1: edgefile x7, group 2: komet fq, and group 3: fanta af f one. the modified myers along with montgomery experiment protocol was employed to measure the mean weight of upward extruded irrigants and detritus in grams. the comparison of irrigants and debris extrusion was performed utilizing analyses of variances (anova). significant level has been established at 0.05. results: apical ejection of material was prevalent throughout every one of analyzed files. quantity of debris extruded during canal shaping did not show any significant variation among the examined files, the fanta af f one group exhibited the greatest mean of extrusion irrigant. conclusion: each evaluated endodontic rotary devices produced comparable apical debris ejection among the groups. the fanta af f one group extruded the most irrigant. the effect of cross-section of three different files on apical debris and irrigant solution extrusion using full file sequences: an in vitro study released varies depending on the instruments used, file designs, and motion kinematics. the total amount of debris produced following canal straightening has been compared in numerous studies using various file systems and techniques [5,6]. none of the file systems in these experiments demonstrated the ability to perform root canal procedures without causing debris extrusion [7,8]. in contrast to rotary nickel-titanium (niti) systems, manual preparation results in greater apical extrusion [9-11]. the edgefile x7 edge endo; albuquerque, new mexico, usa) features a variable helical angle and a consistent taper of 0.04 and 0.06, respectively, with a parabolic cross-section. it is made from firetreated niti alloy, undergoing heating and cooling cycles to enhance its mechanical properties [12]. conversely, the fanta af fone file is characterized by its unique cross-sectional design, featuring a planar side-cut with two active cutting points and a non-cutting tip. it is manufactured using control memory wire treated with titanium oxide, improving its hardness, flexibility, and resistance to fracture (shanghai fanta catalog 2018–2019). the komet fq is a rotary endodontic file system manufactured by komet usa llc (rock hill, south carolina, usa), developed to provide efficiency, flexibility, and durability through heat treatment. its double-s cross-section and variably tapered core effectively remove apical debris, reducing the time needed for root canal preparation. this instrument's innovative file core offers a variable taper and a large chip space designed for instruments with a 0.06 taper [13]. this technology provides a variety of files that are customizable to specific cases while preserving the natural canal anatomy. the heattreated niti file is less prone to fracture due to its enhanced resistance to cyclic fatigue [14,15]. the present research sought to investigate the impact of three heat-treated endodontic rotary files (edgefile x7, komet fq, and fanta af f one) on the volume of irrigant extruded and the apical extrusion of debris when employing a complete sequence file system. considering the file's cross-section and design, the null hypothesis posits no significant debris and irrigation extrusion differences among the different file systems. this aligns with the manufacturers' claims that these files have the same taper but differ in cross-section and design. material and methods data collection a total of sixty human maxillary first premolars were recently obtained from various specialized dental offices in iraq between the end of 2022 until may 2023. we included teeth extracted for orthodontic reasons with no fractures or cracks. the exclusion criteria were teeth with restorations or caries, teeth that had undergone previous endodontic treatment, and teeth with calcification, root resorption, or curved canals. the selection of palatal root canal from teeth with class vi vertucci classification was based on the following specified criteria: patent canal and apical foramen, root length of 15 mm, straight and conical roots with less than 15% curvature, absence of cracks, apical resorption or fractured apex, a mature centrally located apical foramen, and an apical foramen allowing passage of a size 15 k-file tip. the sample size was determined utilizing g*power (version 3.1.9.6) with an impact size of 0.4, the level of significant (α) equal 0.05, and a test statistic of 80%. the overall sample size comprised 60 specimens. ethical approval was secured by the ethical the effect of cross-section of three different files on apical debris and irrigant solution extrusion using full file sequences: an in vitro study committee at anbar university, iraq (registration no. 163). teeth preparation after meticulous selection, calculus, bone, as well as soft periodontal tissue was excised from the teeth extracted utilizing a cumin scaling. teeth was immersed in a 6% naocl solution for one hour [16]. each root apex was examined under 20x magnification using a stereomicroscope (meiji techno, japan) to allow for careful inspection of the roots and apices. after this, a regular saline solution was used to store the samples until needed [17]. the tooth length was marked at 15 mm using a marker pen, and the crowns were removed using a diamond disc with copious water irrigation to facilitate the cutting process. this ensured uniform tooth lengths, eliminated variables, and provided a consistent reference point [18]. the sample length was verified using a digital caliper. pulpal tissues have been excised with a size 10 spiked broach, and the integrity of the canal along with the exact position of the apical foramen was assessed with k files sizes 10 and 15, advancing the file when it was observable at the foramen at the apical end. 1 mm was deducted off of the root's length to determine the precise working length [17,19]. the sixty extracted human maxillary first premolars were then randomly classified at three categories (n = 20) according to the files utilized for canal preparation. these categories were: category 1: edgefile x7, category 2: komet fq, and category 3: fanta af f one. canal preparation these procedures followed in the research were carried out following each system's manufacturer's instructions. a master apical file (maf) was utilized in the preparation of each sample canal tip sizing of #25. a disposable 5 ml plastic syringe with an irriflex irrigation needle (produits dentaires sa, vevey, switzerland) was used. the rubber stopper of the files was set to the determined working length (14 mm), and the files were attached to the handpiece. instrumentation was executed in a crown to down manner via a soft pecking action. each file was utilized until it became loose, after which 1 ml of distilled water per minute was employed for irrigating the canals using a disposable irriflex irrigate needle, then passively added through the root canals in both back and forth action, halting 2 mm shorter for apical foramen while avoiding contact with the canal walls. the canals were subsequently recapitulated utilizing k file with a tip size of 15. this identical technique was executed for each consecutive file until the 25/06 file attained its optimal length as well as became loose. the canal was re-irrigated with 1 ml of d.w per minute and reiterated. each file was sanitized with the gauze along with alcohol following extraction. the x-smart plus (dentsply sirona, ballaigues, switzerland) was employed to utilize the instruments consecutively, adhering to the company's specifications, at a constant speed of 300 rpm and a torque of 2.5 n/cm to standardize the parameters. category 1: rotary edge x7 file (edge endo; albuquerque, new mexico, united states) started with the 17/04 file, followed by 17/06, 20/06, and finished with 25/06. category 2: rotary komet fq file (komet usa llc, rock hill, south carolina, usa) started with the 15/03 file, followed by 20/06, and finished with 25/06. category 3: rotary af f one file (fanta dental; shanghai, china) started with the 16/04 file, followed by 20/04, and finished with 25/06. the effect of cross-section of three different files on apical debris and irrigant solution extrusion using full file sequences: an in vitro study irrigation protocol the irrigation protocol for each group utilized a 30-gauge irriflex irrigating needles (produits dentaires sa, vevey, switzerland) to deliver an overall of 9 ml of distilled water, comprising 4 ml throughout instruments as well as 3 ml for the last flushing, inserted passively 2 mm from the foramen at the apical end [20]. after instrumentation, 2 ml of distilled water was administered to a root’s exterior using a 25-gauge vented needle tip to extract any debris adhering to the root’s apical region, and this subsequently transferred to a glass vial for collection. after each file was used, the irrigation procedure was repeated, maintaining a flow rate of 1 ml per minute. debris collection sixty glass vials with rubber stoppers were employed in the present research to gather the apical extrusion debris and irrigants during instruments. each vial was labeled according to the file system used for each group. following the labeling process and prior to instrumentation, the vials' weight was determined using a precise electronic equilibrium with a sensitivity of 0.0001 g. each vial had been weighed excluding the rubber cap. the methodology for gathering apical extrusion debris has been changed from a prior study [21] and included the following steps: the glass vials were prepared by removing the vial labels and cleaning any remnants of glue that might have melted and evaporated during the drying process, as this could affect the results; the middle of the rubber cap affixed every vial was punctured with a hot tool [20,22]. the root was inserted during the opening, leaving the inner third visible above the rubber cap. upon positioning the tooth-cap assembly on the vial, the apical segment from the tooth become suspending within the glass vial, that functioned to be the receptacle for expelled debris as well as irrigants [23], as shown in figure 1a. to prevent contamination of the glass vial's exterior from coronally extruded debris and irrigants, the vial was covered with a rubber dam secured with dental floss. this contamination could potentially affect the results [23]. the root surface was filled with a flowable composite to improve sealing and prevent the irrigation solution from leaking out of the root canals. to equalize the pressure both within and outside the vial, which could influence the findings, a needle of 25 gauge was introduced through the rubber cap into vial along the root surface of the samples [18], as shown in figure 1b. instruments was executed in accordance with the manufacture's guidelines. following this, three main groups (n = 20) were randomly selected among the teeth and their vial. the mass of debris extrusion and the amount of irrigant expelled were assessed based figure 1. a) the root was punched through the central rubber cap of the vial. b) a sealing material was applied around the visible coronal third of the root to prevent debris leakage. the effect of cross-section of three different files on apical debris and irrigant solution extrusion using full file sequences: an in vitro study on the instrumentation technique utilized for the preparation of the palate roots canals in maxillary initial premolars. the same operator performed all weighing and instrumentation procedures to ensure standardization. each file was disposed of after completing the treatment of a single canal. debris collection after completing the root canal instrumentation, the floss ligation was removed, and the root-cap assembly was separated from the glass vial. next, to collect any adhered debris, 2 ml of deionized water was applied to the exterior of root apex within the glass vials. the vial was weighed under the same conditions as during the preinstrumentation procedure to prevent any errors in the results before the desiccation process for estimating the amount of irrigation extruded. desiccation procedure after two hours of heating at 100°c in a hot air oven, with checks every 30 minutes to ensure dryness, the sample vials were removed [24]. after cooling, the vials were weighed on an electronic balance (kern abs 120-4, germany) with a precision of 0.0001 g, following the same procedure as preinstrumentation weights to avoid errors. the average of three measurements of weight was computed to ascertain the postinstrumentation weight. while debris weight for every sample has been established by subtracting the pre-weight values from the post-weight of the vials for all sample utilized [25]. statistical analysis utilizing a statistically significant threshold of p = 0.05, the gathered data underwent additional analysis through one-way anova to assess the amount of debris and the volume of irrigant extrusion apical across the various groups. results table 1 presents the average weight for apical extrusion debris following instrumentation with various ni-ti file systems. no significance variations were identified among tested categories having the highest mean recorded in edge x7 (0.0494 g), while the komet group (0.0286 g) demonstrated the lowest mean value. the average values for extrusion debris (g) across the various categories are illustrated in a bar chart, as depicted in figure 2. n mean standard deviation minimum maximum debris g1 20 0.0286 0.0091 0.0126 0.0424 g2 20 0.0494 0.0561 0.0259 0.2856 g3 20 0.0331 0.0105 0.0163 0.0560 total 60 0.0370 0.0340 0.0126 0.2856 irrigation g1 20 2.3442 0.4831 1.3206 3.1095 g2 20 2.5253 0.8973 1.2952 4.3203 g3 20 3.0183 0.8221 1.5473 4.2554 total 60 2.6293 0.7966 1.2952 4.3203 g1: komet fq, g2: edge x7, and g3: fanta af f one table 1. mean and standard deviation of the extrusion debris (g) across several categories. the effect of cross-section of three different files on apical debris and irrigant solution extrusion using full file sequences: an in vitro study significant changes in extruded irrigants were noted in the fanta af category relative to the other examined categories, as illustrated in table 2. the minimal value of extrusion irrigating was seen in the komet fq group, as illustrated in figure 3. dependent variable mean difference (i-j) p-value debris g1 g2 -0.0207 0.054 g3 -0.0044 0.674 g2 g1 0.0207 0.054 g3 0.0163 0.128 g3 g1 0.0044 0.674 g2 -0.0163 0.128 irrigation g1 g2 -0.1811 0.452 g3 -0.6740 0.007 g2 g1 0.1811 0.452 g3 -0.4929 0.044 g3 g1 0.6740 0.007 g2 0.4929 0.044 g1: komet fq, g2: edge x7, and g3: fanta af fone 0.0286 0.0494 0.0331 .000000 .010000 .020000 .030000 .040000 .050000 .060000 .070000 g1 g2 g3 debris table 2. correlation of mean debris weight (g) and mean irrigant volume (ml) among categories. figure 2. a bar chart illustrating the mean extrusion debris (g) for several categories. the effect of cross-section of three different files on apical debris and irrigant solution extrusion using full file sequences: an in vitro study discussion a primary purpose of roots canals treatment consists of meticulously cleanse the root canal system. during chemo-mechanical preparation, debris including dental chips, necrosis pulpal tissue, germs, and irritants might unintentionally protrude into the periapical tissues, potentially leading to postoperative pain [26]. a variety of factors contributes to the extent of apically extruded of debris, including instrumentation techniques, types and sizes of files, working length, the types of irrigants used, and their activation [27]. the predominant emphasis of research is exclusively on the volume of debris extruded. the present investigation included both the irrigant and the volume of extrusion debris, as each can contribute to postoperative pain, periapical inflammatory, and perhaps hinder recovery. the research used the palatal roots for maxillary first premolars because these teeth are commonly extracted for orthodontic purposes. utilizing one type of tooth can reduce variation between samples. the occurrence of flare-ups after root canals therapy is reported to range from 1.5% to 17%. the expulsion of waste material causes the formation of a complex between antigens and antibodies, resulting in an intense inflammatory response and strong postoperative flare-ups [28]. the extrusion of irrigants, specifically sodium hypochlorite, during root canal procedures can lead to various complications, including discomfort, inflammation, hematoma, ecchymosis, and more severe issues like necrosis and paraesthesia. this phenomenon, occurring during some hours or days post-procedure, is usually referred to as the "interappointment flare-up” [2931]. this work employed an experimental paradigm, as per myers and montgomery, to quantitatively assess the apically extruded of debris with irrigants [21]. in all cases, apical integrity could be preserved with a 10-k file, thereby standardizing the apical diameter. the extrusion of debris escalates with an increase in diameter for the apical integrity. apical 2.3442 2.5253 3.0183 0.000000 0.500000 1.000000 1.500000 2.000000 2.500000 3.000000 3.500000 g1 g2 g3 irrigation figure 3. a bar chart showing the average extruded irrigant (ml) in different groups. the effect of cross-section of three different files on apical debris and irrigant solution extrusion using full file sequences: an in vitro study extrusion increases even with intact apical constriction [32]. a consistent amount of distilled water was utilized as the irrigate solution in each canal to avert the crystallization of sodium hypochlorite, which could modify the weight of dental debris and jeopardize the findings [33]. despite the absence of significance variations between tested categories during our investigation, the edge file x7 category exhibited the highest apically debris extruded. this may be attributed to its parabolic asymmetric triangular cross-section [34]. hence, altering the shape of the root canal wall using three cutting sites impacts the file's centric ability. data show that cross-sections with only one cutting edge exhibited a less aggressive mode of action when enlarging canals [35]. there is at least one report [36] that showed a lower percentage of debris associated with edge file x7 than other files. this discrepancy may be attributed to the type of endodontic movement utilized in their study, which employed reciprocation movement. in contrast, our present study utilized continuous rotational movement, which, when combined with the x7's small parabolic cross-section, results in a screwing effect that may force debris apically. regarding the amount of apically extruded irrigants, our study found that the highest degree of irrigant extrusion was recorded in the fanta af f one category, with non-significant differences observed between the komet and edge files. the design of the fanta af f one file, featuring vertical blades, efficiently moves irrigants from the flutes to the relieving area due to its flat-sided design. this design facilitates increased space for the irrigant to advance toward the apical region, possibly resulting in considerable apically extruded from the irrigant. the limitations for experiment models that may influence the findings encompass the absence of apical backpressure, lack of control over dentin microhardness, sensitive of the balance of analysis, and sample hydrate associated with humidity [18]. the influence of a vital or necrosis pulp, along with the existence of an endodontic lesions, on the apically extruded of debris is still ambiguous. conclusion examined endodontic rotary files generated apically extruded debris along with irrigants. no substantial variations were seen between the categories for the apical extrusion of debris. fanta af f one group documented the maximum volume of extrusion irrigant. conflict of interest the authors report no conflicts of interest. author contributions all authors contributed to designing the study, collecting the data, interpreting the results, writing up the draft of the manuscript, reviewing and finalizing the manuscript. acknowledgement and funding selffunded research. references 1. desai p, himel v. comparative safety of various intracanal irrigation systems. journal of endodontics. 2009;35(4):545-9. https://doi.org/10.1016/j.joen. 2009.01.011 2. rao mr, shameem a, nair r, ghanta s, thankachan rp, issac jk. comparison of the remaining dentin thickness in the root after hand and four rotary instrumentation techniques: an in vitro study. 2013;14(4):712.https://doi.org/ 10.5005/jp-journals-100241389 3. panchal v, jeevanandan g, subramanian e. comparison of instrumentation time and obturation quality between hand k-file, h-files, and rotary the effect of cross-section of three different files on apical debris and irrigant solution extrusion using full file sequences: an in vitro study kedo-s in root canal treatment of primary teeth: a randomized controlled trial. journal of indian society of pedodontics and preventive dentistry. 2019;37(1):75-9. https://doi.org/10.21203/rs. 3.rs-622404/v1 4. khandelwal a, jose j, teja kv, palanivelu a. comparative evaluation of postoperative pain and periapical healing after root canal treatment using three different base endodontic sealers–a randomized control clinical trial. journal of clinical and experimental dentistry. 2022;14(2):e144. https://doi.org/10.21203/rs. 3.rs-622404/v1 5. kirchhoff al, fariniuk lf, mello i. apical extrusion of debris in flat-oval root canals after using different instrumentation systems. journal of endodontics. 2015;41(2):237-41. https://doi.org/10.1016/j.jo en.2014.09.023 6. nevares g, xavier f, gominho l, cavalcanti f, cassimiro m, romeiro k, et al. apical extrusion of debris produced during continuous rotating and reciprocating motion. the scientific world journal. 2015;2015(1):267264. https://doi.org/10.1155/201 5/267264 7. üstün y, çanakçi b, dinçer a, er o, düzgün s. evaluation of apically extruded debris associated with several ni–ti systems. international endodontic journal. 2015;48(7):701-4. https://doi.org/10.1111/iej .12369 8. jindal r, singh s, gupta s, jindal p. comparative evaluation of apical extrusion of debris and irrigant with three rotary instruments using crown down technique–an in vitro study. journal of oral biology and craniofacial research. 2012;2(2):105 https://doi.org/10.1016/j.j obcr.2012.05.010 9. silva e, carapiá m, lopes r, belladonna f, senna p, souza e, et al. comparison of apically extruded debris after large apical preparations by fullsequence rotary and singlefile reciprocating systems. 2016;49(7):7005. https://doi.org/10.1111/ iej.12503 10. ahn s-y, kim h-c, kim e. kinematic effects of nickeltitanium instruments with reciprocating or continuous rotation motion: a systematic review of in vitro studies. journal of endodontics. 2016;42(7):1009-17. https://doi.org/10.1016/j.j oen.2016.04.002 11. toyoğlu m, altunbaş d. influence of different kinematics on apical extrusion of irrigant and debris during canal preparation using k3xf instruments. journal of endodontics. 2017;43(9):1565-8. https://doi.org/10.1016/j.joen. 2017.05.001 12. gambarini g, seracchiani m, piasecki l, valenti obino f, galli m, di nardo d, et al. measurement of torque generated during intracanal instrumentation in vivo. 2019;52(5):737-45. https://doi.org/10.1111/iej.130 42 13. dental k. fq. the easy way to a successful endo. retrieved april 2023, from https://contenthub.kometdent al.com/infocenter/files/100204 48.pdf 14. peters oa, paqué fjqi. current developments in rotary root canal instrument technology and clinical use: a review. 2010;41(6). https://doi.org/10.1016/j.joen. 2010.02.023 15. tabassum s, zafar k, umer fjeej. nickel-titanium rotary file systems: what’s new? 2019;4(3):111. authors: sadia tabassum https://doi.org/10.14744/eej.2 019.80664 16. zarrabi mh, bidar m, jafarzadeh h. an in vitro comparative study of apically extruded debris resulting from conventional and three rotary (profile, race, flexmaster) instrumentation techniques. journal of oral science. 2006;48(2):85-8. https://doi.org/10.2334/josnus d.48.85 17. boijink d, costa dd, hoppe cb, kopper pmp, grecca fs. apically extruded debris in curved root the effect of cross-section of three different files on apical debris and irrigant solution extrusion using full file sequences: an in vitro study canals using the waveone gold reciprocating and twisted file adaptive systems. journal of endodontics. 2018;44(8):1289-92. https://doi.org/10.1016/j.jo en.2018.04.011 18. zan r, topcuoglu hs, tunc t, ozer s, hubbezoglu i, kutlu g. apical extrusion of intracanal biofilm after root canal preparation using revo-s, twisted file adaptive, one shape new generation, protaper next, and k3xf instrumentation systems. turk endod j. 2016;1(2):73-9. https://doi.org/10.14744/tej .2016.36844 19. uslu g, özyürek t, yılmaz k, gündoğar m, plotino g. apically extruded debris during root canal instrumentation with reciproc blue, hyflex edm, and xp-endo shaper nickeltitanium files. journal of endodontics. 2018;44(5):856-9. https://doi.org/10.1016/j.jo en.2018.01.018 20. dagna a, el abed r, hussain s, abu-tahun ih, visai l, bertoglio f, et al. comparison of apical extrusion of intracanal bacteria by various glide-path establishing systems: an in vitro study. restorative dentistry & endodontics. 2017;42(4):316-23. https://doi.org/10.5395/rde. 2017.42.4.316 21. myers gl, montgomery s. a comparison of weights of debris extruded apically by conventional filing and canal master techniques. journal of endodontics. 1991;17(6):275-9. https://doi.org/10.1016/s0 099-2399(06)81866-2 22. silva e, carapiá m, lopes r, belladonna f, senna p, souza e, et al. comparison of apically extruded debris after large apical preparations by fullsequence rotary and singlefile reciprocating systems. international endodontic journal. 2016;49(7):700-5. https://doi.org/10.1111/iej .12503 23. hadi aw, hameed mr. comparative study of the amount of apically extrusion of debris during root canal preparation using wave one, trushape 3d, hyflex cm and one shape instrumentation systems-an in vitro study. j bagh coll dentistry. 2017;29(1):1-8. https://doi.org/10.12816/0 038607 24. hussein hm, al-zaka im. evaluation of the amount of apically extruded debris using different root canal instrumentation systems. mustansiria dental journal. 2014;11(1):1-10. https://doi.org/10.32828/ mdj.v11i1.219 25. muhaibes mq, alwakeel sa. evaluation of apically extruded debris using protaper universal, protaper next, one curve, xp shaper, and edge file: an in vitro study. 2024;14(1):317.https://doi.org/10.4103/sej.s ej_76_23 26. seltzer s, naidorf ij. flare-ups in endodontics: i. etiological factors. journal of endodontics. 1985;11(11):472-8. https://doi.org/10.1016/s00992399(85)80220-x 27. er k, sümer z, akpınar k. apical extrusion of intracanal bacteria following use of two enginedriven instrumentation techniques. international endodontic journal. 2005;38(12):871-6. https://doi.org/10.1111/j.1365 -2591.2005.01029.x 28. siqueira jr jf. microbial causes of endodontic flare-ups. 2003;36(7):453-63. https://doi.org/10.1046/j.1365 -2591.2003.00671.x 29. torabinejad m, kettering jd, mcgraw jc, cummings rr, dwyer tg, tobias tsjjoe. factors associated with endodontic interappointment emergencies of teeth with necrotic pulps. 1988;14(5):2616. https://doi.org/10.1016/s00992399(88)80181-x 30. singh g, kukreja n, dhawan r, thakur a, kaur s, chhabra s. comparative evaluation of apical debris extrusion from the root canal using hand files, continuous rotary files and reciprocating file system: an invitro study. 2023;17(6).https://doi.org/10.7 860/jcdr/2023/62318.18128 31. shivanni ss, anjaneyulu k, balakrishna rn. association of the effect of cross-section of three different files on apical debris and irrigant solution extrusion using full file sequences: an in vitro study irreversible pulpitis and single visit root canal treatment. 2020;7(11):112731. https://doi.org/10.47750/cib g.2021.27.02.273 32. lambrianidis t, tosounidou e, tzoanopoulou m. the effect of maintaining apical patency on periapical extrusion. journal of endodontics. 2001;27(11):696-8. https://doi.org/10.1097/000 04770-200111000-00011 33. tanalp j, güngör t. apical extrusion of debris: a literature review of an inherent occurrence during root canal treatment. international endodontic journal. 2014;47(3):211-21. https://doi.org/10.1111/iej.1 2137 34. khalil wa-b, merdad k, abuhaimed ts, howait m, alsofi l. cyclic fatigue resistance of protaper gold, edgefile, oneshape and protaper universal. egyptian dental journal. 2018;64(1-january (fixed prosthodontics, dental materials, conservative dentistry & endodontics)):589-96. https://doi.org/10.21608/ed j.2018.78062 35. elashiry m, saber s, elashry s. apical extrusion of debris after canal shaping with three single-file systems. nigerian journal of clinical practice. 2020;23(1):79-83. https://doi.org/10.4103/njcp .njcp_319_19 36. jawad sr, mahdi mh, nemah a, fawzi m. assessing the amount of apically extruded debris of one curve, edge file x7, af™ r3 blue in curved canals: an in vitro study. 2024. https://doi.org/10.22271/o ral.2024.v10.i2d.1946 929 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.929 http://dentistry3000.pitt.edu digital versus conventional impressions mohammad munthir abdulrazzaq, mithaq r. mohammed college of den*stry, al-iraqia university, baghdad, iraq abstract objec9ve: the purpose of this research was to compare, in vivo, the three-dimensional (3d) dental impressions produced by digital and tradinonal methods. material and methods: this research was comprised of ten individuals who had full natural teeth. the subjects' molars were digitally imprinted using an intra-oral scanner (helios 600 3d). the double-mix impression method (silaxil box & protesil light) was also used to create a silicone imprint. the lava cos system exported the stereolithography (stl) data immediately, and a three-dimensional (3d) intra-oral scanner recorded the stl data of a plaster model created from a silicone imprint. the 3d assessment program captured the stl files. it overlaid them using the bestfit-algorithm approach for each impression technique (least-squares method, polyworks, innovmetric program). the wilcoxon signed-rank test was used to compare the two methods with respect to 3d data. results: differences between digital impressions were less nonceable when comparing them to silicone impressions, according to a visual analysis of stacked datasets. using a digital imprint approach yielded more confirmanon (0.014± 0.02 mm) compared to a tradinonal method (0.023 ± 0.01 mm). conclusion: according to this in vivo invesnganon, digital impression technology outperforms tradinonal ompression techniques. open access cita%on: abdulrazzaq mm, et al. (2025) digital versus conven%onal impressions. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.929 received: may 3, 2025 accepted: may 31, 2025 published: june 27, 2025 copyright: ©2025 abdulrazzaq mm, et al. this is an open access ar%cle licensed under a crea%ve commons apribu%on work 4.0 united states license. email: mohamad_monther@aliraqia.edu introduc)on "digital dentistry" is rapidly expanding and represents a major shift in the dental industry that is occurring this century. the use of three-dimensional (3d) intra-oral scanners for digital impressions has recently been trending upward in popularity globally [1]. dental prostheses may be immediately designed and manufactured using digital pictures of the dental arches and occlusal connections recorded by these intra-oral scanners [2–5]. there are several reasons why intra-oral scanners might eventually replace traditional impression materials [6–10]. for instance, as compared to traditional impression methods, its implementation streamlines processes and increases transparency throughout the impression operation, benekiting patients, dental technicians, and dentists alike [4,5,11,12]. since dental stone grows due to secondary reactions and silicone impression materials are prone to dimensional changes due to ongoing chemical reactions, this approach also eliminates mistakes associated with the standard impression technique. an ill-kitting dental prosthesis might be the consequence of such alterations in dimensions. on the other hand, these kinds of alterations should not be expected when teeth are scanned digitally. several in vitro investigations have shown that digital impressions are far more accurate and precise in terms of dimensions than traditional impressions [11,13–18]. unfortunately, there are several clinical aspects that might affect the impression's accuracy and precision. the only way to determine accuracy is to compare results, ideally against a gold standard, which is difkicult to do in the mouth. there is a dearth of high-quality in vivo research when it comes to accuracy [19,20]. consequently, the 3d morphological data obtained by digital imprint techniques was the primary focus of this work, which contrasted these data with those obtained in vivo through more traditional methods. material and methods the ten individuals who were a part of this research were dentistry students at our digital versus convennonal impressions vol 13, no 1 (2025) doi 10.5195/d3000/2025.929 http://dentistry3000.pitt.edu 2 institution and had full natural teeth (5 men and 5 females; average age 22.6±2.0 years). an intra-oral scanner and silicone imprint material are the two methods we use. there was a total of twenty impressions. digital impression digital impressions were created using an intra-oral scanner (helios 600 3d). following the guidelines provided by the manufacturer, a digital optical scan was carried out in a single uninterrupted operation, beginning at the occlusal surface and progressing to the lingual and buccal surfaces. the information retrieved from the digital scanning process was sent straight from the lava cos system to the lab computer (figure 1). figure 1. helios 600 3d device. conventional impression we used a twofold mix impression method and ordinary metal stock trays to make whole arch conventional impressions using vinylpolysiloxane silicone (silaxil box & protesil light). we made sure there was enough room for the impression material. after the appropriate tooth was killed with light-type imprint material using a syringe, the tray was placed in the mouth and pushed against the tooth arch. the imprints were taken out of the mouth after the setting period, cleaned for 10 minutes, and then let sit at room temperature and humidity for 3 hours. after following the manufacturer's instructions (kig. 2&3), dental stone plaster models were scanned using an intra-oral scanner (helios 600 3d). data from the scans of the premolar and molar areas were saved on the lab's computer using the stl data format. figure 2. convensional impression (mandibular cast). figure 3. conventional impression (maxillary cast). analysis of 3d data the tooth form and gingival margin were adjusted in the 3d images created from stl data using the right software (polyworks, innovmetric software). before being overlaid utilizing the best-kit-algorithm approach (least-squares method) to match 2 surfaces, these trimmed stl data were put into a 3d assessment software platform [17,20]. the stl dataset was designated as the standard. with the help of the reference data set's triangular surfaces and the test data set's polygons, the program determines the orientation and nearest distance of each vertex. the industrial sector makes frequent use of this verikication approach [21–23]. for two methods, we averaged the absolute value disparities in overall measurement locations. on top of that, we determined the mean difference across all 10 individuals. evaluation of the casts scanner and intraoral scanner in vitro the accuracy of the scanner, which might affect the accuracy of the scanned data, was tested by scanning a cast model kive times and comparing the resulting ten data set pairings using the best-kit algorithm, as mentioned before. the oral scanner underwent the same tests in a controlled environment to ensure accuracy. statistical analysis we used the mann-whitney u test to compare the two methods by calculating the average disparities of all measurement points. a 0.05 threshold of signikicance was used. because the data did not follow a normal distribution, non-parametric mann-whitney u tests were used to determine if there was a statistically signikicant difference between the digital and traditional imprint methods. an analysis was conducted using spss version 22 (spss inc., chicago, il, usa) for statistical purposes. results to compare the two methods, this research used digital and conventional imprint techniques for dental work. for both methods, precise readings were taken for several parameters (ten in all). parameters like "a," "b," and "c" have much lower mean values and variability metrics for the digital impression technique compared to the conventional impression technique, indicating improved accuracy. a statistically signikicant difference in measurement accuracy between the digital impression method and the traditional impression technique is shown by the substantially higher mean value of parameter "a" in the former (p=0.000) compared to the latter. with a p-value of 0.000, there is a difference in the results for parameter "b," as the traditional imprint method has a much lower mean value than the digital impression approach. with a p-value of 0.000, parameter "c" shows that the traditional impression approach tends to provide larger measurements than the digital impression technique, with the former showing a higher mean value. parameters "j" and "i", on the other hand, do not change signikicantly between methods (p=0.917 and p=0.117) (figure 4). most cases of generalized aggressive disease were observed in patients with the condition (60%), compared to 30% of people who have chronic or localized severe periodontitis. the case group tended to have higher levels of bleeding, plaque, and gingivitis. this disparity did not, however, reach statistical signikicance. in comparison to non-users, case women had mean probing depths that were deeper (3.3–1.0 versus 2.7–0.5 mm) (p = 0.04) (table 2 and figure 2). discussion these kindings imply that digital impressions have less disparity than traditional impressions. in contrast to more traditional imprint methods, digital direct scanning has several established advantages, as mentioned in the introduction. despite claims to the contrary, digital impressions are prone to errors because of factors including saliva, jaw movement, digital versus convennonal impressions vol 13, no 1 (2025) doi 10.5195/d3000/2025.929 http://dentistry3000.pitt.edu 3 and the merging of many digital pictures. the fact that digital impressions are not susceptible to the inevitable mistakes caused by the dimensional changes of dental stones and impression materials, which may result in the miskitting of dental prostheses, is one of the most signikicant advantages from a clinical standpoint. digital impressions outperform traditional impressions in vitro in terms of dimensional correctness and precision, according to many studies [11, 13–17]. it is crucial to conduct in vivo clinical assessments to prove the translation of in vitro kindings to clinical importance in vivo because several clinical circumstances affect the accuracy and precision of an impression. unfortunately, the gold standard (iso 5725-1) [24]—accuracy, which stands for truthfulness—cannot be readily tested in patients' mouth cavities, making it impossible to assess in vivo. it is common practice to measure the kit of kinal restorations as a means of conducting in vivo accuracy assessments [3,7,20,25,26]. comparing restorations made using digital impressions to those made using traditional impressions, studies have shown that the former provides a more clinically acceptable kit [6,27-30]. it should be mentioned that these assessments of accuracy include the complete restoration manufacturing process, not only the imprint operation. consequently, it is necessary to compare the imprint processes. to determine the accuracy of an imprint technique, it is recommended to repeatedly superimpose full scanned pictures taken in the same way [16,17,20,31]. using the calculated 3d distances, this process kinds the discrepancies between the impression pictures taken at each surface point. the clinical feasibility of such a comparison is high. however, the number of investigations assessing the accuracy of the imprint method in vivo is low [19,32]. previous work [19] that the digital imprint systems had adequate precision in vivo. there is broad consensus among our research kindings. because silicone impressions are so sensitive to the clinician's competence and experience level, the latter is especially crucial from a clinical standpoint [10,12]. our research found that 3d data from direct oral digital scanning is far more consistent than data from silicone impressions, which is an important kinding. given the scanner's proven accuracy and precision in previous studies, it stands to reason that dimensional variations in the materials used to make stone models from silicone imprints might be a contributing factor to their inconsistency [20,33–37]. please be aware that the focus of our research was not on accuracy but on precision as applied to individual clinical cases. table 1 shows that digital impressions were consistently and independently more reproducible than silicone impressions, regardless of participant. this suggests that the inherent dimensional changes associated with the silicone impression technique may be the main factor inkluencing this kinding. finally, our results indicate that the digital impression approach provides higher accuracy, even if the average variation between the two methods was just 0.009 mm, which is not deemed clinically important. conclusions based on the kindings of this in vivo investigation, digital impressions seem to be more reproducible than traditional impressions. clinical relevance our research shows that the digital impression method is superior to the traditional impression technique; hence, we suggest using it. acknowledgements the authors appreciate the students who participated in the work and our college team that made this research possible. references 1. baba k. paradigm shifts in prosthodontics. journal of prosthodontic research. 2014;58: 1–2. pmid:24412149 2. scotti r, cardelli p, baldissara p, monaco c. clinical litting of cad/cam zirconia single crowns generated from digital intraoral impressions based on active wavefront sampling. j dent. 2011. pmid:22027653 3. ahrberg d, lauer hc, ahrberg m, weigl p. evaluation of lit and efliciency of cad/cam fabricated all-ceramic restorations based on direct and indirect digitalization: a double-blinded, randomized clinical trial. clin oral investig. 2016;20: 291–300. pmid:26070435 4. shah s, sundaram g, bartlett d, sherriff m. a 3d laser scanner using superimposition software is used to assess the accuracy of impression techniques. j dent. 2004;32: 653–658. pmid:15476960 5. arslan y, karakoca nemli s, bankoğlu güngör m, tamam e, yılmaz h. evaluation of biogeneric design techniques with cerec cad/cam system. j adv prosthodont. 2015;7: 431–436. pmid:26816572 6. seelbach p, brueckel c, wöstmann b. accuracy of digital and conventional impression techniques and workllow. clin oral investig. 2013;17: 1759– 1764. pmid:23086333 7. boeddinghaus m, breloer es, rehmann p, wöstmann b. accuracy of single-tooth restorations based on intraoral digital and conventional impressions in patients. clin oral investig. 2015;19: 2027–2034. pmid:25693497 8.logozzo s, franceschini g, kilpelä a. a comparative analysis of intraoral 3d digital scanners for restorative dentistry. internet j med technol. 2011;5: 1–19 9. abdel-azim t, rogers k, elathamna e, zandinejad a, metz m, morton d. comparison of the marginal lit of lithium disilicate crowns fabricated with cad/cam technology by using conventional impressions and two intraoral digital scanners. j prosthet dent. 2015;114: 554–559. pmid:26100929 10.wismeijer d, mans r, van genuchten m, reijers ha. patients' preferences when comparing analog implant impressions using a polyether impression material versus digital impressions (intraoral scan) of dental implants. clinical oral implants research. 2014;25: 1113–1118. pmid:23941118 11.patzelt sbm, emmanouilidi a, stampf s, strub jr, att w. accuracy of full-arch scans using intraoral scanners. clin oral investig. 2014;18: 1687–1694. pmid:24240949 12. yuzbasioglu e, kurt h, turunc r, bilir h. comparison of digital and conventional impression techniques: evaluation of patient's perception, treatment comfort, effectiveness and clinical outcomes. bmc oral health. biomed central; 2014;14: 10. pmid:24479892 13.van der meer wj, andriessen fs, wismeijer d, ren y. application of intra-oral dental scanners in the digital workllow of implantology. glogauer m, editor. plos one. 2012;7: e43312. pmid:22937030 14. karl m, graef f, schubinski p, taylor t. effect of intraoral scanning on the passivity of lit of implant-supported lixed dental prostheses. quintessence int. 2012;43: 555–562. pmid:22670250 15. schaefer o, decker m, wittstock f, kuepper h, guentsch a. impact of digital impression techniques on the adaption of ceramic partial crowns in vitro. j dent. 2014;42: 677–683. pmid:24508541 16. yang x, lv p, liu y, si w, feng h. accuracy of digital impressions and fitness of single crowns based on digital impressions. materials. 2015;8: 3945–3957. 17.lee sj, betensky ra, gianneschi ge, gallucci go. accuracy of digital versus conventional implant impressions. clinical oral implants research. 2015;26: 715–719. pmid:24720423 18. cho s-h, schaefer o, thompson ga, guentsch a. comparison of accuracy and reproducibility of casts made by digital and conventional methods. j prosthet dent. 2015;113: 310–315. pmid:25682531 19.ender a, attin t, mehl a. in vivo precision of conventional and digital methods of obtaining complete-arch dental impressions. j prosthet dent. 2016;115: 313–320. pmid:26548890 20. ender a, mehl a. accuracy of complete-arch dental impressions: a new method of measuring trueness and precision. j prosthet dent. 2013;109: 121–128. pmid:23395338 21. kwon sw, liapi ka, haas ct, bosche f. algorithms for litting cylindrical objects to sparse range point clouds for rapid workspace modeling. 20th international symposium …. 2003. 22. cho y-k, kwon s-w. rapid geometric modeling for construction automation. ksce j civ engineering. ksce j of civ engineering. 2003;7: 225–231. 23. valero e, adan a, cerrada c. automatic construction of 3d basic-semantic models of inhabited interiors using laser scanners and rfid sensors. sensors. molecular diversity preservation international; 2012;12: 5705–5724. pmid:22778609 digital versus convennonal impressions vol 13, no 1 (2025) doi 10.5195/d3000/2025.929 http://dentistry3000.pitt.edu 4 24. ting-shu s, jian s. intraoral digital impression technique: a review. j prosthodont. 2015;24: 313–321. pmid:25220390 25. syrek a, reich g, ranftl d, klein c, cerny b, brodesser j. clinical evaluation of all-ceramic crowns fabricated from intraoral digital impressions based on the principle of active wavefront sampling. j dent. 2010;38: 553–559. pmid:20381576 26. kim s-y, kim m-j, han j-s, yeo i-s, lim y-j, kwon h-b. accuracy of dies captured by an intraoral digital impression system using parallel confocal imaging. int j prosthodont. 2013;26: 161– 163. pmid:23476911 27. su t-s, sun j. comparison of marginal and internal lit of 3-unit ceramic lixed dental prostheses made with either a conventional or digital impression. j prosthet dent. 2016. pmid:27061628 28. berrendero s, salido mp, valverde a, ferreira a, pradıés g. inlluence of conventional and digital intraoral impressions on the lit of cad/cam-fabricated all-ceramic crowns. clin oral investig. 2016. pmid:26800669 29.kollmuss m, kist s, goeke je, hickel r, huth kc. comparison of chairside and laboratory cad/cam to conventional produced all-ceramic crowns regarding morphology, occlusion, and aesthetics. clin oral investig. 2016;20: 791–797. pmid:26245275 30. brawek pk, wolfart s, endres l, kirsten a, reich s. the clinical accuracy of single crowns exclusively fabricated by digital workllow—the comparison of two systems. clin oral investig. 2013;17: 2119–2125. pmid:23371756 31. güth j-f, keul c, stimmelmayr m, beuer f, edelhoff d. accuracy of digital models obtained by direct and indirect data capturing. clin oral investig. 2013;17: 1201–1208. pmid:22847854 32.ender a, zimmermann m, attin t, mehl a. in vivo precision of conventional and digital methods for obtaining quadrant dental impressions. clin oral investig. 2015. pmid:26547869 33.lee w-s, park j-k, kim j-h, kim h-y, kim w-c, yu c-h. a new approach to accuracy verilication of 3d surface models: an analysis of point cloud coordinates. journal of prosthodontic research. 2016;60: 98–105. pmid:26603682 34. sousa mvs, vasconcelos ec, janson g, garib d, pinzan a. accuracy and reproducibility of 3-dimensional digital model measurements. am j orthod dentofacial orthop. 2012;142: 269–273. pmid:22858338 35. hayashi k, sachdeva auc, saitoh s, lee s-p, kubota t, mizoguchi i. assessment of the accuracy and reliability of new 3-dimensional scanning devices. am j orthod dentofacial orthop. 2013;144: 619–625. pmid:24075671 36.barreto ms, faber j, vogel cj, araujo tm. reliability of digital orthodontic setups. angle orthod. 2016;86: 255–259. pmid:26042573 37. asquith j. dental arch relationships on threedimensional digital study models and conventional plaster study models for patients with unilateral cleft lip and palate. cleft palate-craniofacial journal. 2012;49: 530–534. pmid:21214319 figure 4. conventional and digital imprint method mean values. 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 a b c d e f g h i j conventional impression technique digital impression technique 892 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.892 http://dentistry3000.pitt.edu apical foramen positional variability and its minimal distance from the anatomical apex in premolars a cbct-based analy.c study maysaloon s. saeed1 1college of medicine, university of duhok, iraq abstract objec1ves: this study aimed to determine the posi1on of the af in rela1on to the root surfaces of human permanent premolar teeth in the region using cone-beam computed tomography (cbct) imaging. materials and methods: a descrip1ve cross-sec1onal study was conducted using cbct images of 206 pa1ents from the iraqi kurdistan region. mul1planar cbct scans were analyzed to determine the af posi1on rela1ve to the root canal surface of the premolars. the mean distance between the af and anatomical apex was measured and compared between quadrants and sexes. sta1s1cal analysis was performed using fisher’s chi-square and ttests, with a significance threshold set at p = 0.05. results: the central af loca1on was the most prevalent in premolars, followed by distal and mesial loca1ons. however, the central and mesial posi1ons were the most prevalent in the mandibular right second premolar. the mean af-anatomic apex distance for the first premolars on the right side was 0.6205 mm. for the second premolars, the distances were 0.6205 mm in males and 0.5854 mm in females. no significant varia1ons were observed based on the parameters outlined in the methodology. conclusion: the apical foramen was predominantly located centrally in the premolars, followed by the distal posi1on. to enhance the success rate of root canal therapy, a minimum of 1 mm safety margin from the anatomic apex in premolars is recommended to improve treatment outcomes. open access cita%on: maysaloon ss. (2025) apical foramen posi%onal variability and its minimal distance from the anatomical apex in premolars: a cbct-based analy%c study. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.892 received: march 30, 2025 accepted: may 14, 2025 published: june 10, 2025 copyright: ©2025 maysaloon ss. this is an open access ar%cle licensed under a crea%ve commons auribu%on work 4.0 united states license. email: maysaloonshaman@gmail.com introduc)on complete cleaning and shaping of the root canals, along with a three-dimensional sealed root canal system (rcs) filling, are considered the most essential and widely practiced endodontic procedures worldwide [1]. accurate morphological knowledge of the apical region is crucial, as instrumentation and filling of root canals are based to a significant extent on this information [2,3]. understanding the apical area and morphology of the tooth root canal is a complex and critical aspect that clinical practitioners must consider when making decisions during endodontic treatment [3]. numerous factors contribute to the variations found in studies on root canals and apical foramina (af), including ethnicity, age, and sex. a successful endodontic procedure commences with a comprehensive assessment of the root canals and their anatomical variability [1-3]. apical foramen posifonal variability and its minimal distance from the anatomical apex in premolars vol 1, no 1 (2012) doi 10.5195/d3000/2025.892 http://dentistry3000.pitt.edu 2 cone beam computed tomography (cbct) is a valuable 3d orthogonal imaging tool for the maxillofacial skeleton and its internal structures. it utilizes a lower dose of radiation compared to conventional ct [4] and has been demonstrated to be more accurate than digital x-rays in identifying rcs. it precisely determines root canal morphology and af location in relation to the root surfaces, which requires exact identification during access preparation, and reduces errors and failures in endodontic treatments by eliminating the issue of root superimposition from neighboring anatomical structures. the precise assessment of the af distance has proven to be a highly beneficial tool in clinical dental practice and must be taken into consideration during root canal procedures. complete debridement of these regions through mechanical instrumentation is impractical, and necrotic tissue remnants and microorganisms in the apical portion affect post-treatment outcomes [5]. cbct images provide insight into the spatial resolution of anatomical variations and enable clinicians to visualize any necessary access modifications for treatment. from an anatomical and clinical perspective, the narrowest part of the root canal is the apical constriction or minor foramen. from this point, the canal gradually widens and terminates at the af or major foramen. the cementodentinal junction is the histological landmark where the pulp ends and the periodontal ligament begins. it is expected to be located at the apical constriction, but its position can be irregular (figure 1). the af is the main opening to the apical portion of the root canal and constitutes the site where endodontic filling is performed [6]. in numerous instances, the terms “apex” and “af” have been used interchangeably. the anatomical apex refers to the anatomical terminus of the root, as observed on radiography. in contrast, the af is the main opening of the root canal in relation to the apex. the af can be in mesial, distal, buccal, and lingual positions [7]. recent studies have reported that in over 60% of canals, the af is not located at the apex. furthermore, the distance between the af and radiographic apex can vary by up to 3 mm [8]. this apical deviation of the foramen is attributed to aging and cementum deposition. additionally, the degree of deviation varied according to tooth type. over the past three decades, various methods, such as clearing, modeling, and histologic and radiographic techniques, have been employed to assess the structure of the root canal. recently, the application of cbct has increased significantly. cbct can provide 3d observations of anatomical structures and pathological conditions [9]. material and methods the study protocol was approved by the institutional ethics committee of the college of dentistry, university of duhok, kurdistan region, iraq. the case records of 696 premolars from 206 patients (102 males and 104 females) who underwent cbct scans between november 2021 and september 2024 were retrieved from the databases of three private dental imaging centers located in three governorates in the kurdistan region of iraq. in this retrospective study, cbct images of permanent premolar teeth from duhok, erbil, and sulaymaniyah were included. to obtain a representative sample of the kurdish population in this region, the main public dental clinic in the capital of these governorates was selected as the data collection setting. cbct images were obtained using a newtom giano (verona, italy) cbct system. two types of software were used for image analysis and evaluation: villa 3d planner (italy) for treatment planning and anatomage i in vivo dental viewer (italy) for 3d visualization. the machines were operated at 90 kvp and 10ma. the field of view was 8 × 8 cm, and the voxel size was 75 µm. images in sections of 0.5 mm with intervals of 1 mm in the coronal and sagittal planes in terms of the 10 mm were prepared using nnt software for appropriate examination of the teeth. in the axial view, only the lower one-third of the roots was investigated, and the roots were examined horizontally (parallel to the occlusal surface) so that they passed through the upper sections of the teeth and were parallel to the sagittal plane. in the first mode, images were cross-sectional or in the sagittal view, enabling examination of the buccal and lingual surfaces. in the second mode, the coronal view of the mesiodistal teeth was investigated. it is noteworthy that the af may be positioned mesiodistally or buccolingually relative to the outer surface of the tooth. consequently, in both the coronal and sagittal views, the distance between the af and the perpendicular and tangential lines on the radiographic apex (in 10 mm) was measured using nnt software. furthermore, the distance from the af to the anatomic apex was observed in the axial, sagittal, and coronal planes. healthy teeth with no previous endodontic treatments, restorations, or root resorption were chosen as inclusion criteria. teeth with open apices (not fully formed), root resorption, or calcification (partial or complete radiographic obliteration of the pulp chamber and root canals), as well as teeth with previous endodontic treatment, were excluded. healthy teeth with no prior treatment or restoration were included. the images were evaluated, and the data were captured using microsoft excel 2016. the data were subsequently exported to the statistical package for the social sciences (spss version 26, ibm corp., il, and usa). chi-square test was used to compare proportions. fisher’s exact test was used instead of the chi-square test when the expected value of more than 20% of the cells of the table was <5. kolmogorov–smirnov test, unpaired “t”, and paired “t” tests were used for sample comparison. the level of signikicance was set at 0.05. the results were presented using spss software, and the outcomes were represented in the form of tables and bar charts. results six hundred and ninety-six premolars that met the specikic inclusion criteria and were from individuals between the ages of 18 and 60 were included in this study. the sample comprised 102 males (49.51%) and 104 females (50.49%), as presented in (table 1). table 1. prevalence of af locations in maxillary premolar teeth in both genders. tooth type location male (%) female (%) total (%) right ufpt central 58.3 51.1 54.7 distal 37.5 42.2 39.8 mesial 4.2 6.7 5.5 right uspt central 69.2 66.7 68.0 distal 28.2 31.1 29.6 mesial 2.6 2.2 2.4 apical foramen posifonal variability and its minimal distance from the anatomical apex in premolars vol 1, no 1 (2012) doi 10.5195/d3000/2025.892 http://dentistry3000.pitt.edu 3 left ufpt central 53.2 47.9 50.5 distal 44.7 52.1 48.4 mesial 2.1 0.0 1.1 left uspt central 54.1 63.4 58.7 distal 40.5 29.3 34.9 mesial 5.4 7.3 6.4 for the right maxillary kirst premolar teeth (r.ufpt), the majority of apical foramina in both males and females were centrally located, accounting for 58.3% and 51.1%, respectively. the second most prevalent location was distal in both males and females, with percentages of 37.5% and 42.2%, respectively. the least common location was mesial, occurring in 4.2% of males and 6.7% of females. for the left maxillary kirst premolar teeth (l.ufpt), the distribution of the apical foramina was relatively similar between males and females. the most frequent location was central, accounting for 53.2% in males and 47.9% in females. the second most common location was distal, observed in 44.7% of males and 52.1% of females. a minimal percentage of apical foramina was mesially located in males (2.1%). for the right maxillary second premolar teeth (r.uspt), the majority of apical foramina in both males and females were centrally positioned, occurring in 69.2% and 66.7% of cases, respectively. the second most prevalent location was distal, recorded in 28.2% of males and 31.1% of females. a minimal percentage of apical foramina was mesially located in both males and females (2.6% and 2.2%, respectively). for the left maxillary second premolar teeth (l.uspt), the majority of apical foramina were centrally positioned, observed in 54.1% of males and 63.4% of females. a smaller percentage was distally located in both males and females (40.5% and 29.3%, respectively), while the mesial location was the least common, occurring in 5.4% of males and 7.3% of females (table 2). [refer to the last page for table 2] by fisher’s exact test. **by chi-square test. lfpt: lower first premolar teeth; lspt: lower second premolar teeth. there is a statistically signikicant difference in the location of apical foramina between males and females, for the location of apical foramina in the left kirst premolar teeth. the p-value for this location is 0.067, which is marginally signikicant at the 0.05 level. for the right mandibular kirst premolar teeth (r.lfpt), the majority of apical foramina were centrally located in both males and females, comprising 58.1% and 53.5%, respectively. the second most prevalent location was the distal, occurring in 30.2% of males and 23.3% of females. the least frequent location was mesial, observed in 11.6% of males and 23.3% of females. for the left mandibular kirst premolar teeth (l.lfpt), the majority of apical foramina were centrally positioned, recorded in 48.8% of males and 72.7% of females. a smaller proportion was distally located in both males and females (29.3% and 18.2%, respectively), while the least common location was mesial in both males and females (22% and 9.1%, respectively). the majority of apical foramina in the right mandibular second premolar (r.lspt) were centrally situated in both males and females (65% and 58%, respectively). the second most prevalent location was mesial in both males and females (20% and 26%, respectively), while the least common location was distal (15% and 16%, respectively). for the left mandibular second premolar teeth (l.lspt), the central location was the most prevalent in both males and females, occurring in 75.7% and 68.8% of the cases, respectively. the second most common location was distal, observed in 16.2% of males and 22.9% of females, while the mesial location exhibited the lowest percentage in both males and females (8.1% and 8.3%, respectively). the average (±standard deviation [sd]) distance from the af to the anatomic apex of the kirst and second premolars in males was 0.61–0.62 mm with ± sd of 0.142 and p=0.54 in the right and left quadrants, and for females was 0.58–0.60 mm with ± sd of 0.129 and p=0.22 for both right and left quadrants. according to the t-test, no statistically significant difference was observed in the distance between the af and the anatomic apex of the kirst and second premolars in both the right and left quadrants (p = 0.857). a comparison of the mean distance between the af and anatomic apex of the kirst and second premolars between the right and left sides in both males and females is presented in tables 3 and 4. table 3. comparison of mean distance between apical foramen and anatomic apex of right kirst premolar and right second premolar between males and females (mm). table 4. comparison of mean distance between apical foramen and anatomic apex of left kirst premolar and left second premolar between males and females. tooth type right (mean ± sd) left (mean ± sd) pvalue first premolars 0.62 ± 0.14 0.62 ± 0.14 0.86 second premolars 0.6 ± 0.13 0.59 ± 0.12 0.28 discussion a comprehensive understanding of root anatomy and canal morphology is essential for the efficient execution of biomechanical cleaning and shaping, which is crucial for achieving predictable endodontic outcomes. however, variations in root canal morphology present clinical challenges that may result in unfavorable endodontic treatment outcomes. cbct is an excellent ex vivo and in vivo method for evaluating external and internal root morphology compared with conventional 2d radiography [6-9]. in the present study on the maxillary first premolars (r.ufpt and l.ufpt), the most prevalent deviation in the location of the apical foramina in males and females was central (52.6%), followed by distal (44.1%) and mesial (3.3%). variations were observed between quadrants and genders for these teeth. no cases were recorded for females in the mesial location on the left side, and the distal location was more prevalent on the left side for both sexes than that on the right side. similar results were also reported in a recent study conducted on the brazilian population [9], which indicated that the most common af location in the maxillary first premolars (39.9%) was central. in the present study, the most common location for the maxillary second right and left premolar teeth (r.uspt, l.uspt) was the central position, accounting for 63.4% of cases. this was followed by the distal (32.2 %) and mesial (4.4 %) locations. almost all af locations were consistent in both quadrants and both sexes, except for the distal location in the left upper second premolar tooth (l.uspt), which was more prevalent in males than in females. a similar result was reported in a study conducted on a subpopulation in age group mean ± standard devition males females pvalue first premolars 0.62±0.14 0.59±0.12 0.544 second premolars 0.62±0.14 0.58±0.11 0.204 apical foramen posifonal variability and its minimal distance from the anatomical apex in premolars vol 1, no 1 (2012) doi 10.5195/d3000/2025.892 http://dentistry3000.pitt.edu 4 yemen, which also indicated that the center was the most frequently observed location for the second upper premolars [10]. in a study conducted in brazil [9], it was found that the most common location for af among the second upper premolars was central, accounting for 57.4%. in the present study, the location of the af for the lower first right and left premolars (r.lfpt, l.lfpt) in both sexes was predominantly in the center (58.3%), followed by distal (25.1%) and mesial (16.6%). in the r.lfpt, the distal and central locations were similar between the sexes, but the mesial location was more prevalent in females than in males. however, in the left lfpt, the central location was more prevalent in females than in males, whereas mesial and distal locations were more prevalent in males than in females. these results were corroborated by other studies conducted in the kuwaiti population [11] and brazil [9], where the central location was found to be the most common among the sample studies of the mandibular first premolars. however, a specific subgroup within the iranian population contradicted the present findings. they reported that the most common location for the apical foramina among mandibular first premolars was the distal [12]. significant variations were observed among different populations, which may be attributed to factors such as sex, sample size, ancestry, data collection methods, and study design [13]. for the mandibular right and left second premolars (r.lspt, l.lspt) in our study, both sexes exhibited a predominant central location, accounting for 66.9% of the cases. this was followed by the distal (17.5 %) and mesial (15.6 %) locations. the central location in both quadrants was more prevalent in males than females. on the right side, the mesial and distal locations were approximately equivalent in both sexes. regarding the mesial location, both sexes demonstrated similar percentages on the left side. however, in females, the distal location was more prevalent compared to males. a comparable finding was observed in the yemeni subpopulation, where the center was reported as the most frequent location [10]. furthermore, a similar observation was noted in the brazilian population [9], where the most common location was central, accounting for 42.85% and 50.98% of cases, respectively. the present study determined that the average distance from the af to the anatomic apex of the first and second premolars in males and females was 0.61–0.62 mm (± sd 0.142, p=0.54) and 0.58–0.60 mm (± sd 0.129, p=0.22), respectively. the results of the apex-to-foramen distance measurements in this study were in close concordance with previous findings. burch and hulen [14] reported the af distance to be 0.59 mm in a study of all tooth types, and a study conducted by arora and tewari [15] reported the distance between the af to be in the range of 0.052–2.91 mm. a similar study conducted by naseri et al. [16] revealed that the mean distance between af was found to be in the range of 0.3–0.7 mm, which is consistent with the values calculated in the present study. in another study by martos et al. [17], which was conducted using a stereomicroscope on mandibular molars of a brazilian population, the mean distance from the apex to the af was 0.80 (±0.54) mm. akhlaghi et al. [18] in an ex vivo study on a local iranian population using india ink on mandibular second molars demonstrated that the mean (±sd) distance from the apex of the af was 0.30–0.47 mm. india ink was used in this study, which renders the evaluation method distinct from that of the present study. a study conducted by cheung et al. [19] indicated that the average distance of the af from the anatomic apex at the cshaped mandibular second molars in a chinese population is 0.79–0.89 mm, as determined using micro-ct. these minor variations are, to some extent, attributable to the varying methods employed to measure the distance as well as to the different reference points that were likely utilized. moreover, the other reason for this difference may be due to variations among diverse populations. in a recent study conducted by reda et al. in 2022, they found that one af was the most common among all the premolars and that the mean distance of af from the anatomic apex recorded between 3.40 ± 1.80 and 5.12 ± 1.98 [20]. a study conducted among the chinese population by yang et al. reported an average distance of 1–2 mm within their study sample [21]. this variation in the af distance suggests a potential racial predilection. in a recent comparable study conducted by arsari et al, in 2021 on the anterior maxillary teeth, they observed that the mean apical foramen distance to the anatomical apex in anterior maxillary teeth in males and females was 0.64±0.36 and 0.58±0.32 mm, respectively (p=0.003). this difference was statistically significant according to their statistical indices ,while the mean distance between apical foramen and anatomical apex in central incisor and canine teeth was not significant in males and females (p=0.06, and p=0.25, respectively), but they observed a significant difference concerning the mean interval between apical foramen and lateral apex in lateral incisor teeth of men and women (p=0.02) [22]. in another study [23], mandibular premolars were compared and the mean length from the apical foramen to the apex was 0.59 mm and 0.47 mm, respectively. meanwhile, the non-clinical and clinical mean lengths from the apical constriction to the apex were 0.75 mm and 0.73 mm, respectively. nonetheless, no significant differences were detected between the apical constriction and the apex and apical foramen and apex [23]. conclusions accurate evaluation of apical anatomy using cbct is essential for performing root canal treatment. the most common location for the apical foramina of all premolar teeth was the central af, followed by the distal. the distance between the af and anatomic apex in the mandibular premolars of the kurdistan population was approximately 1 mm. therefore, the extent of obturation should be 1 mm short of the radiographic apex, and the root canal procedure should be terminated at this point. conflict of interest the authors have no conklicts of interest to declare. author contribu)ons as the sole author of this manuscript, m.s. saeed contributed to the conception, design, data collection, analysis, and interpretation of results. m.s. saeed drafted the manuscript, approved the final version, and is responsible for all aspects of the work. funding this research received no specific grant from any funding agency in the public, apical foramen posifonal variability and its minimal distance from the anatomical apex in premolars vol 1, no 1 (2012) doi 10.5195/d3000/2025.892 http://dentistry3000.pitt.edu 5 commercial, or private sectors. acknowledgement we would like to give many thanks to the dentists and technicians who gave us so much. ethical considerations before commencing the study, permission was obtained from the college of dentistry at duhok university. the official paper number was 917, dated 23/11/2021. approval was also obtained from the ethics committee at the directorate general of health in duhok governorate. the official paper number was 4255 on october 20, 2020. references 1. vertucci fj. root canal anatomy of the human permanent teeth. oral surg oral med oral patho l984 ;99: 585–9. 2. de pablo ov, estevez r, sanchezn mp, et al. root anatomy and canal configuration of the permanent mandibular first molar: a systematic review. j endod 2010; 36: 1919–31. 3. chen g, chang yc. effects of liquidand pastetype edta on smear-layer removal during rotary root-canal instrumentation. j dent sci 2011; 6: 41–7. 4. ahmed hma, dummer pmh, editors. endodontic advances and evidence-based clinical guidelines. new jersey: wiley blackwell; 2022. 5. celikten b, orhan k, aksoy u, et al. cone beam ct evaluation of root canal morphology of maxillary and mandibular premolars in a turkish cypriot population. bdj open 2016; 2: 15006. 6. garg n., textbook of endodontics, second edition, jaypee brothers, new delhi, 2010. 7. wolf t, paque f, patyna m, et al. three dimensional analysis of the physiological foramen geometry of maxillary and mandibular molars by means of micro-ct. int j oral sci 2017; 9: 151– 7. 8. wu mk, wesselink pr, walton re. apical terminus location of root canal treatment procedures. oral surg oral med oral pathol oral radiol endod 2000; 89: 99–103. 9. estrela c, couto gs, bueno mr, et al. apical foramen position in relation to proximal root surfaces of human permanent teeth determined by using a new cone-beam computed tomographic software. j endod 2018; 44: 1741–8. 10. senan em, alhadainy ha, genaid tm, et al. root form and canal morphology of maxillary first premolars of a yemeni population. bmc oral health 2018; 18: 94. 11. alenezi dj, al nazhan sa, al maflehi n, et al. root and canal morphology of mandibular premolar teeth in a kuwaiti subpopulation: a cbct clinical study. j int soc prev comm dent 2020; 10; 235–41. 12. hajihassani n, roohi n, madadi k, et al. evaluation of root canal morphology of mandibular first and second premolars using cone beam computed tomography in a defined group of dental patients in iran. scientifica (cairo) 2017; 2017: 1504341. 13. jain a, bahuguna r. root canal morphology of mandibular first premolar in a gujarati population an in vitro study. dent res j 2011; 8; 118– 22. 14. burch jg, hulen s. the relationship of the apical foramen to the anatomic apex of the tooth root. oral surg oral med oral pathol 1972; 34: 262– 8. 15. arora s, tewari s. the morphology of the apical foramen in posterior teeth in a north indian population. int endod j 2009; 42: 930–9. 16. naseri m, ahangari z, momayyez m. evaluation of the distance of apical constriction and radiographic apices in extracted maxillary second premolars using the clearing technique. j dent sch 2012; 30: 95–100. 17. martos j, ferrer luque cm, gonzález rodríguez mp, et al. topographical evaluation of the major apical foramen in permanent human teeth. int endod j 2009; 42: 329–34. 18. akhlaghi nm, abbas fm, mohammadi m, et al. radicular anatomy of permanent mandibular second molars in an iranian population: a preliminary study. dent res j (isfahan) 2016; 13: 362–6. 19. cheung gs, yang j, fan b. morphometric study of the apical anatomy of c shaped root canal systems in mandibularsecond molars. int endod j 2007; 40: 239–46. 20. reda r, zanza a, bhandi s, et al. surgical-anatomical evaluation of mandibular premolars by cbct among the italian population. dent med probl 2022; 59: 209–16. 21. yang h, tian c, li g, et al. a cone-beam computed tomography study of the root canal morphology of mandibular first premolars and the location of root canal orifices and apical foramina in a chinese subpopulation. j endod 2013; 39: 435–8. 22. arsari f, abesi f, gholinia h,soleymani a. evaluation of apical foramen position and distance from the anatomical apex of roots in anterior maxillary teeth by cone -beam computed tomography (cbct) in a selected iranian population. caspian j dent res 2021; 10: 20 – 9. 23. nurulaqmar iwani s, kamaruzaman m, jawami aa. microcomputed tomography (micro-ct) analysis of apical mandibular premolar in relation to clinical sign presentation: an in vitro study. saudi dent j. 2024 jan;36(1):129-133. apical foramen posifonal variability and its minimal distance from the anatomical apex in premolars vol 1, no 1 (2012) doi 10.5195/d3000/2025.892 http://dentistry3000.pitt.edu 6 table 2. prevalence of af locations in mandibular premolar teeth for both genders. tooth type location male no. (%) female no. (%) total no. (%) right lfpt central 50 (58.1) 46 (53.5) 96 (55.8) distal 26 (30.2) 20 (23.3) 46 (26.6) mesial 10 (11.6) 20 (23.3) 30 (17.6) total 86 (100) 86 (100) 172 (100) right lspt central 52 (65) 58 (58) 110 (61.5) distal 12 (15) 16 (16) 28 (15.5) mesial 16 (20) 26 (26) 42 (23) total 80 (100) 100 (100) 180 (100) left lfpt central 40 (48.8) 64 (72.7) 104 (60.7) distal 24 (29.3) 16 (18.2) 40 (23.7) mesial 18 (22.0) 8 (9.1) 26 (15.6) total 82 (100) 88 (100) 170 (100) left lspt central 56 (75.7) 66 (68.8) 122 (72.3) distal 12 (16.2) 22 (22.9) 34 (19.5) mesial 6 (8.1) 8 (8.3) 14 (8.2) total 74 (100) 96 (100) 170 (100) 974 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.974 http://dentistry3000.pitt.edu efficacy of dexamethasone on pain and swelling following extraction of impacted mandibular third molars a randomized controlled trial saber mizher college of den*stry, tikrit university, iraq abstract objec.ve: surgical extracfon of impacted mandibular third molars is a common dental procedure associated with significant postoperafve complicafons, including pain and swelling. dexamethasone, a corfcosteroid known for its anf-inflammatory properfes, has been uflized to mifgate these postoperafve sequelae. this study aimed to evaluate the efficacy of submucosal dexamethasone injecfon in reducing postoperafve pain and swelling compared to a control group receiving normal saline. material and methods: a randomized controlled trial was conducted involving 40 pafents scheduled for surgical extracfon of impacted mandibular third molars. parfcipants were randomly assigned to two groups: the study group received a submucosal injecfon of 8mg dexamethasone, while the control group received an equivalent volume of normal saline. postoperafve pain was assessed using a numeric rafng scale (nrs) at 24 hours, 48 hours, and 7 days post-surgery. facial swelling was measured at the same intervals using standardized landmarks. results: the results indicated that the study group experienced significantly less pain at 24 hours (nrs: 4.28 ± 0.71 vs. 5.8 ± 0.87, p=0.001) and 48 hours (nrs: 3.45 ± 0.75 vs. 4.9 ± 0.99, p=0.001) compared to the control group. addifonally, facial swelling was significantly reduced in the dexamethasone group at 48 hours (3.01% vs. 6.49%, p=0.001). by the 7th postoperafve day, both groups showed similar levels of pain and swelling, indicafng that the effect of dexamethasone was most pronounced in the early postoperafve period. conclusion: submucosal injecfon of dexamethasone is effecfve in minimizing postoperafve pain and swelling following the surgical extracfon of impacted mandibular third molars. the findings support the use of dexamethasone as a standard adjuncfve treatment in oral surgery to enhance pafent recovery during the crifcal early postoperafve phase. further studies are warranted to explore opfmal dosing and long-term outcomes associated with this intervenfon. open access cita%on: mizher s. (2025) efficacy of dexamethasone on pain and swelling following extrac%on of impacted mandibular third molars: a randomized controlled trial. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.974 received: june 28, 2025 accepted: june 30, 2025 published: august 21, 2025 copyright: ©2025 mizher s. this is an open access ar%cle licensed under a crea%ve commons axribu%on work 4.0 united states license. email: xxxxx@esraa.edu.iq introduc)on surgical extracfon of impacted mandibular third molars is a common dental procedure that can result in significant postoperafve complicafons, including pain, swelling, and trismus. these sequelae can lead to discomfort, funcfonal impairment, and a prolonged recovery period for pafents. the management of postoperafve pain and swelling is crucial to improve pafent outcomes and safsfacfon [1,2]. corfcosteroids, such as dexamethasone, have been widely used to reduce inflammafon and alleviate postoperafve symptoms. dexamethasone has potent anf-inflammatory properfes, making it an effecfve agent in reducing swelling and pain. the submucosal route of administrafon has gained popularity due to its targeted acfon, reduced systemic side effects, and ease of administrafon [3,4]. despite the growing evidence supporfng the use of dexamethasone in oral surgery, there is a need for further research to establish its efficacy in reducing postoperafve pain and swelling following impacted mandibular third molar surgery. the opfmal dosage, fming, and route of administrafon of dexamethasone remain unclear, and the current literature is limited by the variability in study designs, sample sizes, and outcome measures [5,6]. this randomized controlled trial aims to evaluate the efficacy of submucosal dexamethasone injecfon in reducing postoperafve pain and swelling following surgical extracfon of impacted mandibular third molars. the study will compare the outcomes of pafents receiving submucosal dexamethasone injecfon with those receiving a normal saline injecfon. the results of this study will provide valuable efficacy of dexamethasone on pain and swelling following extracfon of impacted mandibular third molars vol 13, no 1 (2025) doi 10.5195/d3000.2025.974 http://dentistry3000.pitt.edu 2 insights into the effecfveness of submucosal dexamethasone injecfon in managing postoperafve pain and swelling, ulfmately contribufng to improved pafent care and outcomes in oral surgery. material and methods study design: this randomized controlled trial was conducted from november 4, 2022, to december 15, 2022. the study was approved by the insftufonal review board, and informed consent was obtained from all parfcipants. par.cipants: a total of 40 pafents aged 17 to 40 years, requiring surgical extracfon of impacted mandibular third molars, were enrolled in the study. pafents were randomly assigned to two groups: the study group (n=20) received a submucosal injecfon of 8 mg dexamethasone, while the control group (n=20) received an equivalent volume of normal saline (normon, spain). inclusion criteria included pafents with fully impacted mandibular third molars, while exclusion criteria included those with systemic diseases, allergies to corfcosteroids, or pregnant women [7,8]. surgical procedure: all surgical extracfons were performed under local anesthesia using 20 mg/ml lidocaine with 0.0125 mg/ml epinephrine. a full-thickness two-sided flap was created using a no. 15 blade and scalpel handle no. 3. flap reflecfon was achieved with a periosteal elevator, followed by bone removal using a low-speed surgical handpiece and normal saline irrigafon. the impacted tooth was extracted, and the flap was reposifoned and sutured [9,10]. interven.on: immediately afer the surgical procedure, the study group received a submucosal injecfon of 8 mg dexamethasone (caspian tamin, iran) at the surgical site, while the control group received an equivalent volume of normal saline. the injecfons were administered using a dental syringe (dental syringe, pakistan) to ensure accurate delivery. outcome measures: postoperafve pain was assessed using a numeric rafng scale (nrs) at 24 hours, 48 hours, and 7 days post-surgery. pafents were instructed to rate their pain intensity on a scale from 0 (no pain) to 10 (worst pain imaginable). facial swelling was measured at baseline, 48 hours, and 7 days postoperafvely using standardized landmarks on the face. data analysis: data were analyzed using spss sofware (version 25). descripfve stafsfcs were calculated for demographic data. comparisons between groups for pain scores and swelling measurements were performed using independent t-tests and anova, with a significance level set at p < 0.05. the results were presented as mean ± standard deviafon (sd) [11,12]. results summary of findings the results of this study indicate that submucosal dexamethasone injecfon is effecfve in reducing both postoperafve pain and swelling following the surgical extracfon of impacted mandibular third molars. the most significant benefits were observed within the first 48 hours post-surgery, with both pain and swelling returning to similar levels by the 7th postoperafve day. these findings support the use of submucosal dexamethasone as a standard adjuncfve treatment in oral surgery to enhance pafent recovery during the crifcal early postoperafve phase. demographic data a total of 40 pafents were enrolled in the study, with 20 pafents in the study group receiving submucosal dexamethasone injecfon and 20 pafents in the control group receiving normal saline. the mean age of the pafents was 28.85 years, with a standard deviafon of ±6.95 years. the sex distribufon showed that in the control and intervenfon groups 12 parfcipants were male and eight were female, with no significant difference between the groups (p ≥ 0.05). pain assessment postoperafve pain scores were assessed using the numeric rafng scale (nrs) at 24 hours, 48 hours, and 7 days post-surgery. at 24 hours postoperafvely, the study group reported significantly lower pain scores compared to the control group (4.28 ± 0.71 vs. 5.8 ± 0.87, p=0.001). similarly, at 48 hours, the pain scores remained significantly lower in the study group (3.45 ± 0.75 vs. 4.9 ± 0.99, p=0.001). by the 7th postoperafve day, the pain scores in both groups were comparable, with no stafsfcally significant difference (0.2 ± 0.41 vs. 0.43 ± 0.31, p=0.069). swelling assessment facial swelling was measured at baseline, 48 hours, and 7 days postoperafvely. at 48 hours, the study group exhibited significantly less swelling compared to the control group (3.01% vs. 6.49%, p=0.001). by the 7th postoperafve day, the swelling in both groups was similar, with no stafsfcally significant difference (1.05% vs. 1.26%, p=0.16). adverse effects no serious adverse effects were reported in either group during the study period. minor side effects, such as transient nausea and dizziness, were noted in a few pafents but were not significantly different between the groups. discussion the findings of this randomized controlled trial demonstrate that submucosal dexamethasone injecfon significantly reduces postoperafve pain and swelling following the surgical extracfon of impacted mandibular third molars. the study revealed that pafents receiving dexamethasone reported lower pain scores at 24 and 48 hours postoperafvely compared to the control group, with a notable reducfon in facial swelling at 48 hours. these results align with previous studies that have highlighted the efficacy of corfcosteroids in managing postoperafve inflammafon and discomfort [13,14]. corfcosteroids, parfcularly dexamethasone, are known for their potent anf-inflammatory properfes. they inhibit the release of inflammatory mediators, such as prostaglandins and leukotrienes, which play a crucial role in the development of pain and swelling following surgical trauma [15]. the significant reducfon in pain and swelling observed in this study supports the findings of moraschini et al. (2016), who conducted a meta-analysis indicafng that dexamethasone administrafon [1], parfcularly via the submucosal route, effecfvely mifgates postoperafve symptoms afer third molar surgery [16,17]. conversely, some studies have reported mixed results regarding the efficacy of dexamethasone. for instance, klongnoi et al. (2012) found no significant difference in pain reducfon when comparing dexamethasone to placebo in certain pafent populafons. this discrepancy may be ajributed to variafons in study design, pafent demographics, and the fming of dexamethasone administrafon. this study's focus on the submucosal route may explain the more favorable outcomes, as this method allows for localized delivery of the medicafon, maximizing its anf-inflammatory effects while minimizing systemic exposure [18]. these results also indicate that the benefits of dexamethasone are most pronounced in the early postoperafve period, with pain and swelling levels converging by the seventh day. this finding is consistent with the pharmacokinefcs of dexamethasone, which has a half-life of approximately 36 to 54 hours, suggesfng that its effects diminish as the drug is metabolized [19,20]. these findings contribute to the growing body of evidence supporfng the use of corfcosteroids in oral surgery and highlight the importance of opfmizing postoperafve care to enhance pafent recovery. conclusions this randomized controlled trial demonstrates that submucosal dexamethasone injecfon is an effecfve intervenfon for minimizing postoperafve pain and swelling following the surgical extracfon of impacted mandibular third molars. the results indicate that pafents receiving dexamethasone experienced significantly lower pain scores at 24and 48-hours post-surgery, as well as reduced facial swelling efficacy of dexamethasone on pain and swelling following extracfon of impacted mandibular third molars vol 13, no 1 (2025) doi 10.5195/d3000.2025.974 http://dentistry3000.pitt.edu 3 at the 48-hour mark compared to the control group receiving normal saline. the findings support the use of submucosal dexamethasone as a standard adjuncfve treatment in oral surgery, parfcularly during the crifcal early postoperafve period when pafents are most vulnerable to discomfort and inflammafon. the localized administrafon of dexamethasone not only enhances its efficacy but also minimizes potenfal systemic side effects, making it a favorable opfon for managing postoperafve sequelae. considering these results, it is recommended that dental practitioners consider incorporating submucosal dexamethasone injections into their postoperative care protocols for patients undergoing third molar extractions. further research is reasonable to explore optimal dosing strategies, timing of administration, and longterm outcomes associated with this intervention. overall, the use of submucosal dexamethasone injection represents a valuable advancement in enhancing patient recovery and improving the quality of care in oral and maxillofacial surgery. references 1) moraschini v, hidalgo r, porto barboza e. effect of submucosal injection of dexamethasone after third molar surgery: a meta-analysis of randomized controlled trials. int j oral maxillofac surg. 2016; 45(2):232-40. 2) klongnoi b, kaewpradub p, boonsiriseth k, wongsirichat n. effect of single dose preoperative intramuscular dexamethasone injection on lower impacted third molar surgery. int j oral maxillofac surg. 2012; 41(4):376-9. 3) bamgbose bo, akinwande ja, adeyemo wl, ladeinde al, arotiba gt, ogunlewe mo. effects of co-administered dexamethasone and diclofenac potassium on pain, swelling and trismus following third molar surgery. head face med. 2005; 1:11. 4) giri ky, joshi a, rastogi s, dandriyal r, indra b prasad n, singh h, et al. efaicacy of intravenous dexamethasone administered preoperatively and postoperatively on pain, swelling, and trismus following third molar surgery. a comparative study. oral surgery. 2019; 12 (2):110-7. 5) al-shamiri hm, shawky m, hassanein n. comparative assessment of preoperative versus postoperative dexamethasone on postoperative complications following lower third molar surgical extraction. int j dent. 2017; 2017:1350375. 6) warraich ra, faisal m, rana m, shaheen a, gellrich nc, rana m. evaluation of postoperative discomfort following third molar surgery using submucosal dexamethasone a randomized observer blind prospective study. oral surg oral med oral pathol oral radiol. 2013; 116 (1):16-22. 7) nair rb, rahman nm, ummar m, haaiz ka, issac jk, sameer km. effect of submucosal injection of dexamethasone on postoperative discomfort after third molar surgery: a prospective study. j contemp dent pract. 2013; 14 (3):401-4. 8) o'hare pe, wilson bj, loga mg, ariyawardana a. effect of submucosal dexamethasone injections in the prevention of postoperative pain, trismus, and oedema associated with mandibular third molar surgery: a systematic review and meta-analysis. int j oral maxillofac surg. 2019; 48 (1):1-10. 9) al-badri s, al-harbi f, al-mohaya m, almansour a, al-mutairi a. the effect of dexamethasone on postoperative pain and swelling after third molar surgery: a systematic review and meta-analysis. j oral maxillofac surg. 2020; 78 (4):678-87. 10) chugh a, singh s, mittal y, chugh v. submucosal injection of dexamethasone and methylprednisolone for the control of postoperative sequelae after third molar surgery: randomized controlled trial. int j oral maxillofac surg. 2018; 47 (2):22833. 11) kaur r, kumar ac, garg r, sharma s, rastogi t, gupta vv. early prediction of mandibular third molar eruption/impaction using linear and angular measurements on digital panoramic radiography: a radiographic study. indian j dent. 2016; 7 (2):66-71. 12) siddiqi a, morkel j, zafar s. antibiotic prophylaxis in third molar surgery: a randomized double-blind placebo-controlled clinical trial using split-mouth technique. int j oral maxillofac surg. 2010; 39 (2):107-14. 13) pires wr, bonardi jp, faverani lp, momesso ga, muñoz xmj, silva afm, et al. late mandibular fracture occurring in the postoperative period after third molar removal: systematic review and analysis of 124 cases. int j oral maxillofac surg. 2017; 46 (1):46-53. 14) al-badri s, al-harbi f, al-mohaya m, almansour a, al-mutairi a. the effect of dexamethasone on postoperative pain and swelling after third molar surgery: a systematic review and meta-analysis. j oral maxillofac surg. 2020; 78 (4):678-87. 15) lee y, lai hy, lin pc, lin ys, huang sj, shyr mh. a dose ranging study of dexamethasone for preventing patient-controlled analgesia-related nausea and vomiting: a comparison of droperidol with saline. anesthesia & analgesia. 2004; 98 (4):1066-71. 16) syed kb, alqahtani fhk, mohammad aha, abdullah im, qahtani hsh, hameed ms. assessment of pain, swelling and trismus following impacted third molar surgery using injection dexamethasone submucosally: a prospective, randomized, crossover clinical study. j int oral health. 2017; 9 (3):116-20. 17) polderman j, farhang-razi v, van dieren s, kranke p, de vries jh, hollmann mw, et al. adverse side effects of dexamethasone in surgical patients. cochrane database syst rev. 2018; 2018 (3):cd012123. 18) al-badri s, al-harbi f, al-mohaya m, almansour a, al-mutairi a. the effect of dexamethasone on postoperative pain and swelling after third molar surgery: a systematic review and meta-analysis. j oral maxillofac surg. 2020; 78 (4):678-87. 19) chukwuneke f, onyejiaka n. management of postoperative morbidity after third molar surgery: a review of the literature. niger j med. 2007; 16 (2):107-12. 20) gozali p, boonsiriseth k, kiattavornchareon s, khanijou m, wongsirichat n. decreased post-operative pain using a sublingual injection of dexamethasone (8 mg) in lower third molar surgery. j dent anesth pain med. 2017; 17 (1):47-53. 991 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.991 http://dentistry3000.pitt.edu evaluation of surface roughness of heat cured pmma after immersion in artificial saliva and different solutions shurooq falih altaie1, nihad h. alfuraiji1, rasha mohammed zwyyer2, enas shihab ahmed3 1college of health and medical techniques, middle technical university, baghdad, iraq 2college of health and medical techniques, university of bilad alrafidain, diyala, iraq 3college of den@stry, university of bilad alrafidain, diyala, iraq abstract objecbve: the aim of this in vitro study was to evaluate the surface roughness of heat-cured acrylic resin aler immersion in armficial saliva and different solumons. material and methods: 70-disc shaped specimens 10mm in diameter and 2 mm in thickness were prepared from heat-cured acrylic resin. then, divided into 2 groups. first group consist of 4 groups as follow: (control) immersion in dismlled water, immersion in zamzam water, immersion in diluted thyme oil and immersion in mouth wash (listerine ®) with 10 samples each. the samples were immersed in their respecmve solumon for 8 h per day for one month. the second group consist of 3 groups as follow dismlled water, armficial saliva normal and acidic with 10 samples each. the samples kept in armficial saliva for 16h per day for one month. profilometer was used to evaluate the surface roughness (µm). for analyzing stamsmcally, the obtained data, one-way anova and lsd test were used. p-value<0.05 was considered significant. results: all solumons showed no stamsmcally significant difference on surface roughness of specimens at 8h and 16h for one month. however, the zamzam water group showed posimvely significant difference when immerse 8h for one month. conclusion: the six solumons used in this study none produced increased in surface roughness compared to control group for two mmes of immersion nevertheless, the best result as denture cleanser was obtained in zamzam water which was showed the less value in surface roughness (µm). open access cita%on: altaie sf, et al. (2025) evalua%on of surface roughness of heat cured pmma ader immersion in ar%ficial saliva and different solu%ons. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.991 received: july 15, 2025 accepted: july 31, 2025 published: september 9, 2025 copyright: ©2025 altaie sf, et al. this is an open access ar%cle licensed under a crea%ve commons auribu%on work 4.0 united states license. email: shurooq.falih@mtu.edu.iq introduc)on acrylic resin has been recommended as a denture base material for several years. in addition to having outstanding mechanical qualities, denture base material should ideally gain some important physical characteristics, such as biocompatibility and a strong link with prosthetic teeth [1]. most denture users have poor oral hygiene and struggle to keep their dentures clean. the primary cause of disorders affecting the oral mucosal tissues is unclean dentures. denture wearers were found to have a signidicant correlation between denture stomatitis and inadequate denture hygiene. for better health, especially in elderly people, edentulous individuals must maintain proper dental hygiene and keep their dentures clean [2]. dentures can be cleaned mechanically, chemically, or by combining the two [3].the mechanical cleaning procedure is usually used by patients, and it is insufdicient for cleaning undercut sections of dentures, which might harbor bacteria. furthermore, aged patients with weak motor coordination and handicapped patients with low physical ability and a lack of cooperation may not clear the accumulated biodilm appropriately [4]. the most popular chemical cleaning techniques that have been proposed for prosthesis disinfection are denture cleansers. in addition to being biocompatible, bactericidal, and fungicidal, the best cleaner should also be easy to use and not change the prosthesis's structure in any way. most importantly, it should effectively remove both organic and inorganic deposits [3]. long-term use of denture cleaners can make pmma surfaces rougher [5]. the rough, pitted surfaces of acrylic dentures serve as a breeding ground for bacteria to colonize and build biodilms [3]. the presented in vitro study was conducted to evaluate the effect of artidicial saliva and different solutions(distilled water, zamzam water, diluted thyme oil and mouth evaluamon of surface roughness of heat cured pmma aler immersion in armficial saliva and different solumons vol 13, no 1 (2025) doi 10.5195/d3000.2025.991 http://dentistry3000.pitt.edu 2 wash) on surface roughness of heat-cured acrylic resin material. the null hypothesis tested was that no signidicant differences would be found after immersion in different solutions. material and methods preparation of specimens metal patterns were constructed [10mm in diameter and 2mm in thickness] [6] . the metal patterns were covered with separating media and let to dry after the lower part of the dental dlask was dilled with dental die stone mixed in accordance with the manufacturer's instructions. a layer of separating material was then applied to the whole surface of the stone and the metal patterns after they had been carefully placed to half of their depth and allowed to set. then, the upper part of the dlask was placed on top of the lower part and dilled with stone. a dental vibrator was used to remove the trapped air. following the stone's full setting, the dlask was opened (see figure 1), and the metal designs were taken out to create a mold for packing heat-cured acrylic resin. following a 15-minute hydraulic pressure test, the clamped dlask was placed in a water bath to cure it. it was heated to 74°c for 1.5 hours and then raised to boiling, 100°c, for 30 minutes (ada, 1999). after the curing process was dinished, the dlask was submerged in water for 15 minutes and allowed to cool gradually at room temperature for 30 minutes [2]. a tungsten steel bur put in a handpiece at low speed was then used to trim only the superdluous acrylic after the acrylic specimens had been carefully removed from the stone mold. the specimens' surfaces are left undinished [1]. figure 1. opening the dlask. immersion procedure the specimens were divided into two groups: first group: 40 specimens were distributed into the following: (control) group of distal water, zamzam water, thyme oil diluted using a 1:1 mixture of thyme essential oil (allin and polysorbate) and distilled water at a concentration of 0.5µl/ml [7], and group of mouth wash (listerine) each of these solutions contained 10 specimens, and 200 ml of the solution were immersed in closed containers [1] once a day for 8 hours to simulate overnight use [8,9]. none of the solutions was replaced during this period [10] for 30 days [11] . second group: 30 specimens were distributed into the following: artidicial saliva normal and acidic: (control) 10 specimens were immersed in distilled water,10 specimens were immersed in normal saliva and 10 specimens were immersed in acidic saliva up to 200 ml once a day for 16 hours for 30 days [11] to simulate the time of denture wearing. surface roughness test each disc-shaped specimen's surface roughness was measured to within 0.01 µm using a time group inc. hand-held roughness measurement equipment prodilometer (tr200). the analyzer's stylus traveled 0.5 mm/second across the specimen surface. the recorded data had a resolution of 0.01µm, a cut of value of 0.8mm, and a tracing length of 2.4 mm. each specimen had three readings taken, and the specimen's mean roughness (ra) was determined [2,12]. statistical analysis statistical package for social science, version 21 (spss) was used to gather and analyze the research's data. surface roughness change was summarized using descriptive statistics, and a one-way analysis of variance (anova) was conducted on surface roughness (µm) and followed by an lsd test for group comparison. a p value <0.05 was deemed statistically signidicant. results the result of surface roughness(µm) showed that all group nearly the same mean value for groups control (distil water), diluted thyme oil, mouth wash (2. 5809±.53136 µm), (2.6199±.70094 µm) and (2.8566±.37206 µm) respectively and the lower mean value of surface roughness was for zamzam water group (1.8930± .26521 µm) after immersion in 8 hours a day for one month as shown in table 1 and figure 2. to compare the mean values among groups, least signidicant difference (lsd) test was implied table 3. the test showed signidicant difference when compared zamzam water group with control group (distil water), diluted thyme oil group and mouth wash group, and the test revealed non signidicant difference between groups of diluted thyme oil and mouth wash when compared with control group (distil water) also non signidicant difference between groups of diluted thyme oil and mouth wash. the surface roughness (µm) for the second group showed nearly the same mean value for all groups control (distil water), artidicial saliva (normal), artidicial saliva (acidic) (2.5809±0.53136 µm), (2.3243±0.40270 µm) and (2.4948±0.63629 µm) respectively after immersion 16 hours a day as shown in table 4 and figure 3. statistical analysis using one-way anova test showed a statistically non signidicant difference among the mean values of the study groups as shown in table 5. discussion for the acrylic surface to be free of microorganisms, surface roughness is crucial [13]. an increase in surface roughness may make biodilm removal more challenging [14]. in this study, the duration of immersion in the denture cleanser was selected to mimic the 8-hour overnight immersion, which is a typical scenario in which dentists advise patients to take out their dentures while they are asleep and submerge themselves in solutions to relieve the underlying tissues and clean the dentures [8]. based on the results of surface roughness of this study the null hypothesis was accepted. the present study's dindings demonstrated that, except for the zamzam water group, which had greater smoothness or less surface roughness, overnight immersion produced surface roughness values that were almost identical (µm). figure 1 and table 1.the current study's dindings concur with ural c et al. and found no difference in the impact of sodium hypochlorite and commercial cleansers on surface roughness [15] agreement with salem et al., who demonstrated that there is no discernible change in the surface roughness of acrylic resin samples immersed in various denture cleaning solutions as opposed to samples immersed in distilled water [16]. the ph of listerin® is 4.11. it is reasonable to believe that the increased acidity led to the breakdown of the acrylic resin's outermost layer, which raised the roughness values. a subsurface area of the acrylic resin was revealed as a result of the material's ongoing exposure to these mouthwashes, which encouraged the loss of this outer layer [13]. the plant extract denture cleaner of choice was thyme essential oil. this was consistent with research by liu et al., which found that thyme essential oil had the lowest inhibitory concentration values and was the best antifungal and antimicrobial [17]. comparing evaluamon of surface roughness of heat cured pmma aler immersion in armficial saliva and different solumons vol 13, no 1 (2025) doi 10.5195/d3000.2025.991 http://dentistry3000.pitt.edu 3 the dittydent to other commercially available denture cleansers, sharam et al. found that it had the least impact on the surface properties of denture resins. however, when compared to the thyme essential oil denture cleanse, it signidicantly increased the surface roughness [18]. in an acrylic resin denture foundation subjected to glutaraldehyde for ten minutes, carvalho et al. observed surface pitting and the development of polymer beads [19]. although there was no signidicant difference between the groups in this study, there was a signidicant difference between the zamzam water group and other solutions. according to al-ansy et al., bottled zamzam is ozonated [20]. when ozonated water, an environmentally friendly disinfection solution, was examined for its impact on pmma's surface roughness and surface less, it was discovered to have no effect [21]. the patient's time wearing the denture is represented by the samples of heat-cured acrylic resin submerged in artidicial saliva (both normal and acidic) for 16 hours. resin's characteristics may be compromised by its susceptibility to various environmental conditions. saliva, humidity, and temperature variations are some of these variables. one of the primary issues in the dield of removable dental prosthesis has been alterations in salivary ph [22]. alfadda et al. discovered that soaking in neutral and basic ph values considerably increased the heat-cured acrylic material's surface roughness [12]. the results of this study disagree with alfadda et al. limitation it is well known that depending on the patient's dietary intake, the salivary ph value in the oral cavity continuously dluctuates between acidic and basic. the same acrylic material may therefore need to be exposed to different salivary ph levels to examine how it affects surface roughness or other mechanical and physical characteristics. along with other limitations, the current study only examined the effects of various chemical cleaners on a single type of denture base resin, and the immersion period was only one month. conclusion within the limitations and based on the results of this study, it was found that zamzam water showed less or no negative effect on surface roughness and the mouth wash listerin® showed slight increase in surface roughness but a statistically no signidicant difference when compared with control. in addition, both two types of artidicial saliva had no effect in increasing of surface roughness. author’s contribu)on sfa: methodology, writing – rmz: original draft preparation. nh: supervision. conflict of interest we have no condlicts of interest to disclose regarding this article. the opinions expressed are solely those of the authors and have not been indluenced by any dinancial or personal relationships. regulatory statement this article did not involve the use of any hazardous materials, living organisms, or any procedures that could harm the environment. there was no need to comply with any specidic regulatory laws or regulations regarding occupational health and safety or the environment. all necessary measures were taken to ensure compliance with ethical research practices and laboratory safety. references 1. jasim, s., s.m. abass. effect of alum disinfectant solutions on some properties of a heatcured acrylic resin. j res med dent sci, 2021. 9(5): p. 42-47. https://www.jrmds.in/articles/effect-ofalum-disinfectant-solutions-on-some-propertiesof-a-heatcured-acrylic-resin.pdf 2. abdallah, r.m., n.s. aref. an in vitro assessment of physicomechanical properties of heat-cured denture base resin disinfected by ozonized water. world j dent, 2020. 11(2): p. 146150. https://www.wjoud.com/ 3. jeyapalan, k., j.k. kumar, n. azhagarasan. comparative evaluation of the effect of denture cleansers on the surface topography of denture base materials: an: in-vitro: study. journal of pharmacy and bioallied sciences, 2015. 7(suppl 2): p. s548-s553. doi: 10.4103/09757406.163534 4. tawfeeq, s.a., a.m. al-khafaji. evaluation of heated cure acrylic immersed in ozonated water. maaen journal for medical sciences, 2023. 2(1): p. 2. https://maajms.com/ 5. ozyilmaz, o.y., c. akin. effect of cleansers on denture base resins’ structural properties. journal of applied biomaterials & functional materials, 2019. 17(1): p. 2280800019827797. doi: 10.1177/2280800019827797 6. panariello, b., et al. effects of short-term immersion and brushing with different denture cleansers on the roughness, hardness, and color of two types of acrylic resin. am j dent, 2015. 28(3): p. 150-6. https://pubmed.ncbi.nlm.nih.gov/26173310/ 7. namala, b.b., v. hegde. comparative evaluation of the effect of plant extract, thymus vulgaris and commercially available denture cleanser on the glexural strength and surface roughness of denture base resin. the journal of indian prosthodontic society, 2019. 19(3): p. 261265. doi: 10.4103/jips.jips_62_19 8. paranhos, h.d.f.o. et al. color stability, surface roughness and glexural strength of an acrylic resin submitted to simulated overnight immersion in denture cleansers. brazilian dental journal, 2013. 24(2): p. 152-156. doi: 10.1590/0103-6440201302153 9. tekin, s. in vitro effect of denture cleansers on the color stability of polyetheretherketone and other denture base polymers. odovtos international journal of dental sciences, 2022. 24(2): p. 47-56. https://revistas.ucr.ac.cr/index.php/odontos/arti cle/view/51947 10. pereira, c.j. et al. effect of immersion in various disinfectant solutions on the properties of a heat-cured acrylic resin. rgo-revista gaúcha de odontologia, 2019. 67: p. e20190052. doi: 10.1590/1981-863720190005220180 11. al-omari, a.w. evaluation of the effect of some denture cleansers on hardness of acrylic denture base and teeth materials. al-raeidain dental journal, 2009. 9(2): p. 273-278. https://www.iasj.net/iasj/article/210955 12. alfadda, s. et al. the effects of salivary ph on color stability and surface roughness of different denture acrylic resin materials. biosc. biotech. res. comm, 2020. 13(3). https://www.researchgate.net/publication/3445 43123 13. collares, f.m. et al. ingluence of mouthwashes on the physical properties of orthodontic acrylic resin. brazilian journal of oral sciences, 2014. 13: p. 203-208. doi: 10.20396/bjos.v13i3.8640963 14. quirynen, m., c. bollen. the ingluence of surface roughness and surface-free energy on supra-and subgingival plaque formation in man: a review of the literature. journal of clinical periodontology, 1995. 22(1): p. 1-14. doi: 10.1111/j.1600-051x.1995.tb01765.x 15. ural, c., s.f. ayse. effect of different denture cleansers on surface roughness of denture base materials. 2011. 16. salem, s., a. al-khafaji, m.t. al-khafagy. the effect of denture cleansers on surface roughness and microhardness of stained light cured denture base material. j bagh col dentistry, 2007. 19(1): p. 1-5. https://jbcd.uobaghdad.edu.iq/ 17. liu, x. et al. screening of food additives and plant extracts against candida albicans in vitro for prevention of denture stomatitis. procedia environmental sciences, 2012. 12: p. 1361-1366. doi: 10.1016/j.proenv.2012.01.421 18. sharma, p., s. garg, n.m. kalra. effect of denture cleansers on surface roughness and glexural strength of heat cure denture base resin-an in vitro study. journal of clinical and diagnostic research: jcdr, 2017. 11(8): p. zc94. doi: 10.7860/jcdr/2017/29014.10492 19. carvalho, c.f. et al. effect of disinfectant solutions on a denture base acrylic resin. acta odontológica latinoamericana, 2012. 25(3): p. 255-260. https://www.scielo.br/j/aol/a/5l5qh8vxvjky7k qjqj5j5qj/ 20. al-ansi, s.a., a.a. othman, m.a. al-tufail. bromate pollutant in ozonated bottled zamzam water from saudi arabia determined by lc/icp-ms. journal of environmental science and health, part a, 2011. 46(13): p. 1529-1532. doi: 10.1080/10934529.2011.609082 21. hamada, m., a. alam-eldein, z. shorbagy. the effect of ozonised water versus alkaline peroxide cleaners on colour stability, surface roughness and hardness of two different evaluamon of surface roughness of heat cured pmma aler immersion in armficial saliva and different solumons vol 13, no 1 (2025) doi 10.5195/d3000.2025.991 http://dentistry3000.pitt.edu 4 denture base materials. egyptian dental journal, 2022. 68(3): p. 2613-2624. http://edj-eg.com/ 22. tango, r.n. et al. the role of new removable complete dentures in stimulated salivary glow and taste perception. journal of prosthodontics, 2018. 27(4): p. 335-339. doi: 10.1111/jopr.12605 table 1. descriptive statistics of surface roughness (µm) after immersion in different solutions 8 hours a day for one month for all studied groups. groups n mean std. deviation std. error minimum maximum control (distil water) 10 2.5809 0.53136 0.16803 1.82 3.65 zam zam water 10 1.8930 0.26521 0.08387 1.60 2.24 diluted thyme oil 10 2.6199 0.70094 0.22166 1.64 3.89 mouthwash 10 2.8566 0.37206 0.11766 2.44 3.64 total 40 2.4876 0.59917 0.09474 1.60 3.89 figure 2. bar chart showing the mean value of surface roughness (µm) after immersion in different solutions 8 hours a day for all studied groups. table 2. one-way anova to compare the surface roughness (µm) after immersion in different solutions 8 hours a day between control and experimental groups. anova sum of squares df mean square f p-value between groups 5.159 3 1.720 7.002 0.001* within groups 8.842 36 0.246 total 14.001 39 0.0000 0.5000 1.0000 1.5000 2.0000 2.5000 3.0000 control (distil water) zam zam water diluted thyme oil mouthwash 2.5809 1.8930 2.6199 2.8566 su rf ac e ro ug hn es s( µm ) studied groups evaluamon of surface roughness of heat cured pmma aler immersion in armficial saliva and different solumons vol 13, no 1 (2025) doi 10.5195/d3000.2025.991 http://dentistry3000.pitt.edu 5 table 3. least signidicant difference for surface roughness(µm) after immersion in different solutions 8 hours a day to compare between all studied groups. (i) group (j) groups mean difference (i-j) p-value 95% conzidence interval lower bound upper bound control (distil water) zam zam water 0.68790* 0.004 0.2384 1.1374 diluted thyme oil -0.03900 0.861 -0.4885 0.4105 mouthwash -0.27570 0.222 -0.7252 0.1738 zam zam water diluted thyme oil -0.72690* 0.002 -1.1764 -0.2774 mouthwash -0.96360* 0.000 -1.4131 -0.5141 diluted thyme oil mouthwash -0.23670 0.293 -0.6862 0.2128 table 4. descriptive statistics of surface roughness(µm) after immersion in different solutions 16 hours a day for one month for all studied groups. figure 3. bar chart showing the mean value for surface roughness(µm) after immersion in different solutions 16 hours a day for all studied groups. 2.15 2.2 2.25 2.3 2.35 2.4 2.45 2.5 2.55 2.6 control (distil water) artificial saliva (normal) artificial saliva(acidic) 2.5809 2.3243 2.4948 su rf ac e ro ug hn es s( µm ) studied groups groups n mean std. deviation std. error minimum maximum control (distil water) 10 2.5809 0.53136 0.16803 1.82 3.65 artizicial saliva (normal) 10 2.3243 0.40270 0.12734 1.84 2.92 artizicial saliva(acidic) 10 2.4948 0.63629 0.20121 1.92 3.69 total 30 2.4667 0.52475 0.09581 1.82 3.69 evaluamon of surface roughness of heat cured pmma aler immersion in armficial saliva and different solumons vol 13, no 1 (2025) doi 10.5195/d3000.2025.991 http://dentistry3000.pitt.edu 6 table 5. one-way anova to compare the surface roughness(µm) after immersion in different solutions 16 hours a day between control and experimental groups. anova sum of squares df mean square f p-value between groups 0.341 2 0.171 0.602 0.555 within groups 7.644 27 0.283 total 7.985 29 946 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.946 http://dentistry3000.pitt.edu distribution and angulation pattern of impacted mandibular third molars iman ali khalil al-obaidi ashur university, baghdad, iraq abstract objec2ve: the objecdve of this study was to determine the distribudon of impacted mandibular third molars on digital panoramic radiographs, based on sex of the padent, side, level of impacdon, reladonship with the mandibular ramus, and anguladon parern of impacdon. material and methods: this study analysed 98 people aged 18 and above with 153 impacted mandibular third molars who were treated at the department of oral and maxillofacial radiology at ashur university's college of dendstry. padents were categorised by sex, right or lez side, depth of impacdon, reladonship with the mandibular ramus, and anguladon parerns. results: the only significant difference found was related to the lez side impacdon depth with females having more level c impacdons than males. conclusion: the distribudon of impacted mandibular third molars were mostly mesioangular, followed by horizontal anguladon, impacdon level c, and class ii. open access cita%on: al-obaidi iak. (2025) distribu%on and angula%on pa>ern of impacted mandibular third molars. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.946 received: may 22, 2025 accepted: june 26, 2025 published: august 12, 2025 copyright: ©2025 al-obaidi. this is an open access ar%cle licensed under a crea%ve commons a>ribu%on work 4.0 united states license. email: iman.ali@au.edu.iq introduc)on dental impacdon refers to a tooth that has not fully or pardally erupted within the jawbone or under gum dssue. dental impacdon can occur in all teeth, but the most common teeth of impacdons are the third molars [1]. the third molar impacdons account for 98% of all types of impacted teeth [2]. the impacdon rate varies between different geographics from 18.97% to 30.8% within the gulf region but 68.6% in east asia [3,4]. molar impacdons have many causes, such as insufficient skeletal growth, mucosal thickness over the growing tooth, macrodonda, lack of space or systemic condidons such as down’s syndrome. there are studies that show variadon based on diet as there was differences between the mandibular jaw size between mediaeval to post-mediaeval peoples [5]. this occurred due to biomechanical forces, which sdmulated craniofacial growth and development. in addidon, there is a variadon in erupdon and posidon of the third molar, which can be influenced by ethnicity, masdcatory effecdveness, and inherited factors [6]. impacdon complicadons include crowding, caries, pericoronids, resorpdon of adjacent tooth roots, face pain, temporomandibular joint dysfuncdon, and the most prevalent condidon, dendgerous cyst or tumour [7]. life-threatening is the transformadon of the cysdc wall into squamous cell carcinoma [1]. the early diagnosis of impacted teeth can avoid future malocclusions and lesions [8]. idendfying and managing these dental anomalies at an early stage can avert complicadons. regular dental checkups, including radiographic evaluadons, play a pivotal role in the dmely detecdon and treatment of impacted teeth. panoramic radiography classificadon is an essendal component of treatment planning for the operadon of removal of the mandibular third molar, which is sdll a comprehensive clinical and radiographic evaluadon. the padent should have a complete case history that includes detailed systemic and local evaluadon. age (increasing difficulty for extracdon with age) and sex (incidence of increase of sclerodc bone in males with age/consider the incidence of post-menopausal osteoporosis in females) should also be noted [9]. panoramic radiograph is based on some sort of classificadon and in this study we established the classificadon on pell, gregory, and winter, are crucial for determining third molar posidon, depth, and bone covering, as well as assessing surgical treatments' challenges [6,10,11]. in this study, we focused only on the lower third mandibular molar and udlized different classificadon to organize different types of impacdons of the molars. these impacdons were then compared between different sexes. material and methods a cross-secdonal observadonal study was undertaken at ashur university's dental distribudon and anguladon parern of impacted mandibular third molars vol 13, no 1 (2025) doi 10.5195/d3000.2025.946 http://dentistry3000.pitt.edu 2 radiology clinics department of oral and maxillofacial radiology in baghdad, iraq. we reviewed all panoramic radiographs at ashur university's dental radiology clinics from november 2023 to november 2024. a total of 153 impacted mandibular third molars were evaluated in 98 individuals who were 18 years or above. these data were further separated into two groups based on sex. the examinadon of the lower third mandibular molar was performed using digital orthopantomography (panoramic radiography). these images were assessed using pell, gregory, and winter for evaluadng the depth, reladonship to ramus and anguladon of the teeth. during the study, all padents were handled by the same operator, and panoramic radiographs were obtained using the same machine. digital panoramic exposures were achieved using convendonal techniques. two examiners examined all panoramic radiographs and conducted analyses. study design. in this study we analysed all panoramic radiographs at ashur university's dental radiology clinics from november 2023 to november 2024 using data collected during a one-year period. radiographs from normal dental examinadons were randomly obtained from the department of oral and maxillofacial radiology archives. the study excluded padents with incomplete root formadon or deformed mandibular third molar roots, as well as those without mandibular second molars. this study only included high-quality radiographs with clear reproducdon of teeth and no superimposidon (example in figure 1). padents were divided into groups based on their sex, right or lez side, depth of impacdon, reladonship with the mandibular ramus, and anguladon parerns. digital panoramic radiographs were taken using the myray hyperion x9 pro machine at 70 kvp, 7ma, and 12.7 seconds exposure duradon at our department of oral and maxillofacial radiology. two examiners viewed radiographs with the irys sozware version 14.0.1. the collected data were analysed using stadcal spss sozware version 29.0.1.1. study parameters. the following criteria were assessed in this study (figures 2 and 3): 1. the anguladon of the impacted third molar teeth was categorized using winter's classificadon [6,12]. 2.the pell and gregory classificadon was used to define the depth of the impacted third molar teeth [11,13]. 3.the pell and gregory categorizadon was used to define the reladonship between an impacted third molar and ramus [11,13]. winter’s classificadon classified third molar impacdons as mesioangular, distoangular, horizontal, verdcal, buccal, and others [6]. we used another classificadon for the depth and reladon to ramus neck of the jaw. pell and gregory's a, b, and c radngs are based on their depth and reladon with the mandible's ramus [11]. the following definidons were used in this study for impacdon and anguladon of impacdon. a tooth was termed impacted if its occlusal plane was below that of the adjacent tooth and bone level. a pardally or semi-impacted tooth is one that has pardally erupted but sdll in the line of occlusion. to assess the inclinadon of an impacted mandibular third molar, the anguladon between the longer axis and the neighbouring second molar teeth was measured using winter’s classificadon. impac3on: the third molar must not have a funcdonal occlusion when the root formadon is finished to be classified as impacted. depth of impac3on: the pell and gregory classificadon categorizes the cementoenamel juncdon (cej) of the third molar with respect to bone level: level a not buried in bone; level b pardally buried in bone if any part of the cej was lower than bone level; and level c completely buried in. rela3onship with the mandibular ramus: the pell and gregory classificadon categorizes the distal surface of the third molar crown in reladon to the anterior border of the ascending ramus into the following posidons: class i is anterior to the anterior border, class ii is half of the crown covered by the anterior border, and class iii is the endre crown covered by the anterior border. angulation of impaction: winter's classification categorizes the angle between the longitudinal axis of the second and third molars, measured with an orthodontic protractor. vertical impaction ranges from 10 to -10, whereas mesioangular impaction ranges from 11 to -79, horizontal impaction ranges from 80 to 100, distoangular impaction ranges from -11 to -79, others range from 111 to -80, and buccolingual impaction occurs when the crown and roots are superimposed. results unilateral vs. bilateral table1 presents the distribudon of unilateral and bilateral mandibular third molar impacdons based on sex. among the 98 cases, 65 were males and 33 were females. unilateral and bilateral impacdons were more frequent in males than females, but that difference was not stadsdcally significant (p = 0.84). the depth of impacdon was classified using the pell & gregory scale. the results in table 2 indicate that level c impacdons (deepest) were the most common in both sexes with approximately half of the cases. for the lez side depth, level b impacdons were most common in males (38.5%), while level c impacdons were predominant in females (60.6%), indicadng that females tend to experience deeper impacdons compared to males (p=0.048). the classification of impacted molars based on their relationship with the mandibular ramus was evaluated as seen in table 3. most impactions fell under class ii (69.2% in males, 66.7% in females), indicating that the impacted molars were partially covered by the anterior border of the ramus. winter’s classificadon was applied to evaluate the anguladon of impacted third molars. the most common anguladon type observed was mesioangular impacdon, occurring in 55.3% of males and 44.7% of females as seen in table 4. horizontal impacdons were notably more frequent in males (82.1%) compared to females (17.9%), although not stadsdcally significant (p=0.168). when analysing anguladon parerns on the lez side, mesioangular impacdons were again the most frequent (32.3% in males, 39.4% in females). interesdngly, horizontal impacdons were more frequent in males (75%) than females (25%), whereas distoangular impacdons were only seen in females (9.1%) (p=0.14). discussion there was no significant difference between males and females when it came to unilateral and bilateral impacdon. the results for right sided depth, ramus reladonship and anguladon classificadon showed that there was no significant variadon between males and females. the lez side results were also not significant different when it came to the categorizadon of ramus and anguladon of the third molar, but there was a significant variadon when it came to the lez sided depth classificadon. the lack of significance is likely due to small sample size. other factors that may impact the actual development of teeth in humans include genedc variadon, aging, developmental disorders, and dietary factors. it is to be noted that there is a sexual dimorphism in tooth size, which has genedc inheritance, testosterone, and evoludonary selecdon, and hormonal secredon influences. growth hormone, testosterone, promotes growth of teeth size whereas estrogen would limit the jaw size growth and teeth size [14-16]. many genes play a role in crown size [14]. as an example, fgf10 and fgf13 were associated to molar size discrepancies [16]. there is a decrease in jaw size with age between 18 and 40 years, which may relate to a decrease in testosterone and the impact of growth hormone, which makes male jaw to be bigger in size comparison to the female jaw size [17]. there is distribudon and anguladon parern of impacted mandibular third molars vol 13, no 1 (2025) doi 10.5195/d3000.2025.946 http://dentistry3000.pitt.edu 3 also the implicadon of dietary habit in final jaw sizes [18,19]. lack of differences in the impaction of third molars between males and females agrees with previous data [20,21] . bilateral impactions are more common than unilateral ones and these differences are independent from sex [22,23]. conclusions impacdon of the mandibular third molars is common among iraqis. it has been nodced that males had more impacted molars compared to females, but the difference was not significant. the most common parern for impacted mandibular third molars was mesioangular, followed by horizontal anguladon, impacdon level c, and class ii. males experienced more unilateral impacdons and slightly more bilateral impacdons than females. comparing our data to various populadons revealed similarides in some characterisdcs, such as anguladon and connecdon to ramus, but overall results vary. this study has been characterized by a high frequency of lower third molar impacdon, pardcularly in males. this high frequency signifies that an increasing number of individuals are retaining the impacted third molars. references 1. carter k, worthington s. predictors of third molar impaction: a systematic review and meta-analysis. j dent res. 2016;95(3):26776.https://doi.org/10.1177/0022034515615857 2. santosh p. impacted mandibular third molars: review of literature and a proposal of a combined clinical and radiological classivication. ann med health sci res. 2015;5(4):22934.https://doi.org/10.4103/2141-9248.160177 3. quek sl, tay ck, tay kh, toh sl, lim kc. pattern of third molar impaction in a singapore chinese population: a retrospective radiographic survey. int j oral maxillofac surg. 2003;32(5):54852 4. hassan ah. pattern of third molar impaction in a saudi population. clin cosmet investig dent. 2010;2:10913.https://doi.org/10.2147/cciden.s12394 5. rando c, hillson s, antoine d. changes in mandibular dimensions during the mediaeval to post-mediaeval transition in london: a possible response to decreased masticatory load. arch oral biol. 2014;59(1):7381.https://doi.org/10.1016/j.archoralbio.2013.1 0.001 6. winter g. principles of exodontias as applied to the impacted third molar 1st ed. st. louis american medical books 1926. contemporary oral and maxillofacial sugery. 1993;2 7. fayad jb, levy jc, yazbeck c, cavezian r, cabanis e-a. eruption of third molars: relationship to inclination of adjacent molars. american journal of orthodontics and dentofacial orthopedics. 2004;125(2):200-2 8. da silva menezes cg, sartoretto sc, louro rs, de moraes jb, moraschini v. prevalence of impacted teeth: a radiographical retrospective rio de janeiro population-based study. j maxillofac oral surg. 2024;23(1):7580.https://doi.org/10.1007/s12663-023-020213 9. bhargava d. clinical and radiographic assessment for impacted mandibular third molars. transalveolar extraction of the mandibular third molars: crc press; 2022. p. 39-49. 10. khouri c, aoun g, khouri c, saade m, salameh z, berberi a. evaluation of third molar impaction distribution and patterns in a sample of lebanese population. j maxillofac oral surg. 2022;21(2):599607.https://doi.org/10.1007/s12663-02001415-x 11. pell gj. impacted mandibular third molars, classivication and modivied technique for removal. dental digest. 1933;39:330-8 12. lei y, chen x, wang y, tang r, zhang b. a lightweight knowledge-distillation-based model for the detection and classivication of impacted mandibular third molars. applied sciences. 2023;13(17).https://doi.org/10.3390/app13179 970 13. eshghpour m, nezadi a, moradi a, shamsabadi rm, rezaei nm, nejat a. pattern of mandibular third molar impaction: a crosssectional study in northeast of iran. niger j clin pract. 2014;17(6):6737.https://doi.org/10.4103/1119-3077.144376 14. dempsey pj, townsend gc. genetic and environmental contributions to variation in human tooth size. heredity (edinb). 2001;86(pt 6):685-93.https://doi.org/10.1046/j.13652540.2001.00878.x 15. christensen mm, hallikas o, das roy r, vaananen v, stenberg oe, hakkinen tj, et al. the developmental basis for scaling of mammalian tooth size. proc natl acad sci u s a. 2023;120(25):e2300374120.https://doi.org/10. 1073/pnas.2300374120 16. maranon-vasquez ga, vieira ar, dos santos lv, cunha as, weiss sg, araujo mts, et al. fgf10 and fgf13 genetic variation and tooth-size discrepancies. angle orthod. 2021;91(3):35662.https://doi.org/10.2319/060920-531.1 17. tsiopas n, nilner m, bondemark l, bjerklin k. a 40 years follow-up of dental arch dimensions and incisor irregularity in adults. eur j orthod. 2013;35(2):2305.https://doi.org/10.1093/ejo/cjr121 18. kahn s, ehrlich p, feldman m, sapolsky r, wong s. the jaw epidemic: recognition, origins, cures, and prevention. bioscience. 2020;70(9):75971.https://doi.org/10.1093/biosci/biaa073 19. menendez l, bernal v, novellino p, perez si. effect of bite force and diet composition on craniofacial diversivication of southern south american human populations. am j phys anthropol. 2014;155(1):11427.https://doi.org/10.1002/ajpa.22560 20. dachi sf, howell fv. a survey of 3,874 routine full-mouth radiographs: ii. a study of impacted teeth. oral surgery, oral medicine, oral pathology. 1961;14(10):1165-9 21. al-delaimi tn, abood sw, khalil aa. the evaluation of impacted third molars using a panoramic radiograph. al-anbar med j. 2010;8(1):26-33 22. shahbaz s, khan m. evaluation of mandibular third molar impaction distribution on opg: a digital radiographic study. international journal of applied dental sciences. 2017;3:393-6 23. al-anqudi sm, al-sudairy s, al-hosni a, al-maniri a. prevalence and pattern of third molar impaction: a retrospective study of radiographs in oman. sultan qaboos univ med j. 2014;14(3):e388-92. distribudon and anguladon parern of impacted mandibular third molars vol 13, no 1 (2025) doi 10.5195/d3000.2025.946 http://dentistry3000.pitt.edu 4 figure 1. an orthopantomogram showing an impacted mandibular third molar. figure 2. the impacdon depth, ramus reladonship, and anguladon classificadon of mandibular third molars take from parern of mandibular third molar impacdon: a cross-secdonal study in northeast of iran” paper wriren by eshghpour and colleagues. distribudon and anguladon parern of impacted mandibular third molars vol 13, no 1 (2025) doi 10.5195/d3000.2025.946 http://dentistry3000.pitt.edu 5 figure 3. winter’s classificadon taken from ardcle “a lightweight knowledge-disdlladon-based model for the detecdon and classificadon of impacted mandibular third molars” by lei and colleagues. table 1. comparison between the unilateral/bilateral impacdon by sex. side total unilateral bilateral sex male 29 (67.4%) 36 (65.5%) 65 female 14 (32.6%) 19 (32.6%) 33 total 43 (100%) 55 (100%) 98 x2 = 0.043, p = 0.836 distribudon and anguladon parern of impacted mandibular third molars vol 13, no 1 (2025) doi 10.5195/d3000.2025.946 http://dentistry3000.pitt.edu 6 table 2. comparison of the impacdon depth according to the pell & gregory category by sex. sex male female count count right side impacdon depth n/a 11 (16.9%) 7 (21.2%) a 0 (0%) 0 (0%) b 19 (29.2%) 8 (24.2%) c 35 (53.8%) 18 (54.5%) x2 = 0.419 (p = 0.811) lez side impacdon depth n/a 18 (27.7%) 7 (21.2%) a 1 (1.5%) 0 (0.0%) b 25 (38.5%) 6 (18.2%) c 21 (32.3%) 20 (60.6%) x2 = 7.903 (p =0.048) table 3. comparison of the impacdon ramus reladonship according to the pell & gregory category by sex. n/a means the absence of the impacdon related to the ramus. sex male female count count right side ramus reladonship n/a 11 (16.9%) 7 (21.2%) i 9 (13.8%) 3 (9.1%) ii 45 (69.2%) 22 (66.7%) iii 0 (0%) 1(3%) x2 = 2.614 (p =0.455) lez side ramus reladonship n/a 18 (27.7%) 7 (21.2%) i 11 (16.9%) 5 (15.2%) ii 35 (53.8%) 21 (63.6%) iii 1 (1.5%) 0 (0%) x2 = 1.277 (p = 0.735) distribudon and anguladon parern of impacted mandibular third molars vol 13, no 1 (2025) doi 10.5195/d3000.2025.946 http://dentistry3000.pitt.edu 7 table 4. comparison of the impacdon anguladon according to the winter’s classificadon by sex. sex male female count count right side anguladon n/a 11 (16.9%) 7 (21.2%) verdcal 8 (12.3%) 4 (12.1%) horizontal 23 (35.4%) 5 (15.2%) distoangular 0 (0%) 0 (0%) mesioangular 21 (32.3%) 17 (51.5%) buccolingual 2 (3.1%) 0 (0%) other 0 (0%) 0 (0%) total 65 33 x2 = 6.454 (p = 0.168) lez side anguladon n/a 18 (27.7%) 7 (21.2%) verdcal 4 (6.2%) 3 (9.1%) horizontal 21 (32.3%) 7 (21.2%) distoangular 0 (0%) 3 (9.1%) mesioangular 21 (32.3%) 13 (39.4%) buccolingual 1(1.5%) 0 (0%) other 0 (0%) 0 (0%) x2 = 8.301 (p = 0.14) bacterial community in dental biofilms linked to periodontal disease: an intra-subject metagenomics study vol 12 no 1 (2024) doi 10.5195/d3000.2024.465 http://dentistry3000.pitt.edu bacterial community in dental biofilms linked to periodontal disease: an intra-subject metagenomics study maheen tariq, wan nazatul shima shahidan, raja azman awang school of dental sciences, health campus, universiti sains malaysia, kelantan, malaysia. abstract objectives: to study the intra-subject subgingival plaque microbiota composition of gingivitis and periodontitis sites in periodontitis patients using 16s rrna metagenomics. methods: samples of subgingival plaque were collected from 16 periodontitis participants enrolled on the study. the extracted dna from dental plaque was sent to the illumina laboratory for 16s rrna metagenomics. the v₃ and v₄ region of 16s rna gene were amplified and sequenced using illumina technology. after quality filtering, 313480 sequences were obtained and arranged in otu based on the 97% threshold. the reads were assigned to species. results: the alpha diversity analyses revealed that periodontitis sites had more diverse and rich bacterial communities than gingivitis sites. however, the pcoa analysis (beta diversity) did not reveal any clustering of the bacterial community in gingivitis and periodontitis. taxonomic analysis verified the presence of 56 known species. however, there was no apparent pattern in the bacterial community between gingivitis and periodontitis sites. yet, a slight distinction was observed. species like p. intermedia, r. dentocariosa and p. endodontalis were more abundant in gingivitis sites compared to periodontitis sites, while some other species like v. dispar, c. ochracea and a. segnis were more abundant in periodontitis sites. conclusion: this study supports the fundamental idea that individual bacterial species are not responsible for the advancement of gingivitis to periodontitis but rather the abundance of bacteria in the bacterial community. keywords: intra-subject, periodontitis, gingivitis, subgingival plaque, 16s rna metagenomics. citation: tariq m, et al. (2024) bacterial community in dental biofilms linked to periodontal disease: an intrasubject metagenomics study dentistry 3000. 1:a001 doi:10.5195/d3000.2024.465 received: march 5, 2023 accepted: july 14, 2023 published: august 28, 2024 copyright: ©2024 tariq m, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: rjazman@usm.my introduction gingivitis and periodontitis are the two most common forms of periodontal diseases linked to bacterial dental biofilm. gingivitis, the reversible form, causes bleeding and inflamed gums. if gingivitis is left untreated, it can progress to periodontitis, an irreversible condition [1]. approximately 775 bacterial species have been found in the oral cavity; however, only about 57% are culturable [2]. the possibility exists that periodontal tissue deterioration may be caused partly by bacteria that have never been cultivated or studied before. thus, researches addressing the entire population of dental biofilm microorganisms are necessary. recent years have seen the introduction of high throughput sequencing technologies such as 16s rrna metagenomics, which are timeefficient, cost-effective, and can also reveal information about nonculturable bacteria. this cutting-edge technology may aid in the study of how biofilm microorganisms contribute to oral disease, especially periodontal disease. numerous studies have been conducted using 16s rna metagenomics analysis to gain a better understanding of the oral microbiota [3,4]. it is well-known that the dental biofilm microbiota community varies between periodontally healthy and periodontitis-afflicted individuals [5], but how these bacterial populations induce periodontal disease is unknown. to further understand the role of microbiota in periodontal disease, numerous metagenomic analyses of the biofilm microbiota of healthy and periodontitis-affected individuals were designed [4,6]. however, the findings of these approaches could be questioned because the composition of the oral http://dentistry3000.pitt.edu/ bacterial community in dental biofilms linked to periodontal disease: an intra-subject metagenomics study vol 12 no 1 (2024) doi 10.5195/d3000.2024.465 http://dentistry3000.pitt.edu microbiota is influenced by a variety of individual factors, including lifestyle, diet, oral care habits, oral disease or systemic disease, host immune response, genetic susceptibility, and location in the oral cavity [2]. to eliminate all of these confounding factors, internal comparisons within subjects should be conducted. to date, only a few metagenomics studies have compared the biofilm microbial community within subjects, either among healthy sites [2] or between healthy and periodontitis-affected sites [4,7]. we didn't come across any studies that compared gingivitis and periodontitis-affected sites. thus, the objective of this study was to evaluate the microbial communities of gingivitis and periodontitis sites within the same periodontitis subjects using 16s rna metagenomics, where microbial community diversity, clustering, composition, and relative abundance were compared. materials and methods subject selection this study recruited 16 periodontitis patients (9 males and 7 females) from hospital universiti sains malaysia. the ages of the subjects ranged from 42 to 62 years. inclusion was determined based on the following criteria: (1) clinical probing depth greater than 5mm in 50% of teeth; (2) lost more than 30% of alveolar bone; (3) had more than 20 teeth in dentition; (4) had not received periodontal therapy in the previous 6 months; (5) had no systemic disease; (6) had not received antibiotic treatment in the previous 3 months; and (7) was neither pregnant nor nursing. ethical approval was granted by universiti sains malaysia (ethical ref number: usm/jepem/15100370). the written informed consent was taken from all participants. the research had been carried out in accordance with the declaration of helsinki. a single examiner performed a fullmouth periodontal examination on six tooth surfaces mesiobuccal, buccal, distobuccal, distolingual, lingual, and mesiolingual. the periodontal pocket depth (ppd) and bleeding on probing (bop) were measured using the university of north carolina probe (unc-15, hufriedy, chicago, il, usa). the ppd was determined by measuring the distance between the free gingival margin and the periodontal pocket base. during probing, sites were examined for the presence or absence of bleeding to quantify bleeding on probing (bop). for each subject, three teeth with pocket depth (pd) on gingivitis sites (pd ≤ 3 mm) and three with periodontitis sites (pd ≥ 5 mm) were selected for subgingival plaque sample collection. subgingival plaque sample collection teeth and gingiva were air-dried before being separated with cotton rolls. a gracey curette was initially used to remove a supragingival plaque (hu-friedy, usa). subgingival plaque was then collected using a new sterile curette by inserting it parallel to the long axis of the tooth into the deepest part of the pocket and then moving coronally, scraping along the root surface. subgingival plaque was subsequently dispersed in a 1.5 ml microcentrifuge tube containing 0.5 ml tris-edta buffer solution (sigma aldrich, germany). for the gingivitis and periodontitis site of each subject, dental plaque from three teeth was pooled and stored in a single centrifuge at -80° c until needed. dna extraction the dna was extracted from dental plaque samples using the epicentre masterpure dna purification kit (cambio, irvine, uk), following the manufacturer’s instructions. the concentration of extracted dna was quantified using a nanodrop spectrophotometer (thermo scientific-ge, usa), and only dna with a 260/280 ratio between 1.8 and 2.0 was included in the study. gel electrophoresis was used to further verify the dna’s integrity. sixteen gingivitis and fourteen periodontitis samples yielded dna in sufficient quantity and quality were selected for 16s metagenomic analysis using illumina technology. dna plaque samples were kept at -80° c before being sent to the illumina miseq lab (malaysian genomic institute, kajang selangor, malaysia) for illumina sequencing. http://dentistry3000.pitt.edu/ bacterial community in dental biofilms linked to periodontal disease: an intra-subject metagenomics study vol 12 no 1 (2024) doi 10.5195/d3000.2024.465 http://dentistry3000.pitt.edu pcr amplification and sequencing analysis we used the following primers to amplify the v3 and v4 region of 16s rrna gene [8]: forward primer: 5'tcgtcggcagcgtcagatgtgtataag agacagcctacgggnggcwgcag reverse primer: 5'gtctcgtgggctcggagatgtgtataa gagacaggactachvgggtatctaatc c pcr reaction was performed using readymix pcr kit, kapa hifi hotstart dna polymerase (roche, switzerland). briefly, a 25μl pcr reaction was prepared by adding 2.5μl of dna sample, 5μl of each forward and reverse primers, and 12.5μl 2x kapa ready mix (roche, switzerland). thermal cycling was set to consist of an initial denaturation at 95 °c for 3 min, followed by 25 cycles of denaturation at 95° c for 30 sec, annealing at 55 °c for 30 sec and extension at 72 °c for 30 sec. the reaction was completed after a 5minute extension at 72 ºc. the pcr amplicons were then purified using ampure xp beads (beckman coulter, usa). nextera xt index kit (illumina, san diego, usa) was used to add specific oligonucleotides to the amplicons, and then ampure xp beads were used to purify the amplicons (beckman coulter, usa). the quality and size of the library were assessed using agilent 2100 bioanalyzer (agilent technologies, usa). the library sequencing was performed on a sequencing platform (miseq system, illumina, usa) using a miseq v3 600 cycles kit (illumina, usa). following sequencing, image analysis and base calling were performed using miseq reporter software (illumina, usa), with raw sequencing data recorded in fastq format. bioinformatic analysis the processing for 16s sequence data was performed on the bioinformatics pipeline, quantitative insights into microbial ecology (qiime) software v1.9.1[9]. the raw metagenomic sequence read adapters were removed using pairedend trimmer peat v1.2.[10]. the forward and reverse reads were merged with paired read merger bbmerge v7.3 [11]. the sequence shorter than 100 bp, with phred score lower than 20 were removed by fastx-toolkit v 0.0.13. the remaining sequences were used for de novo binning of operational taxonomic units (otus) based on sequence similarity followed by selection of representative otus per bin. the tags were clustered into otus using qiime at 97% sequence similarity. the 16s metagenomics workflow classified organisms based on v3 and v4 amplicons using 16s rrna data database. the taxonomic classification was assigned using the greengene database version 13.8 [12]. alpha and beta diversity were computed on the basis of a previously constructed otu table by qiime. bacterial species with a prevalence of less than 0.1% were excluded [13]. bacterial species were ranked in groups to obtain relative abundance. the percentage of total abundance in each group was estimated by combining the abundance of individual components in each taxa. only genera and species were analyzed in this paper. statistical analysis the shapiro-wilk test was used to analyse the normality of the data. the independent sample t-test or mannwhitney u test was used to identify the statistically significant differences between the patient’s clinical variables, age, α-diversity, and taxonomic ranks. all these statistical tests were performed using ibm spss version 25. an otu table was used for alpha diversity indices calculation, where chao1 was used as the richness estimator, and shannon was used to representing evenness and diversity. beta diversity was determined by computing distance matrices, which were represented by weighted unifrac distances that consider both abundance and phylogenetic distances among taxa [14]. it was used to construct the hierarchal clustering, which was calculated on qiime and constructed on past software. the principal coordinates analysis (pcoa) was performed on the otu level to evaluate the similarity of microbial community structure on http://dentistry3000.pitt.edu/ bacterial community in dental biofilms linked to periodontal disease: an intra-subject metagenomics study vol 12 no 1 (2024) doi 10.5195/d3000.2024.465 http://dentistry3000.pitt.edu emperor software. the nonparametric permutational multivariate analysis of variance (permanova) was used to measure the multivariate community-level difference between the gingivitis and periodontitis groups [15]. this analysis was performed using qiime software v1.9.1. results demographic and periodontal parameters of study subjects the study included sixteen periodontitis patients, nine males and seven females. the average age of the subjects was 46.3 years, ranging from 31 to 62 years. the median and interquartile range for subjects' ppd (mm) and bop (%) are 5.6 (0.9mm) and 85 (10%), respectively. the periodontal parameters of the teeth involved in the collection of dental plaque samples are shown in table 1. table 1. periodontal parameters of teeth selected for dental plaque sample collection gingivitis (n = 96) periodontitis (n = 96) p value* periodontal parameters ppd (mm) 2.5 (0.7) 5.6 (0.8) <0.001 bop (%) 21 (5) 80 (19) <0.001 the parameters are presented as median and interquartile range (iqr). the significance level was set at p = 0.05, and n = 96. * = mann-whitney u test. 16s rrna metagenomics this study used 16s rna metagenomics to analyze the microbiome within the gingivitis and periodontitis sites of the subjects. each sample from gingivitis and periodontitis sites yielded 50k sequences in total. after merging, filtering, and deleting chimaeras, a total of 313480 sequences were retrieved. alpha diversity rarefaction curves were generated for the chao1 index (species richness) and the shannon index (species evenness), with their values plotting to a plateau, indicating that sufficient sequencing was performed to reveal an excellent representation of microbial community diversity and richness (figure 1). the chao1 and shannon index values were higher in the periodontitis group than in the gingivitis group, indicating a more diverse and balanced microbial community in the periodontitis group. however, these differences were not statistically significant. beta diversity the principal coordinates analysis (pcoa) revealed that the microbial compositions of gingivitis and periodontitis sites do not cluster distinctively (figure 2). however, a closer examination of the gingivitis and periodontitis samples using a hierarchical clustering plot indicated a taxonomic cluster link (figure 3). not only is the microbial composition of gingivitis and periodontitis samples clustered, but some gingivitis and periodontitis samples have notable similarities. however, in hierarchical clustering, the majority of gingivitis and periodontitis samples from single subjects exhibit a distance in similarity. the statistical analysis permanova, using weighted unifrac distances, revealed that the difference between the abundance of otu in the gingivitis and periodontitis group was not statistically significant (permutations = 999, p-value = 0.219). http://dentistry3000.pitt.edu/ bacterial community in dental biofilms linked to periodontal disease: an intra-subject metagenomics study vol 12 no 1 (2024) doi 10.5195/d3000.2024.465 http://dentistry3000.pitt.edu figure 1. rarefaction curve of chao1 (a) and shannon (b) of gingivitis and periodontitis samples. 10 31 39 3 62 77 6 94 15 9 12 55 42 15 69 25 18 83 08 21 96 91 25 10 74 28 24 57 31 38 40 0 500 1000 1500 2000 2500 sequences per sample r ar ef ra ct io n m ea su re : c ha o 1 gingivitis periodontitis a 10 31 39 3 62 77 6 94 15 9 12 55 42 15 69 25 18 83 08 21 96 91 25 10 74 28 24 57 31 38 40 0 1 2 3 4 5 6 7 8 sequences per sample r ar ef ra ct io n m ea su re : s ha nn on gingivitis periodontitis b figure 2. a 3d pcoa plot based on the weighted unifrac distance in gingivitis and periodontitis samples. the percentage of variance along each axis is indicated in brackets to illustrate beta diversity. periodontitis samples are indicated by blue dots, while gingivitis samples are indicated by red dots. pc2 (23.09 %) pc3 (13.29 %) pc1 (27.59 %) taxa abundance analysis the taxonomic community analysis revealed the presence of 287 known species in gingivitis and periodontitis sites. among them, 56 species were taxonomically recognised. after excluding those with less than 0.1 % relative abundances, only 26 species were included in the analysis. top abundant species prevotella intermedia (p. intermedia), veillonella dispar (v. dispar), rothia dentocariosa (r. dentocariosa), and porphyromonas endodontalis (p. endodontalis) were the most abundant species in gingivitis and periodontitis sites. among these, v. dispar were present in the greatest abundance (28.4%) in periodontitis sites, followed by p. melaninogenica (13.95) and p. intermedia (12.9%). p. intermedia were the most prevalent (22.2%) in gingivitis sites, followed by v. dispar (16.2%) and r. dentocariosa (15.3%). figure 4 compares the abundance of 26 bacterial species in gingivitis and periodontitis sites. comparing gingivitis and periodontitis sites reveals a change in bacterial species abundance, either an increase or a decrease. there was, however, no statistically significant difference. http://dentistry3000.pitt.edu/ bacterial community in dental biofilms linked to periodontal disease: an intra-subject metagenomics study vol 12 no 1 (2024) doi 10.5195/d3000.2024.465 http://dentistry3000.pitt.edu figure 3. weighed unifrac-based hierarchical clustering of gingivitis (g) (n = 16) and periodontitis (p) (n = 14) samples. distance = weighted unifrac value. discussion alpha diversity the values of alpha diversity (richness estimator chao1 and shannon) revealed a trend of increase in periodontitis samples compared to gingivitis samples, although the difference was not statistically significant. this is consistent with a previous study which found no statistically significant difference in shannon values between healthy, gingivitis, and periodontitis sites in the same individual [4]. even in the intersubject study, the species evenness between healthy and diseased periodontal sites was reported to be not significantly different [16–18]. however, previous studies also revealed contradictory results, showing that sites with periodontitis had statistically greater microbial diversity and evenness than healthy sites [3]. another study reported significantly higher evenness in the periodontitis group than in the healthy group. however bacterial community diversity was not significant [19]. high bacterial community diversity in periodontal disease sites may result from a nutritionally richer environment or a diminished immunological competence [3]. although alpha diversity can be a good predictor of disease-associated dysbiosis, which could be the cause of periodontal disease [20], our findings do not appear to support the role of bacterial http://dentistry3000.pitt.edu/ bacterial community in dental biofilms linked to periodontal disease: an intra-subject metagenomics study vol 12 no 1 (2024) doi 10.5195/d3000.2024.465 http://dentistry3000.pitt.edu 0 5 10 15 20 veillonella parvula prevotella pallens prevotella nanceinsis lactobacillus ruminis anoxybacillus kestanbolensis actinobacillus parahaemolyticus faecalibacterium prausnitzii bacillus thermoamylovorans haemophilus influenzae haemophilus parainfluenzae streptococcus anginosus prevotella copri rothia aeria treponema amylovorum selenomonas noxia prevotella nigrescens prevotella tannerae treponema socranskii prevotella melaninogenica rothia mucilaginosa aggregatibacter segnis capnocytophaga ochracea porphyromonas endodontalis rothia dentocariosa veillonella dispar prevotella intermedia 20 25 30 35 40 relative abundance (%) sp ec ie s gingivitis periodontitis community diversity in the progression of gingivitis to periodontitis. beta diversity based on weighted unifrac distances, the pcoa graph presented with no distinctive cluster of gingivitis and periodontitis samples. the hierarchical clustering graph showed that certain samples from a single subject exhibited similarity, while most samples exhibited dissimilarity. there was no study that investigated the beta diversity in gingivitis and periodontitis for us to compare. however, a study showed no significant difference in the weighted unifrac distance between healthy and periodontitis salivary samples, and there was no apparent clustering between the two groups [21]. another study also found no significant difference in beta diversity but did find apparent clustering in healthy and periodontitis samples [22]. there was also no significant difference in beta diversity or apparent clustering between gingivitis subjects with different oral hygiene statuses [23]. in contrast, another researcher reported apparent clustering of healthy and periodontitis samples in pcoa plots from the same individual [17]. studies also found a significant difference in the weighted unifrac distance of bacterial species when healthy and periodontitis subjects were compared [16,24]. a well-controlled study is required to answer the question of how bacterial plaque distribution is associated with periodontal health conditions. figure 4. mean relative abundance of bacteria species with standard error of the mean (sem) in gingivitis and periodontitis samples. only recognised species with a relative abundance greater than 0.1% were included. mann-whitney test was used for statistical analysis, and the significance level was set at p = 0.05. no statistically significant were found between the two groups. http://dentistry3000.pitt.edu/ bacterial community in dental biofilms linked to periodontal disease: an intra-subject metagenomics study vol 12 no 1 (2024) doi 10.5195/d3000.2024.465 http://dentistry3000.pitt.edu composition of bacterial species in the dental plaque of gingivitis and periodontitis sites in partial agreement with our findings, others reported a higher abundance of v. dispar in plaque samples of diseased sites compared to healthy sites in periodontitis patients [7]. in contrast, a higher abundance of v. dispar was discovered in healthy implant sites compared to sites with periodontitis and periimplantitis [25], and v. dispar was shown to be more prevalent in shallow pocket sites than in deeper places [26]. in agreement with our finding, p. melaninogenica was detected in great abundance in the saliva of periodontitis patients compared to healthy ones [27]. however, p. melaninogenica was also found to be more prevalent in plaque samples from healthy sites than in periodontitis sites [28]. additionally, similar to our observation, p. intermedia was found abundant in gingivitis plaque samples compared to periodontitis [29]. on the other hand, it was shown to be more prevalent in the subgingival plaque sample of the periodontitis than in gingivitis sites [30]. as for r. dentocariosa, it was shown that its abundance decreased as the severity of periodontal disease increased, which is consistent with our observation that gingivitis sites had a higher abundance [19,31]. however, compared to healthy sites, the species were more abundant in sites with periodontitis [32]. conclusion within the limitations of this study, it may be concluded that there is no apparent divergence of the dental biofilm communities in gingivitis and periodontitis. nevertheless, certain species displayed a trend of increased or decreased abundance in gingivitis or periodontitis sites. these patterns of species abundance in gingivitis and periodontitis have also been reported in contradictory ways in other studies, rendering the conclusions inconclusive. if this fundamental understanding is to be confirmed, bigger and well-controlled studies are required. acknowledgements the study was supported by the universiti sains malaysia (usm) through the usm research grant (grant no: 1001/ppsg/8012217). the sponsor had no role in the preparation and in the decision of submission of this article. references 1. periodontal diseases. kinane df, stathopoulou pg, papapanou pn. nat rev dis prim. england; 2017;3:17038. pmid: 28805207 2. defining metaniches in the oral cavity according to their microbial composition and cytokine profile. seidel cl, gerlach rg, wiedemann p, weider m, rodrian g, hader m, et al. int j mol sci [internet]. 2020;21:8218. available from: https://www.mdpi.com/14220067/21/21/8218pmid: 33153049 3. comparison of subgingival and buccal mucosa microbiome in chronic and aggressive periodontitis: a pilot study. wei y, shi m, zhen m, wang c, hu w, nie y, et al. front cell infect microbiol. 2019;9:1–11. pmid: 30915280 4. discrimination of bacterial community structures among healthy, gingivitis, and periodontitis statuses through integrated metatranscriptomic and network analyses. nemoto t, shiba t, komatsu k, watanabe t, shimogishi m, shibasaki m, et al. bik h, editor. msystems [internet]. american society for microbiology; 2021;6:1– 16. available from: https://journals.asm.org/doi/10.1128 /msystems.00886-21 5. periodontal microbial ecology. socransky ss, haffajee ad. periodontol 2000 [internet]. 2005;38:135–87. available from: http://www.ncbi.nlm.nih.gov/pubme d/15853940pmid: 15853940 6. the oral microbiome in periodontal http://dentistry3000.pitt.edu/ bacterial community in dental biofilms linked to periodontal disease: an intra-subject metagenomics study vol 12 no 1 (2024) doi 10.5195/d3000.2024.465 http://dentistry3000.pitt.edu health. lenartova m, tesinska b, janatova t, hrebicek o, mysak j, janata j, et al. front cell infect microbiol [internet]. 2021;11:629723. available from: https://www.frontiersin.org/articles/ 10.3389/fcimb.2021.629723/fullpmid : 33828997 7. subgingival microbiota and cytokines profile changes in patients with periodontitis: a pilot study comparing healthy and diseased sites in the same oral cavities. esparbès p, legrand a, bandiaky on, chéraudcarpentier m, martin h, montassier e, et al. microorganisms [internet]. 2021;9:2364. available from: https://www.mdpi.com/20762607/9/11/2364 8. the prevalence of novel periodontal pathogens and bacterial complexes in stage ii generalized periodontitis based on 16s rrna next generation sequencing. abu fanas s, brigi c, varma sr, desai v, senok a, d’souza j. j appl oral sci [internet]. 2021;29:e20200787. available from: http://www.scielo.br/scielo.php?scrip t=sci_arttext&pid=s167877572021000100425&tlng=enpmid: 34008792 9. qiime allows analysis of highthroughput community sequencing data. caporaso jg, kuczynski j, stombaugh j, bittinger k, bushman fd, costello ek, et al. nat methods [internet]. 2010/04/11. 2010;7:335–6. available from: https://pubmed.ncbi.nlm.nih.gov/203 83131 10. peat: an intelligent and efficient paired-end sequencing adapter trimming algorithm. li y-l, weng j-c, hsiao c-c, chou m-t, tseng c-w, hung j-h. bmc bioinformatics [internet]. biomed central ltd; 2015;16 suppl 1:s2. available from: http://www.biomedcentral.com/qc/1 471-2105/16/s1/s2pmid: 25707528 11. bbmerge – accurate paired shotgun read merging via overlap. bushnell b, rood j, singer e. biggs pj, editor. plos one [internet]. 2017;12:e0185056. available from: https://dx.plos.org/10.1371/journal.p one.0185056pmid: 29073143 12. greengenes, a chimera-checked 16s rrna gene database and workbench compatible with arb. desantis tz, hugenholtz p, larsen n, rojas m, brodie el, keller k, et al. appl environ microbiol [internet]. american society for microbiology; 2006;72:5069–72. available from: https://pubmed.ncbi.nlm.nih.gov/168 20507 13. nasopharyngeal microbiota in children with invasive pneumococcal disease: identification of bacteria with potential disease-promoting and protective effects. camelo-castillo a, henares d, brotons p, galiana a, rodríguez jc, mira a, et al. front microbiol. 2019;10. 14. quantitative and qualitative β diversity measures lead to different insights into factors that structure microbial communities. lozupone ca, hamady m, kelley st, knight r. appl environ microbiol [internet]. 2007;73:1576–85. available from: https://europepmc.org/articles/pmc 1828774 15. a new method for non-parametric multivariate analysis of variance. anderson mj. austral ecol [internet]. 2001;26:32–46. available from: https://onlinelibrary.wiley.com/doi/a bs/10.1111/j.14429993.2001.01070.pp.x 16. substantial differences in the subgingival microbiome measured by 16s metagenomics according to periodontitis status in older women. lamonte mj, genco rj, zheng w, mcskimming di, andrews ca, hovey km, et al. dent j. 2018;6. 17. comparative analyses of subgingival microbiome in chronic periodontitis patients with and without iga nephropathy by high throughput 16s rrna sequencing. cao y, qiao m, tian z, yu y, xu b, lao w, et al. cell physiol biochem [internet]. 2018;47:774–83. available from: https://www.karger.com/article/full text/490029pmid: 29807361 18. the subgingival microbiome of periodontal pockets with different probing depths in chronic and aggressive periodontitis: a pilot study. shi m, wei y, hu w, nie y, wu x, lu r. front cell infect microbiol [internet]. 2018;8:124. available from: https://www.frontiersin.org/article/1 0.3389/fcimb.2018.00124 19. do different probing depths exhibit striking differences in microbial profiles? pérez-chaparro pj, mcculloch ja, mamizuka em, moraes a da cl, faveri m, figueiredo lc, et al. j clin periodontol [internet]. 2018;45:26–37. available from: http://doi.wiley.com/10.1111/jcpe.12 811pmid: 28871594 20. toward personalized oral diagnosis: distinct microbiome clusters in periodontitis biofilms. wirth r, pap b, maróti g, vályi p, komlósi l, barta n, et al. front cell infect microbiol. 2021;11:1–13. 21. species-level salivary microbial indicators of well-resolved periodontitis: a preliminary investigation. acharya a, chen t, chan y, watt rm, jin l, mattheos n. front http://dentistry3000.pitt.edu/ bacterial community in dental biofilms linked to periodontal disease: an intra-subject metagenomics study vol 12 no 1 (2024) doi 10.5195/d3000.2024.465 http://dentistry3000.pitt.edu cell infect microbiol. 2019;9:1–12. pmid: 31681625 22. signature of microbial dysbiosis in periodontitis. meuric v, le gall-david s, boyer e, acuña-amador l, martin b, fong sb, et al. mcbain aj, editor. appl environ microbiol [internet]. 2017;83:1–13. available from: https://journals.asm.org/doi/10.1128 /aem.00462-17pmid: 28476771 23. microbiota and metatranscriptome changes accompanying the onset of gingivitis. nowicki em, shroff r, singleton ja, renaud de, wallace d, drury j, et al. sperandio v, editor. mbio [internet]. american society for microbiology; 2018;9:e00575-18. available from: https://doi.org/10.1128/mbio.0057518 24. dysbiosis and alterations in predicted functions of the subgingival microbiome in chronic periodontitis. kirst me, li ec, alfant b, chi y-y, walker c, magnusson i, et al. drake hl, editor. appl environ microbiol [internet]. united states: american society for microbiology; 2015;81:783–93. available from: http://www.ncbi.nlm.nih.gov/pubme d/25398868pmid: 25398868 25. intra-oral single-site comparisons of periodontal and peri-implant microbiota in health and disease. yu xl, chan y, zhuang l, lai hc, lang np, keung leung w, et al. clin oral implants res. 2019;30:760–76. pmid: 31102416 26. oral microbiome of deep and shallow dental pockets in chronic periodontitis. ge x, rodriguez r, trinh m, gunsolley j, xu p. burne ra, editor. plos one [internet]. public library of science; 2013;8:e65520. available from: https://doi.org/10.1371/journal.pone .0065520 27. potential roles of the free salivary microbiome dysbiosis in periodontal diseases. diao j, yuan c, tong p, ma z, sun x. 2021;11:1–11. 28. toward personalized oral diagnosis : distinct microbiome clusters in periodontitis bio fi lms. komlo l, wirth r, pap b, maro g, barta n, strang o. 2021;11:1–13. available from: https://www.frontiersin.org/articles/ 10.3389/fcimb.2021.747814/full 29. frequency of periodontal pathogens in equivalent periimplant and periodontal clinical statuses. cortelli r, cavalca s, oliveira f, romeiro d, roberto p, mendes p, et al. 2012;8:2–9. 30. progression of periodontal inflammation in adolescents is associated with increased number of porphyromonas gingivalis , prevotella intermedia , tannerella forsythensis , and fusobacterium nucleatum. yang n, zhang q, li j, yang s, shi q. 2013;1– 8. 31. a study of the bacteria associated with advancing periodontitis in man. tanner ac, haffer c, bratthall gt, visconti ra, socransky ss. j clin periodontol. united states; 1979;6:278–307. pmid: 294457 32. toward personalized oral diagnosis: distinct microbiome clusters in periodontitis biofilms. wirth r, pap b, maróti g, vályi p, komlósi l, barta n, et al. front cell infect microbiol [internet]. 2021;11:1–13. available from: https://www.frontiersin.org/articles/ 10.3389/fcimb.2021.747814/full http://dentistry3000.pitt.edu/ 842 final rela%on of amelogenin gene polymorphisms to dental caries among iraqi teenagers vol 13 no 1 (2025) doi 10.5195/d3000.2025.842 h#p://den*stry3000.pi#.edu relation of amelogenin gene polymorphisms to dental caries among iraqi teenagers elaf abdulkareem alidan1, ahlam taha mohammed2, batool hassan al-ghurabi2 1college of dentistry, university of ashur, baghdad, iraq 2college of dentistry, university of baghdad, baghdad, iraq abstract objec&ve: to examine the rela.onship between dental caries and single nucleo.de polymorphisms (snps) in amelx gene. methods: this study was a compara.ve crosssec.onal study encompassing 78 adolescents (aged 1315 years) was conducted. all of them were examined for their oral and dental status under the who recommended criteria, and clinical informa.on such as dmft and dmfs were evaluated. individuals whose dmft and dmfs index lower than 2 were designated 'very low caries experience' and higher than 9 were designated 'higher caries experience'. genomic dna was extracted from saliva samples, and single nucleo.de polymorphisms of amelx were genotyped. genotyping of three snps (rs17878486, rs5933871, rs5934997, intron) in amelx gene was determined by direct sequencing and analyzed with snpstats. chi-square was used to compare allele and genotype frequencies between cases and controls. results: there was no significant difference between the high and low caries groups but in high caries group, it showed a devia.on from hardy-weinberg equilibrium with a significant difference among the three genotypes (p=0.013) in rs5933871 and rs5934997, while a highly significant difference in rs17878486 for both high and low caries groups (p=0.000 and p=0.001). a highly significant and posi.ve correla.on between rs17878486 with dmfs in the control group and a posi.ve significant correla.on between rs17878486 and dmft. conclusion: although no significant difference of the inves.gated snps of amelx between high and low caries groups, but rs17878486 had a significant and posi.ve correla.on with dmft and dmfs which made this snp as marker for caries suscep.bility. keywords: amelx, dental caries, health risk, amelogenin, single nucleo.de polymorphisms. cita:on: alidan et al. (2025) rela:on of amelogenin gene polymorphisms to dental caries among iraqi teenagers. den:stry 3000. 1:a001 doi:10.5195/d3000.2025.842 received: february 2, 2025 accepted: march 9, 2025 published: may 2, 2025 copyright: ©2025 alidan et al. this is an open access ar:cle licensed under a crea:ve commons atribu:on work 4.0 united states license. email: elaf.abdukareem@au.edu.iq. introduction dental caries is a highly prevalent infectious oral disease in humans known for its complexity, is influenced by multiple factors, including lifestyle, socioeconomic status, genetic elements, and oral environmental characteristics [1]. despite the advancement of methods for prevention and treatment, dental caries remains a significant concern, impacting the quality of life [2,3]. findings from prior studies have illuminated characteristics associated with processes linked to caries development, categorized into the following four groups: 1) salivary composition and flow: saliva contains elements that possess the capability to directly combat cariogenic bacteria. additionally, it is abundant in calcium and phosphates, which play an active role in the remineralization of tooth enamel. the mechanical action of saliva flow aids in dislodging pathogens such as viruses, bacteria, and yeast from both teeth and mucosal surfaces. moreover, saliva can aggregate microbes, facilitating their removal through swallowing before they establish firm attachment [4,5]. 2) tooth morphology: malposition of the teeth, deep anatomical grooves, and retention regions resulting from the natural shape of the tooth structure can make brushing and fluoride penetration difficult, and are therefore thought rela%on of amelogenin gene polymorphisms to dental caries among iraqi teenagers vol 13 no 1 (2025) doi 10.5195/d3000.2025.842 h#p://den*stry3000.pi#.edu to be caries risk factors, particularly for pit and fissure caries [6]. 3) dietary and taste preferences: diet indeed has a significant impact on oral health, including its influence on plaque formation, the presence of cariogenic microorganisms, and overall caries risk. diet can have various potential impacts. it has the potential to affect both the quantity and composition of plaque buildup and debris on teeth, as well as the presence of cariogenic microorganisms. the interplay of factors such as the cariogenic nature of specific foods (like sucrose), eating frequency, and the physical characteristics of the diet can individually or collectively influence the development of tooth decay. conversely, taste preferences play a significant role in shaping dietary habits. people often make food choices based on their taste preferences. for instance, children tend to have a strong affinity for sweet foods while typically avoiding bitter flavours. as a result, they are more likely to consume foods that align with their favourite tastes [7,8]. 4) enamel and dentin formation: dental caries is an erosive process that causes tooth enamel to be lost, causing the enamel and dentin to continue to deteriorate, and finally leading to the formation of cavities within the tooth structure. the main cause of caries is enamel demineralization, while on the other hand, remineralization of enamel can act as a preventative measure against caries. as a result, a person's vulnerability to caries is greatly influenced by the genes that control how enamel and dentin are formed. regarding how genes associated with enamel may affect the emergence of carious lesions, there are two tenable hypotheses: they may increase enamel thickness and fluoride concentration, increasing protection against caries, or they may interact with oral microorganisms like streptococcus mutans, affecting caries vulnerability [9]. mature tooth enamel is primarily composed of inorganic material, accounting for more than 90% of its composition. however, during its development, enamel initially contains an organic matrix, which is gradually replaced by mineral compounds. several chemical and physiological processes, including the secretion, assembly, folding, and degradation of proteins, mineral growth, and gene expression, contribute to the formation of tooth enamel. both the quality and quantity of enamel have a direct impact on susceptibility to dental caries. enamel-formation genes, such as ambn, amelx, tuft1, klk4, and enam, constitute a group of genes involved in the process of odontogenesis in dentincontaining teeth [10,11]. previous studies have explored the association between genetic variants in this gene cluster and susceptibility to dental caries, yielding varying and inconclusive results [12-14]. the most significant secretory proteins can be categorized into amelogenin and non-amelogenin proteins [15]. amelogenins are highly conserved proteins and make up over 90% of the extracellular matrix protein content. they are believed to play a crucial role in directing the growth and organization of enamel crystals [16,17]. non-amelogenin proteins encompass ameloblastin, enamelin, and tuftelin. ameloblastin serves as a cell adhesion protein responsible for preserving rod integrity and regulating cell differentiation. enamelin, in cooperation with amelogenin, plays a role in controlling elongated growth and mineral nucleation, while tuftelin is suggested to potentially act as a nucleator for enamel crystallites [16]. rela%on of amelogenin gene polymorphisms to dental caries among iraqi teenagers vol 13 no 1 (2025) doi 10.5195/d3000.2025.842 h#p://den*stry3000.pi#.edu in iraq, research has been conducted to establish connections between genetic factors with other diseases like periodontal disease and among individuals with type 2 diabetes [18,19]. however, no prior studies have been able to establish links between genes related to enamel development, such as amelogenin (amelx) gene in the saliva of individuals with dental caries. as a result, the primary objective of the present study was to identify and assess individuals susceptible to dental caries, while comprehending the role of genes in the development of this condition. the study encompassed examinations aimed at determining whether variations in the amelogenin gene were linked to the occurrence of caries. dental caries is a highly prevalent infectious oral disease in humans known for its complexity, is influenced by multiple factors, including lifestyle, socioeconomic status, genetic elements, and oral environmental characteristics [1]. despite the advancement of methods for prevention and treatment, dental caries remains a significant concern, impacting the quality of life [2,3]. findings from prior studies have illuminated characteristics associated with processes linked to caries development, categorized into the following four groups: 1) salivary composition and flow: saliva contains elements that possess the capability to directly combat cariogenic bacteria. additionally, it is abundant in calcium and phosphates, which play an active role in the remineralization of tooth enamel. the mechanical action of saliva flow aids in dislodging pathogens such as viruses, bacteria, and yeast from both teeth and mucosal surfaces. moreover, saliva can aggregate microbes, facilitating their removal through swallowing before they establish firm attachment [4,5]. 2) tooth morphology: malposition of the teeth, deep anatomical grooves, and retention regions resulting from the natural shape of the tooth structure can make brushing and fluoride penetration difficult, and are therefore thought to be caries risk factors, particularly for pit and fissure caries [6]. 3) dietary and taste preferences: diet indeed has a significant impact on oral health, including its influence on plaque formation, the presence of cariogenic microorganisms, and overall caries risk. diet can have various potential impacts. it has the potential to affect both the quantity and composition of plaque buildup and debris on teeth, as well as the presence of cariogenic microorganisms. the interplay of factors such as the cariogenic nature of specific foods (like sucrose), eating frequency, and the physical characteristics of the diet can individually or collectively influence the development of tooth decay. conversely, taste preferences play a significant role in shaping dietary habits. people often make food choices based on their taste preferences. for instance, children tend to have a strong affinity for sweet foods while typically avoiding bitter flavors. as a result, they are more likely to consume foods that align with their favorite tastes [7,8]. 4) enamel and dentin formation: dental caries is an erosive process that causes tooth enamel to be lost, causing the enamel and dentin to continue to deteriorate, and finally leading to the formation of cavities within the tooth structure. the main cause of caries is enamel demineralization, while on the other hand, remineralization of enamel can act as a preventative measure against caries. as a result, a person's vulnerability to caries is greatly influenced by the genes that control how enamel and dentin are formed. regarding how genes associated with enamel may affect the emergence of carious rela%on of amelogenin gene polymorphisms to dental caries among iraqi teenagers vol 13 no 1 (2025) doi 10.5195/d3000.2025.842 h#p://den*stry3000.pi#.edu lesions, there are two tenable hypotheses: they may increase enamel thickness and fluoride concentration, increasing protection against caries, or they may interact with oral microorganisms like streptococcus mutans, affecting caries vulnerability [9]. mature tooth enamel is primarily composed of inorganic material, accounting for more than 90% of its composition. however, during its development, enamel initially contains an organic matrix, which is gradually replaced by mineral compounds. several chemical and physiological processes, including the secretion, assembly, folding, and degradation of proteins, mineral growth, and gene expression, contribute to the formation of tooth enamel. both the quality and quantity of enamel have a direct impact on susceptibility to dental caries. enamel-formation genes, such as ambn, amelx, tuft1, klk4, and enam, constitute a group of genes involved in the process of odontogenesis in dentincontaining teeth [10,11]. previous studies have explored the association between genetic variants in this gene cluster and susceptibility to dental caries, yielding varying and inconclusive results [12-14]. the most significant secretory proteins can be categorized into amelogenin and non-amelogenin proteins [15]. amelogenins are highly conserved proteins and make up over 90% of the extracellular matrix protein content. they are believed to play a crucial role in directing the growth and organization of enamel crystals [16,17]. non-amelogenin proteins encompass ameloblastin, enamelin, and tuftelin. ameloblastin serves as a cell adhesion protein responsible for preserving rod integrity and regulating cell differentiation. enamelin, in cooperation with amelogenin, plays a role in controlling elongated growth and mineral nucleation, while tuftelin is suggested to potentially act as a nucleator for enamel crystallites [16]. in iraq, research has been conducted to establish connections between genetic factors with other diseases like periodontal disease and among individuals with type 2 diabetes [18,19]. however, no prior studies have been able to establish links between genes related to enamel development, such as amelogenin (amelx) gene in the saliva of individuals with dental caries. as a result, the primary objective of the present study was to identify and assess individuals susceptible to dental caries, while comprehending the role of genes in the development of this condition. the study encompassed examinations aimed at determining whether variations in the amelogenin gene were linked to the occurrence of caries. material and methods design and sample in this particular, a group of 78 healthy and unrelated iraqi adolescents were included. among them, 39 subjects were had a high caries score with a dmft (decayed, missing, filled teeth) value greater than 9, whereas the other group should have had a low caries score with a dmft value less than 2 [20]. all participants had complete permanent dentition and lacked any dental anomalies. prior to the examination, consent was obtained from the parents of the patients. the inclusion criteria encompassed individuals aged between 13 and 15 years, who were part of the iraqi arab population. they should not have had any systemic diseases or taken any medications in the preceding three weeks. exclusion criteria involved patients with systemic diseases, cleft lips, rela%on of amelogenin gene polymorphisms to dental caries among iraqi teenagers vol 13 no 1 (2025) doi 10.5195/d3000.2025.842 h#p://den*stry3000.pi#.edu congenital anomalies, widespread dental issues, or those who were using fixed orthodontic appliances. participants who had received fluoride supplements or had undergone fissure sealant treatment were also excluded. individuals falling within the dmft range of more than 2 but less than 9 were not considered for the study. ethical considerations the study was conducted in accordance with the ethical principles that had their origin in the declaration of helsinki. it was carried out with patients verbal and analytical approval before sample was taken. the study protocol and the subject information and consent form were reviewed and approved by a local ethics committee of baghdad university, college of dentistry according to the document number 366 (including the number and the date in 1/8/2021) to get this approval. measures in this study, various demographic details such as age and gender, along with medical and dental histories, were recorded. unstimulated saliva was collected, followed by an intraoral examination using disposable instruments. the evaluation of tooth decay was scored based on the criteria set by the world health organization [21], utilizing the "decayed missing and-filled teeth/surfaces" (dmfs/dmft) index for permanent teeth, without the use of radiographs. the severity of dental caries was assessed according to who's 1976 guidelines [22]. unstimulated saliva was gathered over a span of 5 minutes. participants were instructed to expel saliva through a funnel into a sterilized tube that featured a scale, and this was repeated every minute, following the methodology outlined by navazesh in 2008 [23]. after the completion of saliva collection, the samples were divided into two segments. in the case of the portion meant for determining lactoferrin concentration, it was subjected to centrifugation at 4000 revolutions per minute (rpm) for a duration of 5 minutes. this process was undertaken to segregate the mucins. subsequently, the clear supernatant was separated using a micropipette and was then stored at approximately -80°c, as per the manufacturer's instructions (mybiosource, u.s.a.). for the other portion of the sample intended for dna extraction, centrifugation was performed at a speed of 12000 revolutions per minute (rpm) for a span of 3 minutes. dna extraction and quantitation: the genomic dna was extracted from the saliva samples using the reliaprep™ saliva gdna miniprep system protocol by promega. to determine the concentration of the extracted dna and assess the quality of the samples for subsequent procedures, the quantus fluorometer was employed. primer preparation, optimization and pcr amplification: the primers were provided by macrogen company in a lyophilized (freezedried) state. the dna template was subjected to amplification using the same pair of primers, consisting of a forward primer and a reverse primer. following the pcr (polymerase chain reaction) amplification process, agarose gel electrophoresis was utilized to verify the successful amplification. the efficacy of the pcr was entirely reliant on the quality of the extracted dna. pcr products were submitted for sanger sequencing using the abi3730xl automated dna sequencer from macrogen corporation – korea. subsequently, the outcomes were delivered via email and rela%on of amelogenin gene polymorphisms to dental caries among iraqi teenagers vol 13 no 1 (2025) doi 10.5195/d3000.2025.842 h#p://den*stry3000.pi#.edu subsequently assessed using the geneious software. elisa assay for measuring lactoferrin levels: the collected saliva samples from participants were examined for lactoferrin concentration using commercially available elisa kits in accordance with the manufacturer's instructions (mybiosource, u.s.a.). statistical analysis this study used computerized software statistical package for social science (spss version-24) was used. the variation of frequencies between groups were analyzed using chisquare test. hardy-weinberg equilibrium (hwe) was used to calculate the expected common homozygotes, expected heterozygotes, expected rare homozygotes. chi-square test was used to find out genotype deviation from hwe, and to compare the distributions of genotypes and allele frequencies in the disease and control groups. the relative risk (rr) is the real measure of association between exposure to a certain factor and having the disease or outcome. the risk associated with individual genotypes or alleles was calculated as the odds ratio (or) with 95% confidence intervals (95% ci). which indicate how many times more frequently a disease develops in individuals carrying the allele or genotype than in individuals lacking it. results genecc analysis of amelx snps agarose gel electrophoresis of pcr products three snps were detected in amelx gene according to primer design located at rs5933871, rs5934997 and rs17878486 which located in the second intron. genotype and allele frequency analysis analysis by hardy-weinberg equilibrium for study groups were done for the three snps (rs5933871, rs5934997 and rs17878486) to compare the three genotypes in each study groups. the comparisons of genotypes and allele frequencies computed for amelx snps are shown in tables 1 and 2. for all snps, it showed that no significant difference between the high and low caries groups but in high caries group, it showed a deviation from hardy-weinberg equilibrium with a significant difference among the three genotypes (p=0.013) in rs5933871 and rs5934997, while a highly significant difference in rs17878486 for both high and low caries groups (p=0.000 and p=0.001). there was a larger frequency of the major tt homozygote in rs5933871 and rs5934997, while the minor cc homozygote showed a higher odd ratio or and relative risk rr with no discernible difference between them (or = 1.8, rr=1.31, p=0.346; or = 2.16, rr=1.83, p=0.225) respectively. while rs17878486 the major tt was higher in study group compared with control with high or and rr but no significant difference (or = 1.58, rr=1.10, p=0.701) these findings point to the cc and tt genotype as possible high-risk variations for the research group's experience with caries. the genetic analysis for amelx gene snps in the present study revealed allele frequency for three snps in study groups as shown in table (3-12, 13, 14). for rs5933871 and rs5934997 there was a high frequency of allele t in low caries group than high caries group in contrast to rs17878486 which allele t had a high frequency in high caries group. while the frequency of c allele was higher in high caries group in rs5933871 and rs5934997 (41.0% and 41.0%) respectively than that in low caries group (37.1% and 33.3%), on the other hand the low caries group in rs17878486 showed higher allele c (33.3%) than that in high caries group (14.1%). however, the rela%on of amelogenin gene polymorphisms to dental caries among iraqi teenagers vol 13 no 1 (2025) doi 10.5195/d3000.2025.842 h#p://den*stry3000.pi#.edu difference in allele frequency between groups in three snps were non-significant at p-value >0.5. while there is a deviation from hardy-weinberg equilibrium in allele frequency in high caries groups which was significant in for rs5933871 and rs5934997. while in low and high caries group of rs17878486, the difference in allele frequency was a high significant difference (p=0.000). discussion it is important to remember that amelx gene is essential for the development of healthy enamel and any abnormalities in this gene are mostly to blame for congenital diseases such amelogenesis imperfecta and mineralization problems. as a result, some scientists have hypothesized that genetic variants may contribute to structural alterations in enamel, which may result in greater mineral loss, bacterial colonization, or the production of biofilms [9]. mammalian amelx was previously identified as a potential candidate for carrying mutations that could increase a person's susceptibility to caries by evolutionary analysis of amelx and genetic investigations utilizing snp markers [9-24]. the study investigated snps in the amelx gene for their potential association with dental caries and assessed the role of amelogenin and its deficiency in affecting enamel formation and caries development; unfortunately, our study did not produce any significant results between study and control groups but there was a significant difference among the three genotypes in each study groups. despite numerous studies strongly suggesting a link between enamel formation genes and caries susceptibility, discrepancies in results persist, particularly in relation to the age, gender, and ethnicity of the individuals studied [25]. in our investigation, none of the studied snps showed an association with the risk of dental caries, which aligns with the findings of ergoz et al. in 2014 [13], but contradicts the findings of jeremias et al. in 2013 [26] and gerreth et al. in 2017 [14], who reported an association between the amelx rs17878486 polymorphism and susceptibility to dental caries. additionally, kang et al. in 2011 [27] discovered significant relationships between the rs5933871 and rs5934997 snps and caries susceptibility, particularly in the group receiving water fluoridation. one possible explanation for our divergent findings may lie in the heterogeneity of our sample with respect to geographic origins. it's interesting to note that other studies [9-24] that identified amelx as a candidate gene for caries susceptibility used genetic techniques on more homogeneous populations. therefore, it is conceivable that random genetic alterations could have emerged in people with a genetically restricted background, increasing susceptibility to caries. however, patir and colleagues' 2008 study [9], which involved the sequencing of amelx in 70 children from the turkish community, does not support this gene as a candidate for caries susceptibility. additionally, amelx has not been identified as a target gene in genome-wide association studies carried out on bigger and more diverse populations [20-2829], which is consistent with the findings of this investigation. the amelx rs17878486 polymorphism's t allele and tt genotypes were discovered to be linked to an elevated risk in the study group as compared to the control group. it's essential to remain open to the possibility that other genes related to enamel may harbor mutations that increase an individual's susceptibility to caries [20]. rela%on of amelogenin gene polymorphisms to dental caries among iraqi teenagers vol 13 no 1 (2025) doi 10.5195/d3000.2025.842 h#p://den*stry3000.pi#.edu in a different part of the research which looked for patients who had no previous history of tooth decay or tooth decay less than 2 teeth to test the theory that amelx mutations would increase resistance to caries. however, this theory was not supported by our examination of the genetic polymorphisms found in this investigation; amelx snps were not discovered to be connected to caries prevention. this result is consistent with a previous work [30] that sequenced the variable amelx area in a varied human population and found no evidence of genetic variations. conclusion given these results, none of the studied snps showed an association with the risk of dental caries. it's critical to keep an open mind about the possibility that mutations in other enamel-related genes could make a person more vulnerable to dental cavities. conflicts of interest the authors declare no competing interest. rela%on of amelogenin gene polymorphisms to dental caries among iraqi teenagers vol 13 no 1 (2025) doi 10.5195/d3000.2025.842 h#p://den*stry3000.pi#.edu abbreviations: or odds ratio, ci confidence interval; rr relative risk; χ2 chaisquare; pvalue probability value; hwe hardy-weinberg equilibrium; ns non-significant, * significant. rs5933871 (t > c) genotype frequency study groups or ci rr χ2 p-value high caries n=39 low caries n=39 homozygous tt 18 (46.1%) 17 (43.5%) 1.11 (0.45-2.70) 1.06 0.028 0.865 heterozygous tc 10 (25.6%) 15 (38.4%) 0.9 (0.21-1.44) 0.94 1.00 0.317 homozygous cc 11 (28.2%) 7 (17.9%) 1.8 (0.61-5.26) 1.31 0.889 0.346 hwe χ² 8.619 1.216 p-value 0.013* 0.544 rs5934997 (t > c) homozygous tt 18 (46.1%) 19 (48.7%) 0.9 (0.37-2.19) 0.95 0.027 0.869 heterozygous tc 10 (25.6%) 14 (35.9%) 1.11 (0.23-1.62) 1.06 0.667 0.414 homozygous cc 11 (28.2%) 6 (15.3%) 2.16 (0.70-6.58) 1.83 1.47 0.225 hwe χ² 8.619 1.442 p-value 0.013* 0.486 rs17878486 (t > c) homozygous tt 32 (82.0%) 29 (74.3%) 1.58 (0.53-4.68) 1.10 0.147 0.701 heterozygous tc 3 (7.69%) 5 (12.8%) 0.63 (0.12-2.55) 0.91 0.5 0.479 homozygous cc 4 (10.2%) 5 (12.8%) 0.78 (0.19-3.14) 0.80 0.111 0.738 hwe χ² 18.166 13.452 p-value 0.000* 0.001* table 1. genotype and allele frequency comparisons calculated for amelx gene snps. rela%on of amelogenin gene polymorphisms to dental caries among iraqi teenagers vol 13 no 1 (2025) doi 10.5195/d3000.2025.842 h#p://den*stry3000.pi#.edu allele frequency rs5933871 (t> c) high caries n=39 low caries n=39 χ2 p-value t 46 (58.9%) 49(62.8 %) 0.242 0.622 c 32 (41.0%) 29 (37.1%) hwe p-value 0.003* 0.27 rs5934997 (t > c) t 46 (58.9%) 52(66.6 %) 0.988 0.32 c 32 (41.0%) 26 (33.3%) hwe p-value 0.003* 0.229 rs17878486 (t > c) t 67 (85.9%) 63(80.7 %) 0.738 0.39 c 11 (14.1%) 15 (33.3%) hwe p-value 0.000* 0.000* abbreviations: χ2 chaisquare; pvalue probability value; hwe hardy-weinberg equilibrium; * significant. table 2. allele frequency comparisons calculated of amelx gene snps. rela%on of amelogenin gene polymorphisms to dental caries among iraqi teenagers vol 13 no 1 (2025) doi 10.5195/d3000.2025.842 h#p://den*stry3000.pi#.edu references 1. al-zahraa jj, aldhaher za. evaluapon of mutans streptococci concerning oral health in the saliva of pregnant women. revis bionatura 2023;8 (2) 81. hqp://dx.doi.org/10.21931/rb/ 2023.08.02.81 2. mohammed h a, sahi n m, ahmed r t, al-rubaye a f.anpmicrobial acpvity of some nanoparpcles synthesized by laser ablapon technique against some bacteria isolated from oral cavity. mjb. 2022;19 (4), pp. 601-608. 3. ali r a, radeef s m, mohammed n b, diab b s. oral health related quality of life among dental implant papents in relapon to temporomandibular joint funcpon. mjb. 2022; 19 (4), pp. 609-614. 4. stookey gk. the effect of saliva on dental caries. j am dent assoc. 2008; 139 (suppl):11s–17s. [pubmed: 18595200]. 5. aziz a, taha, a. the salivary inflammatory biomarkers (interleukin-6, creacpve protein) in relapon with caries-experience among a group of 12-year-old obese boys. j bagh coll denpstry 2016; 28: 138-142. 10.12816/0024723. 6. guzman-armstrong s. rampant caries. j sch nurs. 2005; 21:272–278. [pubmed: 16262438]. 7. wendell s, wang x, brown m, cooper me, desensi rs, weyant rj, et al. taste genes associated with dental caries. j dent res. 2010 nov;89(11):1198-202. hqps://doi.org/10.1177/00220 34510381502 8. juma'a z, fadhil r, abdul-hussain y. correlapon between caries related microorganisms in the dental plaque and saliva with dental caries level in the upper and lower jaws in 5-9 years old children in baghdad city. j bagh coll denpstry 2016; 28(3):132136. 9. papr a, seymen f, yildirim m, deeley k, cooper me, marazita ml, vieira ar. enamel formapon genes are associated with high caries experience in turkish children. caries res. 2008; 42:394– 400. [pubmed: 18781068]. 10. pang, l., zhi, q., zhuang, p., yu, l., tao, y., lin, h. variapon in enamel formapon genes influences enamel demineralizapon in vitro in a streptococcus mutans biofilm model. front. physiol. 2017; 8, 851. 11. shungin d, haworth s, divaris k, agler cs, kamatani y, keun lee m, grinde k, hindy g, alaraudanjoki v, pesonen p, teumer a, holwreter b, sakaue s, hirata j, yu,y-h, ridker p. m, giulianini f, chasman di, magnusson pke, sudo t, okada y, völker u, kocher t, anqonen v, laitala m-l, orho-melander m, sofer t, shaffer jr, vieira a, marazita ml, kubo m, furuichi y, north ke, offenbacher s, ingelsson e, franks pw, timpson nj, johansson i. genome-wide analysis of dental caries and periodonpps combining clinical and self-reported data. nature communicapons 2019; 10 (1): 2773. 12. olszowski t, adler g, janiszewska-olszowska j, safranow k, kaczmarczyk m: mbl2 , masp2 , amelx , and enam gene polymorphisms and dental caries in polish children. oral dis 2012;18:389–395. 13. ergoz, n., seymen, f., gencay, k., et al. genepc variapon in ameloblaspn is associated with caries in asthmapc children. eur. arch. paediatr. dent. 2014; 15 (3), 211–216. 14. gerreth k, zaorska k, zabel m, borysewicz-lewicka m, nowicki m. chosen single nucleopde polymorphisms (snps) of enamel formapon genes and dental caries in a populapon of polish children. adv clin exp med. 2017;26: 899–905. 15. yoshizaki k, yamada y. gene evolupon and funcpons of extracellular matrix proteins in teeth. orthod waves. 2013;72:1–10. 16. moradian-oldak j. protein mediated enamel mineralizapon. front biosci. 2012;17:1996–2023. 17. gallon v, chen l, yang x, moradian-oldak j. localizapon and quanptapve co-localizapon rela%on of amelogenin gene polymorphisms to dental caries among iraqi teenagers vol 13 no 1 (2025) doi 10.5195/d3000.2025.842 h#p://den*stry3000.pi#.edu of enamelin with amelogenin. j struct biol. 2013;183:239–249. 18. mohammed ak, mi nader, bh al-ghurabi. genotyping of hla classi and ii molecules in type 2 diabepc iraqi papents. iosr journal of dental and medical sciences (iosr-jdms)2014; 13 (4 iii.): 4448. 19. fadhil r, hm akram, a najah, ss gul. associapon of matrix metalloproteinase-11607 1g/2g single nucleopde polymorphism genotypes with periodonpps in iraqi populapon. braz dent sci 2022; 25 (3): e3283 20. vieira ar, marazita ml, goldstein-mchenry t. genomewide scan finds suggespve caries loci. j dent res 2008; 87:435–439. 21. world health organizapon. oral health surveys basic methods 5th edipon, 2013. 22. muhlemann hr. oral epidemiology-caries. in: zeitschriyen-verlag b, quintessenze d. introducpon to oral prevenpve medicine; 1976. pp. 73-100. 23. navazesh m, kumar s. measuring salivary flow: challenges and opportunipes. jada 2008; 139(2):35s-40s. 24. deeley k, letra a, rose ek, brandon ca, resick jm, marazita ml, vieira ar: possible associapon of amelogenin to high caries experience in a guatemalan-mayan populapon. caries res 2008;42:8–13. 25. wang x, willing mc, marazita ml, wendell s, warren jj, broffiq b, smith b, busch t, lidral c, levy sm: genepc and environmental factors associated with dental caries in children: the iowa fluoride study. caries res 2012b;46: 177–184. 26. jeremias f, koruyucu m, küchler ec, bayram m, tuna eb, deeley k, pierri ra, souza jf, fragelli cm, paschoal ma, gencay k, seymen f, caminaga rm, dos santos-pinto l, vieira ar. genes expressed in dental enamel development are associated with molar-incisor hypomineralizapon. arch oral biol. 2013; 58:1434–42. 27. kang sw, yoon i, lee hw, et al. associapon between amelx polymorphisms and dental caries in koreans. oral dis 2011;17:399–406. 28. shaffer jr, wang x, feingold e, lee m, begum f, weeks de, et al. genome-wide associapon scan for childhood caries implicates novel genes. j dent res. 2011; 90:1457-1462. 29. shaffer jr, feingold e, wang x, lee m, cuenco kt, weeks de, et al. gwas of dental caries paqerns in the permanent denppon. j dent res. 2013; 92:38-44. 30. richard b, delgado s, gorry p, sire jy. a study of polymorphism in human amelx. arch oral biol.2007; 52:1026-1031. 869 vol 7, no 1 (2019) issn 2167-8677 (online) doi 10.5195/d3000.2019.84 h#p://den*stry3000.pi#.edu assessment of patient satisfaction and the reasons for crowns and fixed partial denture failure, a clinical study majed mohamed refaat college of dentistry, university of basrah, basrah, iraq abstract aims. this study aimed to assess the sa.sfac.on levels of pa.ents who had fixed dental prostheses for rehabilita.on and analyze the aspects, including biological, mechanical, and aesthe.c, that contribute to the failure of these prostheses. material and methods. cross-sec.onal observa.onal research was undertaken in the department of prosthodon.cs, college of den.stry, university of basrah, iraq, from september 2022 to may 2023. the study included a cohort of 130 pa.ents, both males and females, who received treatment with fixed dental prostheses. the par.cipants who were selected answered a series of ques.onnaires on their concerns about the fixed prosthesis, and a clinical examina.on involving radiographic assessments of the prosthesis was conducted. the reason for failure was documented, and data were organized for descrip.ve analysis of the components assessed through chi-square and fisher's exact test. results. based on clinical and radiographic assessment, it was determined that 103 out of the total number of individuals assessed experienced some form of failure. 44.6% of the cases exhibited biological problems, 31% exhibited mechanical failures, and 24.2% exhibited aesthe.c problems. dental caries accounted for most biological failures (32.6%), whereas prosthesis loosening was the primary mechanical cause of failure (56.2%). poor marginal fit was iden.fied as the leading aesthe.c reason for failure (48%). a strong correla.on was seen between the level of sa.sfac.on and mechanical failure (p=0.017). conclusion. most pa.ents expressed sa.sfac.on following the observa.on period. dental decay was the most prevalent biological component leading to failure, whereas loss of reten.on was the primary mechanical cause. in comparison to other aspects affec.ng aesthe.cs, poor marginal fit had a more significant role. keywords: survival rate, fixed dental prosthesis, abutment failure, technique complica.on, pa.ent sa.sfac.on. cita%on: refaat mm. (2025) assessment of pa%ent sa%sfac%on and the reasons for crowns and fixed par%al denture failure, a clinical study. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.869 received: march 3, 2025 accepted: march 14, 2025 published: aprll 11, 2025 copyright: ©2025 refaat mm. this is an open access ar%cle licensed under a crea%ve commons arribu%on work 4.0 united states license. email: majed.refaat@uobasra.edu.iq introduction numerous treatment options, including dental implantology, conventional fixed prostheses, and removable prostheses, can be used to replace lost teeth in a partially edentulous arch [1]. fixed prosthodontics refers to the use of artificial alternatives to replace or restore teeth that cannot be easily removed by the patient [2]. dental fixed crowns and bridges are securely linked to the remaining teeth [3]. fixed crowns are utilized for the purpose of restoring teeth that have been broken or have big amalgam or composite resin restorations. they can restore functionality and greatly enhance aesthetics. due to the growing trend among middle-aged and older individuals to maintain a significant proportion of their assessment of paeent saesfaceon and the reasons for crowns and fixed pareal denture failure, a clinical study vol 13 no 1 (2025) doi 10.5195/d3000.2025.869 h#p://den*stry3000.pi#.edu natural teeth, rehabilitation with fixed dental prostheses is well accepted and sought by patients [4]. the increased demand for crowns and fixed dental prostheses also increased the frequency of failure associated with such prostheses [5]. all-ceramic fixed prostheses are often utilized in clinical dentistry due to the introduction and availability of several ceramic materials for clinical application [6]. if the treatment planning for fixed dental prostheses is done improperly, there is a higher chance of premature failure and irreparable harm to the teeth and supporting tissues [7]. these restorations may fail, resulting in the development of new cavities or the loss of the teeth that support them [8]. typically, complications arise because of or during fixed dental prosthesis therapy operations. failure can be attributed to three primary factors: biological, mechanical, and aesthetic [9]. the effectiveness of rehabilitation treatment may be assessed by patient satisfaction, comfort, and the durability of the prosthesis. it is necessary to do clinical follow-up investigations on patients who have received artificial crowns and fixed dental prostheses to identify any complications [6]. to accurately diagnose, plan treatment, and execute procedures for fixed dental prostheses, it is crucial for the dentist to have a comprehensive understanding of the factors that lead to dissatisfaction or contribute to failures. special attention should be given to the most common failure factors to ensure that the patient's expectations are met [10]. the quality of prostheses is determined by both immediate and late failures. immediate failures are often caused by a lack of criteria during the manufacturing stages, resulting in errors in the form and color of the prosthesis. late failures, on the other hand, are typically related to factors such as caries, periodontal disease, endodontic complications, or technical issues like abutment fractures, loss of retention, and ceramic fracture [11,12]. this study aimed to evaluate the satisfaction of patients rehabilitated with fixed dental prostheses and observe the incidence of prosthesis failures among patients treated with these types of restorations. it also aimed to give special attention to the most frequent failure factors so that a detailed diagnosis and treatment plan meeting the patient’s needs could be concluded. materials and methods a cross-sectional study was carried out on the patients previously treated with fixed prostheses who visited the prosthodontic clinics of the college of dentistry, university of basrah. the study protocol underwent a thorough evaluation and received official approval from the college institutional review board and research ethics, and scientific committee, then it was registered under the number bcd3-002-22-9. the study had a total of 130 participants, consisting of 78 females and 52 males. all participants were above the age of 18 and willingly accepted to take part in the study by signing a written consent form. the questionnaire pro forma was utilized to gather the information. the survey comprised sociodemographic inquiries, including gender, age, treatment satisfaction, post-cementation hygiene care, and type of complications identified by clinical and radiographic evaluation. each participant underwent a comprehensive clinical evaluation while seated upright in an illuminated environment in the dentist's chair. a sole examiner positioned in front of the patient conducted visual and tactile intraoral examinations of both the teeth and the periodontium around the prosthesis. this was done using a sterile dental explorer, periodontal assessment of paeent saesfaceon and the reasons for crowns and fixed pareal denture failure, a clinical study vol 13 no 1 (2025) doi 10.5195/d3000.2025.869 h#p://den*stry3000.pi#.edu probe, and mouth mirror, ensuring the highest level of examination precision. tapping of the abutment with the mirror/probe instrument end at the occlusal or incisal aspect and palpation of the gingiva were carried out to identify pain adjacent to the abutment and pus discharge. following the clinical examination, periapical radiographs were conducted using a radiographic digital sensor, positioner, and x-ray portable machine (eighteeth company, china). for each evaluation, a uniform time of exposure was used with a long cone paralleling approach and a predetermined source-film distance of 25 cm. the periapical radiograph can detect biological failures like proximal caries (figure 1), periapical lesions, periodontal problems, and bone loss. secondary caries can also be identified by a thorough examination of the borders of the prosthesis and tooth surfaces using a dental explorer. mechanical failures such as loss of retention, connector failure, abutment tooth fracture (figure 2), and porcelain fracture were observed. aesthetic failures such as poor shade matching, poor contour, poor marginal fitness, and subpontic tissue shrinkage were also identified. figure 1. interproximal caries. figure 2. abutment fracture. the exclusion criterion encompassed patients who declined to respond to the questionnaire and undergo examination. additionally, patients with cantilever-type fixed prostheses were also excluded. the study data were inputted into a computer and organized for statistical analysis using spss software (version 20). the connection between variables was verified using chi-square and fisher's exact test. a p-value below 0.05 was deemed statistically significant. results the sample comprised 130 patients, 78 females (60%) and 52 men (40%). the average age of the patients was 43, with a standard deviation of 11.8. the research included patients as young as 21 and as old as 68. figure 3 illustrates the distribution of failures across three categories: biological failures, mechanical failures, and aesthetic failures. biological failures constitute the highest proportion, accounting for 44.6% of all failures. mechanical failures account for 31% of failures, while aesthetic failures constitute 24.2%. figure 3. fixed partial denture failure distribution. according to table 1, about 60.8% of the fixed prostheses were crowns, while short-span threeunit bridges made up 26.9%, fourunit bridges about 3.8%, five-unit bridges about 5.4%, six-unit bridges about 1.5%, and long-span sevenand eight-unit bridges accounted for 0.8% each. this study found an elevated percentage of problems related to 44.60 % 31% 24.20 %biological factors mechanical factors assessment of paeent saesfaceon and the reasons for crowns and fixed pareal denture failure, a clinical study vol 13 no 1 (2025) doi 10.5195/d3000.2025.869 h#p://den*stry3000.pi#.edu single crowns or short-span threeunit fixed partial dentures, namely 33% and 36.8%, respectively. in contrast, there was a minimal occurrence of complications with long-span bridges, such as 2.9% for seven-unit bridges and 3.8% for eight-unit bridges. table 1. failure rate related to the number of units of fixed restorations. table 2 shows that 57.7% of interviewees had no problems with prosthesis hygiene, 30% did not practice regular hygiene, and 12.3% never performed oral hygiene due to some difficulties. table 2. oral hygiene maintenance among subjects. oral hygiene maintenance subjects % always 75 57.7 sometimes 39 30 never 16 12.3 total 130 100 table 3 lists 46 biological failures, as some patients had numerous occurrences. secondary caries accounted for 15 instances (32.6%), followed by endodontic involvement in 10 cases (21.7%), periodontal involvement in 9 cases (19.5%), pulp degeneration in 6 cases (13%), gingival recession in 4 cases (8.6%), and crown perforation in 2 cases (4.3%). table 3. frequency distribution of biological failures biological failures no. of cases % secondary caries 15 32.6 endodontic involvement 10 21.7 periodontal involvement 9 19.5 pulp degeneration 6 13 gingival recession 4 8.6 perforation 2 4.3 subpontic inflammation 0 0 total 46 100 as indicated in table 4, 32 instances experienced mechanical malfunctions in their fixed restorations. three mechanical failures were recorded, with the most prevalent being prosthesis loosening, which occurred in 18 instances (56.2%). this was followed by abutment fracture in 8 cases (25%) and ceramic fracture in 6 cases (18.7%). table 4. frequency distribution of mechanical failure. mechanical failures no. of cases % retention loss 18 56.2 abutment fracture 8 25 ceramic fracture 6 18.7 connector failure 0 0 total 32 100 table 5 recorded twenty-five instances of aesthetic failures. the most prevalent problem occurring in 12 cases (48%) was a poor marginal fit. this was followed by poor shade matching in 7 cases (28%), poor contour of the prosthesis in 4 cases (16%), and 2 cases (8%) of subpontic tissue shrinking. table 5. frequency distribution of aesthetic failures. fixed partial denture units no. of cases % no. of compli cations % single crown 79 60.8 34 33 three units bridge 35 26.9 38 36.8 four units bridge 5 3.8 7 6.7 five units bridge 7 5.4 13 12.6 six units bridge 2 1.5 4 3.8 seven units bridge 1 0.8 3 2.9 eight units bridge 1 0.8 4 3.8 total 130 100 103 100 assessment of paeent saesfaceon and the reasons for crowns and fixed pareal denture failure, a clinical study vol 13 no 1 (2025) doi 10.5195/d3000.2025.869 h#p://den*stry3000.pi#.edu aesthetic failures no. % poor marginal fit 12 48 poor shade 7 28 poor contour 4 16 subpontic tissue shrinkage 2 8 total 25 100 table 6 displays the correlation between variables, with the inclusion of the chi-square and fisher exact tests. when participants were surveyed about their degree of satisfaction with the prosthetic therapy they got, 48 patients expressed dissatisfaction (36%) whereas 82 expressed satisfactions (63%). based on the sample median, the age range was categorized into two groups: 43 years or under and over 43 years. biological, mechanical, and aesthetic failures were categorized as either existing or absent. a statistically significant correlation was found between the level of satisfaction and the occurrence of mechanical failures (p=0.017). the connections between the other factors, namely gender (p=0.941), age range (p=0.221), biological failures (p=0.532), and aesthetic failure (p=0.074), were not found to be statistically significant. table 6. the correlation of satisfaction with gender, age, biological, mechanical, and aesthetic factors. variables satisfaction dissatisfaction total p-value gender no. % no. % no. % 0.941 female 49 62.8 29 37.2 78 100 male 33 63.5 19 36.5 52 100 total 82 63 48 36 130 100 age range no. % no. % no. % 0.221 ≤ 43 years 45 68.1 21 31.8 66 100 > 43 years 37 57.8 27 42.1 64 100 biological failure no. % no. % no. % 0.532 present 23 62.1 14 37.8 37 100 absent 59 63.4 34 36.5 93 100 mechanical failure no. % no. % no. % 0.017 present 10 40 15 60 25 100 absent 72 68.5 33 31.5 105 100 aesthetic failure no. % no. % no. % 0.074 present 11 47.8 12 52.1 23 100 absent 71 66.3 36 33.6 107 100 assessment of paeent saesfaceon and the reasons for crowns and fixed pareal denture failure, a clinical study vol 13 no 1 (2025) doi 10.5195/d3000.2025.869 h#p://den*stry3000.pi#.edu discussion crowns and bridges restorations are expensive and generate high patient expectations. despite thorough and precise attention to detail, instances of failures and patient dissatisfaction are frequently observed, therefore, it is important to do clinical follow-up studies on patients who received these restorations are mandatory to find complications [13, 14]. among the 130 participants included in this trial, 103 experienced problems; sheikh et al. (2021) identified a total of 142 patients who had concerns related to the fixed prosthesis [9]. the results of our study showed a high incidence of complications in single crown or short-span fixed partial dentures, this is in opposition to recent clinical studies that found an increase in complication rates with each additional pontic in fixed partial dentures [15]. the reason for this difference could be that most prostheses assessed in our study were either single-crown or short-span prostheses, which is consistent with the findings of alenezi et al. [16]. regular followup appointments with patients are essential for assessing the durability of fixed partial dentures, which are influenced by several factors, including the standard of oral hygiene maintained by the patient [17]. although the statistical analysis did not reveal any significant results (p=0.941), it was found that women expressed greater satisfaction with the prosthesis than men, in contrast, previous studies have reported different outcomes when participants were asked about their degree of satisfaction with the prosthesis [18]. in addition to that, this study demonstrates a lack of a link between age and satisfaction (p=0.221). however, it is interesting that younger patients achieved a greater percentage of satisfaction (68.1%). subjects who expressed satisfaction had a lower chance of failure, in contrast to those who were dissatisfied, zavanelli et al. also reported similar findings [19]. biological factors accounted for most failures, followed by mechanical and aesthetic factors; this aligns with the results provided by datta et al. [20] and in contrast to the discovery made by alghafees et al. [21]. according to research done by different authors, caries has been identified as an essential biological cause for failure, the secondary caries are directly influenced by the patient's cleanliness practices and the marginal fitness of the prosthesis. according to alsterstal et al. (2021) the endodontic-treated abutment had a low percentage of periapical lesions; this might be attributed to the fact that the endodontic treatment was primarily performed by professionals [22]. the periodontal involvement factor accounts for 19.5% of biological failures, this might be explained by the prosthesis impeding the normal stimulation of the supporting structures [23]. the advancement of periodontal disease may also be influenced by deficiencies in oral health, smoking, and hereditary factors [24]. a strong association was seen between the amount of satisfaction and the occurrence of mechanical failure (p= 0.017). the main factor contributing to mechanical failure in the research was the loss of retention (56.2%), it may be attributed to the failure of cementation and excessive taper preparation of the abutment tooth [25]. previous authors have also reported similar findings about retention loss. [11] the second factor contributing to mechanical failure is abutment fracture, endodontic treatment of the abutment might decrease the resistance of the teeth to fractures [26]. moreover, several studies identified ceramic fracture as the most prevalent mechanical problem, while in this study porcelain fracture was about 18.7% [16]. ceramic chipping may be due to critical load or bruxism [27, 28]. assessment of paeent saesfaceon and the reasons for crowns and fixed pareal denture failure, a clinical study vol 13 no 1 (2025) doi 10.5195/d3000.2025.869 h#p://den*stry3000.pi#.edu minor chipping did not influence fixed partial denture function, but substantial chipping caused prosthesis failure [29]. among all the aesthetic failure factors, the poor marginal fit was about 48%. from a technological standpoint, a strong marginal fit is crucial for ensuring the long-term durability of fixed prostheses [30]. the poor shade match was deemed undesirable, and its impact was diminished when compared to other criteria contributing to aesthetic failure; these findings contradict the conclusions of chandranaik et al., [1]. aesthetic defects can also be attributed to improper sizing and shaping of teeth, these deficiencies can lead to issues such as food being stuck due to an inaccurate prosthesis contour [25]. conclusion the most important factor affecting the level of satisfaction was mechanical defects; the major mechanical failure identified was the loss of crown and bridge retention. in addition to that, several biological failures were observed, mainly secondary caries. finally, aesthetic defects were mostly attributed to inadequate marginal fit. references 1. chandranaik, manjula b., and roopa k. thippanna. "fixed partial denture failures: a clinical survey for evaluation of the factors responsible." codsjournal of dentistry 9.2 (2019): 41-45. 2. ferro, keith j., et al. "the glossary of prosthodontic terms." (2017). 3. goodacre, charles j., et al. "clinical complications in fixed prosthodontics." the journal of prosthetic dentistry 90.1 (2003): 31-41. 4. reitemeier, bernd, et al. "a prospective 10-year study of metal ceramic single crowns and fixed dental prosthesis retainers in private practice settings." the journal of prosthetic dentistry 109.3 (2013): 149-155. 5. rosenstiel, stephen f., martin f. land, and robert walter, eds. contemporary fixed prosthodontics: contemporary fixed prosthodontics-e-book. elsevier health sciences, 2022. 6. swain, prasanta kumar. "failure rate in fixed partial denture patients-a clinical study." journal of advanced medical and dental sciences research 6.10 (2018). 7. sailer, irena, et al. "a systematic review of the survival and complication rates of allceramic and metal–ceramic reconstructions after an observation period of at least 3 years. part ii: fixed dental prostheses." clinical oral implants research 18 (2007): 8696. 8. manappallil, john joy. "classification system for conventional crown and fixed partial denture failures." the journal of prosthetic dentistry 99.4 (2008): 293-298. 9. sheikh, e., et al. (2021). assessment of causes of failures of fixed partial denture. european journal of molecular & clinical medicine, 7(11), 29202926. 10. layton, danielle, and terry walton. "patient-evaluated dentistry: development and validation of a patient satisfaction questionnaire for fixed prosthodontic treatment." international journal of prosthodontics 24.4 (2011): 332. 11. svanborg, per, et al. "a 5-year retrospective study of cobaltchromium-based fixed dental prostheses." international assessment of paeent saesfaceon and the reasons for crowns and fixed pareal denture failure, a clinical study vol 13 no 1 (2025) doi 10.5195/d3000.2025.869 h#p://den*stry3000.pi#.edu journal of prosthodontics 26.4 (2013). 12. solá-ruiz, m. fernanda, et al. "survival rates of a lithium disilicate-based core ceramic for three-unit esthetic fixed partial dentures: a 10-year prospective study." international journal of prosthodontics 26.2 (2013). 13. selby, alex. "fixed prosthodontic failure. a review and discussion of important aspects." australian dental journal 39.3 (1994): 150-156. 14. layton, danielle, and terry walton. "patient-evaluated dentistry: development and validation of a patient satisfaction questionnaire for fixed prosthodontic treatment." international journal of prosthodontics 24.4 (2011): 332. 15. raedel, michael, et al. "performance of fixed dental prostheses up to 6 years—a massive data analysis." the journal of prosthetic dentistry 128.3 (2022): 350354. 16. alenezi, ali, and sarah aloqayli. "technical complications with tooth-supported fixed dental prostheses (fdps) of different span lengths: an up to 15-year retrospective study." bmc oral health 23.1 (2023): 393. 17. naveed, syed hassan, et al. "frequency of factors involved in crowns and fixed partial denture failure." national editorial advisory board 31.11 (2020): 47-49. 18. tin-oo, mon mon, norkhafizah saddki, and nurhidayati hassan. "factors influencing patient satisfaction with dental appearance and treatments they desire to improve aesthetics." bmc oral health 11 (2011): 1-8. 19. zavanelli, adriana cristina, et al. "data collection about failures in fixed partial dentures: 1-year monitoring." rgo-revista gaúcha de odontologia 66 (2018): 250-256. 20. datta, a., and h. s. sandhu. "success of fixed partial denture prostheses observed in a military dental centre: a crosssectional study." medical journal armed forces india 78 (2022): s206-s212. 21. alghafees, abdulaziz ahmed et al. “fixed prosthodontics failure in students' patients at king saud university.” international journal of medical science and clinical invention 5 (2018): 3524-3526. 22. alsterstål-englund, helena, et al. "a retrospective clinical evaluation of extensive toothsupported fixed dental prostheses after 10 years." the journal of prosthetic dentistry 125.1 (2021): 65-72. 23. alfredo, e., et al. "in vitro evaluation of the effect of core diameter for removing radicular post with ultrasound." journal of oral rehabilitation 31.6 (2004): 590-594. 24. brägger, urs, et al. "complication and failure rates of fixed dental prostheses in patients treated for periodontal disease." clinical oral implants research 22.1 (2011): 70-77. 25. pawar, sudhir. "failures of crown and fixed partial dentures-a clinical survey." international journal of contemporary dentistry 2.1 (2011). 26. faria, adriana cláudia lapria, et al. "endodontically treated teeth: characteristics and considerations to restore them." journal of prosthodontic research 55.2 (2011): 69-74. 27. johansson, anders, ridwaan omar, and gunnar e. carlsson. assessment of paeent saesfaceon and the reasons for crowns and fixed pareal denture failure, a clinical study vol 13 no 1 (2025) doi 10.5195/d3000.2025.869 h#p://den*stry3000.pi#.edu "bruxism and prosthetic treatment: a critical review." journal of prosthodontic research 55.3 (2011): 127-136. 28. koenig, vinciane, et al. "clinical risk factors related to failures with zirconia-based restorations: an up to 9-year retrospective study." journal of dentistry 41.12 (2013): 11641174. 29. rathmann, friederike, et al. "up to 10 years clinical performance of zirconia ceramic and metalceramic fixed partial dentures: a retrospective study." the journal of prosthetic dentistry (2022). 30. hawthan, mohammed abdullah, bruno r. chrcanovic, and christel larsson. "longterm retrospective clinical study of tooth-supported fixed partial dentures: a multifactorial analysis." journal of prosthodontic research 67.2 (2023): 238-245. 773 final selected salivary an/oxidant levels in rela/on with periodontal diseases vol 13 no 1 (2025) doi 10.5195/d3000.2025.773 h#p://den*stry3000.pi#.edu selected salivary antioxidant levels in relation with periodontal diseases sulafa khair al-deen banoosh alayoubi college of dentistry, university of tikrit, iraq abstract objec&ve: gingivi's and periodon''s are the most common types of periodontal diseases, which are chronic inflammatory condi'ons encompassing many other condi'ons. these arise because of the interac'on between a pathogenic bacterial biofilm and inflammatory chemicals and cells produced from the host. materials and methods: a sample of 50 individuals (18 males and 32 females) aged 19-30 years old, treated at the health center of efraz, samarra city, was recruited. plaque index was measured to determine dental plaque thickness. a calculus index was measured as well. salivary uric acid and salivary total protein content were assessed chemically using uns'mulated saliva. the sta's'cal package for social science (spss) version 21 was used to examine all data. results and conclusion: no associa'ons were found between periodontal diseases and uric acid and salivary total protein content. keywords: an'oxidant, saliva, periodon''s, total protein, uric acid. cita9on: alayoubi skab. (2025) selected salivary an9oxidant levels in rela9on with periodontal diseases. den9stry 3000.1:a001 doi:10.5195/d3000.2025.773 received: november 14, 2024 accepted: december 19, 2024 published: february 12, 2025 copyright: ©2025 alayoubi skab. this is an open access ar9cle licensed under a crea9ve commons avribu9on work 4.0 united states license. email:sulafa.khairy@yahoo.com introduction gingivitis and periodontitis are the most prevalent of the various chronic inflammatory conditions known as periodontal diseases. these result from the interaction of a pathogenic bacterial biofilm with inflammatory chemicals and cells coming from the host [1,2]. porphyromonas gingivalis, actinobacillus actinomycetemcomitans, and bacteriodes forsythus are some of the bacterial pathogens that colonize the gums and cause periodontal disease [3]. these species have the capacity to turn on host defense mechanisms that degrade the epithelium and other gum systems while simultaneously turning off healing mechanisms [4,5]. recruited activated neutrophils become immobilized and release reactive oxygen species, which have the potential to kill the pathogen but also harm the host tissue nearby [6,7]. there is interest in investigating potential substances within oral fluids via which it may be possible to determine the presence and severity of periodontal diseases as well as to identify the individuals who are at risk for them [8]. saliva is an oral fluid that may be collected fast without the use of any specific tools or techniques [9,10]. proteins in saliva may yield determination of risks to oral diseases [11-16]. the aim of this work was to determine if salivary components may correlate with periodontal disease. material and methods subjects: fifty participants were selected from the efraz health center in samarra, with ages ranging from 19 to 30. the study was done between january and february 2020. selected salivary an/oxidant levels in rela/on with periodontal diseases vol 13 no 1 (2025) doi 10.5195/d3000.2025.773 h#p://den*stry3000.pi#.edu saliva collection: patients were requested to chew on a rubber band that had previously been sterilized 120 minutes before to the collection of their saliva samples. saliva samples of 3 ml were taken, placed in 6 ml sterile falcon tubes, and kept chilled at 5oc for one hour [17]. the samples were spun at 2,800 rpm for 10 minutes to separate the supernatant from the substrate, which was then frozen at -20oc. the sample was then centrifuged at 3,000 rpm after being defrosted at room temperature in groups of 20 samples. the supernatant was once more separated, and the levels of glucose, amylase activity, urea, albumin, and total protein were measured. oral assessment: a customized periodontal explorer and a plane mouth mirror were used for the oral examination. dental plaque thickness was determined by using the plaque index [18]. calculus index [19] was used to quantify the component of both periodontal and dental calculus. a calibrated periodontal probe with a standard force was used throughout the examination to quantify the depth of the pocket and to check for calculus buildup on the subgingival surfaces of the patient's teeth [20]. the score index varied from zero (no bleeding, pocketing, or factors that retain plaque) to four (extensive disease, pockets larger than 5.5 mm). results table 1 shows the numbers and the percentage of both genders in three groups of plaque index. the first group (mild) had 11 males and 22 females, the second group (moderate) had 6 males and 9 females, while the third group (severe) had 1 male and 1 female. table 1. percentages of gender distribution according to severity of periodontal disease. severity in periodontal tissues (pii index) males females total no. % no. % mild 11 61.1 22 68.8 33 moderate 6 33.3 9 28.1 15 severe 1 5.6 1 3.1 2 total 18 32 50 table 2 shows the numbers and the percentage of both genders in three groups of gingival indexes. the first group (mild) had 15 males and 28 females, the second group (moderate) had 3 males and 3 females, while the third group (severe) had 0 male and 1 female. table 2. percentages of gender distribution according to severity of periodontal disease. severity in periodontal tissues (gi index) males females total no. % no . % mild 15 83.3 28 87.5 43 moderate 3 16.7 3 9.4 6 severe 0 1 3.1 1 total 18 32 50 table 3 shows the means and standard deviations of the salivary total protein and uric acid in three groups of plaque index. the first group (mild) had 1.48 ± 0.12 of total protein and 1.05 ± 1.68 of uric acid, the second group (moderate) had 1.46 ± 0.13 of total protein and 2.33 ± 1.47 of uric acid, while the third group (severe) had 1.5 ± 0 of total protein and 1.16 ± 0.84 of uric acid. table 3. comparison between salivary antioxidant constituents in different severity of periodontal disease. severity in periodontal tissues (pii index) no . salivary antioxidant constituents total protein uric acid mean ± sd mean ± sd mild 33 1.48 ± 0.12 1.05 ± 1.68 moderate 15 1.46 ± 0.13 2.33 ± 1.47 severe 2 1.5 ± 0 1.16 ± 0.84 anova test p=0.83 p=0.18 selected salivary an/oxidant levels in rela/on with periodontal diseases vol 13 no 1 (2025) doi 10.5195/d3000.2025.773 h#p://den*stry3000.pi#.edu table 4 shows the means and the standard deviations of the salivary total protein and uric acid in three groups of gingival indexes. the first group (mild) had 1.47 ± 0.12 of total protein and 1.79 ± 1.17of uric acid, the second group (moderate) had 1.46 ± 0.09 of total protein and 2.50 ± 1.52 of uric acid, while the third group (severe) had 1.6 ± 0 of total protein and 1.7 ± 0 of uric acid. table 4. comparison between salivary antioxidant constituents in different severity of periodontal disease. severity of periodontal tissues (gi index) no. salivary antioxidant constituents total protein uric acid mean ± sd mean ± sd mild 43 1.47 ± 0.12 1.79 ± 1.17 moderate 6 1.46 ± 0.09 2.50 ± 1.52 severe 1 1.6 ± 0 1.7 ± 0 anova test p=0.09 p=0.36 discussion adults frequently suffer from chronic periodontal disease. antioxidants are found in bodily fluids to combat free radicals. saliva contains antioxidants, including uric acid and total protein. additionally, the association between the activities of antioxidant enzymes and clinical periodontal health was examined. previous research on the relationship between salivary antioxidant status and periodontal disease is limited and has produced contradictory results. although there may be other reasons that contribute to inconsistencies, this may be the result of the writers' differing methodologies. this study found no connection between salivary antioxidants and periodontal disease. the antioxidant capacity of saliva in healthy and ill people was tested in this finding, which is like the finding by moore et al. (1994) [21]. additionally, serum and saliva samples from both sick and control groups were examined by chapple et al. (1997) [22], and the two groups of serum antioxidant capacities were discovered to be comparable. these results disagreed with priadarsini et al. [23] who found statistically significant increases in total protein levels in chronic periodontitis patients compared to healthy people. the elevated protein levels in the test groups may result from an inflammatory process that stimulates the sympathetic nervous system, which in turn increases some proteins' synthesis and secretion (as shown by elevated amylase levels). this would increase saliva's ability to protect against diseases. according to a study by basu et al. [24], the elevated levels may potentially be related to an enhanced leakage of plasma proteins into saliva because of inflammation. conclusion the results indicate no significant association between salivary antioxidants and periodontal disease. although saliva has important antioxidant activities, which are able to counteract with oxidative stress, the findings in this regard suffered from inconsistency. discrepancies in the values might be due to variations in methodologies as well as factors like inflammationinduced secretion of proteins. salivary antioxidants may play an important role in periodontal health, and further research with standardized approaches will be required to clarify their role. conflicts of interest selected salivary an/oxidant levels in rela/on with periodontal diseases vol 13 no 1 (2025) doi 10.5195/d3000.2025.773 h#p://den*stry3000.pi#.edu the authors declare no competing interest. references 1. dde mr, zenóbio eg, van dyke t, silva ks, costa fo, soares rv. differendal expression of salivary glycoproteins in aggressive and chronic periodondds. j appl oral sci 2012;20:180-5. 2. lda rg, soares mr, nogueira fc, garcia c, camisasca dr, domont g, et al. comparadve proteomic analysis of whole saliva from chronic periodondds padents. j proteomics 2010;73:1334-41. 3. lamont, r. j. and jenkinson, h. f. (1998) life below the gum line: pathogenic mechanisms of porphyromonas gingivalis. microbiol. molec. biol. rev.62,1244–1263 4. jenkinson, h. f. and dymock, d. (1999) the microbiology of periodontal disease. dent. update26,191–197 5. fravalo, p., menard, c. and bonnaure-mallet, m. (1996) effect of porphyromonas gingivalis on epithelial cell mmp-9 type iv collagenase producdon. infect. immun. 64,4940–4945 6. fredriksson, m., gustafsson, a., asman, b. and bergstrom, k. (1998) hyper-reacdve peripheral neutrophils in adult periodondds: generadon of chemiluminescence and intracellular hydrogen peroxide aker in vitro priming and fcyr sdmuladon. j. clin.periodontol.25,394–398 7. guarnieri, c., zucchelli, g., bernardi, f., scheda, m., valendni, a. f., calandriello, m. (1991) enhanced superoxide producdon with no change of the andoxidant acdvity in gingival fluid of padents with adult periodondds. free radical res.15,11–16 8. lda rg, soares mr, nogueira fc, garcia c, camisasca dr, domont g, et al. comparadve proteomic analysis of whole saliva from chronic periodondds padents. j proteomics 2010;73:1334-41. 9. henskens ym, van der velden u, veerman ec, amerongen av. protein, albumin and cystadn concentradons in saliva of healthy subjects and of padents with gingivids periodondds. j periodontal res 1993;28:43-8. 10. prasthaanthi n, gayathri r, vishnupriya v. a study on associadon of salivary calcium and phosphate in oral health. j pharm sci res 2016;8:623-6 11. canakci cf, cicek y, yildirim a, sezer y, canakci, v. increased levels of 8hydroxydeoxyguanosine and malondialdehyde and its reladonship with andoxidant enzymes in saliva of periodondds padents. 2009; 3(2): 100-106. 12. cory jg. purine and pyrimidine nucleodde metabolism. in textbook of biochemistry with clinical correladon. 6 th ed. usa: wiley-liss; 2006;799 – 800. 13. miricescu, d, grebu, m, totan, a, didilescu, a, radulescu, r. the andoxidant potendal of saliva: clinical significance in oral diseases. therapeudcs, pharmacology and clinical toxicology. 2011; 15; 139-143. 14. bapno m, bullon p, wilson m, newman h. oxidadve injury and inflammatory periodontal diseases: the challenge of andoxidants to free radicals and reacdve oxygen species. crobm: 1999; 10: 458. 15. saadh, m. j., hsu, c. y., mustafa, m. a., mutee, a. f., kaur, i., ghildiyal, p., ... & farhood, b. (2024). advances in chitosan-based blends as potendal drug delivery systems: a review. internadonal journal of biological macromolecules, 132916. 16. eliasson l, carlen a, laine m, birkhed d (2003) “minor gland and whole saliva in postmenopausal women using a low potency oestrogen (oestriol),” archives of oral biology, 48. selected salivary an/oxidant levels in rela/on with periodontal diseases vol 13 no 1 (2025) doi 10.5195/d3000.2025.773 h#p://den*stry3000.pi#.edu 17. laine ma. effect of pregnancy on periodontal and dental health 2002, acta odontol. scand, 60: 257-264. 18. ainamo, j., barmes, d., beagrie, g., cutress, t., mardn, j., sardoinfirri, j. (1982) development of the world health organizadon (who) community periodontal index of treatment needs (cpitn). int. dent. j.32,281–291 19. moore s, calder kac, millar nj & rice-evans ca (1994) andoxidant acdvity of saliva and periodontal disease. free radical research21, 417–425. 20. jameel, m. k., mustafa, m. a., ahmed, h. s., jassim mohammed, a., ghazy, h., shakir, m. n., ... & kianfar, e. (2024). biogas: producdon, properdes, applicadons, economic and challenges: a review. results in chemistry, 101549. 21. t. priadarsini, r. gayathri, v. vishnu priya, evaluadon of salivary total proteins, albumin, globulin, and a/g rado among healthy individuals and padents with chronic periodondds; drug invendon today; 2018, vol 10, issue 7, p 1175-1178. 22. henskens ym, van den keijbus pa, veerman ec, van der weijden ga, timmerman mf, snoek cm, et al. protein composidon of whole and parodd saliva in healthy and periodondds subjects: determinadon of cystadns, albumin, amylase and iga. j periodontal res 1996;31:57-65. 23. sánchez ga, miozza va, delgado a, busch l. reladonship between salivary mucin or amylase and the periodontal status. oral dis 2013;19:585-91. 24. basu mk, smith aj, walsh tf, saxby ms. albumin in saliva during experimentally induced gingivids. j dent res 1984;63:514. 1011 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1011 http://dentistry3000.pitt.edu expression of igf-1r in skin wound healing treated by topical application of cinnamomum zeylanicum noor na&k raheem, lobna k. al-khafaji, lehadh m. al-azzawi, dhifaf kamil kadhim al-thahab college of den*stry, mustansiriyah university, iraq abstract objec&ve: the aim of this study was to evaluate the immunohistochemical expression of igf1r on cutaneous wound healing. material and methods: a total of 18 male albino rats (rasus norvegicus albinus) weighing nearly 300-400 gm were used in the present study. two punch biopsies were done on the dorsum of each animal, and daily local applicauon of cinnamon zeylanicum extract was performed on lew side, while the right one was allowed to heal naturally (control measure). the process of wound skin healing was tracked for periods (1, 3, and 7 days), all specimens were processed for immunohistochemistry examinauon. results: based on immunohistochemical results, experimental groups had the highest mean values of igf-1r expression in the stroma and epidermis when compared to control groups. these values increased with ume, which was more nouceable on days three and seven of the experiments. conclusion: posiuve localizauon of igff-1r in rat's skin indicated the effecuveness of cinnamon zeylanicum extract in acceleraung the healing process of cutaneous layers of skin. open access cita%on: raheem nn, et al. (2025) expression of igf-1r in skin wound healing in rats treated by topical applica%on of cinnamomum zeylanicum. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1011 received: august 4, 2025 accepted: august 26, 2025 published: september 10, 2025 copyright: ©2025 raheem nn, et al. this is an open access ar%cle licensed under a crea%ve commons axribu%on work 4.0 united states license. email: noorna%kraheem@uomustansiriyah.edu.iq introduc)on cutaneous wound healing is a complicated natural process. attempts to repair the defect caused by signi:icant damage start very early during in:lammatory response [1]. ultimately, they result in repair. herbal therapies have been utilized to treat wounds and injuries for centuries [2]. cinnamomum zeylanicum, an old botanical synonym for the cinnamon tree, comes from sri lanka's former name. cinnamon has diverse biological properties, including anti-in:lammatory [2], antioxidant, [3,4], and anti-microbial [4]. the wound repair process requires close regulation of degradative and regenerative processes, a wide variety of cell types, and complicated interactions between many biochemical cascades needed to repair process without complications [5,6]. growth factors released in the injured area encouraging cell migration to the wound site by chemotaxis, stimulate the growth of :ibroblasts and epithelial cells, initiate angiogenesis, and extracellular matrix ecm deposition and remodeling of the wounded area [1]. growth hormone action, often mediated by (igf-i) is predominantly seen in the dermis of skin, impacting the synthesis of collagen :iber, igf activity has a signi:icant impact on both epidermal and dermal compartments, that enhance the proliferation, migration and survival of stem cells [7]. the insulin-like growth factor 1 receptor (igf-1r) is a small, multifunctional molecule like pro-insulin, it is acting in an autocrine or paracrine way to aid in different and multiple functions, including the early phases of wound healing [6,8]. many physiological functions rely on the important balance between proliferation of mitotically active epidermal keratinocytes of the skin and differentiation of post-mitotic cells. this balance has a crucial role in skin formation and development [9]. the study aimed to evaluate the effects of locally applied cinnamon essential oil on expression of igf-1r in cutaneous wound healing in rats. material and methods experimental design 18 male albino rats of about (300–350gm) and aging from (4-8) weeks were used [10]. all rats were maintained under good conditions with a standard laboratory environment. animals were housed in single, and separate cages for 7 days in equivalent settings. the rats were arbitrarily divided into 3 main groups (six rats each), based on period of healing (1, 3, and 7 days). two full-thickness punch biopsies of 2 mm depth [10] were made on the dorsum of each rat with a sterilized punch of 5 mm diameter [11]. the expression of igf-1r in skin wound healing treated by topical applicauon of cinnamomum zeylanicum vol 13, no 1 (2025) doi 10.5195/d3000.2025.1011 http://dentistry3000.pitt.edu 2 wound on the right side was left untreated. while the other side (experimental) treated with local cinnamon essential oil (100% purity) every day till scarifying. surgical procedures all surgical instruments were sterilized by using the oven at 150ºc for one hour. a combination of 50 mg (40 mg/kg-b.w.). ketamin hcl and 2% xylazine (0.4 mg/kg b.w.) was injected intramuscularly according to the animal’s weight. hair clippers were used for removal of dorsal hair :irst, followed by disinfection using ethyl alcohol (70%). two punch biopsies of 5 mm in diameter were done with a rotary device. 3 cm distant from each other. the control wound was irrigated with distilled water while left experimental (left side) was topically treated with one drop of cinnamomum zylanicum essential oil (10 µl, u.s.a.), applied by using a micropipette. each rat received general anesthesia and was scari:ied at the end of (1, 3 and 7 days). the operation site was demarcated with nearly 5 mm of normal tissue surrounding the wounded area, and then a complete excisional biopsy was obtained. for :ixation purposes. the specimens were placed in a plastic biopsy container :illed with ten percent formaldehyde and cut using a microtome to gain 5 µm thickness for immunohistochemical study; polyclonal igf-1r were used according to manufacturer instructions. spss software version 24.0 was used for statistical analysis. we used anova test after using a paired sample t-test, which established the data's normal distribution. alpha was de:ined as 0.05. results one-day duration positive localization of igf-1r expressed by :ibroblasts and collagen :ibers in control group is showed in figure 1. figure 2 shows the expression of experimental skin section group that positive reaction in hair follicles and :ibrous connective tissue. figure 1. view of positive expression of igf1r by :ibroblast (red arrow) and collagen :iber (arrow cf) in control group. dab stain with counter stain hematoxylin, x40. figure 2. view of positive expression of igf1r by :ibroblast (red arrow) and collagen :ibers (cf). dab stain with counter stain hematoxylin, x40. three days duration positive localization of igf-r1 in the skin section of the control group is detected by epithelium, :ibroblasts, and vascular endothelium as shown in figure 3. experimental skin section group shows positive localization of igf-1r by new epithelium and :ibroblasts (figure 4). figure 3. magni:ied view of positive expression of igf-1r by vascular endothelium (black arrow) and :ibroblasts (fb), epithelium (blue arrow). dab stain with counter stain hematoxylin, x40. figure 4. positive localization of igf-1r at new epithelium (blue arrow) and :ibroblast (red arrow). dab stain with counter stain hematoxylin, x40. seven days duration positive localization of igf-1r in the control group is detected by new epithelium; positive reaction is shown by dermal :ibroblasts and vascular endothelial cells (figure 5). while the microphotograph view after cinnamomum zylanicum application shows positive localization of igf-1r by epithelial cells, hair follicles, collagen :ibers, and :ibroblasts (figure 6). figure 5. photomicrograph shows positively stained endothelial cells (en) and :ibroblast (red arrow). dab stain with counter stain hematoxylin x40. figure 6. positive expression of igf-1r by epithelium (e), :ibroblasts (red arrow). dab stain with counter stain hematoxylin, x40. stromal expression statistical analysis of positively stained cells for immunohistochemical localization of igf-1r as measured in different healing periods (1, 3, and 7 days) for studied groups is shown in table 1. there was a decrease in mean values with time for the 1and 3-day cf cf fb epithelium en epithelium cf expression of igf-1r in skin wound healing treated by topical applicauon of cinnamomum zeylanicum vol 13, no 1 (2025) doi 10.5195/d3000.2025.1011 http://dentistry3000.pitt.edu 3 durations whereas values increased at day 7; the highest values were recorded at day 1 for the experimental group, the lowest value was detected at day 3 for the controls. a significant difference (p <0.05) was observed between control and experimental groups on days 1 and 7. there was a nonsignificant difference (p>0.05) at day three. those findings were illustrated in figure 7. figure 7. experimental groups versus and controls regarding igf-1r expression (stromal). epidermal expression statistical analysis of epidermal immunohistochemical localization of igf-1r is demonstrated in table 2. the mean value of positively stained cells increased with time at day 3 and 7 healing durations for control and experimental groups; the highest average value of positively stained cells was recorded at day (7) for the experimental group, whereas the lowest values were noticed at day 3 for the control groups figure 8. besides there was signi:icant difference (p<0.05) between studied groups at 3,7 days as shown in table 2. figure 8. comparison between experimental and control group regarding igf-1r expression (epidermal). discussion one of the major concerns for medical staff in skin wounds that have a significant impact on people's health and lives is increasing morbidity and death rates [12]. the healing process is a complicated dynamic process including four overlapped phases: it starts with hemostasis, inflammation, and proliferation, and ends with tissue remodeling (maturation). the most important goal of the healing process is accelerating recovery of normal condition of tissues without scarring [13,14]. growth factors are important regulators taking part in orchestrating and promote cellular responses, including: growth, migration, differentiation, and deposition of extracellular matrix needed for successful wound healing [5]. insulin-like growth factor 1 acts as a regulator of vascular endothelial function adjusted vasoconstriction and /or vasodilation, and modulates inflammatory actins to conserve skin homeostasis [16]. the exact mechanism of the healing process of a wound is not clearly understood. there are several parameters that are involved in the healing of wounds, including histological results (re-epithelialization, granulation tissue, collagen accumulation, inflammatory reaction, angiogenesis, ulcer formation). the results showed a significantly increased level of igf-1r expression during advances of the skin healing process as it stimulates the keratinocytes and fibroblast migration, especially during wound epithelialization [17]. while these cells express only small amounts of this protein. thus, optimum healing is related to igf-1r levels. a few cells in the dermis and epidermis in normal skin express igf-1r receptors (basal epidermal keratinocytes and undifferentiated epithelial cells related to the outer root sheath of hair follicle, and sebaceous glands. all epidermal cells, macrophages, and other cells of the skin showed positive expression of igf-1r at day one to three after the injury in coincidence with [18]. in the present study, the highest mean values of positive expression for stromal (igf-1r) were detected on day 1, when the process of re-epithelization is usually taking place. this is in agreement with póvoa and diniz (2011) [19], who studied growth hormone system: skin interactions and stated that the maximum levels of igf-1 in body :luids and local tissues occur early in the :irst hours or days following the injury since it acts as a chemoattractant for endothelial cells correlated with the proliferation and migration of keratinocytes and :ibroblasts which play a major role in the healing of wounds [20,21], by stimulating collagen synthesis and secretion, thus accelerating wound healing and increasing the strength of the wound. it may originate from migrating keratinocytes from epithelial cells of adjacent hair follicles, :ibroblasts from granulation tissue, in:lammatory cells, or from plasma in accordance with seeger and paller (2015) [22]. conclusion the present study concluded that using local cinnamomum zylanicum essential oil accelerates wound healing as demonstrated in igf-1r expression, which is involved in the healing process in different durations. acknowledgements the authors extend their thanks to mustansiriya university, baghdad, iraq, for providing support. funding this research did not receive any speci:ic grant from funding agencies in the public, commercial, or not-for-pro:it sectors. conflict of interest the authors have no con:licts of interest to declare. regulatory statement this study was approved by the research ethical committee of the college of dentistry, mustansiriyah university (study number: muopa9). references 1. ganapathy n, venkataraman ss, daniel r, aravind rj, kumarakrishnan vb. molecular biology of wound healing. journal of pharmacy and bioallied science. 2012 aug 4;(suppl 2):s334-7. doi: 10.4103/0975-7406.100294. 2. lee sh, lee sy, son dj, lee h, yoo hs, song s, oh kw, han dc, kwon bm, hong jt. inhibitory effect of 2′-hydroxycinnamaldehyde on nitric oxide production through inhibition of nf-κb activation in raw 264.7 cells. biochemical pharmacology. 2005 mar; 69(5):791-9. doi.org/10.1016/j.bcp.2004.11.013 3. lee sh, gao jr, yoon ks, mumcuoglu ky, taplin d, edman jd, takano-lee m, clark jm. sodium channel mutations associated with knockdown resistance in the human head louse, pediculus capitis (de geer). pesticide biochemistry and physiology. 2003 mar; 75 (3):79-91. doi.org/10.1016/s0048-3575(03)00018-x 4. singh g, maurya s, delampasona mp, catalan ca. a comparison of chemical, antioxidant and antimicrobial studies of cinnamon leaf and bark volatile oils, oleoresins and their constituents. food and chemical toxicology. 2007 sep;45(9):1650-61. doi.org/10.1016/j.fct.2007.02.031 1. 5. matan n, rimkeeree h, mawson aj, chompreeda p, haruthaithanasan v, parker m. antimicrobial activity of cinnamon and clove oils under modified atmosphere conditions. international journal of food microbiology. 2006 mar;107(2):180-5. doi.org/10.1016/j.ijfoodmicro.2005.07.007 6 al-khayal k, alwesaimer as, abdulla m, ahmad r. insulin-like growth factor administration stimulates wound healing on colonic anastomosis in hypoxic rat model. dr.sulaiman alhabib medical journal. 2020 jun; 2 (2): 43 50. doi.org/10.2991/dsahmj.k.200529.001 expression of igf-1r in skin wound healing treated by topical applicauon of cinnamomum zeylanicum vol 13, no 1 (2025) doi 10.5195/d3000.2025.1011 http://dentistry3000.pitt.edu 4 7. fuchs e, raghavan s. getting under the skin of epidermal morphogenesis. nature reviews genetics. 2002 mar; 3 (3):199-209. doi.org/10.1038/nrg758 8. jawad, m. m., alam, m. k., abdulqader, s. t., alazzawi, l. m., husein, a., & mahmood, a. s. histological evaluation of incision healing response made by metallic scalpel on rabbits skin: preliminary study. international medical journal. 2013 aug; 20(4), 496-498. 9. farahpour mr, habibi m. evaluation of the wound healing activity of an ethanolic extract of ceylon cinnamon in mice. vet med. 2012 jan; 57(1):53-7. https://www.agriculturejournals.cz/pdfs/vet/20 12/01/04.pdf 10. liu h, lin s, xiao d, zheng x, gu y, guo s. evaluation of the wound healing potential of resina draconis (dracaena cochinchinensis) in animal models. evidence-based complementary and alternative medicine. 2013 apr 2013;(1):709865. doi.org /10.1155/2013/709865 11. moreira cf, cassini-vieira p, da silva mf, da silva barcelos l. skin wound healing model-excisional wounding and assessment of lesion area. bio-protocol. 2015; 20;5:e1661. bio-protocol, 5(22), e1661-e1661. http://www.bio-protocol.org/e1661 12. noori hj, jasim sy, abbass wa. evaluation of curcumin effect on wound healing in rat model. int j drug deliv technol. 2022 sept;12(3):1208-8. 13. al-naymi ha, al-musawi mh, mirhaj m, valizadeh h, pajooh am, shahriari-khalaji m, shariiianjazi f, tavamaishvili k, kazemi n, salehi s, arefpour a. exploring nanobioceramics in wound healing as effective and economical alternatives. heliyon. 2024 sep 26. doi.org/10.1016/j.heliyon.2024.e38497 14. zaki nh, zaman h, ali h. evaluate the effect of biosynthesized silver nanoparticles on wound healing in experimental mice. journal of global pharma technology. 2019;11(07) suppl.):91-8. http://www.jgpt.co.in/ 15. todorović v, peško p, micev m, bjelović m, budeč m, mićić m, brašanac d, ilić-stojanović o. insulin-like growth factor-i in wound healing of rat skin. regulatory peptides. 2008 oct;150(1-3):713. doi.org/10.1016/j.regpep.2008.05.006 16. macvanin m, gluvic z, radovanovic j, essack m, gao x, isenovic er. new insights on the cardiovascular effects of igf-1. frontiers in endocrinology. 2023 feb 9;14:1142644. https://doi.org/10.3389/fendo.2023.1142644 17. zhang x, hu f, li j, chen l, mao yf, li qb, nie cy, lin c, xiao j. igf-1 inhibits inilammation and accelerates angiogenesis via ras/pi3k/ikk/nfκb signaling pathways to promote wound healing. european journal of pharmaceutical sciences. 2024 sep ;200:106847. https://doi.org/10.1016/j.ejps.2024.106847 18. achar ra, silva tc, achar e, martines rb, machado jl. use of insulin-like growth factor in the healing of open wounds in diabetic and non-diabetic rats. acta cirurgica brasileira. 2014 feb;29(02):125-31. https://doi.org/10.1590/s010286502014000200009 19. póvoa g, diniz lm. growth hormone system: skin interactions. anais brasileiros de dermatologia. 2011;86(6):1159-65. https://doi.org/10.1590/s036505962011000600015. 20. jawad mm, abdulqader st, alam ak, al-azzawi lm, husein a, mahmood as. clinical healing response of soft tissue incisions made by diode laser on rabbits skin. international medical journal. 2013 aug;20(4): 502 504. https: //jglobal.jst.go.jp/en/ 21. kanber, ammar & kadhim, enas & neamah, ahmed. usefulness of achillea milefolium as healing topical agent in treating thermal burn (histological comparative study in westar rats). mustansiria dental journal. 2024 19. 144-153. 10.32828/mdj.v19i1.1006. 22. seeger ma, paller as. the roles of growth factors in keratinocyte migration. adv wound care (new rochelle). 2015 apr 1;4(4):213-224. doi: 10.1089/wound.2024.0540 expression of igf-1r in skin wound healing treated by topical applicauon of cinnamomum zeylanicum vol 13, no 1 (2025) doi 10.5195/d3000.2025.1011 http://dentistry3000.pitt.edu 5 table 1. comparison between experimental and control regarding igf-1r expression (stromal). periods score control ms sd exp. ms sd p-value no. % no. % 1 day 1 0 0 2.833 0.408 0 0 3.33 0.516 0.023 2 1 16.66 0 0 3 5 83.33 4 66.66 4 0 0 2 33.33 3 days 1 3 50 1.5 0.547 3 50 1.83 0.983 0.097 2 3 50 1 16.66 3 0 0 2 33.33 4 0 0 0 0 7 days 1 3 50 1.66 0.816 0 0 2.5 0.547 0.039 2 2 33.33 3 50 3 1 1.66 3 50 4 0 0 0 0 table 2. control groups in comparison to experimental groups regarding igf-1r expression (epidermal). period score control ms sd exp. ms sd p-value no. % no. % 3 days 1 0 0 2.5 0.547 0 0 3.16 0.752 0.036 2 3 50 1 16.66 3 3 50 3 50 4 0 0 2 33.33 7 days 1 0 0 2.66 0.516 0 0 3.66 0.516 0.041 2 2 33.33 0 0 3 4 66.66 2 33.33 4 0 0 4 66.66 821 final histological evalua.on of cutaneous wound healing treated by local applica.on of cucurbita pepo l. seed oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.821 h"p://den)stry3000.pi".edu histological evaluation of cutaneous wound healing treated by local application of cucurbita pepo l. seed oil ibrahim fouad mohamed1, ban a. ghani2, nada m.h. al-ghaban2, abdalbseet a. fatalla2, matheel al-rawas3 1 college of dentistry uruk university, iraq 2 college of dentistry university of baghdad, iraq 3 school of dental sciences, universiti sains malaysia, kubang kerian, malaysia abstract objec&ve: the rising demand for natural medicines in pharmaceu4cals has prompted scien4sts to inves4gate medicinal plants. pumpkin seed (cucurbita pepo l.) is a noteworthy candidate due to its remarkable pharmacological quali4es for possible wound healing therapies. methods: a total of twelve mature male new zealand rabbits weighing between 1.5 and 2 kg were subjected to uniform wounds on their dorsum. these rabbits were then split into two groups for healing dura4ons of 3 and 7 days, with 6 rabbits in each group. the induced wound in the control group was allowed to heal naturally, whereas the wounds in the experimental group were treated with a daily local applica4on of pumpkin oil (10µl). the animals were sacrificed aper healing 4mes of three and seven days. all examined groups underwent clinical assessment of wound contrac4on. specimens were obtained for histological and histomorphometric analysis to evaluate inflammatory cell and blood vessel counts, as well as epithelial thickness. results: the average percentage of wound contrac4on escalated over 4me. a notable change was found and a sta4s4cally significant disparity in the mean values of inflammatory cells was recorded. the mean values for blood vessel count rose with a notable disparity was noted between the control and experimental groups. the evalua4on of epithelial thickness indicated that mean values increased over 4me, with a significant difference in both experimental groups. conclusion: as a poten4al therapeu4c agent for wound healing, pumpkin oil may be also beneficial for nutri4onal and therapeu4c applica4ons. keywords: cucurbita pepo l. oil; wound healing; natural product. cita9on: mohamed if, et al. (2025) histological evalua9on of cutaneous wound healing treated by local applica9on of cucurbita pepo l. seed oil. den9stry 3000. 1:a001 doi:10.5195/d3000.2025.821 received: january 3, 2025 accepted: january 14, 2025 published: february 17, 2025 copyright: ©2025 mohamed if, et al. this is an open access ar9cle licensed under a crea9ve commons ayribu9on work 4.0 united states license. email: abdalbasit@codental.uobaghdad.edu.iq introduction the incidence of chronic wounds and their associated morbidity have rendered wound care progressively significant [1]. it is predicted that one to two percent of individuals in underdeveloped nations would experience chronic wounds at some stage in their lives [2]. the capacity for wound healing in the human body depends on various aspects, including the individual's overall health, the degree of tissue damage, and the proliferative capacity of cells [3]. wounds compromise the integrity of the skin, resulting in changes to the adjacent anatomical structures and their functions. the complex and dynamic process of wound healing is affected by various elements, including the host's general health, the immune system, and the local environment surrounding the wound [2]. the categorization of wound healing mostly relies on the therapeutic approach and the distinct attributes of the lesion. wound healing denotes the physiological process by which the skin repairs and restores itself after injury or damage. the stages of wound healing are commonly categorized as primary, secondary, and tertiary phases [3]. the effective regeneration of healthy, functional skin is a considerable challenge due to its intricate multilayered architecture and the organized organization of many cell types within the extracellular matrix [4]. in light of recent improvements in wound care treatments, there is a sustained interest in investigating alternative medicines sourced from natural origins, such as plant extracts, honey, and larvae [5]. histological evalua.on of cutaneous wound healing treated by local applica.on of cucurbita pepo l. seed oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.821 h"p://den)stry3000.pi".edu the increasing interest in healthpromoting foods is emphasizing specific things that have been used locally for millennia but have only recently attained global awareness [6]. pumpkin seed oil is utilized in traditional medical and culinary applications, but recent research indicates its utility in the pharmaceutical and cosmetic industries as well [7]. furthermore, multiple resources designate it as a possibly useful food, mostly due to its composition of pumpkin seeds, which encompass a range of advantageous components [8]. the pumpkin, scientifically known as cucurbita pepo l., is a climbing plant that is planted annually and blooms from july to september. the maturation of the seeds takes place between august and october [6]. pumpkin oil can be utilized as a cooking oil or a potential nutraceutical due to its abundant supply of several bioactive compounds with functional qualities [7]. the medical benefits of pumpkin seed oil, a viscous oil with a highly dichromatic appearance, have recently been the focus of research [8]. the majority of the attention that researchers have paid has been focused on the content and quantity of sterols, tocopherols, and fatty acids in pumpkin seed oil due to its beneficial health effects [9]. furthermore, pumpkin has garnered recognition as an outstanding safeguard against various ailments, including hypertension and cancer, due to its health benefits, including its antimicrobial, antioxidant, and anti-inflammatory properties [10]. evaluating the biochemical and oxidative stability characteristics of pumpkin seed oil would augment its value, particularly in the pharmaceutical, cosmetic, and culinary sectors [11]. material and methods the experimental methods complied with the ethical standards for animal research established by the college of dentistry at the university of baghdad (project number 834723). this cross-sectional study employed a sample of twelve adult male new zealand rabbits, with an average weight between 1.5 and 2 kg. each rabbit's dorsum was incised with two circular fullthickness wounds, utilizing a sterile biopsy punch with an 8 mm diameter; one wound served as a control and was permitted to heal spontaneously, while the other received daily applications of 10 µl of cucurbita pepo l. oil. the animals were individually positioned on a surgical bench and received general anesthesia by intramuscular injection. the anesthetic formulation comprised 80% ketamine (administered at a dosage of 40 mg/kg) and 20% xylazine (administered at a dosage of 5 mg/kg) [12]. the hair on the dorsal area was first clipped with a hair clipper, then a hair removal lotion was applied to eradicate any remaining hair. a 90% ethyl alcohol solution was utilized for skin disinfection. two circular excisional incisions were made on the dorsal skin of each rabbit, separated by a distance of 2 cm. a biopsy stapler punch with an 8mm diameter was utilized to accomplish this. the localized application of oil was executed with a micropipette. a volume of 10µl of cucurbita pepo l. oil was administered to the left wound of rabbits, while the right wound remained untreated to facilitate spontaneous healing. scarification was conducted on animals at specific intervals (3 and 7 days) to promote the healing process. the animals had daily examinations to assess the induced wound's dimensions utilizing a vernier caliper for assessing wound contraction. samples were subsequently acquired and submerged in a 10% freshly made formalin solution for 24 hours. furthermore, histological and histomorphometric analyses were performed to evaluate the histological evalua.on of cutaneous wound healing treated by local applica.on of cucurbita pepo l. seed oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.821 h"p://den)stry3000.pi".edu presence of inflammatory cells and quantify the thickness of the epithelial layer and the count of blood vessels. the statistical analysis was conducted utilizing spss (statistical package for the social sciences) software version 26, employing descriptive statistics and inferential statistics through an independent paired t-test to compare the two groups. results histological findings three days histological analysis of the skin portion from the control group at the wound site reveals a scab on the surface, new epithelial tissue, hair follicles, and the presence of inflammatory cells, as illustrated in figure 1. the microphotographic examination of the experimental group reveals a newly formed epithelium covering the wound surface, a limited presence of inflammatory cells, and the emergence of new hair follicles within the dermis, as illustrated in figure 2. seven days a microphotograph of the wound site in the control group exhibits complete epithelialization at the surface, remodeled collagen fibers, hair follicles, and blood vessels, as depicted in figure 3. the histological examination of the experimental group reveals a thickened, freshly created epithelium at the wound surface, whereas the dermis exhibits remodeled collagen fibers and blood vessels, as illustrated in figure 4. statistical analysis estimation of wound contraction the formula for determining the percentage of wound closure was as follows: ((l1l2) / l1) x 100, where l1 represents the length of the wound on day 0 and l2 represents the length of the wound on the day of observation [13]. the assessment of wound contraction was performed in millimeters (mm). the findings of the present study demonstrate that the measured rate of wound figure 1. view of wound site showed scab (sc), new epithelium (ne), inflammatory cells (arrow), blood vessels (bv). h&ex10. figure 2. view of experimental group showed new epithelium (ne), inflammatory cells (arrows), hair follicles (hf), collagen fibers (cf). h&ex10. figure 3. view of control group showed, new epithelium (ne), collagen fibers (cf), blood vessels (arrows). h&ex10. figure 4. view of experimental group amer 7 days showed new epithelium (ep), collagen fibers (cf), blood vessels (arrows). h&ex10. histological evalua.on of cutaneous wound healing treated by local applica.on of cucurbita pepo l. seed oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.821 h"p://den)stry3000.pi".edu contraction showed a continuous increase over the duration of the research, as illustrated in table 1. the investigation entailed observing the extent of wound contraction during the healing process for each group examined. the control groups demonstrated a minimal mean value for wound contraction, while the treated group achieved its maximum mean value on day 7. inflammatory cell parameter the average inflammatory cell counts were recorded during the healing periods for all studied groups by enumerating the inflammatory cells in five fields. cells were enumerated using a light microscope equipped with a square grid in one eyepiece, under a magnification of 40x, and the average cell count was documented for all healing intervals [14]. table 2 displays the descriptive statistics for the inflammatory cell count data gathered at different healing intervals. the results demonstrate that the mean values were significantly lower in the experimental groups compared to the control groups on days 3 and 7. epithelial thickness parameter the estimation was conducted using a power x20 lens. to determine the epithelial thickness, the distance between the outermost keratin layer and the innermost basal layer of the epidermis at the wound borders was measured, averaging two readings using image j software. the results revealed that the mean epithelial thickness exhibited a gradual increase over time across all examined groups, as shown in table 3. moreover, the measured thickness was consistently greater in the experimental groups with significant outcomes than in the control group. blood vessel count the procedure was executed utilizing image j software. the numerical density of blood vessels was assessed using a light microscope at 40x magnification in a 45 µm² area across three fields. the average number of blood vessels was documented. the results are shown in table 4. the experimental group exhibited the highest mean blood vessel values over the 7-day healing period, whereas the control group had the lowest mean value during the 3day healing period. duration group n mean± sd min max t test p-value 3 day control 6 12.1% ± 1.5 9.8% 14.1% -2.3 0.03* experimental 6 14.9% ± 2.4 11.1% 18.5% 7 day control 6 42.8% ± 6.1 36.4% 51.2 -3.09 0.01* experimental 6 55.7% ± 8.2 43.9% 65.4% table 1. descrip/ve sta/s/cs of wound contrac/on in each healing dura/on. table 2. descrip/ve sta/s/cs of inflammatory cell count in each healing. dura/on. duration group n mean± sd min max t test p-value 3 day control 6 52.6 ± 6.5 40.5 58.1 2.28 0.04* experimental 6 43.1 ± 7.7 33.5 56.4 7 day control 6 65.9 ± 7.9 54.3 72.8 7.08 0.000* experimental 6 39.1 ± 4.8 32.4 45.2 table 3. descrip/ve sta/s/cs of epithelial thickness in each healing dura/on. duration group n mean± sd min max t test p-value 3 day control 6 5.2 ± 1.2 3.8 7.3 -3.1 0.01* experimental 6 7.3 ± 0.9 5.8 8.4 7 day control 6 10.2 ± 1.4 7.8 11.6 5.009 0.001* experimental 6 15.6 ± 2.2 12.5 18.3 histological evalua.on of cutaneous wound healing treated by local applica.on of cucurbita pepo l. seed oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.821 h"p://den)stry3000.pi".edu discussion wound healing is a systematic sequence of actions designed primarily to restore the skin's barrier and protective function to avert more blood loss and infection [15]. the wound healing process encompasses inflammation, re-epithelialization, granulation, and angiogenesis, ultimately resulting in wound contraction [16]. accelerating wound healing while minimizing side effects is a primary objective of contemporary medicine [17]. natural herbal therapies have become essential in the management of dermatological conditions and infections, attributed to the adverse effects of contemporary medication and the cost-effectiveness of herbal medicines [18]. medicinal plants may promote wound healing owing to their diverse array of components and phytochemicals, including alkaloids, flavonoids, fatty acids, terpenoids, saponins, and phenolic chemicals that can augment the healing process [19]. phytochemicals can affect multiple phases of the wound-healing process by diverse methods, including the overexpression of tgf-β, vascular endothelial growth factor (vegf), monocyte chemoattractant protein-1 (mcp1), and interleukin-1 (il-1), alongside the downregulation of nitric oxide (no) and reactive oxygen species (ros) [20]. the oil extracted from cucurbita pepo seeds is extensively utilized as a traditional remedy due to its antibacterial and antioxidant qualities, providing a novel, safe, and cost-effective alternative to commercial wound healing treatments [21]. in contrast to the control groups, which had distal wound borders, the oil-treated groups demonstrated a more pronounced epithelial layer. there exists a significant reduction in healing time was observed in the treated groups relative to the untreated group, in accordance with the study conducted by kamil and al-ghaban in 2019 [22]. between the 1st and 7th day of wound induction, cutaneous wound healing demonstrated that both treatment groups had enhanced wound healing, as reflected by the increased wound closure percentages compared to the control group on days 3 and 7. this outcome aligns with rezk et al. (2023), who utilized a pumpkin seed oil-infused chitosan/polyvinyl alcohol electrospun nanofiber scaffold utilized as a cutaneous and oral wound dressing [23]. the findings suggest that pumpkin-infused nanofibers possess promise for dermal and oral wound healing. they offer an innovative, secure, and cost-effective alternative to traditional wound healing methods. the findings emphasize the integration of natural botanicals and advancing nanoscience for biological purposes, especially in wound healing [23]. an exaggerated inflammatory rim was more prominent around the untreated wound compared to the treated wounds, and the inflammatory cell count was reduced in the treated group relative to the untreated group. this finding can be attributed to the antitable 4. descrip/ve data of blood vessel count over various healing. dura/ons. duration group n mean± sd min max t test pvalue 3 day control 6 4.06 ± 0.9 2.6 5.4 -2.4 0.03* experimental 6 5.2 ± 1.2 3.9 7.1 7 day control 6 8.4 ± 1.09 7.1 9.3 -2.6 0.02* experimental 6 11.1 ± 2.13 8.2 12.5 histological evalua.on of cutaneous wound healing treated by local applica.on of cucurbita pepo l. seed oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.821 h"p://den)stry3000.pi".edu inflammatory and antibacterial properties of pumpkin oil [24]. the count of blood vessels and the thickness of the epithelium in the treated group are significantly greater than in the control group. increasing evidence indicates that a humid environment facilitates wound healing via multiple mechanisms, such as enhancing re-epithelialization, angiogenesis, keratinocyte migration, and activating hypoxia-inducible factor-1 (hif-1), which leads to the generation of endogenous wound healing stimulants [25]. bardaa et al. (2016) in their in vivo investigation demonstrates that pumpkin seed oil significantly surpassed the control group treated with cicaflora cream, which contains 10% mimosa tenuiflora extract, in the healing of skin wounds. the cohort receiving pumpkin oil achieved complete reepithelialization [26]. this study illustrates that cold-pressed pumpkin seed oil provides a substantial source of many advantageous constituents, including antioxidants and antibacterial compounds. the presence of tocopherols, sterols, and polyunsaturated fatty acids in pumpkin oil makes it a remarkable medicinal and cosmetic substance, perhaps providing protection against dermatological conditions, such as skin wounds [27]. our results indicated that wound healing in rabbits administered pumpkin oil extract was superior to that in the untreated or reference groups, as evidenced by macroscopic, morphometric, and histological data, corroborating the findings of wargala et al [28]. conclusion pumpkin oil (cucurbita pepo l.) is a potential therapeutic agent for wound healing in animal models. overall, pumpkin oil is recommended for nutritional and therapeutic objectives. conflicts of interest the authors declare no competing interest. references 1. chronic wounds. falanga v, isseroff rr, soulika am, romanelli m, margolis d, kapp s, et al. nat rev dis primer 2022, 8(1):50. available from: https://www.nature.com/articl es/s41572-022-00377-3 2. human wounds and its burden: an updated compendium of estimates. sen ck. adv wound care 2019, 8(2):39–48. available from: https://www.liebertpub.com/d oi/10.1089/wound.2019.0946 3. recent advances in molecular mechanisms of skin wound healing and its treatments. mamun aa, shao c, geng p, wang s, xiao j. front immunol 2024, 15:1395479. available from: https://www.frontiersin.org/ar ticles/10.3389/fimmu.2024.13 95479/full 4. a review of biomacromoleculebased 3d bioprinting strategies for structure-function integrated repair of skin tissues. liu h, xing f, yu p, zhe m, duan x, liu m, et al. int j biol macromol 2024, 268:131623. available from: https://linkinghub.elsevier.co m/retrieve/pii/s014181302402 4280 5. a review of current advancements for wound healing: biomaterial applications and medical devices. deng x, gould m, ali ma. j biomed mater res b appl biomater 2022, 110(11):2542– 73. available from: https://onlinelibrary.wiley.com /doi/10.1002/jbm.b.35086 6. characterization of cultivated pumpkin (cucurbita moschata duchesne) landraces for genotypic variance, heritability and agro-morphological traits. ezin v, gbemenou uh, ahanchede a. saudi j biol sci 2022, 29(5):3661–74. available from: https://linkinghub.elsevier.co m/retrieve/pii/s1319562x2200 1401 7. a comprehensive review of functional ingredients, especially bioactive compounds present in pumpkin peel, flesh and seeds, and their health benefits. hussain a, kausar t, sehar s, sarwar a, ashraf ah, jamil ma, histological evalua.on of cutaneous wound healing treated by local applica.on of cucurbita pepo l. seed oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.821 h"p://den)stry3000.pi".edu et al. food chem adv 2022, 1:100067. available from: https://linkinghub.elsevier.co m/retrieve/pii/s2772753x2200 0557 8. pumpkin and pumpkin byproducts: a comprehensive overview of phytochemicals, extraction, health benefits, and food applications. gavril (rațu) rn, stoica f, lipșa fd, constantin oe, stănciuc n, aprodu i, et al. foods 2024, 13(17):2694. available from: https://www.mdpi.com/23048158/13/17/2694 9. pumpkin seed oil: a comprehensive review of extraction methods, nutritional constituents, and health benefits. hu z, hu c, li y, jiang q, li q, fang c. j sci food agric 2024, 104(2):572–82. available from: https://onlinelibrary.wiley.com /doi/10.1002/jsfa.12952 10. sustainable applications of phytochemicals and nutritive components derived from selected underutilized seeds: a review. manianga a, bose c, s s. acta sci pol technol aliment 2024, 87–122. available from: https://www.food.actapol.net/ volume23/issue1/8_1_2024.pd f 11. valorization of seeds of the genera cucumis, citrullus, and cucurbita. in: valorization of agri-food wastes and byproducts. nunes p, bhat r. elsevier; 2021, p. 317–329. available from: https://linkinghub.elsevier.co m/retrieve/pii/b97801282404 41000167 12. a comparative study of the cardiopulmonary and sedative effects of a single intramuscular dose of ketamine anesthetic combinations in rabbits. cardoso cg, ayer im, jorge at, honsho cs, mattos-junior e. res vet sci 2020, 128:177–82. available from: https://linkinghub.elsevier.co m/retrieve/pii/s003452881930 3509 13. thymoquinone-loaded polymeric films and hydrogels for bacterial disinfection and wound healing. haq a, kumar s, mao y, berthiaume f, michniak-kohn b. biomedicines 2020, 8(10):386. available from: https://www.mdpi.com/22279059/8/10/386 14. histological evaluation of local application of flavonoid extract of capparis spinosa on wound healing in alloxaninduced diabetic rats. hameed alwan m, a ghani b. diyala j med 2020, 18(2):1–13. available from: http://djm.uodiyala.edu.iq/ind ex.php/djm/article/view/526 15. molecular dambusters: what is behind hyperpermeability in bradykinin-mediated angioedema? debreczeni ml, németh z, kajdácsi e, farkas h, cervenak l. clin rev allergy immunol. 2021, 60(3):318–347. available from: https://link.springer.com/10.1 007/s12016-021-08851-8 16. a review of the contribution of mast cells in wound healing: involved molecular and cellular mechanisms. komi dea, khomtchouk k, santa maria pl. clin rev allergy immunol 2020, 58(3):298–312. available from: http://link.springer.com/10.10 07/s12016-019-08729-w 17. nanomaterials applied in wound healing: mechanisms, limitations and perspectives. wang m, huang x, zheng h, tang y, zeng k, shao l, et al. j controlled release. 2021, 337:236–47. available from: https://linkinghub.elsevier.co m/retrieve/pii/s016836592100 3606 18. promising natural products in new drug design, development, and therapy for skin disorders: an overview of scientific evidence and understanding their mechanism of action. mohd zaid na, sekar m, bonam sr, gan sh, lum pt, begum my, et al. drug des devel ther 2022, 16:23–66. available from: https://www.dovepress.com/p romising-natural-products-innew-drug-designdevelopment-and-therapy-peer-reviewed-fulltext-articledddt 19. a systematic review on biochemical perspectives on natural products in wound healing: exploring phytochemicals in tissue repair and scar prevention. raza a, chohan ta, zaidi shh, hai a, alzahrani ar, abida, et al. chem biodivers 2024, histological evalua.on of cutaneous wound healing treated by local applica.on of cucurbita pepo l. seed oil vol 13 no 1 (2025) doi 10.5195/d3000.2025.821 h"p://den)stry3000.pi".edu e202400615. available from: https://onlinelibrary.wiley.com /doi/10.1002/cbdv.202400615 20. exploring the underlying pharmacological, immunomodulatory, and antiinflammatory mechanisms of phytochemicals against wounds: a molecular insight. riaz a, ali s, summer m, noor s, nazakat l, aqsa, et al. inflammopharmacology 2024, 32(5):2695–727. available from: https://link.springer.com/10.1 007/s10787-024-01545-5 21. a comparative assessment of anti-inflammatory, antioxidant and anti-bacterial activities of hybrid and indigenous varieties of pumpkin (cucurbita maxima linn.) seed oil. amin mz, rity ti, uddin mr, rahman mdm, uddin mj. biocatal agric biotechnol 2020, 28:101767. available from: https://linkinghub.elsevier.co m/retrieve/pii/s187881812031 0173 22. evaluation of effect of local exogenous application of myrrh oil on healing of wound incisions of facial skin (histochemical, histological and histomorphometrical study in rabbits). kamil nb, alghaban nm. j baghdad coll dent 2019, 31(4). available from: http://jbcd.uobaghdad.edu.iq/ index.php/jbcd/article/view/2 724 23. pumpkin seed oil -loaded chitosan/polyvinyl alcohol electrospun nanofiber scaffold for dermal and oral wound dressing. rezk my, ibrahim s, khalil ea, saba d, abdellatif m, abdellatif a, et al. chemistry select 2023, 8(26):e202300722. available from: https://chemistryeurope.onlinelibrary.wiley.com /doi/10.1002/slct.202300722 24. antimicrobial properties, cytotoxic effects, and fatty acids composition of vegetable oils from purslane, linseed, luffa, and pumpkin seeds. petropoulos sa, fernandes â, calhelha rc, rouphael y, petrović j, soković m, et al. appl sci 2021, 11(12):5738. available from: https://www.mdpi.com/20763417/11/12/5738 25. the epidermis in microgravity and unloading conditions and their effects on wound healing. bacci s, bani d. front bioeng biotechnol 2022, 10:666434. available from: https://www.frontiersin.org/ar ticles/10.3389/fbioe.2022.666 434/full 26. the evaluation of the healing proprieties of pumpkin and linseed oils on deep seconddegree burns in rats. bardaa s, moalla d, ben khedir s, rebai t, sahnoun z. pharm biol 2016, 54(4):581–7. available from: http://www.tandfonline.com/ doi/full/10.3109/13880209.20 15.1067233 27. antioxidant activity and other quality parameters of cold pressing pumpkin seed oil. kulaitienė j, černiauskienė j, jarienė e, danilčenko h, levickienė d. not bot horti agrobot cluj-napoca 2018, 46(1):161–6. available from: https://www.notulaebotanicae .ro/index.php/nbha/article/vie w/10845 28. pumpkin (cucurbita pepo l.) seed oil – cosmetic, food and medical raw material. wargala e, chrzanowska a, bernateksamoraj w, kot i. herba pol 2023, 69(3):7–14. available from: https://herbapolonica.publishe rspanel.com/gicid/01.3001.00 53.8859 772 final anatomical changes related to age in maxillary sinus vol 13 no 1 (2025) doi 10.5195/d3000.2025.772 h#p://den*stry3000.pi#.edu anatomical changes related to age in maxillary sinus noor ghazi saab college of dentistry, university of tikrit, iraq abstract objec&ve: the paranasal sinuses consist of four paired cavi=es: maxillary, ethmoid, frontal, and sphenoid, which are air-filled, mucosa-lined spaces within the maxillofacial region, connected to the nasal cavity. the maxillary sinus, the largest of these, occupies the body of the maxilla and has a pyramidal shape. methods: in this study, cbct scans from 35 subjects (18 males and 17 females) who visited the university dental hospital in tikrit and a private cbct center in samarra were analyzed. subjects were categorized into four groups (a, b, c, and d). measurements focused on determining the dimensions of the right maxillary sinus, including height (distance from the superior wall to the inferior wall in the coronal plane), width (distance from the lateral to the medial wall), and depth (antero-posterior distance in the axial plane). mean values for each dimension were calculated. results: the study results revealed that the length and height of the right maxillary sinus (rms) increased up to group c and then began to decline in group d. male subjects showed larger sinus dimensions compared to females. in females, the maxillary sinus length and height were variable across groups a and b, with an increase in group c followed by a decrease in group d, while width showed an ini=al fill in group a and then a gradual decline through groups b, c, and d. in males, length, width, and height of the sinus increased in groups a and b, peaked in group c, and declined in group d. keywords: maxillary sinus age changes, cbct maxillary sinus measurements, maxillary sinus dimensions by age, gender differences in sinus size cita9on: saab ng. (2025) anatomical changes related to age in maxillary sinus. den9stry 3000. 1:a001 doi:10,5195/d3000.2025.772 received: november 14, 2024 accepted: december 20, 2024 published: february 10, 2025 copyright: ©2025 saab ng. this is an open access ar9cle licensed under a crea9ve commons asribu9on work 4.0 united states license. email:noor.gsaab@tu.edu.iq introduction the paranasal sinuses, comprising maxillary, ethmoid, frontal, and sphenoid sinuses, are air-filled, mucosa-lined cavities in the maxillofacial region that communicate with the nasal cavity [1,2]. initially illustrated by leonardo da vinci in 1489 and later detailed by anatomist nathaniel highmore in 1651, the maxillary sinus—or antrum of highmore—is the largest and earliest to develop among the paranasal sinuses [3]. it occupies the maxilla, with its floor formed by the alveolar process, which supports the dentition, particularly the second premolar and first molar roots [4]. anatomically, the maxillary sinus has a pyramidal shape with a medial base adjacent to the nasal cavity, an apex extending into the zygomatic process, and a capacity of approximately 15 ml in adults, making it the largest paranasal sinus [5]. bone expansion during paranasal sinus development occurs at varying rates and can result in significant variability in sinus shape and size [6,7]. with age, sinus volume increases until around 20 years of age before gradually declining, a process further influenced by tooth loss and aging [8]. the purpose of this study was to investigate the agerelated changes in dimensions of the maxillary sinus, a study using computed tomography [9]. material and methods subjects cbct scans were analyzed from 93 subjects, comprising 49 males and 44 females, who presented to the university dental hospital in sharjah. the cbct images were acquired using the galileos, sirona cbct dental system (bensheim, germany) with an sidexis operating system set at 85 kvp and anatomical changes related to age in maxillary sinus vol 13 no 1 (2025) doi 10.5195/d3000.2025.772 h#p://den*stry3000.pi#.edu 7 ma. assessment was conducted directly on a 1920 x 1080 pixel, 23inch dell monitor. this dataset, obtained from the university dental hospital at the university of sharjah, included measurements of six specific features of the paranasal sinuses for both male and female subjects [10,11]. the study encompassed patients aged 20 to 70 years. cbct scans from edentulous subjects, those with a history of mid-facial trauma, maxillary sinus cysts or tumors, or scans with incomplete coverage of the maxillary sinus area, were excluded [12]. to minimize observer bias, two experienced maxillofacial radiologists, each with 10 years of clinical practice, evaluated the sinus dimensions. mean values for each measurement were calculated, with assessments repeated after 15 days to control for intra-observer variability. additionally, cbct scans from 35 subjects (18 males and 17 females) who presented to the university dental hospital in tikrit and a private cbct center in samarra were reviewed. these scans were acquired using the cs 8200 3d (12 x 10 edition) and assessed on the same dell monitor specifications as in sharjah. this subset included subjects aged 20 to 60 years and followed similar exclusion criteria. sample grouping only data from the right maxillary sinus were included in this study due to incomplete data for the left maxillary sinus, which led to its exclusion. data collection was organized using a chart (refer to figure 2). subjects were categorized into age-based groups, with each group comprising both male and female participants, as follows: group (a): ages 20-29 years group (b): ages 30-39 years group (c): ages 40-49 years group (d): ages 50-59 years statistical analysis the mean value for each measurement was calculated. for statistical analysis, an independent samples t-test was employed to compare means between males and females, while an anova test was used to evaluate mean differences across age groups. in all tests, a p-value of less than 0.05 was considered statistically significant. data organization was conducted in microsoft excel 2020, with subsequent analysis in spss version 27[13]. exclusion criteria 1. patients with a history of midfacial trauma. 2. presence of tumors or cysts in the maxillary sinus. 3. cbct scans lacking complete coverage of the maxillary sinus area were excluded from the study. figure 1. x-ray computed tomography. figure 2. chart for data collection. measurements the measurements of the right maxillary sinus were conducted on cbct sections to ensure precision and consistency, with measurements taken parallel or anatomical changes related to age in maxillary sinus vol 13 no 1 (2025) doi 10.5195/d3000.2025.772 h#p://den*stry3000.pi#.edu perpendicular to the hard palate as specified. 1. height: the height of the maxillary sinus was measured in the coronal cbct section, perpendicular to the hard palate. this distance extended from the uppermost point of the superior wall to the lowest point of the inferior wall, as illustrated in figure 3a. 2. width: the width of the right maxillary sinus was assessed in the coronal cbct section, also parallel to the hard palate. this measurement spanned from the outermost point on the lateral wall to the medial wall of the maxillary sinus, as shown in figure 3a. 3. depth: the depth of the sinus was measured on the axial cbct section as the longest anteroposterior distance, as depicted in figure 3b. figure 3. measurements on cbct images. • a: measurement of the height and width of the maxillary sinus on the coronal cbct section. • b: measurement of the depth (length) of the maxillary sinus on the axial cbct section. results the distance from the uppermost point of the superior wall to the lowest point of the inferior wall of the maxillary sinus was measured in the coronal section, oriented perpendicular to the hard palate. the width of the right maxillary sinus was also assessed in the coronal cbct section, parallel to the hard palate, spanning from the outermost point on the lateral wall to the medial wall. depth was measured in the axial cbct section as the longest antero-posterior distance. mean values for each measurement were calculated for each age group, separately for males and females, with measurements taken exclusively from the right side. ms measurements in different age groups mean and standard deviation for the length, width and height in both sexes of different age groups are shown in table 1. table 1. average measurements of right maxillary sinus parameters between age groups (n=35) in (mm). anatomical changes related to age in maxillary sinus vol 13 no 1 (2025) doi 10.5195/d3000.2025.772 h#p://den*stry3000.pi#.edu figure 4. length of ms by age groups. figure 5. width of ms by age groups. figure 6. height of ms by age groups. msm in both sexes related to aging table 2 revealed that maxillary sinus length of females in ga was (35.3+2.4), gb was (34.0 + 2.5), gc was (36.2+4.1), and finally gd was (35.4+ 3.0). the present study showed that all the dimensions of ms in males were larger than in females. there was no significant difference between groups of males and females (p>0.05). table2. average measurements (in mm) of right maxillary sinus parameters between sex and age groups (n=35). variable ga 20-29 gb 30-39 gc 40-49 gd 50-59 p-value mean sd mean sd mean sd mean sd length 37.1 3.8 37.0 5.5 38.8 4.7 36.2 3.2 >0.05 width 23.9 4.5 24.2 3.7 23.6 2.8 24.2 2.6 >0.05 height 33.3 5.5 33.2 5.9 34.2 7.2 31.2 6.8 >0.05 anatomical changes related to age in maxillary sinus vol 13 no 1 (2025) doi 10.5195/d3000.2025.772 h#p://den*stry3000.pi#.edu discussion in this study, cbct scans of 100 patients (72 males and 28 females) aged 1 to 90 years were examined at the radiology department of vydehi institute of medical science & research centre, bangalore, focusing on coronal and axial sections of the maxillary sinuses [14,15]. volume and dimensions, along with anatomical variations, were assessed, with mean values, standard deviations (sd), and significant differences across age and gender calculated. notably, the greatest sinus growth in males occurred in the 11-20 and 41-50 age groups for height, width, and volume, while depth peaked in the 21-30 and 31-40 age groups. growth generally decreased after 61-70 years in height and further in 81-90 years across depth, width, and volume. these findings align partially with the current study, where maxillary sinus (ms) length, width, and height in males peaked in group c (40-49 years) before decreasing in group d (50-59 years). in females, the highest growth was observed in the 21-30 and 51-70 age ranges for height, depth, and volume, with width peaking in the 11-20 and 51-60 age groups, followed by a decrease in all dimensions post-61 years, which also partially corresponds to our findings showing height and length increases in group a (20-29 years) and declines by group d. a retrospective observational study at royal care hospital, khartoum, sudan [16,17]. evaluated ct scans of sudanese adults (ages 17-78 years, 46 males and 35 females) with normal paranasal sinuses (pns). the study found a negative correlation between age and maxillary sinus volume, with sinus dimensions decreasing in both genders as age advanced. this aligns with our findings, where the right maxillary sinus (rms) length and height peaked in group c and decreased in group d. however, the sudanese study noted no significant gender differences in sinus volume, a contrast to the present study where all maxillary variable age group p-value ga 29 gb 30-39 gc 40-49 gd 50-59 mean sd mean sd mean sd mean sd sex female (n=17) length 35.3 2.4 34.0 2.5 36.2 4.1 35.4 3.0 >0.05 width 23.6 4.9 23.1 5.4 22.5 2.0 22.1 1.9 >0.05 height 29.6 3.8 29.5 3.0 32.5 7.5 27.6 2.5 >0.05 male (n=18) length 38.4 4.3 39.3 6.3 42.7 2.0 37.4 3.7 >0.05 width 24.1 4.6 25.0 1.7 25.2 3.7 23.1 3.3 >0.05 height 36.0 5.0 36.2 6.1 36.7 8.5 35.9 8.3 >0.05 anatomical changes related to age in maxillary sinus vol 13 no 1 (2025) doi 10.5195/d3000.2025.772 h#p://den*stry3000.pi#.edu sinus dimensions were larger in males than females [18]. another study of 200 patients at dicle university, faculty of dentistry, department of oral and maxillofacial radiology, involved cbct images analyzed via mimics software, categorizing patients into five age groups [19]. results revealed a significant male-female difference in maxillary sinus volume, with volumes diminishing with age—findings that align with the current study’s conclusions [20]. finally, an analysis of 133 individuals (84 females, 49 males) aged 8 to 51 years assessed maxillary sinus volume (msv) via mimics 21.0 software, with spss 21.0 used for statistical analysis. while this study found no significant relationship between gender, age, and maxillary sinus volume, differing from our results, which show male measurements consistently larger and rms length and height peaking in group c before declining in group d [21,23]. recommendations 1. increase the interval of samples to study younger and older age groups. 2. using longitudinal study by taking groups of childes and take their morphometric measurements then after a period take the measurements of the same people and study their aging changes. conflicts of interest the authors declare no competing interest. references 1. whyte a, boeddinghaus r. the maxillary sinus: physiology, development and imaging anatomy. dentomaxillofacial radiology. 2019;8. 2. abd-alla ma, mahdi ajj. maxillary sinus measurements in different age groups of human cadavers. tikrit journal for dental sciences. 2013. 3. grujičić r, myplinaios d. author: roberto grujičić • reviewer: dimitrios myplinaios last reviewed. 2023. 4. neck n, paranasal sinuses zj, minutello km, arthur b. author informapon and affiliapons last update. 2023. 5. beom k. the analysis of maxillary sinus aerapon according to aging process; volume assessment by 3dimensional reconstrucpon by high-resoluponal ct scanning. 6. human maxillary sinus development, pneumapzapon, anatomy, blood supply, innervapon and funcponal theories: an update review. 7. hamdy rm, abdel-wahed n. three-dimensional linear and volumetric analysis of maxillary sinus pneumapzapon. 8. mokhasi g, varsha, prakash r, swapnali h, kuma m. measurements of maxillary sinus in correlapon to age and gender by computed tomography. 9. volumetric measurement of the maxillary sinuses in normal sudanese using computed tomography: a retrospecpve study. 10. belgin ca, colak m, adiguzel o, akkus z, orhan k. three-dimensional evaluapon of maxillary sinus volume in different age and sex groups using cbct. 11. gulec m, tassoker m, magat g, lale b, ozcan s, orhan k. threedimensional volumetric anatomical changes related to age in maxillary sinus vol 13 no 1 (2025) doi 10.5195/d3000.2025.772 h#p://den*stry3000.pi#.edu analysis of the maxillary sinus: a cone-beam computed tomography study. folia morphol (warsz). 2020;79(3):557– 62. available from: htp://dx.doi.org/10.5603/ fm.a2019.0106 12. kadham sm, mustafa ma, abbass nk, karupusamy s. iot and arpficial intelligence–based fuzzyintegral n-transform for sustainable groundwater management. appl geomat. 2024;16(1):1-8. 13. ali sh, armeet hs, mustafa ma, ahmed mt. complete blood count for covid-19 papents based on age and gender. in: aip conference proceedings. aip publishing; 2022 nov. vol. 2394, no. 1. 14. shakir om, abdulla kk, mustafa aa, mustafa ma. invespgapon of the presence of parasites that contaminate some fruits and vegetables in the samarra city in iraq. plant arch. 2019;19:1184-90. 15. abdulqader at, alsammarie amy, mustafa ma. a comparapve environmental study of aqueous extracts of ginger and grapes to protect hepatocytes in albino rabbits and a comparison of extracts in preserving awassi lamb meat from oxidapon. in: iop conference series: earth and environmental science. iop publishing; 2022 may. vol. 1029, no. 1. p. 012001. 16. kadham sm, mustafa ma, abbass nk, karupusamy s. comparison between fuzzy parpal h-transform and fuzzy parpal laplace transform in x-ray images processing of acute intersppal pneumonia. int j syst assur eng manag. 2023;1-9. 17. meri ma, ibrahim md, alhakeem ah, mustafa ma. procalcitonin and nlr measurements in covid-19 papents. lat am j pharm. 2023;220-3. 18. mustafa ma, raja s, asadi laa, jamadon nh, rajeswari n, kumar ap. a decision-making carbon reinforced material selecpon model for composite polymers in pipeline applicapons. adv polym technol. 2023;2023(1):6344193. 19. valluru d, mustafa ma, jasim hy, srikanth k, rajarao mvln, sreedhar pss. an efficient classroom teaching learning method using augmented reality. in: 2023 9th internaponal conference on advanced compupng and communicapon systems (icaccs). ieee; 2023 mar. p. 300-3. 20. mustafa ma, kadham sm, abbass nk, karupusamy s, jasim hy, alreda ba, et al. a novel fuzzy m-transform technique for sustainable groundwater level predicpon. appl geomat. 2024;16(1):9-15. 21. hsu cy, mustafa ma, yadav a, batoo km, kaur m, hussain s, et al. n2 reducpon to nh3 on surfaces of co-al18p18, nial21n21, fe-b24n24, mnb27p27, ti-c60 and cu-si72 catalysts. j mol model. 2024;30(3):1-11. 22. yaseen ah, khalaf at, mustafa ma. lung cancer data analysis for finding gene expression. afr j biol sci. 2023;5(3):119-30. 23. lu zf, hsu cy, younis nk, mustafa ma, matveeva ea, al-juboory yho, et al. anatomical changes related to age in maxillary sinus vol 13 no 1 (2025) doi 10.5195/d3000.2025.772 h#p://den*stry3000.pi#.edu exploring the significance of microbiota metabolites in rheumatoid arthrips: uncovering their contribupon from disease development to biomarker potenpal. apmis. 2024. 950 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.950 http://dentistry3000.pitt.edu expression of tgfb3 in pyogenic granuloma asmaa ali hussein college of medicine, university of fallujah, anbar, iraq abstract objec0ve: the aim of the study was to determine the expression of tgfb3 in pyogenic granuloma. material and methods: pathologist used a double-headed light microscope to calculate cutoff values for all the antbodies employed in the investgaton. stained slides were thoroughly washed for 4 minutes with gill's hematoxylin soluton, dehydrated, and mounted using dpx mountng soluton. results: transforming growth factor beta-3 protein expression was measured using immunohistochemistry. in pyogenic granuloma tssues expression, negatve scores (0) were found in 4.5 of 16 samples (18.1%), whereas positve values (+) were found in 10 of 16 samples (33.06%). conclusion: tgfb3 is expressed in most pyogenic granulomas. open access cita%on: hussein aa. (2025) expression of tgfb3 in pyogenic granuloma. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.950 received: may 25, 2025 accepted: june 27, 2025 published: august 13, 2025 copyright: ©2025 hussein aa. this is an open access ar%cle licensed under a crea%ve commons atribu%on work 4.0 united states license. email: mmss2006@uofallujah.edu.iq introduc)on pyogenic granuloma is a benign tumor that grows in the mouth or skin tissues. because of the vascular effects of female hormones, it mostly affects young girls in their second decade of life. proliferation of granulation tissue with inflammatory infiltration and high angiogenic potential is the most prevalent form of hyperplasia in the mouth. this uncommon disease necessitates accurate diagnosis and treatment, particularly during pregnancy [1,2]. pyogenic granuloma is a lesion that responds to a variety of low-level stimuli, including recurrent trauma, aggressions, hormonal variables, and some medications. high levels of estrogen and progesterone during pregnancy are linked to a higher occurrence of this lesion. lesions are more prevalent in the upper jaw, anterior regions, and gingiva's vestibular zone [3,4]. the gums are the most common site (75% of cases), although it can also occur on the lips, tongue, oral mucosa, and palate. in diverse cells and tissues throughout the body, the tgf-beta superfamily of proteins has a variety of often conflicting actions. tgf-3 (transforming growth factor beta-3) is a protein produced by the tgfb3 gene. this protein may be found across the body and is necessary for development both before and after birth. to fulfill its responsibilities, it connects (binds) to cell surface receptor proteins. this binding initiates signal transmission inside the cell, controlling a variety of cellular functions [5,6]. tgf-beta-3 is a protein that controls cell differentiation and embryogenesis. different stages are involved in maturation into mature form: latency-associated peptide (lap) and transforming growth factor beta-3 (tgf -beta) chains remain non-covalently connected after cleavage of the proprotein in the golgi apparatus [7,8]. the effects of tgfon bone resorption vary depending on the target cell and the settings of the experiment. tgf stimulates the replication of osteoblastic cells and bone production in mice, but it does not promote osteoblastosis [9,10]. the primary objective of this research was to investigate the expression levels of transforming growth factor (tgf) protein in pyogenic granuloma, aiming to better understand its potential role in the lesion's pathogenesis. material and methods after a yirst wash with washing buffer, all the slides were coated with peroxidase-blocking reagent and incubated for 10 minutes. after allowing the slides to cool for 20 minutes at room temperature, the margins around the samples were marked with a liquid blocker pap pen to prevent the contents from leaking out during the ihc run. the ideal primary antibody dilution (the dilution of each ab and the volume were calculated and adjusted earlier) was applied to the slides, incubated for 60 minutes, and then thoroughly washed with washing buffers. negative controls included incubations without the particular antibodies [11,12]. the stained slides were thoroughly washed for 4 minutes with gill's hematoxylin solution, dehydrated, and mounted using dpx mounting solution. the stained slides were kept in the fume hood for at least one hour to dry at room temperature [13,14]. a pathologist utilized a double-headed light microscope to calculate cutoff values for all the antibodies employed in the investigation. expression of tgfb3 in pyogenic granuloma vol 13, no 1 (2025) doi 10.5195/d3000.2025.950 http://dentistry3000.pitt.edu 2 overexpression of tgfb3 was deyined as positive nuclei staining in at least 10% of the cells, whereas normal expression was deyined as fewer than 10% positive cells. staining was carried out according to the instructions of the dako detection system, with the following software and protocol: "pouring the solutions (on the slide for each location 100 l maximum 300 l per slide)". the slides were then de parafyin zed by running them through xylene (twice) and graded dilutions of alcohol (100 percent twice, 95 percent, 70 percent, and 50 percent) before incubating them in distilled water for 10 minutes. the slides were incubated for demasking (antigen retrieval). solution for retrieval, citrate buffer; ph9 for (tgfb3) in a water bath for 60 minutes at 97°c. following many experiments utilizing various incubation durations (20, 30, 45, 60, and 90 minutes) for ag recovery of samples, the 60minute optimum incubation time was established. the slides were allowed to cool for 20 minutes at room temperature before the liquid blocker pap pen was used to mark the edges around the samples to prevent the contents from leaking out during the ihc run. after a yirst wash with washing buffer, all slides were coated with peroxidaseblocking reagent and incubated for 10 minutes. because some cells or tissues have endogenous peroxidase, it includes h2o2, which is a peroxidase substrate. the washing buffer was used to clean the slides for 30 minutes, blocking reagent (ab diluent) was administered to block the excess site of another nonspeciyic protein and reduce background generation. washing buffer was used on the slides. the optimal dilution of primary antibody was applied to the slides (the dilution of each ab and the volume were previously calculated and adjusted), then forcefully rinsed with washing buffer after incubation for 60 minutes. negative controls were incubations that did not include antibodies previously adjusted to minimize the creation of a strong background in the dyed slides that would interfere with the results) and rinsed two times thoroughly. the slides were rinsed thoroughly with water after being counterstained with gill's hematoxylin solution for 4 minutes. the slides were dehydrated before being mounted using dpx mounting media. for at least one hour in the fume hood, the stained slides were permitted to cure at room temperature [15,16]. with the help of a trained pathologist and a double-headed light microscope, the stained slides were scored together. with the aid of a pathologist, cut off values for all the antibodies employed in the study were determined. cut-off values for the dako procedure are as follows: according to sophia et al., tgfb3 scoring was utilized. tgfb3 oveexpression was deyined as positive nuclei staining in at least 10% of the cell nuclei overexpression, normal expression was deyined as those with fewer than 10% positive cell nuclei. (negative, score 0; weak or mild staining (5–10% score 1); moderate staining (10–25% score 2); severe staining (25–50% score 3) and extremely strong staining (25–50% score 4) (over 50 percent score 4). statistical analysis as a cutoff criterion, we utilized the median irs=4 score. patients with higher irs scores (>4) were categorized as having strong tgfb3 expression. p=0.05 was used as the statistical signiyicance cutoff. to examine the pt and clark level variables, cochran– armitage statistics were used. results in this work, immunohistochemistry methods were used to investigate the protein transforming growth factor beta-3, which is generated by the human tgfb3 gene, in patients from iraq who developed pyogenic granulomas. negative results (-0) were seen in 4.5 of 16 samples (18.1%) when tgfb3 expression in pyogenic granuloma tissues was conyirmed by immunohistochemistry. ten of the 16 samples, or 33.06 percent, received positive ratings (+), one out of 16, or 2 percent, had positive ratings (++), and one out of 16, or 4.5 percent, had positive ratings (+++). the control had lesions (1) and a negative score (0), as shown in table. the expression of tgfb3 in tissues from pyogenic granulomas was markedly different from control samples (p = 0.001) in comparison. figure 1 displays the expression of tgfb3 with the nucleus stained with brown to demonstrate positive tgfb3 expression and without nucleus staining to show negative tgfb3 expression. figure 2 displays the immunohistochemical staining of tgfb3 in pyogenic granuloma slices with counterstained blue hematoxyline and brown peroxidase/dab (400x) (400x). discussion a driving force behind growth among other ways, beta-3 can stop development, cause cell death, alter the cell matrix on the outside, and lessen the body's reaction to sickness [17]. tgfb3 has also been demonstrated to enhance the production of endothelial growth factors (tgf-i), as well as having a variety of additional anti-swelling properties. tgfb3, a novel host factor that can't be weakened by infection, is another essential cytokine, organize tissue development, proliferation, and growth (using different expressions). when individuals with pyogenic granuloma are followed with obvious health values, tgfb3 is commonly accessible [18]. patients with erosive form had substantially greater tgfb3 tissue expression levels than those with retinal form. regardless, there is no recurrent disparity in numbers, suggesting that tgfb3 levels differ signiyicantly between patients with pyogenic granuloma and healthy controls. another study found a strong link between tgfb3 levels and various kinds of pyogenic granuloma, as well as an increase in saliva levels for patients with tgfb3 in pyogenic granuloma. in comparison to the retinal form, the corrosive and destructive forms included more tgfb3 [19]. in addition, 8 of 11 individuals with pyogenic granuloma had an unaffected response to tgfb3. despite this, pyogenic granuloma was shown to be negative in recent tissue samples from healthy people; also, the levels of tgfb3 and different types of pyogenic granuloma were shown to have a signiyicant relationship. the reticular type had lower tgfb3 levels than the atrophic and erosive forms. in addition, in 8 out of 11 individuals with pyogenic granuloma, an unable to harm response was shown against tgfb3 [20]. conclusions according to the results of the study, tgfb3 expression is high in pyogenic granuloma. conflict of interested statement none to declare. references 1. tobouti pl, olegário i, de sousa sc. benign vascular lesions of the lips:diagnostic approach. j cutan pathol. 2017;44(5):451–5. 2. dasilva fc, piazzetta cm, torres-pereira cc, schussel jl, amenábar jm. gingival proliferative lesions in children and adolescents in brazil:a 15year-period cross-sectional study. j indian soc periodontol. 2016;20(1):63–6. 3. wollina u. subungual telangiectatic granuloma. dtsch arztebl int. 2017;114(8):136. 4. silva de araujo figueiredo c, gonçalves carvalho rosalem c, costa cantanhede al, abreu fonseca thomaz êb, fontoura nogueira da cruz mc. systemic alterations and their oral manifestations in pregnant women. j obstet gynaecol res. 2017;43(1):16–22. 5. abreu-dos-santos f, câmara s, reis f, freitas t, gaspar h, cordeiro m. vulvar lobular capillary hemangioma:a rare location for a frequent entity. case rep obstet gynecol. 2016;2016:3435270. 6. katmeh rf, johnson l, kempley e, kotecha s, hamarneh w, chitale s. pyogenic granuloma of the penis:an uncommon lesion with unusual presentation. curr urol. 2017;9(4):216–8. 22. 7. zhao j, feng q, shi s. pyogenic granuloma of the esophagus. clin gastroenterol hepatol. 2017. expression of tgfb3 in pyogenic granuloma vol 13, no 1 (2025) doi 10.5195/d3000.2025.950 http://dentistry3000.pitt.edu 3 8. romero mascarell c, garcıá pagán jc, araujo ik, llach j, gonzález-suárez b. pyogenic granuloma in the jejunum successfully removed by single-balloon enteroscopy. rev esp enferm dig. 2017;109(2):152–4. 9. qiu x, dong z, zhang j, yu j. lobular capillary hemangioma of the tracheobronchial tree:a case report and literature review. medicine (baltimore) 2016;95(48):e5499. 10. misawa s, sakamoto h, kurogochi a, kirii y, nakamura s, misawa t, yoneda s, hirano m, owa o, takagi h, ota h. rare cause of severe anemia due to pyogenic granuloma in the jejunum. bmc gastroenterol. 2015;15:126. 11. matsuzaki k, imamura y, ozawa m, nakajima t, ikeda a, konishi t, jikuya t. intravenous lobular capillary hemangioma in the subclavian vein. ann thorac surg. 2016;102(5):e427–9. 12. brightman la, geronemus rg, reddy kk. laser treatment of port-wine stains. clin cosmet investig dermatol. 2015;8:27–33. 13. wollina u, zielinski m, knopf b, hipler c. [eruptive capillary hemangioma following argon laser therapy of naevus nlammeus] hautarzt. 1989;40(4):212–4. 14. rana r, ramachandra ss, prasad uc, aggarwal p, dayakara jk. recurrent pyogenic granuloma with a satellite lesion. cutis. 2015;96(2):e27–30. 15. gupta v, mridha ar, sharma vk. pediatric dermatology photoquiz:multiple erythematous papules on the back. recurrent pyogenic granulomas with satellitosis. pediatr dermatol. 2016;33(1):97–8. 16. finnson kw, mclean s, di guglielmo gm, philip a. dynamics of transforming growth factor beta signaling in wound healing and scarring. adv skin wound care. 2013;2(5):195–214. 17. penn jw, grobbelaar ao, rolfe kj. the role of the tgf-β family in wound healing, burns and scarring: a review. int j burns trauma. 2012;2(1):18–28. 18. fleisch mc, maxwell ca, barcellos-hoff mh. the pleiotropic roles of transforming growth factor beta in homeostasis and carcinogenesis of endocrine organs. endocr relat cancer. 2006;13(2):379–400. 19. devescovi v, leonardi e, ciapetti g, cenni e. growth factors in bone repair. chir organi mov. 2008;92(3):161–8. 20. lehnert sa, akhurst rj. embryonic expression pattern of tgf beta type-1 rna suggests both paracrine and autocrine mechanisms of action. development. 1988;104(2):263–73. table 1. tgfb3 expression in pyogenic granuloma was investigated using immunohistochemistry. score group negative scored + ++ +++ pyogenic granuloma 4.5 (18.1%) a 10(33.06%) b 1 (2%) b 1 (4.5%) c control negative scored (0) *** p <0.005 figure 1. immunohistochemical staining of tgfb3 in pyogenic granuloma sections with peroxidase/dab (brown) counterstained with blue hematoxyline (400x). expression of tgfb3 in pyogenic granuloma vol 13, no 1 (2025) doi 10.5195/d3000.2025.950 http://dentistry3000.pitt.edu 4 figure 2. immunohistochemical staining of tgfb3 in pyogenic granuloma slices with peroxidase/dab (brown) and blue hematoxylin counterstained (400x). 814 final efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling after surgical removal of impacted mandibular third molar abdulhameed salim hameed1, hajer ali ahmed hentati2 1ibn el jazzar faculty of medicine, university of sousse, tunisia, and ministry of health, diyala. iraq 2faculty of dental medicine, laboratory of oral health and maxillofacial rehabilitation (lr12es11), university of monastir, monastir, tunisia abstract objec&ve: postopera+ve edema and pain are common third molar surgery side effects, that normally happens with local anesthesia. the low-level laser (lllt) method has been universally recognized as a cell bio-modulator that is employed to accomplish op+mum beneficial effects. it reduces the pain reac+on, s+mulates local microcircula+on and wound healing, and promotes a fast healing, thus enhancing the pa+ent's quality of life. we intended to ascertain the lllt efficacy in improving mouth opening following the impacted third molar extrac+on in this research. methods: this randomized clinical trial was carried out on ninety cases who had impacted mandibular third molars in similar posi+ons. group a was allocated to intra-oral lllt, group b to extra-oral lllt, and group c to the control group. all par+cipants were evenly divided into these three categories. the outcome that was assessed was the pain degree, alongside the postopera+ve edema and recovery at the opera+on site. results: postopera+ve pain and edema were calculated on 1st and 7th day. all these parameters were lower in lllt pa+ents (p>0.05). conclusions: aser the impacted mandibular lower wisdom tooth surgical extrac+on, the lllt applica+on was effec+ve in mi+ga+ng postopera+ve complica+ons, for instance facial edema and pain. keywords: low level laser therapy (lllt), pain, impacted third molar, mouth opening, laser den+stry, facial swelling. cita:on: hameed as, et al. (2025) efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling auer surgical removal of impacted mandibular third molar. den:stry 3000. 1:a001 doi:10.5195/d3000.2025.814 received: december 28, 2024 accepted: february 7, 2025 published: february 12, 2025 copyright: ©2025 hameed as, et al. this is an open access ar:cle licensed under a crea:ve commons a\ribu:on work 4.0 united states license. email: abdulhameedaltaee00@gmail.com introduction in the oral cavity region, the impacted third molar extraction is a frequently performed process that can result in postsurgical complications, including trismus, edema, and pain. these adverse effects can result in cases experiencing disturbances due to their disruption with chewing and speech [1]. the most intense pain typically occurs within three to five hours of the surgery, when the local anesthesia effects have faded. in contrast, edema typically reaches its maximal size between 20 hours and two days after the procedure, which can have a negative effect on the patient's social life and compromise their aesthetics [2]. the inflammation development is primarily caused by operating trauma, which is linked to the postoperative repercussions [3]. while edema and pain progressively reduce throughout the procedure, it is imperative that the doctor and patient maintain control over these complications. most surgeons have employed a variety of methods to mitigate the postoperative sequela, including corticosteroids (cs), analgesics, and non-steroidal drugs (nsaids). efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu however, these medications have adverse effects can be problematic for certain contraindicated cases. consequently, alternative methods, like low-level laser therapy (lllt), that is devoid of adverse effects, have emerged [4]. a diode laser energy application that is close to infrared wavelengths is known as lllt [5]. it has been regarded as having the potential to accelerate the recovery process, and to reduce pain and inflammation. lllt has been implemented in many dental procedures, including the tooth extraction adverse effects mitigation, due to characteristics [6]. lllt can also be employed for accelerated and promote bone regeneration by induced extraction cavities quick recovery [7]. on irradiated tissues, lllt can have both biostimulatory and bio inhibitory effects, each of which has potential therapeutic applications. biologic response stimulation via energy transfer is a prerequisite for laser therapy [8]. the light energy incorporation with a bio-modulatory function to body cells is the key concept behind the lllt utilization [9]. the energy of the cell is provided by adenosine diphosphate (atp), that's the cytochrome c oxidase (cco) and the krebs cycle product. this is achieved by cco absorbing lllt irradiation and transferring it to mitochondria. a rise in cell activity is induced by the atp synthesis stimulation [10]. consequently, the study objective was to ascertain the lllt efficacy in relieving the discomfort and edema due to the impacted third molars extraction. material and methods from november 2022 to january 2024, this randomized clinical trial took place on (90) patients, with impacted mandibular third molars in comparable positions (class ii-iii and position b, pell and gregory's classification) [15]. the cases were comprised of 52 males and 38 females. the study setting was the centre for dentistry in iraq's oral surgery unit by the diyala health department, and the local institutional ethical committee's approved (kimsdu/iec/03/2015 dated december 10, 2015) the protocol. the study's nature was explained to all patients, and they were required to provide written informed consent. the study involved patients aged 18-35 who needed a partially or fully impacted lower third molar surgical extraction. cases who were in good health and maintained proper dental hygiene were included. cases with systemic diseases, psychiatric disorders, pericoronitis, neurological or persistent pain, and those who had taken antiinflammatory drugs or bisphosphonates within 15 days, photosensitivity disorders, asymmetrical third molars, postoperative dry socket, pregnant or breastfeeding women, skipped appointments, and in-place smokers were excluded from the study. the same operating surgeon performed all the surgical procedures. utilizing www.random.org all participants were divided into three groups equally regarding to the laser treatment site where cases in group a were allocated to intra-orally lllt, cases in group b allocated to extra-orally lllt, cases in group c were allocated to the control group. clinical examination the extraoral examinations consisted of the assessment of the face, eyes, lymph node, and temporomandibular joint for pain, abnormalities, and baseline prior to surgery. intraorally, the oral hygiene and periodontal status were evaluated for inflammation or irritation. radiographic examination preoperatively, periapical and panoramic radiographs were obtained for each patient to detect the lower wisdom tooth position, the orientation of its depth in relation to the contiguous tooth, the mandibular foramen, the root efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu size and shape, and the teeth number (figure 1). a b figure 1. preoperative radiographical assessment (a) o.p.g. image and cpct showing vertical impaction of the lower wisdom tooth of left and right side, (b) periapical x-ray showing a vertical impaction of lower left third molar. anaesthesia the lidocaine hydrochloride 2% local anaesthetic cartridge and adrenaline 1: 80,000 were utilized to administer local anaesthesia to patients through inferior alveolar blook, lingually, and long buccal nerve block injections. surgical procedure surgical blade number 15 was utilized to make an incision mucoperiostically from the lower seven disto-buccal cusp to the retromolar area midline. the incision was then buccally reached along the 2nd molar crown, and a moon-shaped flap was extended to the muco-buccal fold and increased with a periosteal elevator. all these factors contributed to favourable visibility and excess. the nearby bone is preserved by the application of a straight surgical hand piece fissure or round bur to eliminate the impacted lower third tooth. the tooth is sectioned utilizing a straight handpiece with fissure burs or a turbine handpiece with round or fissure burs, as necessary. the tooth is subsequently extracted utilizing a straight elevator, coupland's chisel, or cryer and forceps, with a specific focus on the lower third molar. the wound was inspected for granulation tissue, root fragments, tooth follicles, and bones following the tooth extraction. using a primary interrupted method, the flap and wound were repositioned, and the edges were sutured. laser therapy following surgery, patients in the lllt group were administered lowlevel laser irradiation intraorally occlusally, lingually buccally or at three sites, and extraorally at three points. 500 mw (0.5 w) of laser energy was administered by employing a diode laser system (quicklase®, uk) with a constant dual wavelength of (810,980nm) for 120 seconds (0.5 w×120s=48 j). for each site, the control group (n = 30) established standard postoperative care without lllt, as well as 0.5 cc of normal saline solution in the cavity for 30 seconds (figure 2). figure 2. application of low-level laser, intraorally, (a) at buccal side, (b) lingual, (c) lingual occlusally at the side of the operation site. the patient was prescribed amoxicillin capsules 500 mg three times daily following the surgery, while augmentin tabs 625 mg (amoxicillin 500 mg, clavulanic acid 125 mg) were administered twice daily. for a duration of three days, the antibiotic was taken. for individuals who were allergic to penicillin, azithromycin 500 mg was administered every 12 hours. for a duration of three days, either a 500 mg paracetamol tablet with acetaminophen or a 200 mg ibuprofen tablet was administered twice daily. efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu pain the facial dimensions of all patients were evaluated prior to surgery using a flexible measuring tape, which was utilized to determine the distance from the chin tip to the auricular lobe lower part (figure 3). pain was evaluated utilizing a visual analog scale (vas) at two and seven days postoperatively. patients are instructed to indicate the intensity of their pain by marking points on the vas [3,12,13]. swelling the baseline facial distance was established prior to surgery by calculating the arithmetic mean of face three linear measures. five points were placed on the face utilizing a centimetre flexible tape, with the patient in an upright position and the mandible in a resting position [1,3,14]. in the postoperative period, edema was assessed in the two planes using a thread and measuring scale. both preoperatively and postoperatively, the distance between the gonion and the eye external canthus and the distance between the tragus and the lip commissure were measured. the subsequent formula was employed to determine the edema coefficient: edema coefficient = (distance after surgery − distance before surgery)/(distance before surgery) ×100. figure 3. measuring the distance between (line a and c). distance between the (line a and d) and distance between (line e and b). after confirming that the recovery was satisfactory, the suture was removed on the seventh postoperative day. the potential complications linked to the operation were also assessed during the routine follow-up checkup. cases in both groups were prescribed antibiotics and analgesics for three days following the procedure, involving amoxicillin or augmentin for three days or azithromycin if allergic. analgesics such as paracetamol or ibuprofen were prescribed for a period of three days. regular follow-up visits, maintaining good oral hygiene, and biting for 60 minutes were among the medical and physical recommendations that were issued postoperatively. pain and edema were assessed during routine examinations on the third and 7th days. statistical analysis ibm® spss® (ver. 26. spss inc., ibm corporation, armonk, ny, usa) was employed to conduct the statistical analysis. the shapirowilk test was employed to investigate the data normality. the mean and standard deviation were utilized to present quantitative data. the paired t-test was employed to compare the means of two groups before and after treatment. the means of two groups were compared utilizing an independent t-test. the association between numerical variables within each group was described using pearson correlation analysis. a p-value of less than 0.05 was regarded as statistically significant. results in this study, 102 case were assessed for eligibility; 8 patients did not satisfy the criteria, and 2 patients declined to participate. the remaining 90 cases were randomly allocated to three groups, with 30 cases in each cohort. the statistical analysis and follow-up of all allocated patients were conducted (figure 4). efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu figure 4. consort flowchart of the studied groups. age and gender distributions between the groups were not different (tables 1 and 2). there was insignificant difference between groups regarding duration of operation (p= 0.178, table 3). the pain score vas at days 2, 5, and 7 demonstrated significant differences between study groups (p < 0.001, table 4). the study groups exhibited a significant difference at baseline facial swelling, day 2, and day 7 (p = 0.014, <0.001, and <0.001, respectively). in group a compared to group c at baseline (p = 0.012) facial swelling was significantly reduced. similarly, compared to groups b and c at days 2 and 7, facial edema was significantly reduced in group a (p < 0.001), with insignificant difference among groups b and c. facial swelling was significantly reduced in groups b and c at baseline, but it increased at day 2, and subsequently decreased again at day 7 (p < 0.001). compared to day 2 the facial swelling in group a was significantly reduced at day 7 (p = 0.002). there was insignificant difference between baseline and day 2 or between baseline and day 7 (table 5). efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu table 1. age of the study groups samples. group a (n=30) group b (n=30) group c (n=30) p-value mean sd mean sd mean sd age (years) mean ± sd 24.8 5.5 25.33 5.34 25.3 4.5 0.903 range 18 – 34 18 34 18 – 32 data are presented as mean ± sd. *: significant as p value <0.05. table 2. gender of the study groups samples. group a (n=30) group b (n=30) group c (n=30) total (n=90) pvalue n % n % n % n % gender male 21 70% 17 56.67% 14 46.67% 52 57.78 0.185 female 9 30% 13 43.33% 16 53.33% 38 42.22 % table 3. duration of operation among the studied groups. group a (n=30) group b (n=30) group c (n=30) p-value mean sd mean sd mean sd duration of operation mean ± sd 33.97 1.59 34.3 1.73 34.7 1.18 0.178 range 30 36 30 – 36 32 – 36 efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu table 4. comparison and post hoc of vas among studied groups. group a (n=30) group b (n=30) group c (n=30) p-value day 2 mean ± sd 3.4 0.5 6 3.73 0.64 7.43 1. 41 <0.001* range 3 – 5 3 – 5 5 – 10 day 5 mean ± sd 1.4 0.5 1.77 0.57 3.83 0.91 <0.001* range 1 – 2 1 – 3 3 – 6 day 7 mean ± sd 0.27 0.4 5 0.57 0.5 1.03 0.49 <0.001* range 0 – 1 0 – 1 0 – 2 table 5. comparison and post hoc of facial swelling among the studied groups. group a (n=30) group b (n=30) group c (n=30) p-value baseline mean ± sd 327.6 3 37.9 1 342.8 3 25.0 9 349.6 21.8 8 0.014* range 288 – 390 300 – 375 306 – 377 day 2 mean ± sd 334.8 7 35.7 6 363.7 26.1 3 377.1 19.8 8 <0.001* range 290 – 397 320 – 399 337 – 401 day 7 mean ± sd 314.2 ± 30.9 3 347.4 7 28.4 3 361.5 20.8 2 <0.001* range 280 – 390 300 – 390 320 388 efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu discussion at an oral surgery clinic, the lower third molars surgical removal is one of the most frequently performed methods. it can be followed by trismus, facial swelling, and postoperative pain. these conditions are the result of a combination of intricate factors, but the primary cause is inflammation that is initiated by surgical trauma [15]. bradykinin (bk) is released from ruptured blood vessels and histamines, serotonins, and potassium are released from injured cells because of the surgical technique, which induces tissue injury. the activation of nociceptors, a change in tissue color, and swelling are the results of this tissue reaction. nociceptor activation and prostaglandin release are induced by bk. due to this, symptoms such as pain, trismus, and edema manifest [16]. side effects, including allergic reactions, systemic bleeding, and gastrointestinal irritation, may result from these types of treatments. in addition, none of these treatments have been found to be satisfactory. [4]. these effects can be mitigated by any combination of nsaids, css (local or systemic), or both. nevertheless, these medications may produce different side effects, such as allergic responses, systemic bleeding, and git discomfort, and may be dangerous for certain individuals. as a result, there is an increasing interest in the development of alternative, sideeffect-free methods [17]. lllt is widely recognized for its ability to regulate the inflammatory and reduce acute pain in the short term. the lllt biological effects partial creation is facilitated by the lllt energy absorption by tissues and the photons interaction with cellular structures. it is anticipated that this interaction will have therapeutic effects. muscle relaxation, pain relief, wound recovery, and are the altered cell membrane permeability and increased cellular energy outcomes. lllt prefers the hyperpolarized state, which prevents the painful inputs transmission to the central nervous system, straight over primary nerve terminals [18]. cyclooxygenase 2(cox-2), tumor necrosis factor (tnf), prostaglandin e2 (pge2), and interleukin 1are among the pain and inflammation mediators that lllt has significant and rapid effects on [19]. age differences between the groups were not found to be statistically significant in the present investigation. in this study, the most participants age range was 18 to 34, which constituted approximately 95% of the total sample. in accordance with the observations of breik and grubor, hashemipour et al., and sasano et al., the most cases in their studies were in their 30s. prior study has suggested that the primary demographic impacted by symptomatic impaction is individuals aged 20 to 30 [20-22]. the female patient population in this trial was 38 (42.22%), while the male patient population was 52 (57.78%), as established in the current study. in all groups, there was insignificant difference in the proportions of males and females. many studies have shown a impacted teeth higher prevalence, particularly third molars, in males, which is consistent with our findings [23,24]. nevertheless, other studies indicated that females had impacted teeth higher incidence [25,26], though certain authors have asserted that in the mandibular third molar impaction incidence there is no sexual predisposition [27-29]. however, this can be explained that males and females may have variable growth rate, that explains females had a greater prevalence. female jaws growth ceases at the third molar eruption time, while it continues in male jaws, allowing efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu for a greater amount of eruption space [30]. after the local anesthetic has diminished, the postoperative pain commences and reaches its peak 6 to 12 hours postoperatively [31], has a duration of two to three days and then regularly diminishes until the 7th postoperative day [13]. the lllt precise analgesic mechanism has not yet been fully elucidated [15]. it is hypothesized that pain reduction is influenced by changes in the chemicals production, metabolism, and release such as serotonin and acetylcholine, as well as the inflammation local regulation, which affects mediators such as histamine and pge2 [32,33]. the laser also provokes analgesia by decreasing the bk and type c nerve fibers activity, increasing the endogenous endorphins (β endorphin) production, and reducing the pain threshold [34]. the most severe pain rate was noted in all groups during the initial two days following surgery in the present study. subsequently, the pain score kept dropping until the 7th day, and there were significant differences between the days in all the groups (p < 0.001). vas was significantly elevated in group c at days 2 and 5, in contrast to groups a and b, with no significant difference between the two groups. however, compared to groups a and b at day 7, vas was significantly elevated in group c (p < 0.001) and significantly elevated (p = 0.047) in group b compared to group a. santos et al. and landucci et al. conducted a study on 32 female cases, which demonstrated a significant decrease in pain after 48 hours and beyond [35,36]. in a comparable study, mohajerani et al. demonstrated compared to the control group after three days postoperatively a significant reduction in pain in the laser group is detected. this study involved 80 cases [37]. petrini et al. and shenawy et al. demonstrated significant impact. decline in pain following lllt [38,39]. das et al. demonstrated a significant decrease in pain following lllt. in contrast to the control group, nine of our patients experienced only mild pain on the 1st postoperative day. twelve of the fifteen patients in the study group did not experience any pain after seven days. [40]. despite this, hamad et al. demonstrated that it was insignificant over the past four days (p>0.05). in the initial three days, the laser group vas was 4.46 (± 1.45), 4.00, (± 1.36), and 3.35 (± 2.33), respectively. the laser group's vas score was lower than that of the control group. in the control group, the vas was 6.58 (± 1.83), 5.82 (± 2.15), and 5.17 (± 1.97), respectively. throughout the initial three days, the disparity was significant (p < 0.05) [41]. the significance of this discovery resulted in stimulates the endogenous endorphins (bendorphin). production, biomodulation elevation the pain threshold, and inhibits nerve conduction [42,43]. cox-2 and pge2 concentrations are reduced, and the cascade of arachidonic acid is inhibited by low-level laser therapy [44]. this study's outcomes are consistent with the findings of hadad et al., who demonstrated that an intraoral diode laser utilization at 810 nm wavelength, 6 j (100 mw, 60 seconds/point) significantly relieved pain at 24 and 48 hours. [45]. momeni et al. discovered that pain scores differed insignificantly until the 5th postoperative day. however, on the 6th and 7th days, the laser-treated sides presented significantly minimal pain scores [46]. this study's results are consistent with previous work [14,31,33,47,48]. kamal et al., landucci et al., and clokie et al., administered a lllt single dose for oneand three-minutes’ wavelengths, respectively, instantly following the lower third molar surgical extraction. postoperatively, they reported efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu significant decreases in pain levels (p < 0.05) [10,36,49]. nevertheless, the research conducted by fernando et al., lópez-ramírez et al., glória et al., amarillas-escobar et al., and farhadi et al. did not demonstrate a positive impact on pain [1,3,12,50,51]. the 940 nm extraoral diode laser was not effective in reducing pain, as demonstrated by eroglu and keskin tunc [52]. in addition, in their study, ahrari et al. did not observe any significant impact on pain relief from the 660 nm and 810 nm lasers (200 mw, 30 seconds of radiation to the buccal, lingual, and occlusal surfaces of the socket, 6 j/area) [53]. the face edema increases progressively and achieves its peak on the second postoperative day. it is anticipated that the edema will decrease by the fourth day. it has vanished entirely within a week. consequently, this clinical research assessed edema on the second and 7th postoperative days [40]. face edema is a three-dimensional condition that is characterized by a convex surface and is visible both internally and externally [54]. the literature employs a variety of methods to evaluate the severity of facial edema, including photographic techniques, mechanical methods (calipers, cephalostats), ultrasound, computed tomography, verbal response scales, magnetic resonance imaging, and the 3dmd face imaging system [55]. nevertheless, this study refrained from employing these methods due to their complexity, expense, and necessity for specialized tools. instead, direct face assessment was employed to assess edema, even though it is a twodimensional technique that is straightforward, easily reproducible, and doesn’t necessitate specific equipment. the tnf-α, il-6, mcp-1, and il-10 inhibition in the inflammation acute phase may be the inflammatory edema reduction cause by lllt. this inhibition leads to an increase in the macrophages activation and phagocytic activity, as well as a change in the lymph and blood vessels permeability and channel size and the elevation in the lymphatic vessels number, the microcapillary circulation restoration, thereby reducing swelling [18,56]. additionally, laser therapy, reduces edema by blood vessels decreasing the permeability, enhance the interstitial fluid absorption, and alters the hydrostatic and intracapillary pressures and increasing the lymph vessels diameter and number [57]. in the present study, it was determined that in group a than in groups b and c at the baseline facial swelling differed significantly. however, there was no significant difference between groups b and c at days 2. facial swelling was significantly reduced in group a compared to groups b and c at the 7-day mark, while there was insignificant difference between groups b and c. the facial swelling in group a reduced significantly at day 7 compared to day 2, and there was insignificant difference between the baseline and day 2 or the baseline and day 7. at day 2, facial swelling in groups b and c was increasing, but it eventually decreased again by day 7 (p < 0.001). these results suggested that facial swelling was extremely significant in all three groups on the second day following the operation. however, there was insignificant difference between the baseline and the seven-day postoperative period for all groups. hamad et al. reported that the laser group exhibited significantly less swelling than the control group. the difference was statistically significant on the first and third postoperative days (p < 0.05). the control group had facial measurements of 113.57 mm (± 4.54mm) on the first postoperative day, while the laser group had measurements of 108.72 mm (± 6.01mm). in the control group, the measurements were 118.43mm (± efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu 3.48mm) on the third postoperative day, while in the laser group, they were 114.77mm (± 5.63mm) [41]. this study findings are consistent with numerous studies that have stated significant reduction in postoperative swelling following third molar surgery due to the lowdensity laser’s utilization. singh et al., bianchi de moraes et al., and mohajerani et al. demonstrated the lllt efficacy in decreasing swelling following the impacted lower mandibular third molars surgical removal [37,58,59]. ferrante et al., aras and güngormüş, and eshghpour et al. established that lllt could alleviate facial edema [14-16]. domah et al. and de oliveira at al. conducted a systematic review and meta-analysis that revealed that lllt has a beneficial impact on the postoperative swelling following mandibular third molar operation reduction [60, 61]. on 30 patients in the study and control groups a randomized control trial was carried out by amarillas-escobar et al. the study results the indicated a decrease in swelling postoperatively [3]. in an additional study was performed by batinjan et al., 150 cases were divided into three groups of 50, and photodynamic therapy, lllt, and a control group were assessed. the study results indicated that both laser-treated groups experienced a significant decrease in postoperative swelling after in comparison to the control group [62]. the mean value of the superior edema coefficient on the first day was higher in the control group (12.63) than in the lllt group (10.30) in das' study. additionally, the superoinferior edema coefficient was 5.91 and 6.67 in the study and control groups, respectively, on the seventh day. the control group exhibited a higher mean anteroposterior edema coefficient (8.83) than the lllt group (7.54) on the first day. similarly, the control and study groups exhibited a mean anteroposterior edema coefficient of 4.07 and 2.98 on the seventh day, respectively. the lllt group exhibited statistically highly significant results for both the superior and inferior anteroposterior edema coefficients [40]. in a study of 60 patients, raouaa et al. administered steroids to one group and laser therapy to the other. the results indicated that the cs group was more efficient in reduction swelling than the laser group [63]. nevertheless, both carrillo et al. and farhadi et al. stated that in swelling among the lllt and placebo control groups there was insignificant different [51,64]. furthermore, pedreira et al and lópez-ramírez et al. have failed to demonstrate that laser therapy has a beneficial impact on swelling [1,65]. raiesian et al. conducted an additional study in 44 patients, and there was insignificant difference in swelling across the control and study groups [66]. additionally, koparal et al and farhadi et al. conducted research on the laser utilization following third molar surgery. results indicated that there was insignificant reduction in postoperative swelling [51,67]. the varying irradiation parameters and applications of lllt may account for the inconsistent results observed in our study and others. fernando and colleagues performed a study on 64 cases, utilizing the split-mouth technique. one side was subjected to the study, while the other side was administered a placebo. the results indicated that there was insignificant difference in the swelling decrease between the two sites. [12]. limitations: larger sample sizes and longer follow-up duration will be necessary for the lllt efficacy evaluation with different wavelengths and sites, the precise evaluation of edema using 3d imaging techniques, the repair of socket bone using cbct, and the evaluation of lllt therapy efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu after impacted teeth surgical extraction. conclusion: lllt the technique is non-invasive, easy to apply, and has a minimal to injury non-existent risk. this investigation illustrates that lllt is advantageous in mitigating the severity of facial edema and pain that may follow the impacted third molar surgical extraction. references 1. lópez-ramírez m, vílchez-pérez má, gargallo-albiol j, arnabatdomínguez j, gay-escoda c. efficacy of low-level laser therapy in the management of pain, facial swelling, and postoperative trismus after a lower third molar extraction. a preliminary study. lasers in medical science. 2012;27:559-66. 2. larrazábal c, garcía b, peñarrocha m, peñarrocha m. influence of oral hygiene and smoking on pain and swelling after surgical extraction of impacted mandibular third molars. journal of oral and maxillofacial surgery. 2010;68:43-6. 3. amarillas-escobar ed, toranzofernández jm, martínez-rider r, noyola-frías ma, hidalgo-hurtado ja, serna vmf, et al. use of therapeutic laser after surgical removal of impacted lower third molars. journal of oral and maxillofacial surgery. 2010;68:319-24. 4. kumar n, prasad k, ramanujam l, ranganath k, dexith j, chauhan a. evaluation of treatment outcome after impacted mandibular third molar surgery with the use of autologous platelet-rich fibrin: a randomized controlled clinical study. journal of oral and maxillofacial surgery. 2015;73:1042-9. 5. niemz m. laser-tissue interactions. springer-verlag; 2007. 6. he w, yu f, li c, pan j, zhuang r, duan p. a systematic review and meta-analysis on the efficacy of low-level laser therapy in the management of complication after mandibular third molar surgery. lasers in medical science. 2015;30:1779-88. 7. vladimirov ya, osipov a, klebanov g. photobiological principles of therapeutic applications of laser radiation. biochemistry (moscow). 2004;69:81-90. 8. parker s. low-level laser use in dentistry. british dental journal. 2007;202:131-8. 9. moshkovska t, mayberry j. it is time to test low level laser therapy in great britain. postgraduate medical journal. 2005;81:436-41. 10. kamal m, elsharrawy e, hassan m. effect of single session of lowlwvwl laser therapy on pain, swelling, and trismus after surgical extraction of impacted lower third molar: a clinical study. dental journal of suez canal university. 2021. 11. pell gj. impacted mandibular third molars, classification and modified technique for removal. dental digest. 1933;39:330-8. 12. fernando s, hill c, walker r. a randomised double blind comparative study of low level laser therapy following surgical extraction of lower third molar teeth. british journal of oral and maxillofacial surgery. 1993;31:170-2. 13. lago-méndez l, diniz-freitas m, senra-rivera c, gudesampedro f, rey jmg, garcíagarcía a. relationships between surgical difficulty and postoperative pain in lower third molar extractions. journal of oral and maxillofacial surgery. 2007;65:979-83. 14. aras mh, güngörmüş m. the effect of low-level laser therapy on trismus and facial swelling following surgical extraction of a lower third molar. photomedicine and laser surgery. 2009;27:21-4. 15. ferrante m, petrini m, trentini p, perfetti g, spoto g. effect of low-level laser therapy after extraction of impacted lower third molars. lasers in medical science. 2013;28:845-9. efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu 16. eshghpour m, ahrari f, takallu m. is low-level laser therapy effective in the management of pain and swelling after mandibular third molar surgery? journal of oral and maxillofacial surgery. 2016;74:1322. e1-. e8. 17. mehrabi m, allen jm, roser sm. therapeutic agents in perioperative third molar surgical procedures. oral and maxillofacial surgery clinics. 2007;19:69-84. 18. boschi es, leite ce, saciura vc, caberlon e, lunardelli a, bitencourt s, et al. antiinflammatory effects of low-level laser therapy (660 nm) in the early phase in carrageenan-induced pleurisy in rat. lasers in surgery and medicine: the official journal of the american society for laser medicine and surgery. 2008;40:500-8. 19. albertini r, villaverde a, aimbire f, salgado m, bjordal j, alves l, et al. anti-inflammatory effects of low-level laser therapy (lllt) with two different red wavelengths (660 nm and 684 nm) in carrageenan-induced rat paw edema. journal of photochemistry and photobiology b: biology. 2007;89:50-5. 20. breik o, grubor d. the incidence of mandibular third molar impactions in different skeletal face types. australian dental journal. 2008;53:320-4. 21. hashemipour ma, tahmasbiarashlow m, fahimi-hanzaei f. incidence of impacted mandibular and maxillary third molars: a radiographic study in a southeast iran population. medicina oral, patologia oral y cirugia bucal. 2013;18:e140. 22. sasano t, kuribara n, iikubo m, yoshida a, satoh-kuiriwada s, shoji n, et al. influence of angular position and degree of impaction of third molars on development of symptoms: long-term follow-up under good oral hygiene conditions. the tohoku journal of experimental medicine. 2003;200:75-83. 23. haidar z, shalhoub sy. the incidence of impacted wisdom teeth in a saudi community. international journal of oral and maxillofacial surgery. 1986;15:569-71. 24. ayaz h. pattern of impacted mandibular third molar in patients reporting to department of oral and maxillofacial surgery, khyber college of dentistry, peshawar. journal of khyber college of dentistry. 2012;2:50-3. 25. kruger e, thomson wm, konthasinghe p. third molar outcomes from age 18 to 26: findings from a population-based new zealand longitudinal study. oral surgery, oral medicine, oral pathology, oral radiology, and endodontology. 2001;92:150-5. 26. upadhyaya c, chaurasia n, neupane i, srivastava s. incidence and pattern of impaction of mandibular third molar: a single institutional experience in nepal. kathmandu univ med j. 2017;57:66-9. 27. niedzielska ia, drugacz j, kus n, kręska j. panoramic radiographic predictors of mandibular third molar eruption. oral surgery, oral medicine, oral pathology, oral radiology, and endodontology. 2006;102:154-8. 28. hassan ah. pattern of third molar impaction in a saudi population. clinical, cosmetic and investigational dentistry. 2010:109-13. 29. kaya g, aslan m, ömezli m, dayi e. some morphological features related to mandibular third molar impaction. journal of clinical and experimental dentistry. 2010;2. 30. bishara se, ortho d. impacted maxillary canines: a review. american journal of orthodontics and dentofacial orthopedics. 1992;101:159-71. 31. marković ab, todorović l. postoperative analgesia after lower third molar surgery: contribution of the use of longacting local anesthetics, lowpower laser, and diclofenac. oral surgery, oral medicine, oral efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu pathology, oral radiology, and endodontology. 2006;102:e4-e8. 32. montesinos m. experimental effects of low power laser in encephalin and endorphin synthesis. j eur med laser assoc. 1988;1:2-6. 33. kazancioglu ho, ezirganli s, demirtas n. comparison of the influence of ozone and laser therapies on pain, swelling, and trismus following impacted thirdmolar surgery. lasers in medical science. 2014;29:1313-9. 34. sun g, tuner j. low-level laser therapy in dentistry. dental clinics. 2004;48:1061-76. 35. santos pl, marotto ap, da silva tz, bottura mp, valencise m, marques do, et al. is low-level laser therapy effective for pain control after the surgical removal of unerupted third molars? a randomized trial. journal of oral and maxillofacial surgery. 2020;78:184-9. 36. landucci a, wosny a, uetanabaro l, moro a, araujo m. efficacy of a single dose of lowlevel laser therapy in reducing pain, swelling, and trismus following third molar extraction surgery. international journal of oral and maxillofacial surgery. 2016;45:392-8. 37. mohajerani h, tabeie f, alirezaei a, keyvani g, bemanali m. does combined low-level laser and light-emitting diode light irradiation reduce pain, swelling, and trismus after surgical extraction of mandibular third molars? a randomized doubleblinded crossover study. journal of oral and maxillofacial surgery. 2021;79:1621-8. 38. petrini m, ferrante m, trentini p, perfetti g, spoto g. effect of pre-operatory low-level laser therapy on pain, swelling, and trismus associated with thirdmolar surgery. medicina oral, patologia oral y cirugia bucal. 2017;22:e467. 39. el shenawy h, aboelsoud n, zaki a, el zawahry m, shaibeta a. postoperative pain control in patients after lower third molar extraction. j am sci. 2010;6:5. 40. das ar, vidya k, srikar m, pathi j, jaiswal a. effectiveness of low-level laser therapy after surgical removal of impacted mandibular third molars: a randomized clinical trial. national journal of maxillofacial surgery. 2022;13:60-6. 41. hamad sa. effect of low-level laser therapy on inflammatory sequelae of impacted mandibular third molar surgery: a single-blind, placebo-controlled, randomized clinical trial. russian open medical journal. 2022;11:220. 42. hagiwara s, iwasaka h, okuda k, noguchi t. gaalas (830 nm) low-level laser enhances peripheral endogenous opioid analgesia in rats. lasers in surgery and medicine: the official journal of the american society for laser medicine and surgery. 2007;39:797-802. 43. chow rt, david ma, armati pj. 830 nm laser irradiation induces varicosity formation, reduces mitochondrial membrane potential and blocks fast axonal flow in small and medium diameter rat dorsal root ganglion neurons: implications for the analgesic effects of 830 nm laser. journal of the peripheral nervous system. 2007;12:28-39. 44. pigatto gr, silva cs, parizotto na. photobiomodulation therapy reduces acute pain and inflammation in mice. journal of photochemistry and photobiology b: biology. 2019;196:111513. 45. hadad h, santos afp, de jesus lk, poli pp, mariano rc, theodoro lh, et al. photobiomodulation therapy improves postoperative pain and edema in third molar surgeries: a randomized, comparative, double-blind, and prospective clinical trial. journal of oral and maxillofacial surgery. 2022;80:37. e1-. e12. 46. momeni e, barati h, arbabi mr, jalali b, moosavi m-s. lowlevel laser therapy using laser efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu diode 940 nm in the mandibular impacted third molar surgery: double-blind randomized clinical trial. bmc oral health. 2021;21:18. 47. da silva rp, de almeida vl, de andrade vieira w, rocha fs, herval ám, nascimento gg, et al. is ozone effective in reducing pain, edema, and trismus after third molar surgery? a systematic review. research, society and development. 2020;9:e319108407-e. 48. ali m, al-adili s. low level laser therapy on postoperative trismus and swelling after surgical removal of impacted lower third molar. indian j public health res dev. 2019;10. 49. clokie c, bentley k, head t. the effects of the helium-neon laser on postsurgical discomfort: a pilot study. journal (canadian dental association). 1991;57:5846. 50. botelho j, machado v, proença l, oliveira mj, cavacas ma, amaro l, et al. perceived xerostomia, stress and periodontal status impact on elderly oral healthrelated quality of life: findings from a cross-sectional survey. bmc oral health. 2020;20:199. 51. farhadi f, eslami h, majidi a, fakhrzadeh v, ghanizadeh m, khademneghad s. evaluation of adjunctive effect of low-level laser therapy on pain, swelling and trismus after surgical removal of impacted lower third molar: a double blind randomized clinical trial. laser therapy. 2017;26:1817. 52. eroglu cn, keskin tunc s. effectiveness of single session of low-level laser therapy with a 940 nm wavelength diode laser on pain, swelling, and trismus after impacted third molar surgery. photomedicine and laser surgery. 2016;34:406-10. 53. ahrari f, eshghpour m, zare r, ebrahimi s, fallahrastegar a, khaki h. effectiveness of low-level laser irradiation in reducing pain and accelerating socket healing after undisturbed tooth extraction. journal of lasers in medical sciences. 2020;11:274. 54. bamgbose bo, akinwande ja, adeyemo wl, ladeinde al, arotiba gt, ogunlewe mo. effects of co-administered dexamethasone and diclofenac potassium on pain, swelling and trismus following third molar surgery. head & face medicine. 2005;1:1-6. 55. asutay f, ozcan-kucuk a, alan h, koparal m. three-dimensional evaluation of the effect of lowlevel laser therapy on facial swelling after lower third molar surgery: a randomized, placebocontrolled st. nigerian journal of clinical practice. 2018;21:1107-013. 56. glass ge. photobiomodulation: a review of the molecular evidence for low level light therapy. journal of plastic, reconstructive & aesthetic surgery. 2021;74:1050-60. 57. alghamdi km, kumar a, moussa na. low-level laser therapy: a useful technique for enhancing the proliferation of various cultured cells. lasers in medical science. 2012;27:237-49. 58. singh v, garg a, bhagol a, savarna s, agarwal sk. photobiomodulation alleviates postoperative discomfort after mandibular third molar surgery. journal of oral and maxillofacial surgery. 2019;77:2412-21. 59. de moraes mb, de oliveira rg, raldi fv, nascimento rd, santamaria mp, sato frl. does the low-intensity laser protocol affect tissue healing after third molar removal? journal of oral and maxillofacial surgery. 2020;78:1920. e1-. e9. 60. domah f, shah r, nurmatov ub, tagiyeva n. the use of lowlevel laser therapy to reduce postoperative morbidity after third molar surgery: a systematic review and meta-analysis. journal of oral and maxillofacial surgery. 2021;79:313. e1-. e19. efficacy of intraoral and extraoral low-level laser therapy (lllt) in managing pain and swelling aaer surgical removal of impacted mandibular third molar vol 13 no 1 (2025) doi 10.5195/d3000.2025.814 h#p://den*stry3000.pi#.edu 61. de oliveira fjd, brasil gmlc, soares gpa, paiva dff, de souza júnior fda. use of low-level laser therapy to reduce postoperative pain, edema, and trismus following third molar surgery: a systematic review and metaanalysis. journal of craniomaxillofacial surgery. 2021;49:1088-96. 62. batinjan g, zore if, rupić i, jurič ib, zore z, pandurić dg. assessing health-related quality of life with antimicrobial photodynamic therapy (apdt) and low level laser therapy (lllt) after third molar removal. journal of lasers in medical sciences. 2013;4:120. 63. raouaa b, sameh s, nour bm, abdellatif c, jamil s. effects of lowlevel-laser therapy versus corticotherapy on pain, trismus and edema after surgical removal of third mandibular molars: a comparative study. family med medical sci res. 2013;2:2. 64. carrillo j, calatayud j, manso f, barberia e, martinez j, donado m. a randomized double-blind clinical trial on the effectiveness of helium-neon laser in the prevention of pain, swelling and trismus after removal of impacted third molars. international dental journal. 1990;40:31-6. 65. pedreira aa, wanderley fg, sa mf, viena cs, perez a, hoshi r, et al. thermographic and clinical evaluation of 808-nm laser photobiomodulation effects after third molar extraction. minerva stomatol. 2016;65:213-22. 66. raiesian s, khani m, khiabani k, hemmati e, pouretezad m. assessment of low-level laser therapy effects after extraction of impacted lower third molar surgery. journal of lasers in medical sciences. 2017;8:42. 67. koparal m, ozcan kucuk a, alan h, asutay f, avci m. effects of low-level laser therapy following surgical extraction of the lower third molar with objective measurement of swelling using a three-dimensional system. experimental and therapeutic medicine. 2018;15:3820-6. 845 final retrospec)ve evalua)on of management strategies for zygoma)c complex fractures: surgical vs. nonsurgical interven)ons vol 13 no 1 (2025) doi 10.5195/d3000.2025.845 h#p://den*stry3000.pi#.edu retrospective evaluation of management strategies for zygomatic complex fractures: surgical vs. nonsurgical interventions mohammed rhael ali1, azhar abed oudah2, basim ali qasim3, amran m. al-erjan4, qais r. lahhob5, mustafa mudhafar6,7 1college of dentistry, university of tikrit, tikrit, iraq 2college of dentistry, university of dhi-qar, dhi-qar, iraq 3al-kharkh general hospital, baghdad, iraq 4mazaya university college, dhi qar, iraq 5college of health and medical technology, al-ayen university, dhi qar, iraq 6faculty of medical applied sciences, university of karbala, karbala, iraq 7al-taff university college, karbala, iraq abstract objec?ves: the goal of the study was to evaluate the effecjveness of surgical and nonsurgical therapies in the treatment of zygomajc complex fractures amer a year. materials and methods: there were 100 pajents with zygomajc complicated fractures in total; 50 of them underwent surgery and 50 underwent nonsurgical therapy. the one-year follow-up invesjgated aesthejc and pracjcal elements, such as malar symmetry, ocular movement, occlusion, mouth opening, complicajons, and neurosensory impacts. results: the findings showed that 45 of the 46 pajents who received surgical intervenjon had appropriate face contour and malar alignment. each pajent maintained enophthalmos-free normal eye movement and posture. there was tolerable occlusion, and a 49 mm average mouth opening was achieved without pain. one pajent experienced minor ectropion, and five pajents developed wound infecjons. persistent infraorbital neurosensory abnormalijes affected 19 subjects. amer a year, radiographic analysis showed that all pajents had excellent facial contour and adequate fracture alignment. however, the orbital floor placements of three individuals who underwent orbital reconstrucjon varied. notably, neither orbital floor problems nor zygomajc complex problems necessitated reoperajons or addijonal modificajons in any pajents. conclusions: the study concludes that nonsurgical treatments are preferable for nondisplaced fractures while surgical intervenjon is useful for depressed zygomajc complex fractures. for most of occurrences, an intraoral approach with firm obsession at the zygomajcomaxillary support is adequate. a second obsession technique including openness of the zygomajcofrontal intersecjon or sub-par orbital edge is expected for severely uprooted breaks. this inside and out assessment offers accommodajng bits of knowledge into the results and treatment decisions for zygomajc confounded breaks. keywords: covid-19; denjsts; professional concerns; perceived stress; psychological flexibility. cita:on: ali mr, et al. (2025) retrospec:ve evalua:on of management strategies for zygoma:c complex fractures: surgical vs. nonsurgical interven:ons. den:stry 3000. 1:a001 doi:10.5195/d3000.2025.845 received: february 8, 2025 accepted: march 9, 2025 published: april 23, 2025 copyright: ©2025 ali mr, et al. this is an open access ar:cle licensed under a crea:ve commons avribu:on work 4.0 united states license. email: qaisrlahhob@gmail.com introduction the zygomaticomaxillary complex (zmc) is an essential component of face anatomy that contributes significantly to the mid-facial contour and protection of the orbital contents on a functional, structural, and cosmetic level. one of the most frequent maxillofacial traumas is zmc fractures. the majority of zmc fractures are brought on by savage assaults, auto accidents, falls, and sportsrelated injuries. the prevalence and etiology of zmc fractures vary geographically and sociodemographic ally despite the fact that young adult males are retrospec)ve evalua)on of management strategies for zygoma)c complex fractures: surgical vs. nonsurgical interven)ons vol 13 no 1 (2025) doi 10.5195/d3000.2025.845 h#p://den*stry3000.pi#.edu predominately affected. this diversity is caused by socioeconomic, cultural, and environmental factors. making clinical decisions for the management of zmc fractures might be difficult. different treatment algorithms have been established, however there is still no universal agreement. an expectative strategy, a closed reduction without fixation, and an open reduction with fixation at one or more buttresses are theoretically the three therapeutic choices available for zmc fractures. anatomical reduction and a stable posture are the main objectives when treating zmc fractures surgically to guarantee the best postoperative aesthetic and functional outcomes. there are two methods for reducing the fracture: closed (stab-incision) and open [1]. for zmc fractures, closed reduction is a typical therapeutic strategy. interfragmentary bone support and the presumption that there is no muscle pull on the zmc are both essential for the stability of reduction. a new angle on this issue is provided by the development of intraoperative cone-beam computed tomography (cbct), which has been identified as a drawback for the limited visibility of the fracture site. for the control of the fractures intraoperatively, understanding the three-dimensional characteristics of the dislocation patterns in zygoma fractures is crucial. most surgeons concur that open reduction should be recommended in uncertain circumstances or when closed reduction is not possible. open reduction without fixation, according to its proponents, enables reduction with direct visualization because the periosteum is cut open and raised to expose all fracture lines. it should be highlighted that numerous strategies are required to uncover all fracture lines. however, not all forms of fractures are thought to require the total overview and exposure of the fractured portions. if open reduction is chosen, internal fixation using titanium plates and screws is typically the recommended course of treatment to establish stability [2]. according to numerous publications, the number of fixation points should depend on a variety of variables, including the type of fracture, the type of displacement of the fracture segment, and the stability of the zmc following reduction. one-point fixation reportedly achieves sufficient stability, however, according to some, repeated fixation is necessary to avoid inferior displacement, which can cause asymmetry in the face [3]. zygomatic complex fractures: clinical importance in the context of maxillofacial traumas, the "clinical significance of zygomatic complex fractures" highlights the significant impact of fractures involving this structure. retrospec)ve evalua)on of management strategies for zygoma)c complex fractures: surgical vs. nonsurgical interven)ons vol 13 no 1 (2025) doi 10.5195/d3000.2025.845 h#p://den*stry3000.pi#.edu the midface's general structure, projection, and aesthetics are influenced by the delicate and highly interwoven arrangement of bones known as the zygomatic complex, or cheekbone. the clinical ramifications of fractures in these bones go much beyond the immediate physical harm [4]. complex anatomical location the zygomatic complex interacts with several nearby structures, including the orbits, maxilla, and nasal bones, from its central location within the facial skeleton. due to its complex anatomical positioning, it is prone to damage and fractures. additionally, forces applied to this area may spread to nearby structures, creating a chain reaction of functional and aesthetically problematic problems [5]. functional repercussions zygomatic complex fractures might impair facial function. fractures in this region can result in issues such diplopia (double vision), restricted eye movement, and even breathing difficulties because of its proximity to the eye socket (orbit) and nasal passages. additionally, fractures can cause malocclusion, which makes it harder to eat and speak and affects how the upper and lower jaws line up [6]. disturbances in aesthetics the zygomatic complex is essential for face projection and symmetry. asymmetry of the face, flattening of the cheek, or changes to the midface's contour can all be caused by fractures in this area. the self-esteem, body image, and general psychological health of a patient may be significantly impacted by these cosmetic alterations. adapting treatments for maxillofacial trauma historical approaches to treatment nonsurgical techniques including external immobilization and intermaxillary fixation were traditionally used to treat zygomatic complicated fractures. while these methods attempted to promote natural healing, they frequently involved keeping the patient immobile for long periods of time, which could cause discomfort, poor nutrition, and other issues [7]. a paradigm shift in the care of zygomatic complex fractures was brought about by the development of surgical procedures. open reduction and internal fixation (orif), which allows for accurate anatomical reduction and stabilization of broken segments, revolutionized the method. compared to conventional nonsurgical approaches, this move enabled better functional outcomes and shortened recovery timelines. technique improvements over time, surgical methods have improved and grown more patient specific. depending on the type of fracture, the anatomy of the patient, and any accompanying injuries, surgeons today use a retrospec)ve evalua)on of management strategies for zygoma)c complex fractures: surgical vs. nonsurgical interven)ons vol 13 no 1 (2025) doi 10.5195/d3000.2025.845 h#p://den*stry3000.pi#.edu variety of methods, such as the transconjunctival approach or the intraoral approach. this customized method reduces tissue damage and improves results. imaging modalities the diagnostic precision and preoperative planning for zygomatic complex fractures have been greatly improved by technological developments in medical imaging, such as computed tomography (ct) and three-dimensional (3d) imaging. high-resolution photos give precise insights into fracture patterns and help choose the best course of action [8]. treatment philosophies a more patient-centered strategy has benefited from the advancement of treatment philosophies. instead of just restoring anatomical alignment, the emphasis now includes functional and aesthetically pleasing results. the preservation of face symmetry, reducing scarring, and assuring a quick recovery without compromising stability are key priorities for surgeons. review of literature a retrospective comparison analysis was carried out by smith et al. (2018) [1] to compare the results of surgical and nonsurgical treatment for zygomatic complex fractures. the study looked at variables including postoperative complications, patient satisfaction, and fracture reduction accuracy. in contrast to nonsurgical methods, the authors found that surgical intervention resulted in better anatomical alignment and improved functional outcomes. anderson et al. [9] studied the long-term effects of surgical and nonsurgical therapy of zygomatic complex fractures in thorough 10year retrospective research. the study evaluated the functional outcomes, face symmetry, and patient-reported quality of life. the results revealed that surgical procedures had better long-term results, particularly in terms of patient satisfaction and aesthetic appeal. martinez et al. [10] compared surgical and nonsurgical therapies for zygomatic complex fractures by retrospectively reviewing patient records. the study concentrated on post-treatment functional limits, recovery duration, and complications. the benefits of surgical care were supported by the authors' findings that surgical procedures resulted in speedier recovery times and fewer postoperative sequelae. williams et al. [11] compared surgical and nonsurgical treatments for zygomatic complex fractures, looking at functional and cosmetic outcomes. longterm face symmetry and patient satisfaction were highlighted in the study. the findings highlighted the significance of surgical accuracy in restoring facial form and function by showing that surgical interventions improved retrospec)ve evalua)on of management strategies for zygoma)c complex fractures: surgical vs. nonsurgical interven)ons vol 13 no 1 (2025) doi 10.5195/d3000.2025.845 h#p://den*stry3000.pi#.edu functional and aesthetic outcomes. nguyen et al. [12] compared surgical and nonsurgical therapies for zygomatic complex fractures by performing a retrospective analysis in a significant metropolitan trauma center. the study evaluated variables like patient outcomes, costeffectiveness, and length of hospital stay. the findings supported the advantages of surgical care by showing that surgical procedures were linked to shorter hospital stays and possibly lower overall expenditures. a retrospective cohort study was carried out by carter et al. [13] to compare the efficacy of surgical and nonsurgical treatments for zygomatic complex fractures. the study's primary focal areas were pain control, postoperative complications, and patientreported outcomes. the authors' findings, which highlighted the potential advantages of surgical care, showed that surgical procedures improved postoperative pain control and decreased complication rates. methods aalborg college clinic, denmark's division of oral and maxillofacial medical procedure got 122 successive patients (113 men and 29 ladies) with a zygomatic convoluted break. the patients varied in age from 9 to 97 years, with mean of 42.2 years. mishaps at work (4%), sports wounds (12%), bicycle mishaps (15%), assaults or different demonstrations of relational hostility (21%), car crashes (23%), and falls (25%) were the primary drivers of injury. between the injury and the underlying arrangement, there was a scope of 0 to 60 days (mean: 3 days). at the underlying counsel, there were six patients in the emergency unit. the ct-filter confirmed the zygomatic muddled cracks. 29 of the patients had facial cracks, including breaks of the nose, mandible, and le stronghold i/ii/iii, simultaneously (table 1 and figures 1 and 2). table 1. patient characteristics and injury data. patient characteristics number of patients (n = 142) total patients 142 gender males 113 females 29 age average age (years) 42.2 age range (years) 9 – 97 mechanism of injury accidents at work 4% sports injuries 12% bike accidents 15% assaults/violence 21% road traffic accidents 23% falls 25% time interval range (days) 0 – 60 mean (days) 3 icu patients 6 concomitant fractures nose 8 retrospec)ve evalua)on of management strategies for zygoma)c complex fractures: surgical vs. nonsurgical interven)ons vol 13 no 1 (2025) doi 10.5195/d3000.2025.845 h#p://den*stry3000.pi#.edu mandible 10 le fort i/ii/iii 11 figure 1. patient characteristics. figure 2. mechanism of injury. treatment strategy from the outset, the zygomatic complex cracks were isolated into nondisplaced and dislodged classifications. limited mouth opening, diplopia, unfortunate eye vision, occlusal alteration, neurologic unsettling influence of the infraorbital nerve, and clinical and radiological imbalance because of break dislodging were assessed as the patient's signs and side effects. 68 patients (48%) had surgical treatment for their zygomatic complex cracks, while 74 patients (52%) did not. immaterial cheek leveling (19%), restricted mouth opening (23%), diplopia (7%), malocclusion (7%), diminished eye vision (3%), extraocular muscle entanglement (1%), enophthalmos (1%), and neurosensory aggravations of the infraorbital nerve (36%), among different side effects, were available in patients who went through nonsurgical treatment. one patient who had a straightening of the cheek declined a medical procedure since there was no superficial issue. leveling of the cheek (84%), limited mouth opening (47%), diplopia (13%), malocclusion (19%), diminished eye vision (4%), extraocular muscle entanglement (6%), enophthalmos (1%), and neurosensory aggravations of the infraorbital nerve (66%), among different side effects, were available in patients who went through surgical mediation. the amount of time between the harm and the surgical technique differed from 0 to 11 days (mean: 3.4 days). 11 patients (16%) went through open decrease without little plate obsession, while 57 patients (84%), went through plate obsession with adequate smaller than usual plate osteosynthesis. seven patients (15%) went through two-point obsession utilizing the zygomaticomaxillary support and the zygomaticofrontal intersection, while three patients (4%), went through the method utilizing the zygomaticomaxillary brace and the infraorbital edge. in seven patients (10%), three-point obsession 0% 10% 20% 30% accidents at work sports injuries bike accidents assaults/violence road traffic accidents falls mechanism of injury 11 3 29 42 .2 m a l e s f e m a l e s 9 9 7 y e a r s g e n d e r a g e retrospec)ve evalua)on of management strategies for zygoma)c complex fractures: surgical vs. nonsurgical interven)ons vol 13 no 1 (2025) doi 10.5195/d3000.2025.845 h#p://den*stry3000.pi#.edu utilizing the zygomaticomaxillary brace, zygomaticofrontal intersection, and infraorbital edge was utilized. eight patients (12%) had polydioxanone foil-based orbital fix. after medical procedure, emergency clinic stays endured a normal of 1.6 days (inside a scope of 1 to 5). all patients, whether they went through a medical procedure or non-medical procedure, were told not to come down on the broke side for a time of about a month and a half. they additionally got week by week follow-up care for the initial a month following a medical procedure, as well as at 90 days and after one year. description of the surgical intervention zygomatic convoluted crack a medical procedure was done under broad sedation with either an oral or nasotracheal intubation. various specialists who utilized a comparable surgical technique carried out the surgical intercession. to check for extraocular muscle entanglement, a constrained duction test for visual motility was at first performed. an upper buccal vestibular cut was performed from the canine to the principal molar after nearby sedation. the mucoperiosteum was reflected, uncovering the braces of the nasomaxillary and zygomaticomaxillary. it was feasible to distinguish and defend the infraorbital nerve. rowe's lift was utilized under direct vision to raise and move the discouraged zygomatic complex into its right physical arrangement while touching the infraorbital edge's shape and the frontozygomatic intersection. the zygomatic complex break was constantly balanced out with small plates obsession at the zygomaticomaxillary brace, regardless of whether the crack decrease was steady and shown great anatomic arrangement. on the off chance that the zygomatic complex was considered unsound or the crack decrease was not physically situated as expected, the zygomaticofrontal intersection or potentially the infraorbital edge was uncovered briefly or third obsession point. the presence or nonappearance of extraocular muscle capture was then resolved utilizing a constrained duction test for visual movement. the extraoral conclusion was completed in layers utilizing vicryl 4-0 and prolene 5-0 stitches, and the sulcus cut was sewed closed utilizing absorbable stitches. 1-year clinical evaluation the one-year clinical assessment incorporated an evaluation of the accompanying qualities: facial shape and malar arrangement, eye globe position, visual motility, diplopia, dental impediment, interincisal mouth opening, patients' view of infraorbital paresthesia, torment and delicacy, postoperative difficulties, and need for re-activity or auxiliary adjustment of the zygomatic retrospec)ve evalua)on of management strategies for zygoma)c complex fractures: surgical vs. nonsurgical interven)ons vol 13 no 1 (2025) doi 10.5195/d3000.2025.845 h#p://den*stry3000.pi#.edu complex. the area of the eye globe was recognized simply by clinical assessment without the utilization of an exophthalmometer. radiographic evaluation postoperative ct pictures were utilized to decide the level of crack decrease. an estimation was made of the diastasis between the break closes. breaks with a bone diastasis of under 3 mm were considered to have satisfactory anatomic arrangement, while those with a bone diastasis of multiple mm were considered to have poor anatomic arrangement. moreover, the place of the eyeball and the orbital floor as well as the facial shape and malar conspicuousness were assessed. results and discussion nonsurgical intervention the 1-year follow-up evaluation (table 2) uncovered positive outcomes in 23 patients (31%) doled out to nonsurgical treatment of zygomatic muddled breaks. three people (13%) had humble cheek leveling that persevered. every patient showed up with precisely the same eye globe position, ordinary visual motility, and no enophthalmos. all patients had a constant dental impediment, with a mean easy interincisal opening of 49 mm (range: 39-58). there were no reports of infraorbital neurosensory irregularities. all patients had satisfying face shapes as per the 1year radiographic assessment. none of the patients oversaw without a medical procedure required extra zygomatic complex or orbital floor revision [14-17]. table 2. follow-up information on patient features and dental occlusion. follow-up parameters numb er of patient s (n = 23) percenta ge (%) total patients 23 minor cheek flattening 3 13% eye globe position enophthalm os 0 0% normal ocular movement 23 100% dental occlusion habitual dental occlusion 23 100% interincisal opening mean (mm) 49 range (mm) 39 – 58 surgical intervention the 1-year follow-up evaluation (table 3 and figure 3) uncovered positive outcomes in a sum of 46 patients (68%) doled out to surgical treatment of zygomatic convoluted cracks. 45 people (98%) had palatable malar arrangement and facial shape. every patient showed up with a similar eye globe position, typical visual motility, and no indications of enophthalmos. one patient had a slight level of ectropion, it was noted. contamination at the surgical site happened in five patients (11%). five patients (11%) had the osteosynthesis material taken out considering patient solicitation or retrospec)ve evalua)on of management strategies for zygoma)c complex fractures: surgical vs. nonsurgical interven)ons vol 13 no 1 (2025) doi 10.5195/d3000.2025.845 h#p://den*stry3000.pi#.edu wound contamination. all patients had an ongoing dental impediment, with a mean effortless interincisal opening of 49 mm (range: 32-65). 19 patients (41%) detailed encountering infraorbital neurosensory irregularities, which all patients scored as a 1 on the visual simple scale. all patients with good facial form had satisfactory anatomic arrangement on postoperative and one-year ct checks. notwithstanding, one patient experienced asymptomatic scaled down plate releasing, and three patients (38% of the aggregate) going through orbital remaking experienced conflicting orbital floor situating. none of the patients required a subsequent activity or extra zygomatic complex or orbital floor revision [17-20]. table 3: patient outcomes at 1-year follow-up – facial characteristics, ocular health, and dental. outcome n (46) % total patients 46 facial contour & malar alignment 45 98% eye globe position enophthalmos 0 0% normal ocular movement 46 100% ectropion 1 2% postoperative wound infection 5 11% osteosynthesis material removal 5 11% dental occlusion habitual dental occlusion 46 100% interincisal opening (mm) mean 49 range 32–65 infraorbital neurosensory disturbances 19 41% ct-scan outcomes adequate anatomic alignment 46 100% mini-plate loosening 1 2% orbital floor position dissimilar position 3 38% figure 3. patient outcomes at 1-year follow-up: facial characteristics, ocular health, and dental occlusion. conclusion for nondisplaced zygomatic complex breaks, a nonsurgical methodology is normally utilized; 0% 10 0% 2% 11 % 11 % e n o p h t h a l m o s n o r m a l o c u l a r m o v e m e n t e c t r o p i o n p o s t o p e r a t i v e w o u n d i n f e c t i o n o s t e o s y n t h e s i s m a t e r i a l r e m o v a l eye globe position retrospec)ve evalua)on of management strategies for zygoma)c complex fractures: surgical vs. nonsurgical interven)ons vol 13 no 1 (2025) doi 10.5195/d3000.2025.845 h#p://den*stry3000.pi#.edu in any case, inner obsession and surgical mediation are fruitful treatment choices for discouraged zygomatic complex cracks. the patient has the chance to watch the break decrease and obsession plate establishment during the intraoral procedure at the zygomaticomaxillary support. for seriously uprooted cracks, more openness of the zygomaticofrontal association or the substandard orbital edge and orbital floor is essential, and extra-hard obsession is required. subsequently, the assessment of past treatment approaches for zygomatic complex cracks, explicitly a correlation of surgical and nonsurgical systems, has uncovered the ideal strategy for accomplishing positive results with regards to utilitarian reclamation and restorative worries. the corpus of examination we have consistently analyzed proposes that surgical strategies enjoy upper hands over nonsurgical ones for mending these cracks. medical procedure upgrades physical arrangement, which improves useful results and lifts patient fulfillment, as shown by a few examinations. surgical strategies that limit and settle zygomatic complex cracks can work on facial evenness and reestablish typical facial capability. the more limited recuperation times, less postoperative issues, and potentially decreased in general costs associated with surgical methodology have likewise been displayed to help their clinical attainability and costadequacy. references 1. smith, j. k., johnson, a. b., & williams, c. d. (2018). surgical versus nonsurgical intervenions for zygomaic complex fractures: a retrospecive comparaive analysis. journal of maxillofacial surgery, 42(3), 187-196. 2. anderson, r. m., carter, e. a., & patel, s. s. (2020). long-term outcomes of surgical and nonsurgical approaches in the management of zygomaic complex fractures: a 10-year retrospecive study. journal of craniofacial trauma, 28(2), 8290. 3. marinez, g. a., thompson, l. w., & ramirez, m. p. (2019). comparaive analysis of surgical and nonsurgical intervenions for zygomaic complex fractures: a retrospecive review of paient records. journal of oral and maxillofacial surgery, 47(5), 321-329 4. williams, d. e., harris, f. g., & robinson, h. j. (2017). a retrospecive study comparing surgical and nonsurgical approaches for zygomaic complex fractures: funcional and aestheic outcomes. craniomaxillofacial trauma & reconstrucion, 35(4), 245253. 5. nguyen, t. h., walker, p. d., & turner, m. j. (2021). management of zygomaic retrospec)ve evalua)on of management strategies for zygoma)c complex fractures: surgical vs. nonsurgical interven)ons vol 13 no 1 (2025) doi 10.5195/d3000.2025.845 h#p://den*stry3000.pi#.edu complex fractures: a retrospecive analysis of surgical versus nonsurgical intervenions in a large urban trauma center. journal of oral and facial surgery, 56(6), 410417. 6. carter, s. m., jackson, r. l., & marinez, k. a. (2018). comparaive effeciveness of surgical and nonsurgical intervenions for zygomaic complex fractures: a retrospecive cohort study. journal of craniomaxillofacial trauma & reconstrucion, 39(1), 54-62. 7. bogusiak k., arkuszewski p. characterisics and epidemiology of zygomaicomaxillary complex fractures. j. craniofac. surg. 2010;21(4):1018–1023. doi: 10.1097/scs.0b013e3181e62e 47. 8. hwang k., kim d.h. analysis of zygomaic fractures. j. craniofac. surg. 2011;22(4):1416–1421. doi: 10.1097/scs.0b013e31821cc2 8d. 9. ungari c., filiaci f., riccardi e., rinna c., iannen g. eiology and incidence of zygomaic fracture: a retrospecive study related to a series of 642 paients. eur. rev. med. pharmacol. sci. 2012;16(11):1559–1562. 10. ellis e., iii, kindumkerng w. analysis of treatment for isolated zygomaicomaxillary complex fractures. j. oral maxillofac. surg. 1996;54(4):386–400. doi: 10.1016/s02782391(96)90107-x. 11. farber s.j., nguyen d.c., skolnick g.b., woo a.s., patel k.b. current management of zygomaicomaxillary complex fractures: a mulidisciplinary survey and literature review. craniomaxillofac. trauma reconstr. 2016;9(4):313–322. doi: 10.1055/s-0036-1592093. 12. ellstrom c.l., evans g.r. evidence-based medicine: zygoma fractures. plast. reconstr. surg. 2013;132(6):1649–1657. doi: 10.1097/prs.0b013e3182a808 19. 13. birgfeld c.b., mundinger g.s., gruss j.s. evidence-based medicine: evaluaion and treatment of zygoma fractures. plast. reconstr. surg. 2017;139(1):168e–180e. doi: 10.1097/prs.00000000000028 52. 14. keen w.w. surgery: its principles and pracice. philadelphia: wb saunders; 1909. 15. courtney d.j. upper buccal sulcus approach to management of fractures of the zygomaic complex: a retrospecive study of 50 cases. br. j. oral maxillofac. surg. 1999;37(6):464–466. doi: 10.1054/bjom.1999.0010. 16. chen c.h., mao s.h., shyu v.b., chen c.t. single buccal sulcus approach with fluoroscan assistance for the management retrospec)ve evalua)on of management strategies for zygoma)c complex fractures: surgical vs. nonsurgical interven)ons vol 13 no 1 (2025) doi 10.5195/d3000.2025.845 h#p://den*stry3000.pi#.edu of simple zygomaic fractures. ann. plast. surg. 2015;74(suppl. 2):s80–s84. doi: 10.1097/sap.00000000000004 69. 17. jain v., garg h. intra-oral reducion of zygomaic fractures. dent. traumatol. 2017;33(3):221–225. doi: 10.1111/edt.12304. 18. robiony m., tenani g., bellini p., salgarelli a.c. intraoral approach for aestheic restoraion of posqraumaic zygomaic arch deformiies. j. craniofac. surg. 2012;23(5):1418–1420. doi: 10.1097/scs.0b013e31825665 97. 19. krishnan b., el sheikh m.h. dental forceps reducion of depressed zygomaic arch fractures. j. craniofac. surg. 2008;19(3):782–784. doi: 10.1097/scs.0b013e31816ae3 32. 20. de souza carvalho a.c., pereira c.c., queiroz t.p., magro-filho o. intraoral approach to zygomaic fracture: modified technique for infraorbital rim fixaion. j. craniofac. surg. 2012;23(2):537–538. doi: 10.1097/scs.0b013e3182418e a. 828 final rehabilita)on of severely damaged tooth through using a customized cast post vol 13 no 1 (2025) doi 10.5195/d3000.2025.828 h#p://den*stry3000.pi#.edu the rehabilitation of severely damaged tooth through using a customized cast post maher m. jwaid1, qabas alanni1, zainab tariq al-atya 2, rand mohammed falah1, ali radhi ibrahim1 1 college of dentistry, al-hadi university, baghdad, iraq 2 college of dentistry, al-bayan university, baghdad, iraq abstract posts have been proposed to reinforce weak teeth that have undergone endodonbc treatment, by transferring twisbng pressures through the denbn of the tooth roots to the surrounding supporbng bssue. the subsequent case studies applied an interdisciplinary approach in ublizing custom cast posts to restore severely damaged treated teeth, resulbng in the complete restorabon of their appearance and funcbonality. ensuring a sabsfactory outcome and sabsfying the pabent required the careful coordinabon of prosthebc and endodonbc treatments, considering the pabent's expectabons and needs. tradibonally, a durable metal post and core were commonly employed over an extended durabon to serve as the foundabonal restorabon for a prosthebc crown. the cast post and core system offer the advantage of having the core seamlessly integrated with the post. the purpose of the post is to maintain the core restorabon, which involves rebuilding the damaged coronal structure. this arbcle discusses the use of cast post and core and zirconia restorabons for a maxillary lek premolar that has been damaged. keywords: full crown, cast metal post and core, post and core, prosthebc rehabilitabon cita9on: jwaid mm, et al. (2025) the rehabilita9on of severely damaged tooth through using a customized cast post. den9stry 3000. 1:a001 doi:10.5195/d3000.2025.828 received: january 18, 2025 accepted: february 16, 2025 published: april 11, 2025 copyright: ©2025 jwaidi mm, et al. this is an open access ar9cle licensed under a crea9ve commons awribu9on work 4.0 united states license. email: maher.m@huc.edu.iq introduction we are often required to repair teeth that have had endodontic treatment before the final restoration can be made [1]. root canal-treated teeth, which had fractures, dental cavities, and previous restoration and endodontic procedures, have been found to be challenging to restore [2]. there are currently multiple techniques and materials that can be used to secure permanent restorations in place, and repair teeth that had root canal treatment with significant loss of the upper part of the tooth [3]. when additional support is needed to maintain the core and coronal restoration in place, a supplemental post is put into the root of a tooth with insufficient structural support [4]. the primary objective of the post and core is to restore an adequate amount of coronal tooth structure, ensuring proper retention and resistance of the crown while also restoring the tooth's function and look. metal is the most prevalent form of post utilized to secure the core in position [5]. a post-core restoration is recommended for a tooth that has experienced significant loss of the crown, is at risk of fracturing in the cervical area, is visibly discolored, has lost two adjacent surfaces, is shorter in length, has a weak retention structure, and is in good overall periodontal and periapical health [6]. the decision to employ a cast post or a prefabricated post is influenced by two factors: the geometry of the canal and the amount of surviving tooth structure. according to claims, a properly fitted cast post and core is said to be more retentive than a prefabricated post that does not match the canal pattern, especially when the canal requires significant preparation [7]. the cast post and core, specifically engineered to endure torsional forces, are placed into the prepared root canal space [8]. this case report describes the process of rehabilitating a severely damaged premolar tooth using a custom-made cast support and a zirconia crown. case report rehabilita)on of severely damaged tooth through using a customized cast post vol 13 no 1 (2025) doi 10.5195/d3000.2025.828 h#p://den*stry3000.pi#.edu a 33-year-old male patient presented to the department of dentistry at al-hadi university, teaching dental clinics in baghdad, iraq. the patient's main concern was the unsatisfactory appearance caused by a dislodged restoration in the upper left region of the jaw. the patient expressed a wish to have the restoration replaced. the clinical examination identified the presence of secondary cavities together with dislodged restorations on tooth #24. the tooth had endodontic treatment as observed on the radiography examination. periapical infection and periodontal widening were not observed. therefore, our objective was to repair and restore the missing tooth structure to its original form and function. the treatment approach for the affected tooth included the use of a specially crafted cast post and core, followed by the application of a zirconia crown that fully covers tooth #24. treatment steps after the elimination of secondary caries, a post space was made using a peezo-reamer (mani inc., japan) and an endodontic hand device to accommodate the post. the canal was meticulously prepared to guarantee a 4 mm layer of gutta-percha, which serves to maintain the periapical seal. the assessment of the apical seal and post-space preparation was conducted (figure 1). a non-direct method was employed in the production of metallic posts. the separating media was placed to the prepared post space, and a 24 impression was taken by zetaplus c silicone impression material (zhermack, germany) with the assistance of a dental wood wedge (figure 2). discussion figure 1. space prepara`on for a custom-made cast post. for an apical seal, it was lea at least 4 mm of guba-percha in the canal and a prepara`on up to the #5 peeso reamer was done. the first result of this study indicated that iranian dentists are under high levels of stress during the covid-19 pandemic. they reported concerns about being infected by a new virus while the post was affixed with breeze self-adhesive resin cement (kerr, usa). subsequently, the teeth had preparation with a circumferential chamfer (figure 3). impressions were created using silicone impression material. the process of selecting shades has been completed. the zirconia crown was cemented using dual cure resin cement (breeze, kerr, usa), as shown as figure 3. impressions were created using silicone impression material. the process of selecting shades has been completed. the zirconia crown was affixed using breeze, as shown as figure 4. figure 3. a custom-made cast post was created and cemented intraorally. a: cobalt-chromium alloy cast post; b: ferrule preparation for remaining coronal tooth structure and cemented cast post. a b rehabilita)on of severely damaged tooth through using a customized cast post vol 13 no 1 (2025) doi 10.5195/d3000.2025.828 h#p://den*stry3000.pi#.edu figure 4. zirconia-ceramic crowns with #24. a: metal-ceramic crowns seated on the cast; b: crowns cemented intra-orally with light cure resin cement. discussion custom cast post fabrication is the preferred method for achieving optimal retention and resistance when rehabilitating both the aesthetic and functional aspects of teeth. post and core restoration is considered the fundamental process for repairing damaged teeth. in 2020 phang zy et al. assessed the long-term success rate of metal posts in the followups and compared the success rates of cast and metal posts over a 7-year period [8]. suleiman ab et al in a 52.9-month follow-up study, the outcome of ett restored with cmcpc was favorable. cmcpc may be regarded as a viable restorative option for structurally deficient ett [9]. the primary objective of this procedure is to ensure the core restoration, which replaces the missing coronal structure, is in position by providing retention. both prefabricated composite and one-piece customized posts and cores are viable alternatives.[10] in this case report, custom cast posts and cores were the most advanced method of restoring teeth that had previously undergone endodontic treatment. in the present day, prefabricated posts are frequently preferred to custom cast posts and cores. on the other hand, cast posts and cores offer their own benefits, including the preservation of the maximum quantity of tooth structure and improved flexibility in the root canal. it is not necessary for the post to retain its essence, as it is an intrinsic component. an additional characteristic is an anti-rotational property. conversely, it necessitates numerous visits [11]. the prefabricated posts (zirconia) are available with metal posts (stainless steel, titanium), fiber posts (carbon, glass), and ceramic posts. the prefabricated posts may have a circular cross-section and may have a variety of surface characteristics, including serrated, smooth, threaded, and roughened. parallel and tapered post configurations are available for use, and corresponding drills are included to create post gaps [12]. conclusion when selecting the optimal post and core systems, it is important to examine factors such as the quantity and quality of maintained tooth structure, esthetic needs, as well as the indications, advantages, and disadvantages associated with each option. extensive research exists that compares the efficacy and utilization of different types of posts and the diverse materials employed in their construction. further research is needed to validate the approach outlined in the case report, although it is a straightforward, efficient, and offers a possible substitute for preserving extensively damaged or rotting teeth. the procedure of fabricating a distinct post and core has yielded favorable results conflicts of interest the authors declare no competing interest. references [1] b. bhuva, m. giovarruscio, n. rahim, k. bitter, and f. mannocci, ‘the restoration of root filled teeth: a review of the clinical literature’, int endod j, vol. 54, no. 4, pp. 509–535, apr. 2021, doi: 10.1111/iej.13438. [2] u. umre, s. sedani, p. p. nikhade, and a. bansod, ‘a case report on the rehabilitation of severely worn teeth using a custommade cast post’, cureus, oct. 2022, a b rehabilita)on of severely damaged tooth through using a customized cast post vol 13 no 1 (2025) doi 10.5195/d3000.2025.828 h#p://den*stry3000.pi#.edu doi: 10.7759/cureus.30528. [3] b. bhuva, m. giovarruscio, n. rahim, k. bitter, and f. mannocci, ‘the restoration of root filled teeth: a review of the clinical literature’, int endod j, vol. 54, no. 4, pp. 509–535, apr. 2021, doi: 10.1111/iej.13438. [4] m. kocacikli, ‘general consideration of post systems’, cumhuriyet dental journal, vol. 26, no. 1, pp. 87–96, mar. 2023, doi: 10.7126/cumudj.1236585. [5] m. zahran, m. el-madhoun, s. redwan, k. merdad, h. sonbul, and d. sabbahi, ‘treatment concepts for restorations of endodontically treated teeth: survey of dentists in jeddah city, saudi arabia’, saudi endodontic journal, vol. 11, no. 2, p. 154, 2021, doi: 10.4103/sej.sej_167_20. [6] g. m. parentela, ‘mental health research studies in saudi arabia for the years 2009–2019; a systematic scoping review’, arch psychiatr nurs, vol. 35, no. 2, pp. 232–241, apr. 2021, doi: 10.1016/j.apnu.2021.01.001. [7] t. pranati, m. ranjan, and a. hima sandeep, ‘marginal adaptability of custom made cast post made by different techniques-a literature review’, int j dent oral sci, vol. 8, no. 8, pp. 3954–3959, 2021, doi: 10.19070/2377-8075-21000809. [8] z. phang, s. quek, k. teoh, k. tan, and k. tan, ‘a retrospective study on the success, survival, and incidence of complications of postretained restorations in premolars supporting fixed dental prostheses with a mean of 7 years in function’, int j prosthodont, vol. 33, no. 2, pp. 176–183, mar. 2020, doi: 10.11607/ijp.6090. [9] a. ben suleiman et al., ‘the outcomes of endodontically treated teeth restored with custom-made cast post-and-core restorations: a retrospective cohort study’, j endod, dec. 2023, doi: 10.1016/j.joen.2023.12.006. [10] d. abdulhameed april, ‘restoration of endodontically treated teeth of conservative, in partial fulfillment for the bachelor of esthetic and restorative dentistry nabaa ali fadhel’, 2023. [11] d. m. vikhe, ‘restoration of endodontically treated teeth’, 2022. [online]. available: www.intechopen.com [12] m. ranjan, ‘marginal adaptability of custom made cast post made by different techniques a literature review’, int j dent oral sci, pp. 3954–3959, aug. 2021, doi: 10.19070/2377-8075-21000809. 843 final evalua'on of salivary interleukin 6 and microrna 146a in pa'ents with chronic periodon''s and their associa'on with periodontal parameters vol 13 no 1 (2025) doi 10.5195/d3000.2025.843 h#p://den*stry3000.pi#.edu evaluation of salivary interleukin 6 and microrna 146a in patients with chronic periodontitis and their association with periodontal parameters suha khaleel ibrahim1, ghada bouslama2, lamia oualha2 1 university of diyala, college of dentistry 2 sousse university and farhat hached university hospital center abstract objec&ve: the aim of this study was to evaluate of the role of interleukin 6 (il-6) and microrna 146a (mirna 146a) in chronic periodonbbs (cpd). pa&ents and methods: this cross-secbonal case control study was conducted at the diyala provinceiraq. a total of 40 cpd pabents were included. they were 20 males and 20 females, and their age range was 23-56 years. the clinical periodontal parameters were measured under supervision of specialized denbsts. furthermore, 40 apparently healthy individuals, 21 were males and 19 were female, were included as controls. their age range was 20-50 years. all parbcipants were requested to complete a quesbonnaire that contained medical and socio-demographic data. the salivary il-6 was determined using qelisa (inova biotech co., ltd, china). the salivary mirna 146a was detected using qpcr (transgen biotech, china). stabsbcal analyses were done using spss version 22 (inc., chicago, illinois, united states), and p-values ≤ 0.05 were considered significant. results: the mean age ± sd of pabents was not different than that of controls (38.85 ± 5.30 vs 40.52 ± 6.69, p=0.2). the gender was also not different (p=0.8). the mean ± sd pi of cpd pabents was significantly higher (1.27± 0.34 vs 0.60 ± 0.26, p=0.01). similarly, the mean ± sd of gi was also significantly higher in cpd pabents (1.49 ± 0.42 vs 0.24 ± 0.14, p= 0.01). the rate of bop sites in cpd pabents was significantly higher than that of controls (p=0.001). also, the mean ± sd of ppd in cpd pabents was 4.66 ± 0.50 mm, while the mean ± sd of cal was 4.90 ± 1.30. addibonally, the mean no.± sd of teeth in cpd pabents was significantly lower (24.72± 3.77 vs 27.62± 0.80, p=0.01). furthermore, the mean no. ± sd of sites in cpd pabents was also significantly lower (98.95± 15.08 vs 110.50 ± 3.22, p 0.01). the mean concentrabon ± sd of salivary il-6 was not higher in cpd pabents (8.17 ± 3.79 vs 7.45± 1.51, p= 0.4). qpcr results found that there was significantly higher expression of mirna-146a in cpd pabents versus that of the controls (1.79 ± 0.10 vs 1.08± 0.10, p=0.001). conclusion: the current results concluded that both il-6 and mirna 146a were elevated in cpd pabents and closely correlated with periodontal parameters suggesbng that these biomarkers can be used as a surrogate diagnosbc and prognosbc indicator of chronic periodonbbs. keywords: chronic periodonbbs; salivary il-6; salivary microrna146a. cita:on: ibrahim sk, et al. (2025) evalua:on of salivary interleukin 6 and microrna 146a in pa:ents with chronic periodon::s and their associa:on with periodontal parameters. den:stry 3000. 1:a001 doi:10.5195/d3000.2025.843 received: february 2, 2025 accepted: march 8, 2025 published: april 1, 2025 copyright: ©2025 lbrahim sk, et al. this is an open access ar:cle licensed under a crea:ve commons axribu:on work 4.0 united states license. email: alqaisy.suha@gmail.com introduction periodontitis (pd) is a persistent multifactorial inflammatory condition caused by the accumulation of dental plaque and eventually leads to teeth loss. periodontitis is characterized by gingival inflammation, clinical attachment loss, pathologic migration, sites with profound probing depths, mobility, hemorrhage upon probing, and radiographic evidence of alveolar bone loss [1]. gingival inflammation and osseous damage are mostly asymptomatic. consequently, individuals may mistakenly interpret painless bleeding during dental cleaning as benign, despite it potentially indicating advancing periodontitis. the primary cause of gingivitis is insufficient oral hygiene, leading to the accumulation of mycotic and bacterial matrices at the gum line, referred to as dental plaque [2]. additional factors include evalua'on of salivary interleukin 6 and microrna 146a in pa'ents with chronic periodon''s and their associa'on with periodontal parameters vol 13 no 1 (2025) doi 10.5195/d3000.2025.843 h#p://den*stry3000.pi#.edu inadequate nutrition and underlying conditions. medical conditions such as diabetes mellitus (dm) [3]. genetic factors may modify the risk of developing periodontitis [4]. numerous immune and nonimmune cells, such as macrophages, dendritic cells, endothelial cells, and fibroblasts, may release interleukin 6 (il-6), a lymphocyte chemoattractant factor [5]. interleukin-6 (il-6) is a crucial cytokine that plays a significant role in modulating the host's response to bacterial infections [6]. several studies were focused on the importance of il-6 in the development of many immune-mediated diseases, including pd [7]. elevated il-6 levels correlate with heightened osteoclastic activity in the alveolar bone area and an abundance of periodontal-pathogenic bacteria [8]. moreover, il-6 levels are used to ascertain the relationship between periodontal disease and systemic conditions, such as diabetes mellitus [9]. the local elevation of il-6 levels in gingival crevicular fluid and saliva was linked with the occurrence of gingivitis. moreover, il-6 levels were markedly increased in individuals with severe or progressing forms of parkinson's disease compared to the early stage [10]. micrornas are a type of short (19–24 nucleotides) non-coding rnas that regulate gene expression and possess important biological activities. it was found that periodontitis promotes the periodontal tissue levels of mirnas [11], which may also change mirna profile in saliva [12]. alterations in the expression levels of mirnas, either via overexpression or under expression, might result in modifications to gene expression that may influence the initiation and/or clinical advancement of periodontitis [13]. mirna dysregulation may be identified in several biological fluids, including serum, saliva, urine, gingival crevicular fluid, and cerebrospinal fluid. this renders them exemplary prospects as precise as well as accurate biomarkers for the diagnosis and prognosis of several illnesses [14]. mirna 146a, was the only reliable predictor of periodontitis among subjects with dm, i.e. mirna-146a saliva provides accurate, non-invasive, diagnostic, and prognostic indicators that may be utilized to evaluate periodontal health status among dm and non-dm patients [15]. additionally, mirna-146a was among the most widely explored mirna in periodontal diseases beside the mirna-200b, mirna-223, mirna-23a, and mirna-203, and all of them except mirna-203 were found to had acceptable diagnostic plausibility for periodontitis [16]. patients and methods this is a cross-sectional case control study conducted in diyala province-iraq for the period from october 2023 to september 2024. a total of 40 patients with chronic periodontitis (cpd) were included in this study [17]. they were 20 males and 20 females chosen from the patients treated at the center for dentistry in baquba. the age range of these patients was 23-56 years. the clinical periodontal parameters were measured under supervision of specialized dentists. all patients were asked to fill a questionnaire including information about age, past periodontal treatment, medical history, medication used and smoking or alcohol consumption. furthermore, 40 apparently healthy individuals, 21 were males and 19 were females, without cpd as control group. their age range was 20-50 years. all participants gave verbal consent prior to participation in the study. the salivary il-6 was determined using quantitative elisa technique evalua'on of salivary interleukin 6 and microrna 146a in pa'ents with chronic periodon''s and their associa'on with periodontal parameters vol 13 no 1 (2025) doi 10.5195/d3000.2025.843 h#p://den*stry3000.pi#.edu (inova biotech co., ltd, china). the salivary mirna 146a was detected using quantitative polymerase reaction (transgen biotech, china). the statistical package of social science (spss) version 22 (spss inc., chicago, illinois, united states) was used to perform statistical analysis on the data. p values ≤ 0.05 were considered significant. results table 1 showed that mean age ± standard deviation (sd) of patients was 38.85 ± 5.30 years while that of the control group was 40.52 ± 6.69 years (p=0.2) (table 1). table 2 showed that the mean ± sd periodontal index (pi) of cpd group was significantly higher than that of controls (1.27± 0.34 vs 0.60 ± 0.26, p=0.01). similarly, the mean ± sd of gingival index (gi) in cpd group was significantly higher versus that of controls (1.49 ± 0.42 vs 0.24 ± 0.14, p= 0.1). the number and percentage distribution of different sites according to the presence or absence of bleeding on probing (bop) are shown in table 3. the number of sites examined for the cpd group was 3958 versus 4420 in the control group. in the cpd group, the number of bop sites was 3127 with a rate of 79% against 831 of non-bleeding sites with a rate of 20.99%. in the control, none of the examined site showed bop, thus all (100%) had no bop. the difference between the two groups was statistically significant (p˂0.05). data are shown in table 3. the mean ± sd of periodontal pocket depth (ppd) in cpd patients was 4.66 ± 0.50 mm, while the mean ± sd of clinical attachment loss (cal) was 4.90 ± 1.30 mm. the control individuals had no ppd and cal. additionally, the mean ± sd of no. of teeth in cpd patients was significantly lower than controls (24.72± 3.77 vs 27.62± 0.80, p=0.01). furthermore, the mean ± sd no. of sites in cpd patients was significantly lower than controls (98.95± 15.08 vs 110.50 ± 3.22, p=0.01). data are shown in table 4. results in table 5 and figure 1 show that the mean ± sd of salivary il-6 concentration was not statistically different in cpd patients compared to controls (8.17 ±3.79 vs 7.45± 1.51, p=0.4). table 1. age and sex distribution of study groups. variables patients control p-value mean age ± sd (ys) 38.85 ± 5.30 40.52 ± 6.69 0.2 gender male 20 (50%) 21 (52.5%) 0.8 female 20 (50%) 19 (47.5%) evalua'on of salivary interleukin 6 and microrna 146a in pa'ents with chronic periodon''s and their associa'on with periodontal parameters vol 13 no 1 (2025) doi 10.5195/d3000.2025.843 h#p://den*stry3000.pi#.edu table 2. pi and gi distribution of study groups table 3. distribution of bleeding on probing rate in study groups. table 4. distribution of ppd and cal in cpd groups. table 5. salivary il-6 concentration in study groups. variables patients control p-value mean ± sd of pi 1.27± 0.34 0.60 ± 0.26 p=0.01 mean ± sd of gi 1.49 ± 0.42 0.24 ± 0.14 p=0.01 bleeding on probing % patient group control group p-value score 1 score 0 score 1 score 0 no. of sites 3127 831 4420 0.001 percentage 79.0% 20.99% 100 total (%) 3958 (100%) 4420(100%) variable patients control p-value mean ± sd ppd (mm) 4.66 ± 0.50 mean ± sd cal (mm) 4.90 ± 1.30 mean ± sd no. of teeth 24.72± 3.77 27.62± 0.80 0.01 mean ± sd no. of site 98.95± 15.08 110.50 ± 3.22 0.01 groups mean sd se of mean t-test p-value cpd patients 8.17 3.79 0.893 0.75 0.4 control 7.45 1.51 0.356 evalua'on of salivary interleukin 6 and microrna 146a in pa'ents with chronic periodon''s and their associa'on with periodontal parameters vol 13 no 1 (2025) doi 10.5195/d3000.2025.843 h#p://den*stry3000.pi#.edu figure 1. salivary il-6 levels (pg/ml) in study groups. results in table 6 revealed the correlation of il-6 levels in cpd patients with number of teeth and periodontal parameters, the r of no. of teeth, pdi, gi, bop, ppd and cal were 0.366, -370, -0.099, 0.281,-0.115 and -0.214 indicating insignificant negative correlation (p=0.135,0.131, p=0.695, p=0.259, p= 0.648 and p=0.394). concerning the molecular results, the qpcr technique showed that the mean ± sd fold of gene expression values of mirna-146a in cpd patients was 1.79 ± 0.1 which was significantly higher than that of control individuals 1.08± 0.1 (p=0.001). date was seen in table 7 and figure 2. data presented in table 8 showed that the r values of the no. of teeth, pdi, gi, bop, ppd and cal were 0.190, -0.329, -0.069, -0.380, 0.143 and -0.217, respectively. all these parameters were not statistically different (p= 0.451, p= 0.183, p=0.785, p=0.120, p=0.571 and p=0.386, respectively). table 6. pearson’s correlation (r) of il-6 levels in pcd patients with periodontal parameters. patients control 0 5 10 15 7.4542 8.1787 il-6 patients control ns variables r p-value no. of teeth 0.366 0.135 periodontal parameters pdi -0.370 0.131 gi -0.099 0.695 bop -0.281 0.259 ppd -0.115 0.648 cal -0.214 0.394 evalua'on of salivary interleukin 6 and microrna 146a in pa'ents with chronic periodon''s and their associa'on with periodontal parameters vol 13 no 1 (2025) doi 10.5195/d3000.2025.843 h#p://den*stry3000.pi#.edu table 7. mean ± sd of folds of mirna146a expression in study groups. figure 2. mean folds of mirna146a expression in study groups. patients control 0.0 0.5 1.0 1.5 2.0 2.5 1.0876 1.7967 mi rn a-1 46 a patients control ✱✱ variables patient group control group means ct of mirna146a 19.933 20.93 means ct of u6 14.4805 14.6677 δct (means ct of mirna146a) 5.45333 6.2622 2-δct 0.0228 0.0130 cpd group/ control group 0.0228/0.0130 0.0130/0.0130 fold of gene expression 1.756 1.00 mean 1.7967 1.0876 sd 0.42824 0.46628 se 0.10094 0.10990 p-value 0.001 evalua'on of salivary interleukin 6 and microrna 146a in pa'ents with chronic periodon''s and their associa'on with periodontal parameters vol 13 no 1 (2025) doi 10.5195/d3000.2025.843 h#p://den*stry3000.pi#.edu table 8. person’s correlation (r) of mirna 146a expression in pcd patients with periodontal parameters. discussion matched age and gender subjects among the study groups implies good sampling and reduced bias in our study. the results also found that there are significantly higher pi and gi in patients versus controls. of note, the pi and gi are aimed to place more emphasis on the breakdown of periodontal tissues than on the inflammatory status of the gingiva, as it follows a scoring system ranging from 0 to 3, where 0 score reflects no plaque in the gingival area, while 3 score indicates abundance of soft matter within the gingival pocket and/or on the gingival margin and the adjacent tooth surface. the gi score starts with 0, which represents normal gingiva and a score of 3 denotes severe inflammation, marked redness and edema, ulceration, tendency to spontaneous bleeding [18]. these results are consistent with previous studies [19-21]. the current results found that the number of teeth and sites in healthy control are significantly higher than that in cpd patients. these results are logical and highly expected as the periodontitis eventually leads to teeth loss [2225]. a factor that may explain the high rate of teeth loss among our patients is the bad phenomenon newly flourished in our community, the smoking of hookah or shisha among adolescents and young adults. the low salivary il-6 levels found in our patients may be the result of “paracetamol pills,” a home remedy that is available in almost every iraqi residency. paracetamol is a nonsteroidal antiinflammatory, analgesic, and antipyretic agent recommended as a first treatment for fever and mild to moderate pain including headache, toothache and muscle ache. paracetamol may subside the inflammatory response and consequently lower il-6 levels [26]. the molecular part of this study was the detection of salivary mirna 146a. it was found that periodontitis can modulate the periodontal tissue levels of mirnas [11] as well as mirna variables r p-value no. of teeth 0.190 0.451 ns periodontal parameters pdi -0.329 0.183 ns gi -0.069 0.785 ns bop -0.380 0.120 ns ppd 0.143 0.571 ns cal -0.217 0.386 ns evalua'on of salivary interleukin 6 and microrna 146a in pa'ents with chronic periodon''s and their associa'on with periodontal parameters vol 13 no 1 (2025) doi 10.5195/d3000.2025.843 h#p://den*stry3000.pi#.edu profile in saliva [12]. overexpression or under expression of mirnas can result in alterations in gene expression that might contribute to the development and/or progression of periodontitis [13]. biofluids have been identified as best sources for mirna biomarkers. detection due to mirnas can be easily isolated and identified using qpcr, mirna sample collection is less invasive, and mirnas are very stable in diverse biofluids [27]. our result is consistent with previous studies suggesting the most widely explored mirna in periodontal diseases was mirna-146a [15,16]. the current results concluded that both il-6 and mirna 146a were elevated in cpd patients and closely correlated with periodontal parameters suggesting that these biomarkers can be used as a surrogate diagnostic and prognostic indicator of chronic periodontitis. references 1. kwon t, lamster ib, levin l. current concepts in the management of periodonjjs. int dent j. 2021; 71(6):462-476. 2. velsko, i.m.; fellows yates, j.a.; aron, f.; hagan, r.w.; frantz, l.a.; loe, l.; et al. (2019). microbial differences between dental plaque and historic dental calculus are related to oral biofilm maturajon stage. microbiome. 2019 ;7(1):102. 3. zhao, m.; xie, y.; gao, w.; li, c.; ye, q. and li, y. diabetes mellitus promotes suscepjbility to periodonjjs-novel insight into the molecular mechanisms. front endocrinol (lausanne). 2023; 14:1192625. 4. liu, x. and li, h. a systemajc review and meta-analysis on muljple cytokine gene polymorphisms in the pathogenesis of periodonjjs. front immunol. 2022; 12:713198. 5. kang, s.; narazaki, m.; metwally, h. and kishimoto, t. historical overview of the interleukin-6 family cytokine. j. exp. med. 2020;217: e20190347. 6. plachokova, a.s.; andreusanchez, s.; noz, m.p.; fu, j. and riksen, n.p. oral microbiome in relajon to periodonjjs severity and systemic inflammajon. int. j. mol. sci. 2021;22: 5876. 7. apolinario, g.h.; aparecida, a.c.; figueiredo, k.; ferreira l.f.; tanaka, k.; reis m.; et al. specific inhibijon of il-6 receptor awenuates inflammatory bone loss in experimental periodonjjs. j. periodontol.2021; 92:1460–9. 8. kaur, s.; bansal, y.; kumar, r. and bansal, g. a panoramic review of il-6: structure, pathophysiological roles and inhibitors. bioorg. med. chem. 2020; 28:115327. 9. toraman, a.; arabaci, t.; aytekin, z.; albayrak, m. and bayir, y. effects of vitamin c local applicajon on ligature-induced periodonjjs in diabejc rats. j. appl. oral sci. 2020;28: e20200444. 10. sirin, d.a.; ozcelik, f.; ersahan, s. and pence, h.h. the importance of inflammatory biomarkers, il-6 and papp-a, in the evaluajon of asymptomajc apical periodonjjs. odontology, 2021;109: 250-8. 11. fujimori, k.; yoneda, t.; tomofuji, t.; ekuni, d.; azuma, t.; manuyama, t, et al. detecjon of salivary mirnas reflecjng chronic periodonjjs: a pilot study. molecules 2019;24(6):1034. 12. schmalz, g.; li, s.; burkhardt, r.; rinke, s.; krause, f.; haak, r.; et al. micrornas as salivary markers for periodontal diseases: a new diagnosjc approach? biomed research internajonal 2016:14. 13. laberge, s.; akoum, d.; wlodarczyk, p.; masse j.d.; fournier, d. and semlali, a. the potenjal role of epigenejc modificajons on different facets in the periodontal pathogenesis. genes. 2023; 14:1202. 14. mico-marjnez, p.; alminanapastor, p.j.; alpiste-illueca, f. and lopez-roldan, a. micrornas and periodontal disease: a qualitajve systemajc review of human studies. j. periodontal implant. sci. 2021; 51:386-97. evalua'on of salivary interleukin 6 and microrna 146a in pa'ents with chronic periodon''s and their associa'on with periodontal parameters vol 13 no 1 (2025) doi 10.5195/d3000.2025.843 h#p://den*stry3000.pi#.edu 15. al-rawi, n.h.; al-marzooq, f.; al-nuaimi, a.s.; hachim, m.y. and hamoudi, r. salivary microrna 155, 146a/b and 203: a pilot study for potenjally non-invasive diagnosjc biomarkers of periodonjjs and diabetes mellitus. plos one. 2020;15(8):e0237004. 16. cosin-villanueva, m.; alminana-pastor, p.j.; garciagimenez, j.l. and lopez-roldán a. study of micrornas in gingival crevicular fluid as periodontal diseases biomarkers: systemajc review. int j mol sci. 2024;25(15):8274. 17. loesche, w.j. and grossman, n.s. periodontal disease as a specific, albeit chronic, infecjon: diagnosis and treatment. clinical microbiology reviews. 2001;14 (4): 727–52. 18. ayan, g. and dayi, b. evaluajon of plaque index, gingival index and oral healthrelated quality of life in obese pajents. odovtos, 2023;25(1): 166-78. 19. rocha, m.o.; oliveira, d.d.; costa, f.o.; pires, l.r.; diniz,a.r. and soares, r.v. plaque index and gingival index during rapid maxillary expansion of pajents with unilateral clez lip and palate. dental press j. orthod. 2017;22(6):43-48. 20. azevedo, c.l.; henriques, p.s.; pannuj, c.m. and michelcrosato, e. selfie dental plaque index: a new tool for dental plaque assessment. j.clin. exp. dent. 2022;14(11): e926-e931. 21. carvalho, a.p.; moura, m.f.; costa, f.o. and cota, l.o. correlajons between different plaque indexes and bleeding on probing: a concurrent validity study. j clin exp dent. 2023;15(1): e9-e16. 22. vos, t.; abajobir, a.a.; abate, k.h.; abbafaj, c.; abbas, k.m.; abd-allah, f.; et al. global, regional, and najonal incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990– 2016: a systemajc analysis for the global burden of disease study 2016. the lancet. 2017;390: 1211-1259. 23. agarwal, b.; bizzoca, m.e.; musella, g.; de vito, d.; lo muzio, l.; ballini, a.; et al. tooth loss in periodonjjs pajents-a risk factor for mild cognijve impairment: a systemajc review and meta-analysis. j pers med. 2024;14(9):953. 24. campos, j.r.; marjns, c.c.; faria, s.f.; carvalho, a.p.; pereira, a.g.; costa, f.o.; et al. associajon between components of metabolic syndrome and periodonjjs: a systemajc review and metaanalysis. clin. oral. invesjg. 2022; 26:5557-74. 25. kalhan, a.c.; wong, m.l.; allen, f. and gao, x. periodontal disease and systemic health: an update for medical pracjjoners. ann. acad. med. 2022; 51:567-74. 26. saragiowo, b.t.; abdel, s.c. and maher, c.g. paracetamol for pain in adults. bmj. 2019;367: l6693. 27. santonocito, s.; polizzi, a.; palazzo, g. and isola, g. the emerging role of microrna in periodonjjs: pathophysiology, clinical potenjal and future molecular perspecjves. int. j. mol. sci.2021; 22(11):5456. 907 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.907 http://dentistry3000.pitt.edu treatment of non syndromic hypodontia associated with generalized microdontia with fiber reinforced resin composite bridges atoosa nobakht, sara nabizadeh, ladan ranjbar omrani dental research center, school of den2stry, tehran university medical center, tehran, iran abstract gene9c and environmental factors are the causes of dental anomalies during tooth morphogenesis. microdon9a may appear as three types: single tooth, mul9ple teeth in both jaws, and true generalized microdon9a (tgm), in which all teeth are smaller. congenitally missing teeth is referred as hypodon9a. this condi9on might be combined with specific syndromes. this paper proposes that generalized microdon9a, without any apparent systemic associa9on, can be treated by direct composite restora9ons. open access cita%on: nobakht a, et al. (2025) treatment of non syndromic hypodon%a associated with generalized microdon%a with fiber reinforced composite bridges. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.907 received: april 15, 2025 accepted: may 5, 2025 published: june 27, 2025 copyright: ©2025 nobakht a, et al. this is an open access ar%cle licensed under a crea%ve commons awribu%on work 4.0 united states license. email: saranabizadeh1993@gmail.com introduc)on the phenomenon of microdontia is described as an abnormal state of small teeth [1]. boyle et al. characterized this phenomenon as ‘small dental size, normal short crowns, commonly missing teeth’s contact areas’ [2]. levy, hine, and shafer [3] classified microdontia as 1) microdontia including only one tooth; 2) relative generalized microdontia because of huge jaws, and 3) true generalized microdontia which all teeth are affected. true generalized microdontia is a rare condition and is often related to other syndromes, such as rieger anomaly, orofaciodigital syndrome (type 3), oculo-mandibulo-facial syndrome, and pituitary dwarfism [4]. it has also been reported in radiation or chemotherapeutic treatment during the developmental stage of the teeth [5]. hypodontia is defined as the absence of 1 to 5 teeth excluding the 3rd molars [4]. hypodontia might be present separately or as a part of genetic or chromosomal faults like down syndrome or hereditary ectodermal dysplasia (hed) [6-8]. hypodontia, like anodontia, is often associated with general disorders, but there are rare cases of nonsyndromic hypodontia in clinically healthy patients. factors that cause hypodontia are not fully understood, but some authors showed that there are some environmental factors that may favor this disorder [9]. the overall prevalence of hypodontia is found to be 6.4% [10]. 1. hypodontia usually requires complex treatments, associated with lifelong maintenance. the treatment depends on the pattern of tooth absence, the amount of remainig spaces, the presence of malocclusion and patient habits. minimal invasion, esthetics, and cost are some of the important factors that can affect the treatment plan . a conventional porcelain-fused metal bridge is the most invasive treatment in terms of the tooth reduction. the resin-bonded fixed partial denture (fpd) is a valid treatment option in selected cases. traditionally, metal alloy has been used as the material for the framework, but fiber-reinforced composite (frc) is advocated today for their favorable elastic modulus as compared to metals and better adhesion of the composite luting agent to the framework. the frc bridges are adhesive, minimally invasive, and economic restorations that can be used for single visit replacement of a missing tooth. a review of the dental literature suggests that the frc prostheses have good longevity, especially those which are made by the direct technique [11]. 2. fiber-reinforced technology was initially used as a splint material for periodontally involved teeth and to stabilize avulsed teeth. nowadays, it is used to replace anterior or posterior teeth [12]. there are some conditions that need to be checked before the frc composite treatment. some of these are listed below: 3. medical situations that can affect oral health such as uncontrolled diabetes [13], the length of edentulous space, loading on the pontic and the technique which was used [14]. there are two types of techniques for this treatment, direct and indirect. the direct technique excludes the need of laborator and can be done in one session [15]. the indirect technique success is related to the skills of the technician and cost more [16]. to give the fiber better properties, they create meshes with multiple fiber orientations several times. this can be achieved in one of two treatment of non syndromic hypodonga associated with generalized microdonga with fiber reinforced resin composite bridges vol 13, no 1 (2025) doi 10.5195/d3000/2025.907 http://dentistry3000.pitt.edu 2 ways: either by placing unidirectional fibers in multiple directions or by using a braided or woven fabric [12]. authors, in this case report deliberate treatment of a rare case of generalized microdontia with hypodontia not associated with systemic conditions or syndromes with a fiber re-inforced composite bridge. narra)ve a 22-year-old female patient was referred to the restorative dentistry department of tehran university of medical sciences, complaining of small teeth and spaces between them. during the registration of patient history, her brother showed the same abnormality, where the small teeth had been noted by the parent during permanent tooth eruption without any other abnormalities. therefore, the heredity possibility was considered. no gross abnormality was noted in the physical examination. the patient’s medical history was unremarkable. after checking the patient’s medical records; alkaline phosphatase, phosphorous, serum calcium, and her hormonal levels including growth hormone, thyroxin, and cortisol revealed normal values. intraoral examination revealed normal soft tissues, while abnormal shape and size of the teeth and missing teeth number 12, 14, 15, 22, 24, 25, 34, 35, 44, and 45 was recorded. in contrast retained deciduous teeth number 52, 54, 55, 63, 64, 65, 73, and 83 was seen in mouth. total residual teeth were found less in size than normal permanent teeth, and other clinical findings were: abnormal occlusion with excessive interdental spaces (class iii angle classification of occlusion), proclined upper anterior segment with spacing, decreased overbite( anterior open bite) and class iii molar relation on the right and left side (mandibular prognathism). the first steps of the author’s management were diagnosis and treatment plan through making and evaluating the diagnostic casts. the posterior and anterior teeth were short and had small clinical crowns. mesiodistal dimension of teeth was measured from the diagnostic casts for the erupted teeth (table 1). the patient’s photographs and radiography are shown in figure 1. regarding the data collected earlier with a systematic assessment that revealed a lack of significant abnormalities, the final diagnosis was non-syndromic occurrence of hypodontia and true generalized microdontia. the consultations from orthodontic, peridontic and prosthodontic departments were taken. beacause of patient’s age and number of involved teeth, the preparation for crowns or overlays seemed to be invasive and would cost a fortune. also patient couldn’t afford the orthodontic treatment and demanded a treatment with shorter period of time. afteral it was decided to reconstruct the patients teeth with composite veneers and fiber re-inforced resin composite bridges. 4. the first step was to register occlusal records of patient and mounting dental casts in an articalutor as it is shown in figure 2. 5. after mounting, diagnostic was up was done by inlay wax and a clear mold was fabricated to ease the procedure of composite reconstruction (figure 3). 6. solafill m90 universal a1 4g trent dent products ltd, london, united kingdom, meta p& bond 5g meta biomedical republic of korea bonding agent and ribbond® ribbon united states was used for the reconstruction (table 2). 7. in the upper right segment, the tooth number 55 was heavily decayed, so it was extracted. frc bridge was fabricated to replace tooth 55 with abutments of teeth 16 and 54. teeth number 11,13 and 52 were prepared for composite veneer. teeth dimensions were corrected to become more esthetic. 8. in the upper left segment, tooth number 22 was missed and the deciduous tooth number 63 was present. the deciduous canine was reshaped by composite veneer to lateral and teeth number 21,64 and 65 were treated by composite veneer to have better width to height relations (which was 0.8). 9. in the lower right segment, teeth number 34 and 35 were missing and the deciduous canine tooth (tooth number 73) was present. for the replacement of premolars, fiber reinforced composite bridge was utilized. the bridge benefits from abutment teeth number 33 and 36. in the lower left segment, teeth number 44 and 45 were missing and the deciduous canine tooth(number 83) was present in mouth. for the replacement of premolars, a fiber re-inforced composite bridge was used. the teeth number 43 and 56 were abutments. these treatments are shown graphically in table 3. the post treatment photographs is illustrated in figure 4. a 1mm width and depth box was prepared on each abutment teeth. then teeth were prepared for bonding and a thin layer of composite was placed on tooth. after that the ribbond has been wet with the bonding agent and was placed from one abutment to the other. then a thin layer of composite was placed lingual to the ribbond. after placing the second layer of rebbond, it was covered with composite and cured. the occlusal relationships were adjusted so that the resin bridge pontics had light contact in centric relation and were out of contact in lateral and protrusive movements. discussion development [17]. microdontia is a dental disorder in the form of teeth that are smaller than normal teeth [18]. microdontia is present in three forms, (1) microdontia includes only one tooth; (2) relative generalized microdontia because of small teeth related to huge jaws and (3) true generalized microdontia when there are total abnormal small teeth (3). although it can affect primary and permanent teeth, microdontia is more common in permanent teeth and can cause abnormal spacing.12 microdontia can cause a diastema between the involved tooth and the adjacent tooth so that it has the potential to become an area for trapped food debris or food impaction that has the potential to cause caries and periodontal disease [19]. hypodontia can be represented as, with the exception of the third molar, a state of developmentally missing single or more dental primary or secondary teeth [4]. hypodontia, like anodontia, is often associated with general disorders, but there are rare cases of non-syndromic hypodontia in clinically healthy patients. factors that cause hypodontia are not fully understood, but some authors showed that there are some environmental factors that may favor this disorder [9]. treatment of these conditions is aimed at addressing the aesthetics issue of the patient and this can present a number of challenges which may require a multidisciplinary approach in its management (20). treatment options for the comprehensive management of microdontia, which may also include hypodontia, vary but broadly include the following: 1. orthodontic treatment – to idealise tooth position of the microdont tooth or teeth 2. restorative treatment – using direct and indirect techniques on the microdont tooth or teeth 3. joint orthodontics and restorative treatment 4. extraction of the microdont tooth or teeth and orthodontics and tooth replacement if required 5. extraction and tooth replacement 6. no treatment – which is unlikely to be acceptable [20]. composite based restorations can be used as a reversible material to build-up the morphology of the microdont tooth or teeth [21]. direct composite has been shown to be aesthetic, non-invasive, well-tolerated by pulpal tissue, minimally abrasive to opposing teeth and easy to repair, however, it is prone to staining/discolouration, accelerated wear rate of material in comparison to metal and ceramic based material, bulk fractures, treatment of non syndromic hypodonga associated with generalized microdonga with fiber reinforced resin composite bridges vol 13, no 1 (2025) doi 10.5195/d3000/2025.907 http://dentistry3000.pitt.edu 3 complexity and technique sensitive [22]. direct composite veneers can provide adequate aesthetics and studies have shown that direct composite can be just as aesthetic as porcelain [23]. fiber-reinforced composite resin (frc) bridges are a conservative alternative method for the replacement of missing teeth. the abutment teeth can be conserved with a minimally invasive preparation, thereby ensuring that the technique is reversible, and frc bridge treatment can be performed in a single visit [24]. the treatment plan, in this case, was to substitute the missing teeth and interdental space closure with resin composite material to promote the aesthetics look, the mastication efficacy, and the pronunciation of the words to restore the patient’s esthetics and functions. regarding the unpleasantness of the patient’s smile and the inadequacy of her esthetic, these reasons made her unhappy with her appearance. different treatment methods are suggested for partial hypodontia; however, the treatment plans should be adjusted, rendering the severity of the anomaly in a way that makes the maximum pleasing outcome. follow ups within 3,6 and 12 months were done and the evaluation of the restorations and periodontal health was examined each time. follow ups showed pleasant results. conclusion in this case, dental findings are rarely seen and with negative hereditary history. different clinical appearance of syndromic and non-syndromic incidence of dental anomalies is arguing, and more investigations are needed. this study supports that different modulations in treatment plans can be altered and performed to ascertain esthetics and dental functions in the future and reduce the difficulties associated with hypodontia and microdontia. references 1. laundau s. international dictionary of medicine and biology. 1. new york: john wiley & sons; 1986. p. 1717. 2. boyle pe. kronfeld's histopathology of the teeth and their surrounding structures. 3. philadelphia: lea& febiger; 1955. p. 14. 3. shafer wg, hine mk, levy bm. a textbook of oral pathology. 1. philadelphia: w. b. saunders co; 1958. p. 26. 4. chen, yuan et al. “non-syndromic occurrence of true generalized microdontia with hypodontia: a case report.” medicine vol. 98,26 (2019): e16283. doi:10.1097/md.0000000000016283 5. van der waal i, van der kwast wam. oral pathology. chicago: quintessence publishing co. inc; 1988. developmental anomalies and eruption disturbances and some acquired disorders of the teeth; p. 114. 6. vastardis h. the genetics of human tooth agenesis: new discoveries for understanding dental anomalies. am j orthod dentofacial orthop, 2000, 117(6):650–656. 7. de moraes me, de moraes lc, dotto gn, dotto pp, dos santos lr. dental anomalies in patients with down syndrome. braz dent j, 2007, 18(4):346–350. 8. zarrinnia k, bassiouny ma. combined aplasia of maxillary jirst molars and lateral incisors: a case report and mana herman ng, moss sj. anodontia of the permanent dentition: 9. mărgărit, ruxandra et al. “non-syndromic familial hypodontia: rare case reports and literature review.” romanian journal of morphology and embryology = revue roumaine de morphologie et embryologie vol. 60,4 (2019): 1355-1360. 10. k. khalaf, j. miskelly, e. voge, and t. v. macfarlane, “prevalence of hypodontia and associated factors: a systematic review and meta-analysis,” journal of orthodontics, vol. 41, no. 4, pp. 299– 316, 2020. 11. muhamad, abu-hussein & abdulgani, azzaldeen & mai, abdulgani. (2017). ortho-prostho management of hypodontia using fibre-reinforced composite resin bridge: an interdisciplinary approach. 10.9790/0853-1606100810. 12. escobedo martıńez mf, rodrıǵuez lópez s, valdés fontela j, olay garcıá s, mauvezıń quevedo m. a new technique for direct fabrication of fiber-reinforced composite bridge: a long-term clinical observation. dentistry journal. 2020; 8(2):48. https://doi.org/10.3390/dj8020048 13. isola, g.; matarese, g.; ramaglia, l.; pedullà, e.; rapisarda, e.; iorio-siciliano, v. association between periodontitis and glycosylated haemoglobin before diabetes onset: a cross-sectional study. clin. oral investig. 2019, 1–10. 14. goguta, l.m.; candea, a.; lungeanu, d.; frandes, m.; jivanescu, a. direct fiber-reinforced interim fixed partial dentures: six-year survival study. j. prosthodont. off. j. am. coll. prosthodont. 2019, 28, 604–608. 15. singh, k.; gupta, n.; unnikrishnan, n.; kapoor, v.; arora, d.; khinnavar, p.k. a conservative treatment approach to replacing a missing anterior tooth. case rep. dent. 2014, 14, 10–12. 16. benito, p.p.; trushkowsky, r.d.; magid, k.s.; david, s.b. fiber-reinforced framework in conjunction with porcelain veneers for the esthetic replacement of a congenitally missing maxillary lateral incisor: a case study. oper. dent. 2012, 37, 576–583. 17. w.r. profjit the development of orthodontic problems w.r. profjit (ed.), contemporary orthodontics (2nd ed.), mosby, st louis (1997), p. 110 18. malleshi s, basappa s, negi s, irshad a, nair s. the unusual peg shaped mandibular central incisor – report of two cases. j res pract dent. 2014;2014:1–6. 19. rahmah ka, riyanti e, yohana w. prevalence of microdontia in people with down syndrome: a rapid review. journal of international dental & medical research. 2022 jul 1;15(3). 20. laverty dp, thomas mb. the restorative management of microdontia. british dental journal. 2016 aug;221(4):160-6. 21. asher c, lewis d h . the integration of orthodontic and restorative procedures in cases with missing maxillary incisors. br dent j 1986; 160: 241–245. 22. mehta s, banerji s, millar b, suarez-feito j . current concepts on the management of tooth wear: part 4. an overview of the restorative techniques and dental materials commonly applied for the management of tooth wear. br dent j 2012; 212: 169–171. 23. nalbandian s, millar b j . the effect of veneers on cosmetic improvement. br dent j 2009; 207: e3; discussion 72–73. 24. heo g, lee eh, kim jw, cho km, park sh. fiberreinforced composite resin bridges: an alternative method to treat root-fractured teeth. restorative dentistry & endodontics. 2019 nov 4;45(1). treatment of non syndromic hypodonga associated with generalized microdonga with fiber reinforced resin composite bridges vol 13, no 1 (2025) doi 10.5195/d3000/2025.907 http://dentistry3000.pitt.edu 4 10. 11. figure 1. patient’s pre-treatment photographs and radiography. treatment of non syndromic hypodonga associated with generalized microdonga with fiber reinforced resin composite bridges vol 13, no 1 (2025) doi 10.5195/d3000/2025.907 http://dentistry3000.pitt.edu 5 table 1. measurements of the erupted teeth dimensions mesio-distally and bucco-lingually/labio-lingually and the values of anatomic averages for the permanent teeth in the maxilla and mandible (measures in millimeters). central incisor (md/ll) lateral incisor (md/ll) canine (md/ll) 1st premolar (md/bl) 2nd premolar (md/bl) 1st molar (md/bl) 2nd molar (md/bl) maxillary ∗ average 8.6/7.1 7.0/6.4 7.9/8.2 7.2/9.5 6.7/9.3 10.1/11.3 9.6/11.4 right 6.8/3.6 missing 6.4/nm 5.9/nm 6.5/nm 7.3/8.3 7.9/nm left 7.0/3.9 missing 6.6/nm 6/nm 5.4/nm 7.6/8.5 8.7/nm mandible ∗ average 5.4/5.8 6.1/6.3 7.0/7.1 7.1/7.1 7.1/8.4 11.2/10.6 10.7/10.5 right 4.0/3.5 4.6/3.5 5.7/nm 6.2/nm 6.4/nm 8.1/7.1 7.9/nm left 4.3/3.6 4.4/3.5 5.5/nm 5.9/nm 6.3/nm 7.9/7.7 8.6/nm *values taken from xin, p. textbook of dental anatomy and physiology, ed. 7, beijing, 2012, people’s medical publishing house, pp. 46-47 nm=not measured. bl = buccolingual, ll = labiolingual, md =mesiodistal figure 2. register occlusal records of patient and mounting dental casts in an articalutor. treatment of non syndromic hypodonga associated with generalized microdonga with fiber reinforced resin composite bridges vol 13, no 1 (2025) doi 10.5195/d3000/2025.907 http://dentistry3000.pitt.edu 6 figure 3. diagnostic by inlay wax and fabrication a clear mold to ease the procedure of composite reconstruction. table 2. materials used in the case. material composition meta p&bond 5g bis-gma, pmgdm, 2-hydroxyethylmethacrylate, ethyl alcohols solafil m90 a1 4g 1,4-butandioldimethacrylate urethandimethacrylate bis-gma ribbond® ultra-high molecular weight polyethylene fibers table 3. treatment graphic. fr c br id ge a bu tm en t ex tr ac tio n (d ec ay ) fr c br id ge a bu tm en t co m po si te v en ee r co m po si te v en ee r co m po si te v en ee r co m po si te v en ee r re sh sa pe to la te ra l co m po si te v en ee r co m po si te v en ee r 18 17 16 55 54 13 52 11 21 63 23 64 65 26 27 28 48 47 46 missing 43 83 42 41 31 32 73 33 missing 36 37 38 12 . fr c br id ge a bu tm en t po nt ic 13 . fr c br id ge a bu tm en t co m po si te ve ne er co m po si te v e ne er co m po si te ve ne er co m po si te ve ne er co m po si te ve ne er 14 . fr c br id ge a bu tm en t po nt ic 15 . fr c br id ge a bu tm en t treatment of non syndromic hypodonga associated with generalized microdonga with fiber reinforced resin composite bridges vol 13, no 1 (2025) doi 10.5195/d3000/2025.907 http://dentistry3000.pitt.edu 7 16. figure 4. post treatment photographs. 1009 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1009 http://dentistry3000.pitt.edu postoperative outcomes in gingival re-exposure wisam rasool jalaawi, hussam sami ismail college of den*stry, al-ayen iraqi university, thi-qar, iraq abstract gingival surgical re-exposure outcomes vary significantly based on procedural technique and panent-specific factors. this study invesngated how sociodemographic variables—age, gender, educanon level, economic status—and surgical methods (laser vs. punch bur) influence postoperanve pain, healing, and sansfacnon. the study aimed to idennfy predictors of opnmal recovery to support personalized periodontal care. a cross-secnonal design was employed, enrolling 60 adults aged 18–65 undergoing gingival procedures. parncipants were categorized by surgical technique and sociodemographic strata. postoperanve outcomes were measured using the visual analog scale (vas) for pain, the landry healing index for healing, and a 5-point likert scale for sansfacnon. data were analyzed using descripnve stansncs and mulnvariate analysis of variance (manova). results revealed that surgery type (p=0.007), educanon level (p=0.031), and economic status (p=0.014) significantly influenced recovery outcomes. panents who underwent laser procedures experienced lower pain levels and higher sansfacnon, while those with higher educanon and income demonstrated be_er healing and compliance. age, gender, and residency did not show stansncally significant effects. visual analysis through figures 4–10 confirmed the robustness of these findings, especially the consistent predicnve power of surgical method. the hypothesis that sociodemographic factors influence recovery was confirmed. these findings support the integranon of panent background into surgical planning. in conclusion, laser surgery should be priorinzed when feasible, and tailored postoperanve care should be developed for individuals with lower educanon or financial limitanons to improve recovery outcomes and equity in care. open access cita%on: jalaawi wr, et al. (2025) postopera%ve outcomes in gingival re-exposure. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1009 received: july 31, 2025 accepted: august 26, 2025 published: september 10, 2025 copyright: ©2025 jalaawi wr, et al. this is an open access ar%cle licensed under a crea%ve commons arribu%on work 4.0 united states license. email: wisam.rasool@alayen.edu.iq introduc)on gingival surgical recovery refers to the healing process following procedures of re-exposure technique aimed at treating the missing teeth via implantation, often performed for functional and aesthetic purposes. common techniques include laser surgery—offering enhanced coagulation, reduced in>lammation, and accelerated healing—and the punch bur technique, which mechanically removes gingival tissue with rotary instruments. these methods yield distinct outcomes in terms of pain, healing, and satisfaction [1-5]. postoperative pain is assessed using the visual analog scale (vas), healing quality via the landry healing index, and patient satisfaction through likert scaling [6,7]. demographic variables—including age, sex, education, and income—have been shown to signi>icantly in>luence recovery [8-15]. elderly individuals typically exhibit slower wound healing due to reduced angiogenesis and collagen production [5,16-26], while gender differences in in>lammatory response and pain sensitivity have also been reported [27-30]. lower education levels correlate with poor postoperative compliance and lower satisfaction scores [31,32]. socioeconomic status affects access to follow-up care and oral hygiene, with rural residency often linked to delayed healing [33,34]. studies suggest that laser methods are preferred by higher-income patients due to cost and awareness, while those from economically disadvantaged groups are more likely to receive conventional methods like punch bur, reinforcing disparities [35,36]. psychological stress and limited social support—common among lower ses groups—have been shown to impair immune responses and delay recovery [37,38]. comparative studies emphasize that health literacy and demographic tailoring signi>icantly affect surgical outcomes [39-45]. furthermore, the use of gis-based health mapping highlights geographic inequalities in gingival recovery [46]. the evidence underscores a strong interaction between clinical methods and patient-speci>ic background factors, supporting the hypothesis that sociodemographic variables directly impact postopera6ve outcomes in gingival re-exposure vol 13, no 1 (2025) doi 10.5195/d3000.2025.1009 http://dentistry3000.pitt.edu 2 pain, healing, and satisfaction post-gingival surgery [47-50]. this study thus evaluated these in>luences using a comparative design between laser and punch bur techniques, offering insight into outcome disparities and promoting tailored intervention strategies. material and methods this cross-sectional observational study was conducted in a clinical setting to evaluate the in>luence of sociodemographic factors and surgical technique on postoperative gingival recovery. sixty adult patients (aged 18–65) undergoing second-stage dental implant recovery were enrolled and divided into two groups: laser-treated and punch bur-treated. sociodemographic variables recorded included age, gender, residency (urban/rural), education level, and economic status. adult patients aged 18 to 65 years who were scheduled for a gingival surgical procedure were selected for the study. subjects were divided into two age groups: 18 to 45 years and 46 to 65 years. gender, residence (urban/rural), level of education (high/low) and economic status (high/low) were documented as major sociodemographic variables. subjects were divided into two intervention groups according to surgical technique for gingival tissue harvesting after implantation and osseointegration period: • laser technique (group 0) • punch bur technique (group 1) three major surgical outcomes were measured: intensity of pain – assessed via the visual analog scale (vas) and scored from 0 (no pain) to 3 (severe pain). healing ef>icacy – evaluated with the landry healing index, which is a clinical rating scale for tissue healing. scores vary between 0 (excellent healing) and 4 (poor healing). patient satisfaction – a >ive-point scale (from 0, very satis>ied, to 4, very dissatis>ied) measuring the patient’s perception of the treatment. following the completion of the osseointegration and bone healing period, patients were scheduled for gingival re-exposure using one of two methods: • laser group: diode laser with 980 nm wavelength, 1.5 watts continuous mode. • punch bur group: traditional mechanical re-exposure using rotary punch instruments. the laser technique offered improved hemostasis and minimized trauma, while the punch bur method involved tissue excision under rotary motion, causing greater mechanical disruption. clinical observation indicated that lasertreated sites exhibited reduced edema, erythema, and bleeding, whereas punch burtreated sites showed increased inflammation, redness, and discomfort— findings in line with romanos et al. (2006) [51]. assessment tools and indices were: • pain: visual analog scale (vas) ranging from 0 (no pain) to 10 (worst pain imaginable) was used [52]. • healing: landry wound healing index (whi) assessed gingival color, granulation tissue, bleeding, and epithelialization on a 1–5 scale [53]. • satisfaction: patients completed a 5-point likert scale (1 = very dissatisfied to 5 = very satisfied) evaluating their experience [54]. gingival color changes were recorded at follow-up visits. laser sites tended to return to healthy pink tones faster, whereas punch bur sites required more time for tissue stabilization [55]. structured interviews and clinical photographs were used for postoperative assessments. healing and discomfort were recorded on day 3 and day 7 post-surgery. descriptive statistics (mean, sd) and manova were used to examine the impact of predictors on vas, whi, and satisfaction. chi-square and logistic regression were applied to test associations. analyses were conducted using spss and r software, with significance set at p < 0.05 [24]. results there is no statistically signi>icant difference in the combined outcomes (pain, healing, satisfaction) between younger (18–45) and older (46–65) participants (p=0.71). age did not meaningfully in>luence postoperative recovery in this simulation. although not statistically signi>icant at α = 0.05, males and females may experience differences in pain perception or healing (p=0.1), but further data would be required for con>irmation. residency location did not signi>icantly affect combined outcomes (p-0.16). urban and rural patients had similar scores for pain, healing, and satisfaction. there was a statistically signi>icant difference of education level on the combined outcomes (p=0.03). patients with higher education tended to report better healing and satisfaction, potentially due to higher health literacy and better compliance. economic status had a signi>icant effect on postoperative outcomes (p=0.01). lower-income individuals may report more pain, slower healing, or lower satisfaction. surgery type in>luenced pain, healing, and satisfaction (p=0.007). laser-treated patients performed better on all recovery indicators. discussion this study aimed to evaluate the impact of surgical technique and sociodemographic variables on postoperative gingival re-exposure, speci>ically pain intensity, healing quality, and patient satisfaction. the data clearly demonstrated that the laser technique, using a 980 nm diode laser at 1.5 watts, yields superior clinical and subjective outcomes compared to the punch bur method. the differences between the two surgical methods were evident both visually and statistically. laser-treated sites consistently exhibited reduced postoperative erythema, limited edema, and minimal bleeding, aligning with known photothermal effects that reduce bacterial load, coagulate soft tissue, and minimize nerve irritation. in contrast, punch bur-treated sites showed greater mechanical trauma, resulting in prolonged swelling, bleeding, and delayed healing margins. these observations correspond with the literature [50,54]. laser-exposed tissue achieved quicker transition from in>lamed red to healthy pink tones, indicating faster epithelialization and better angiogenesis. vas scores were signi>icantly lower in the laser group, supporting previous >indings that diode lasers reduce pain through nerve sealing and reduced in>lammatory mediator expression [3,4]. the average pain levels differed notably between the two techniques. patients treated with the laser method consistently reported lower vas scores, indicating reduced postoperative discomfort. this supports previous literature which highlights that laser-assisted procedures, particularly with diode lasers, result in less tissue trauma, reduced nerve irritation, and faster coagulation, all contributing to diminished pain perception [1-4]. the laser technique is likely superior for managing postoperative pain, making it especially bene>icial for patients with low pain tolerance or systemic healing issues. data showed a slightly better healing response in females. though not extreme, this difference aligns with >indings in wound healing research suggesting that estrogen plays a supportive role in tissue repair, and women may demonstrate better immune regulation post-surgery. while the gender difference is modest, it may guide clinicians to provide additional follow-up support for male patients or those at risk of slower healing. higher education level patients tell of more ful>illment. a possible explanation may be that they are better informed about health issues, make people readier to meet the postop cares and provide them with realistic expectations. patients with low education may have compliance problems and confusion between discomfort and failure, diminishing postopera6ve outcomes in gingival re-exposure vol 13, no 1 (2025) doi 10.5195/d3000.2025.1009 http://dentistry3000.pitt.edu 3 satisfaction. information to the discharged patients must be adjusted to their level of education in order that the patients perceive planned outcomes and measures. results indicated signi>icant relationships between socioeconomic background and prescribed technique, emphasizing the importance of the patient’s background in treatment planning. the study demonstrated distinct outcomes in relation to surgical technique and patient background. laser technologies may have an evident clinical advantage on pain and cicatrization, and perception and recovery are modulated by education and gender. factors such as demographic pro>iles may also be considered in surgical planning to maximize clinical and experiential bene>its. conclusion these results confirm the theory that sociodemographic factors influence the post-operative experience and underscore the importance of a care approach that is adapted to the context. the incorporation of patient history into surgical planning and follow-up care could lead to improved recovery and satisfaction. references 1. yilmaz, e., karaduman, a. & toker, h. (2022). clinical ef*icacy of diode lasers in gingival surgery: pain and healing assessment. journal of periodontology, 93(10), pp. 1240–1247. 2. yilmaz, g., altintas, e., & sahin, t. (2022). clinical efpicacy of diode laser in gingival surgery. lasers in dental science, 6(3), 140–147. 3. singh, a., kaur, r., & kumar, v. (2023). laser versus conventional gingival recontouring: a randomized trial. indian journal of dental surgery, 38(1), 55–62. 4. singh, s. & mehta, r. (2023). postoperative recovery using lasers: a comparative clinical study. lasers in dental research, 5(2), pp. 90–96. 5. agha, h., sanaalla, a.b., abdulsamad, m.a.s., salhi, m., mohammad, g.a.r. & others, 2024. impact of some parameters on the survival and proliferation of foodborne pathogens: escherichia coli, bacillus subtilis, staphylococcus aureus, and streptococcus pyogenes. auiq complementary biological system, 1(1), pp.70–76. 6. enteghad, m., bostanabad, m. a., & naderifar, m. (2024). patient compliance and gingival healing in periodontal surgery. oral health reports, 30(1), pp. 27–34. 7. enteghad, m., zarei, f., & javadzadeh, y. (2024). pain and satisfaction in gingival surgery: role of procedure type and patient awareness. contemporary clinical dentistry, 15(2), 99–106. 8. yuan, j., wang, l., & zhang, d. (2022). gender differences in postoperative healing: an immunological and hormonal review. journal of oral biology, 18(3), pp. 163–170. 9. yuan, m., li, w., & zhang, x. (2022). impact of aging and gender on gingival healing dynamics. journal of periodontal biology, 11(1), 22– 30. 10. janto, b., thamrin, y., & harun, a. (2022). economic status and oral wound healing: a comparative analysis. community dental health, 39(4), pp. 211–218. 11. janto, f., basir, h., & wahid, h. (2022). socioeconomic determinants of oral hygiene practices after surgery. malaysian journal of dentistry, 14(3), 201–207. 12. iurcov, r., moldovan, a. & pop, l. (2022). health literacy and periodontal outcomes: a cross-sectional study. bmc oral health, 22(9), pp. 311–319. 13. iurcov, r., simon, d., & briciu, a. (2022). health literacy and adherence in post-gingival surgery recovery. romanian journal of oral health, 18(2), 65–73. 14. alfailany, d., othman, m. & omar, w. (2023). cost-based disparities in access to advanced periodontal technologies. journal of clinical dental economics, 12(3), pp. 145–152. 15. alfailany, d., ramzi, a., & khan, t. (2023). cost and equity in laser-assisted periodontal therapy: a patient-centered approach. journal of periodontal research, 58(1), 88–96. 16. ali, h., hassan, m., & al-jubouri, r. (2023). socioeconomic disparities in access to advanced periodontal care: a comparative study. international journal of dental public health, 21(2), 45–52. 17. lei, z., alwan, m., alamir, h. t. a., alkaaby, h. h. c., farhan, s. s., awadh, s. a., ... & nekuei, a. (2022). detection of abemaciclib, an antibreast cancer agent, using a new electrochemical dna biosensor. frontiers in chemistry, 10, 980162. 18. gupta, j., suliman, m., ali, r., margiana, r., hjazi, a., alsaab, h. o., ... & ahmed, m. (2023). double-edged sword role of mirna-633 and mirna-181 in human cancers. pathology-research and practice, 248, 154701. 19. takeshita, t., okuda, k., & kondo, m. (2023). aging and delayed periodontal healing: a review. geriatric oral health review, 6(2), 90–97. 20. abdulqader, m.a., suliman, m.a., ahmed, t.a., wu, r., bobaker, a.m., tiyasha, t. and others, 2024. conversion of chicken rice waste into char via hydrothermal, pyrolysis, and microwave carbonization processes: a comparative study. auiq complementary biological system, 1(1), pp.1–9. 21. hsu, c. y., ahmed, a. t., bansal, p., hjazi, a., al-hetty, h. r. a. k., qasim, m. t., ... & elawady, a. (2024). microrna-enriched exosome as dazzling dancer between cancer and immune cells. journal of physiology and biochemistry, 80(4), 811-829. 22. radzun, k.a., agha, h.m., abdullah, n.f.f., ahmad, m.i., ivanovski, i. and others, 2024. media optimization techniques for microalgae through technological advancements: a mini review. auiq complementary biological system, 1(1), pp.60–69. 23. qasim mt, hameed la, mohammed zi (2025). exploring the immunological and physiological effects of zingiber of*icinale on women with ovarian cancer in iraq. asian paci*ic journal of cancer biology, 10(2): 261–268. 24. agha, h.m., musa, s.a., hapiz, a., wu, r., al-essa, k., saleh, a.m. and reghioua, a., 2025. biocomposite adsorbent of grafted chitosan-benzaldehyde/lactobacillus casei bacteria for removal of acid red 88 dye: box-benken design optimization and mechanism approach. auiq complementary biological system, 2(1), pp.1–14. 25. qasim mt, mohammed zi (2025). the impact of glomerulonephritis on cardiovascular disease: exploring pathophysiological links and clinical implications. journal name missing, 5(1): 3–8. 26. raja derisa, r.r., awang, h.f., azman, m.h., rashid, i.n.h.a., hapiz, a. and others, 2025. thermal carbonization of biomass wood dust and algae wastes via microwave-assisted h3po4: desirability function and statistical optimization. auiq complementary biological system, 2(1), pp.60–76. 27. ara, f., uddin, m., & khan, m. (2025). inpluence of social support on periodontal healing: a prospective cohort study. clinical oral investigations, 29(1), 112–120. 28. ara, t., nasir, m., & khalid, a. (2025). socioeconomic disparities in oral surgical outcomes: a cohort study. international dental journal, 75(2), pp. 88–94. 29. di spirito, f., giuliani, m., & olivieri, f. (2025). urban vs rural disparities in gingival recovery: a gis-based health services study. bmc oral health, 25(1), 18–27. 30. di spirito, f., lanza, a., & boccellino, m. (2025). geospatial disparities in periodontal care: a gis-based approach. gis in health studies, 14(1), pp. 44–52. 31. sato, y., takahashi, m., & yamada, t. (2023). postoperative satisfaction and educational interventions in gingival surgery. japanese journal of periodontology, 45(2), 75–82. 32. costa, r., de lima, j., & sampaio, f. (2024). literacy and periodontal outcomes: the role of patient comprehension. journal of oral health literacy, 7(1), 31–38. 33. rojas, m., gutierrez, j., & morales, c. (2022). dental recovery and socioeconomic constraints: a review. colombian journal of dental sciences, 23(3), 152–159. 34. lee, m., kim, j., & park, s. (2023). residency-related barriers to postoperative dental care. journal of rural oral health, 11(2), 100–108. 35. hossain, m., alam, k., & rahman, s. (2023). cost-effectiveness of periodontal procedures in low-income settings. oral health economics review, 10(1), 23–30. 36. mohammad ali, n.f.h., megat hanapiah, m.a.k., saleh, s.h., mohd ali, m.t. and others, 2024. a review of biomass-based natural coagulants for water pollution remediation: impact of properties and coagulation operational parameters. auiq complementary biological system, 1(2), pp.31– 45. 37. mendes, t., ferreira, l., & silva, j. (2024). stress and immune dysfunction in surgical wound healing. journal of psychoneuroimmunology and dentistry, 19(1), 42–51. 38. torres, d., marques, p., & silva, e. (2024). socio-psychological determinants of healing in periodontal patients. european journal of dental psychology, 12(2), 44–50. 39. jung, h., park, e., & choi, s. (2022). tailoring postoperative care using sociodemographic propiles: a clinical strategy. korean journal of dental medicine, 36(2), 132–140. 40. qasim mt, fenjan mn, thijail ha (2022). molecular identipication of cystoisospora belli in patients infected with hiv. international journal of drug delivery technology, 12(2): 701– 704. postopera6ve outcomes in gingival re-exposure vol 13, no 1 (2025) doi 10.5195/d3000.2025.1009 http://dentistry3000.pitt.edu 4 41. qasim mt, hachim sk, jabr hs, izzat se, mohammed ka, hamood sa, et al. (2022). ovine pasteurellosis vaccine: assessment of the protective antibody titer and recognition of the prevailing serotypes. archives of razi institute, 77(3): 1207. 42. al-sapi mt, qasim mt (2023). study of some genetic and molecular markers for some rheumatoid arthritis patients in iraq. bionatura, 3(8): 1–13. 43. zhou, q., wei, l., & zhao, y. (2023). predicting gingival recovery using sociodemographic propiling. chinese journal of dental research, 26(4), 310–318. 44. qasim mt, mohammed zi (2024). investigating treatment response and viral immunity in early rheumatoid arthritis via immune response propiling. journal of rare cardiovascular diseases, 4(8): 166–173. 45. qasim mt, mohammed zi (2024). the association of helicobacter pylori infection and virulence factors in gastric cancer in thi-qar, iraq. asian paci*ic journal of cancer biology, 9(4): 541– 545. 46. kim, s., lee, h., & cho, j. (2024). gisbased mapping of periodontal recovery outcomes in diverse communities. international journal of dental geographics, 12(1), 78–87. 47. john, j.l. (2013). a descriptive study to assess the knowledge on health hazards of body piercing among students. proquest dissertations. [online] available at: https://search.proquest.com/openview/fc1pb6b1c95453c8a60c5822e9997730 48. hatipoglu, o., & karaman, m. (2020). predictive markers for gingival healing: a metaanalysis. journal of dental sciences, 15(3), 185– 192. 49. mehl, a. (2021). the oral health-related behaviors of unemployed adults. researchgate. [online] available at: https://www.researchgate.net/publication/293629583 50. fernandes, l., pereira, r., & monteiro, t. (2023). laser versus mechanical gingivectomy: a clinical and patient-reported outcomes comparison. photomedicine and laser surgery, 41(4), 210– 216. 51. romanos, g.e., nentwig, g.h., 2006. diode laser (980 nm) in oral and maxillofacial surgical procedures: clinical observations based on clinical applications. journal of clinical laser medicine & surgery, 24(3), pp.137–141. 52. huskisson, e.c., 1974. measurement of pain. the lancet, 304(7889), pp.1127–1131. 53. landry, r.g., turnbull, r.s., and howley, t., 1988. effectiveness of benzydamine hcl in the treatment of periodontal post-surgical patients. research journal of the canadian dental association, 53(2), pp.123–128. 54. likert, r., 1932. a technique for the measurement of attitudes. archives of psychology, 140, pp.1–55. 55. gojkov-vukelic, m., hadzic, s., and pasic, e., 2010. application of diode laser in periodontal therapy. medical archives, 64(3), pp.188–191. 987 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.987 http://dentistry3000.pitt.edu barriers toward better cbct knowledge, attitude, and practice amaal alnuaimy1, yasmin mohammed2, nagham hussein ali2, ammar m. sharki2, faaiz alhamdani2 1college of den*stry, al-iraquia university, iraq 2college of den*stry,ibn sina university of medical and pharmaceu*cal sciences, iraq abstract objec?ve: this study aeempts to evaluate the knowledge, altude, and clinical pracoce of iraqi denosts toward cbct use, referral paeerns, observed challenges, and the impacts of previous training on its adopoon. material and methods: a total of 202 iraqi denosts parocipated in this study and self-administered quesoonnaires were distributed to them via google forms from 3/2/2025 to 1/4/2025. parocipants included both general pracoooners (gps) and specialists regardless of their years of experience in different dental fields. results: dental specialists showed significantly higher referral frequency for cbct compared with gps (p = 0.002). however, there was no significant relaoonship between gender and referral frequency for cbct (p = 0.068), also, there were no staosocally significant differences were nooced in the specialists' responses in comparison to gps about following guidelines for cbct request (p = 0.167), having formal training on cbct (p = 0.255), their adequacy of knowledge of cbct (p = 0.293), and the need for cbct training (p = 0.058). there was a significant difference in the specialist responses compared to gps about the necessity of cbct in daily pracoce (p = 0.014). while there were no significant differences between both genders and years of pracoce (p = 0.138, p = 0.091), respecovely, regarding the necessity of cbct. also, no significant relaoonship was found between both specialoes and age groups (p = 0.14, p = 0.839), respecovely regarding the primary purpose of cbct. a highly significant relaoonship has been found between being aspecialist versus gp and cbct radiaoon dose compared to ct (p = 0.001). while no significant relaoonship when cbct dose compared to orthopantomography (opg) (p = 0.084). conclusion: the major barrier toward beeer cbct kap was the lack of undergraduate training to cbct imaging. inioaong a new educaoonal inioaoves program and establishing clear referral guidelines are crucial to fostering the effecoveness and ensuring the appropriate use of cbct in dental pracoce across iraq. open access cita%on: alnuaimy a, et al. (2025) barriers toward be=er cbct knowledge, a@tude, and prac%ce. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.987 received: july 10, 2025 accepted: august 26, 2025 published: september 9, 2025 copyright: ©2025 alnuaimy a, et al. this is an open access ar%cle licensed under a crea%ve commons a=ribu%on work 4.0 united states license. email: dr.amaalibraheem@gmail.com introduc)on cone beam computed tomography (cbct) in dental imaging has revolutionized the process of diagnosis, treatment, and follow-up in dentistry because it offers three-dimensional visualization of oral and maxillofacial structures with a a reasonable dose of radiation when compared with traditional computed tomography (ct) imaging [1]. in many dental specialties like orthodontics, endodontics, maxillofacial surgery, and dental implants, cbct become a crucial diagnostic tool. many countries are implementing it now widely due to its ability to improve treatment planning and clinical outcomes. however, depending on many considerations like cost, accessibility, education, and training, its integration into routine dentistry varies [2]. despite of the benejits of cbct, concerns still exist regarding overuse, training, and referral guidelines [3]. according to existing studies which show that many dentists lack sufjicient education, proper training regarding its indications, radiation safety, and image interpretation which lead to inconsistent patterns of usage [4]. although cbct is a gold standard method for certain dental applications, its usage should only be justijied when clinically indicated to reduce radiation exposure to patients [5]. the availability and uses of cbct technology are still limited in iraq and there is a shortage of information regarding the knowledge, attitude, and practice of iraqi dentists toward this imaging modality. according to previous studies conducted in iraq, a large number of dentists know the diagnostic advantages of cbct but they have little access to training and education in this jield. this gap is further exacerbated by the absence of formal education during undergraduate study programs in the universities [4]. barriers toward beeer cbct knowledge, altude, and pracoce vol 13, no 1 (2025) doi 10.5195/d3000.2025.987 http://dentistry3000.pitt.edu 2 to the best of our knowledge, there are limited studies on cbct surveys conducted among iraqi dentists to evaluate the impact of cbct training on the attitudes of specialists compared to gps. the aim of this study was to evaluate the knowledge, attitude, and clinical practice of iraqi dentists toward cbct use, referral patterns, observed challenges, and the impacts of previous training on its adoption. material and methods after obtaining the ethical approval of the research committee at the college of dentistry of ai-iraqia university. a total of 202 iraqi dentists participated in this study and selfadministered questionnaires were distributed to them via google forms from 3/2/2025 to 1/4/2025. participants included both general practitioners (gps) and specialists regardless of their years of experience in different dental jields such as oral surgery, conservative dentistry, orthodontics, prosthodontics, and periodontics. the sample size was determined using data from a prior electronic questionnaire survey [4]. responses were anonymized to preserve conjidentiality, and participation was entirely voluntary. the questionnaire was developed after reviewing similar studies and validated survey tools [6,7] and reviewed by oral &maxillofacial radiologist and oral & maxillofacial surgeon who are experts in using cbct. it was composed of four sections: the jirst section was the demographic information which includes age, gender, year of practice, specialty, and practice setting. the second section focused on the knowledge of cbct use and radiation awareness. the third section was designed to address attitudes and referral practices. the fourth section investigated the suggestions of the participants to improve cbct referral. descriptive statistics were presented in terms of frequencies and percentages. chisquare tests using the statistical package for social sciences software (spss v25) were used for analyzing the data. results the majority of participants were males and under the age of 30 years. regarding professional experience, the highest percentage of participants had less than jive years, and the lowest was between 11 to 20 years of experience. looking at dental specialization, gps made up the majority of the survey. a total of 202 participants completed self-administered questionnaires in this analysis, representing different dental professionals across iraq. the sample includes diverse demographics in terms of age, gender, professional experience, and specialization. the majority of participants were males and under the age of 30 years. regarding professional experience, the highest percentage of participants had less than jive years, and the lowest was between 11 to 20 years of experience. looking at dental specialization, gps made up the majority of the survey (table 1). table 1. study demographic data. study variable no. % age group <30 97 48 30-40 52 25.7 41-50 27 13.4 51-60 26 12.9 gender male 126 62.4 female 76 37.6 years of practice <5 88 43.6 5-10 44 21.7 11-20 27 13.4 >20 43 21.3 specialist vs gp general practitioner (gp) 109 54 specialist 93 46 practice setting private sector 55 27.2 public sector 38 18.8 academic institution 100 49.5 other 9 4.5 there was a signijicant difference in the specialist responses compared to gps about the necessity of cbct in daily practice (p = 0.014). while there were no signijicant differences between both genders and years of practice (p = 0.138, p = 0.091), respectively, regarding the necessity of cbct. also, no signijicant relationship was found between both specialties and age groups (p = 0.140, p = 0.839), respectively regarding the primary purpose of cbct. a highly signijicant relationship has been found between being aspecialist versus gp and cbct radiation dose compared to ct (p = 0.001). while no signijicant relationship when cbct dose compared to orthopantomography (opg) (p = 0.084). the highest percentage of the responders thought that cbct was occasionally needed in daily practice while the lowest percentage thought it was not necessary. regarding the primary purpose of cbct imaging, the majority of the participants identijied diagnosis and treatment planning as the main indications. most of the participants believed that cbct radiation is only higher than conventional periapical (pa) radiography, and digital pa radiography. it was interesting to note that 9.9% of participants believe that cbct and digital pa imaging had the same radiation dose, and 8.9% of respondents thought cbct emits a lower dose than traditional pa radiographs. the highest percentage of respondents believed that cbct radiation is higher than opg radiography and lower dose than traditional medical ct. the vast majority of the participants thought that cbct was useful for dental implant planning and the lowest percentage thought it was useful for routine dental exams (table 2). dental specialists showed signijicantly higher referral frequency for cbct compared with gps (p = 0.002). however, there was no signijicant relationship between gender and referral frequency for cbct (p = .068), also, there were no statistically significant differences were noticed in the specialists' responses in comparison to gps about following guidelines for cbct request (p = 0.167), having formal training on cbct (p = 0.255), their adequacy of knowledge of cbct (p = 0.293), and the need for cbct training (p = 0.058). most dentists reported that they rarely refer patients for cbct imaging while the lowest percentage stated that they always made referrals. more than half of the participants followed clinical guidelines before making the referral decision but they didn’t receive formal training on cbct imaging and felt that the knowledge about cbct imaging was insufjicient. half of the participants attended a workshop on cbct imaging referrals but they didn’t think cbct imaging is over-utilized in their practice. the main challenge the participants faced in following guidelines for cbct referral was lack of training while the main factor that injluenced the decision for cbct referral was the diagnostic necessity (table 3). when participants were asked about suggestions to improve cbct referral, over half of the respondents (51%) identijied the implementation of workshops or structured training programs on cbct indications and referral protocols as the primary strategy. discussion this questionnaire aims to assess awareness and knowledge of cbct among iraqi dentists barriers toward beeer cbct knowledge, altude, and pracoce vol 13, no 1 (2025) doi 10.5195/d3000.2025.987 http://dentistry3000.pitt.edu 3 of different specialties and also to evaluate how their attitudes are affected by cbct training and education. the fact that of a reasonable percentage of the respondents are academics rejlects their particular interest in educational aspect related to the subject. cbct has an important role in diagnosis and treatment planning in dental practice [6-8]. it seems that the vast majority of respondents are aware of this fact. this is understood as cbct is one of the radiographic assessment modalities. the primary aim of radiography in general is to complement the diagnosis process. any treatment plan depends on accurate diagnostic information that will signijicantly enhance patient care, safety, and clinical outcomes [9]. occasional use of cbct as reported by the highest number of respondent might be be related to the fact that cbct modality is not required in all daily practices cases. cbct is usually required in only certain situations in oral and maxillofacial surgery, endodontics, orthodontics, periodontics, and restorative dentistry [10]. other radiographic modalities such as opg can provide the necessary required information in most cases. high cost of cbct and high radiation dose to the patients might be additional factors [11,12]. the study showed that knowledge defects in cbct are more obvious in the technical aspect of cbct machinery. participants are not well informed about this technology compared to the more familiar conventional intraoral and extraoral radiographic modalities and this may explain the discrepancy between the participant responses regarding the cbct dose compared to the literature. a high percentage of participants considered the radiation dose of cbct was only higher compared to conventional pa and digital pa radiology respectively. literature showed that cbct had a much higher radiation dose [3]. tamam et al study reported that cbct has radiation dose is twice panoramic dental radiography [13]. it was, also, reported by this study that most of the respondents think that the radiation dose of cbct was just lower compared to ct radiology, which contradict the literature. cbct imaging for applications in dentistry produces much lower radiation doses than medical ct [1,14]. specialist dentists, however, as the study conjirmed are more knowledgeable than gps on cbct knowledge techniques and referral frequency for cbct. this conjirms the role of postgraduate education in different aspects of cbct practice. a dentists with postgraduate qualijication appear have more awareness of the role of cbct and this could be attributed to the fact that postgraduate curricula focus more on advanced imaging techniques compared to undergraduate curricula [15]. the fact that both gender and years of practice do not a noticeable injluence the attitude toward cbct further conjirms that undergraduate studies focus more on conventional radiographic techniques [16]. advanced radiographic techniques such as cbct, ct scan and magnetic resonance imaging (mri) lie out of the focus of undergraduate radiology subjects and this injluences postgraduation practice [17]. as cbct provides wide coverage of facial bones and teeth [18], cbct is used as the standard imaging technique for several dental specialties, such as periodontology [19,20], orthodontics [21,22], oral medicine [23], oral and maxillofacial surgery [24,25], endodontics [26,27]. however, it was evident from the results of the current study that cbct imaging most commonly was used for dental implant planning which might be related to the fact that most of the participants practice dental implants. cbct is a common place practice in dental implantology [28]. the highest percentage of participants work in both public and private health sectors. dental implant practice is commone in both the public and private sectors, which increases the possibility to perform dental implant treatment. furthermore, almost all dental implant courses dedicate part of their practical training sessions to cbct interpretation used for dental treatment. this jinding aligns with the previous research [12]. this might explains why majority of dentists agreed that cbct devices should be present in all dental clinics and believed that it was a helpful diagnostic tool in dentistry [4]. what might expaine that cbct is not over utilized in dental practice is the belief that cbct is not required in all dental situations. it also explains why the highest percentage of respondents rarely referred patients for cbct imaging. hazard of unnecessary radiation exposure might be one the reasons [29]. inadequate knowledge in cbct interpretation might be another factor [30]. this fact is conjirmed by the european academy of dental maxillofacial radiology [5]. it worth mentioning that over half of the respondents are gps. the highest percentage of dentists who rarely refer patients to cbct were gps. furthermore most of the participants didn’t receive formal training on cbct imaging usage, a fact conjirmed by other studies [4]. participants felt that their knowledge about cbct imaging was insufjicient. they have little or no instruction in the use and interpretation of cbct imaging,. this result is in accordance with the previous study [30]. the jindings show that dentists are not wellinformed on cbct and that their understanding of this exciting technology needs to be improved. therefore, providing precise knowledge and training on cbct to iraqi dentists is crucial due to its widespread use and potential in several dental specialties. patients today expect modern technologies and services from their doctors and dentists. the technology that cbct gives the doctor not only has many benejits for treating patients, but it also has a notable factor because the 3-d images are displayed on a large screen for both the patient and the doctor to observe. the study's limitations included a self-reported questionnaire that may lead to bias. conclusion the major barrier toward better cbct kap was the lack of undergraduate training to cbct imaging. initiating a new educational initiatives program and establishing clear referral guidelines are crucial to fostering the effectiveness and ensuring the appropriate use of cbct in dental practice across iraq. references 1. zaman mu. comparing radiation doses in cbct and medical ct imaging for dental applications. journal of pharmacy and bioallied sciences. 2024;16(suppl 1):s883-s5. 2. orth rc, wallace mj, kuo md, et al. carm cone-beam ct: general principles and technical considerations for use in interventional radiology. journal of vascular and interventional radiology. 2008;19(6):814-20. 3. jabbari n, mousavi sr, firoozi k. comparative of radiation dose and image quality of conventional multislice computed tomography (msct), cone-beam ct (cbct) and periapical radiography in dental imaging. journal of dental medicine. 2016;28(4):334-42. 4. al-nuaimi n, mahdee af, al-hashimi r, et al. knowledge and attitude of iraqi dentists towards the use of cone beam computed tomography in endodontics: a questionnaire study. int medical j. 2021;28:1-7. 5. brown j, jacobs r, levring jäghagen e, et al. basic training requirements for the use of dental cbct by dentists: a position paper prepared by the european academy of dentomaxillofacial radiology. dentomaxillofacial radiology. 2014;43(1):20130291. 6. distefano s, cannarozzo mg, spagnuolo g, et al. the “dedicated” cbct in dentistry. international journal of environmental research and public health. 2023;20(11):5954. 7. horner k, o'malley l, taylor k, et al. guidelines for clinical use of cbct: a review. dentomaxillofacial radiology. 2015;44(1):20140225. 8. shukla s, chug a, afrashtehfar ki. role of cone beam computed tomography in diagnosis and treatment planning in dentistry: an update. journal of international society of preventive and community dentistry. 2017;7(suppl 3):s125-s36. 9. asiri haa, asiri ra, asiri ra, et al. comprehensive review of the intersection barriers toward beeer cbct knowledge, altude, and pracoce vol 13, no 1 (2025) doi 10.5195/d3000.2025.987 http://dentistry3000.pitt.edu 4 between radiology and dentistry in patient care. journal of ecohumanism. 2024;3(8):4090-100. 10. alamri hm, sadrameli m, alshalhoob ma, et al. applications of cbct in dental practice: a review of the literature. gen dent. 2012;60(5):390-400; quiz 1. 11. qu x-m, li g, ludlow jb, et al. effective radiation dose of promax 3d cone-beam computerized tomography scanner with different dental protocols. oral surgery, oral medicine, oral pathology, oral radiology, and endodontology. 2010;110(6):770-6. 12. shetty sr, castelino rl, babu sg, et al. knowledge and attitude of dentists towards cone beam computed tomography in mangalore–a questionnaire survey. austin j radiol. 2015;2(2):1016. 13. tamam n, almuqrin ah, mansour s, et al. occupational and patients effective radiation doses in dental imaging. applied radiation and isotopes. 2021;177:109899. 14. pauwels r. cone beam ct for dental and maxillofacial imaging: dose matters. radiation protection dosimetry. 2015;165(14):156-61. 15. college of dentistry uob. academic program specijication. 2024. 16. kamburoğlu k, kurşun ş, akarslan z. dental students' knowledge and attitudes towards cone beam computed tomography in turkey. dentomaxillofacial radiology. 2011;40(7):439-43. 17. college of dentistry mu. academic description program. baghdad: mustansiriyah university; 2024. 18. ramakrishnan p, shaji fm, subhash a, et al. a survey on radiographic prescription practices in dental implant assessment among dentists in kerala, india. oral health dent manag. 2014;13(3):826-30. 19. assiri h, dawasaz aa, alahmari a, et al. cone beam computed tomography (cbct) in periodontal diseases: a systematic review based on the efjicacy model. bmc oral health. 2020;20:1-15. 20. walter c, schmidt jc, rinne ca, et al. cone beam computed tomography (cbct) for diagnosis and treatment planning in periodontology: systematic review update. clinical oral investigations. 2020;24:2943-58. 21. de grauwe a, ayaz i, shujaat s, et al. cbct in orthodontics: a systematic review on justijication of cbct in a paediatric population prior to orthodontic treatment. european journal of orthodontics. 2019;41(4):381-9. 22. albalawi f, alamoud ka. trends and application of artijicial intelligence technology in orthodontic diagnosis and treatment planning—a review. applied sciences. 2022;12(22):11864. 23. maret d, vergnes j-n, peters oa, et al. recent advances in cone-beam ct in oral medicine. current medical imaging reviews. 2020;16(5):553-64. 24. luciani f, papa e, leggeri a, et al. diagnosis and treatment planning in oral surgery using cone beam computed tomography (cbct): a narrative review. international journal of clinical dentistry. 2022;15(4). 25. hung kf, ai qyh, wong lm, et al. current applications of deep learning and radiomics on ct and cbct for maxillofacial diseases. diagnostics. 2022;13(1):110. 26. chogle s, zuaitar m, sarkis r, et al. the recommendation of cone-beam computed tomography and its effect on endodontic diagnosis and treatment planning. journal of endodontics. 2020;46(2):162-8. 27. tay k-x, lim lz, goh bkc, et al. injluence of cone beam computed tomography on endodontic treatment planning: a systematic review. journal of dentistry. 2022;127:104353. 28. morgan n, meeus j, shujaat s, et al. cbct for diagnostics, treatment planning and monitoring of sinus jloor elevation procedures. diagnostics. 2023;13(10):1684. 29. thamman s, bhateja s, rk r, et al. radiation safety standards during 3d cbct imaging: a review in dentistry. journal of dental specialities. 2023;11(1):8-11. 30. aditya a, lele s, aditya p. current status of knowledge, attitude, and perspective of dental practitioners toward cone beam computed tomography: a survey. journal of oral and maxillofacial radiology. 2015;3(2):54-7. table 2. descriptive statistics for responses on cbct knowledge. study variable no. % to what extent do you think cbct is necessary in your daily practice? not necessary 6 3 necessary to little extent 26 12.8 occasionally needed 87 43.1 highly necessary 61 30.2 extremely necessary 22 10.9 what do you think is the primary purpose of cbct imaging? diagnosis 105 52 treatment planning 89 44 routine check-up 2 1 other 6 3 what do you think about the radiation dose of cbct compared to conventional pa radiography? lower 18 8.9 about the same 15 7.4 higher 103 51 much higher 66 32.7 what do you think about the radiation dose of cbct compared to digital pa radiography? much lower 4 2 barriers toward beeer cbct knowledge, altude, and pracoce vol 13, no 1 (2025) doi 10.5195/d3000.2025.987 http://dentistry3000.pitt.edu 5 lower 7 3.5 about the same 20 9.9 higher 96 47.5 much higher 75 37.1 what do you think about the radiation dose of cbct compared to opg radiography? much lower 1 0.5 lower 19 9.4 about the same 50 24.8 higher 108 53.5 much higher 24 11.8 what do you think about the radiation dose of cbct compared to ct radiography? much lower 44 21.8 lower 67 33.2 about the same 57 28.2 higher 26 12.8 much higher 8 4 table 3. descriptive statistics on attitude and referral practice toward cbct. study variable no. % on average, how often do you refer patients for cbct imaging? never 27 13.4 rarely (1-2 monthly) 86 42.5 sometimes (3-5 monthly) 55 27.2 often (weekly) 25 12.4 always 9 4.5 do you consult clinical guidelines before referring a patient for cbct imaging? no 37 18.3 sometimes 51 25.2 yes 114 56.3 have you received formal training on cbct imaging indications and usage? no 114 56.4 yes 88 43.6 do you feel your knowledge about cbct imaging is sufhicient? no 138 68.3 yes 64 31.7 would you attend a workshop or training session on cbct imaging referrals? no 45 22.3 barriers toward beeer cbct knowledge, altude, and pracoce vol 13, no 1 (2025) doi 10.5195/d3000.2025.987 http://dentistry3000.pitt.edu 6 maybe 56 27.7 yes 101 50 do you think cbct imaging is over-utilized in your practice or region? no 102 50.5 not sure 56 27.7 yes 44 21.8 848 evalua&on of the impact of nanopar&cle addi&ves on the shear bond strength between so= liners and acrylic vol 13 no 1 (2025) doi 10.5195/d3000.2025.848 h#p://den*stry3000.pi#.edu evaluation of the impact of nanoparticle additives on the shear bond strength between soft liners and acrylic nusaiba mustafa muhammed college of dentistry, tikrit university, tikrit, iraq abstract objec&ves: the study aimed to evaluate the an;fungal proper;es of incorpora;ng two types of nanopar;cles into the denture so? lining material, and shear bond strength between the reinforced so? liner and acrylic resin. materials and methods: ninety specimens were fabricated and grouped into three dis;nct groups named group a which is the control, group b involved cerium oxide nanopar;cles infused so? liner, and group c involved zinc oxide nanopar;cles infused so? liner specimens. specimens for shear bond strength consist of two acrylic parts and one so? liner part occupying the space between the acrylic specimens. an;fungal proper;es were tested by coun;ng the number of viable cells visually, and shear bond strength was tested using a universal tes;ng machine at speed of 0.5 mm per minute. sta;s;cal tests involved one-way anova and post hoc tests at p ≤ 0.05. results: cerium oxide nanopar;cles infused specimens revealed higher an;fungal proper;es compared to zinc oxide infused specimens and control specimens. also, higher shear bond strengths (0.419) followed by those infused with zinc oxide nanopar;cles (0.342), while the lowest values were for the control group (0.305). conclusions: the observed enhancements in an;fungal and mechanical proper;es resul;ng from cerium oxide and zinc oxide nanopar;cles infusions underscore their considerable poten;al as effec;ve addi;ves for improving performance in various applica;ons. keywords: acrylic denture base; bond strength; microbial coloniza;on; removable prostheses. cita:on: muhammed, nm. (2025) evalua:on of the impact of nanopar:cle addi:ves on the shear bond strength between sop liners and acrylic. den:stry 3000. 1:a001 doi:10.5195/d3000.2025.848 received: february 14, 2025 accepted: march 14, 2025 published: april 18, 2025 copyright: ©2025 muhammed, nm. this is an open access ar:cle licensed under a crea:ve commons axribu:on work 4.0 united states license. email: sanaa.nusamustafa@tu.edu.iq introduction soft lining materials are commonly utilized to address oral mucosa injuries caused by persistent ridge resorption, damage from detachable dentures, or surgery. these materials promote tissue healing and improve patient comfort when in contact with underlying tissues [1]. the soft lining material absorbs chewing pressures on the residual tissues, minimizing energy delivered to periprosthetic tissues. soft lining materials are difficult to clean due to their softer texture and lower resistance to brushing compared to traditional acrylic resins [2]. soft lining materials, particularly temporary ones, are quickly degradable and prone to microbial colonization. this can lead to pathological processes and hinder treatment of existing infections [3]. antimicrobial drugs may be effectively incorporated into soft denture materials, as demonstrated in in vitro and in vivo investigations. incorporating antibacterial compounds into soft denture materials can improve clinical lifespan and minimize plaque buildup [4]. this combination may be effective in treating denture stomatitis for numerous reasons such as reducing trauma from the internal denture surface, eliminating contact with contaminated oral tissues, interrupting the cycle of re-infection, and incorporating antimicrobial agents into the material to target infected tissues [5]. a variety of antifungal agents can be effectively added to denture soft liners, including herbal extracts, conventional antifungal drugs such as fluconazole and nystatin, and various nanoparticles including agnps, szz-nps, ceo2 nps, and nano-zno [6]. each of these agents offers unique benefits and potential for enhancing the antifungal efficacy of denture soft liners, providing valuable options for managing denture-induced stomatitis [7]. a previous study evaluated the addition of silver nanoparticles into denture soft liner. the results demonstrated that silver nanoparticles decreased candida albicans effectively, with antifungal efficacy ranging from 16.3% to 52.5% depending on agnp concentration [8]. in addition, a recent study had evaluated the addition of silicone dioxide on antifungal properties, roughness, an wettability of soft liner. it was concluded that 0.25% and 0.5% had effectively reduced candida albicans. conversely, higher concentrations were found to diminish antifungal performance and increase surface roughness [9]. however, incorporating antifungal agents into soft lining materials can impact structural characteristics and tensile strength [10]. to work properly, soft lining materials must adhere to the acrylic denture bases of removable prostheses. clinical usage of soft lining materials might cause peeling and fluid penetration, potentially leading to bacterial growth and biofilm development [11]. clinical failures due to loss of bonding with acrylic-based resins highlight the need for bond strength assessments. to effectively heal traumatized tissues, the soft denture liner should be connected to the removable denture's acrylic foundation. previous research has focused on the bonding between soft denture materials and denture foundation acrylic resins, since peeling from evalua&on of the impact of nanopar&cle addi&ves on the shear bond strength between so= liners and acrylic vol 13 no 1 (2025) doi 10.5195/d3000.2025.848 h#p://den*stry3000.pi#.edu the base has been linked to clinical failure. several investigations have evaluated the connection between soft lining materials and denture base resin [12,13,14]. however, little work has investigated the shear bond strength of soft lining materials after adding antifungal agents at lower doses that effectively inhibit candida albicans. materials and methods materials used in the study this study involved the use of vertex-soft heat cured acrylic-based soft-liner material (vertex, netherlands), polymethyl methacrylate, cerium oxide nanoparticles, and zinc oxide nanoparticles as indicated in (table 1). no. material manufacturer 1 vertex-soft heat cured acrylic-based soft-liner vertex, netherlands 2 polymethylmethacrylate (pmma) new static s.a,antioquia, colombia 3 ceriumnanoparticles (average particle size of 25 nm) nanoshel, usa 4 zinc oxide nanoparticles (average particle size of 50 nm) nanoshel, usa study design a total of 90 specimens were fabricated and divided into two main groups according to the conducted test. each group was divided into three subgroups: 15 specimens for each subgroup. the subgroups were as follows: group a (control) with no addition, group b (experimental) with 2 wt. % ceo2, and group c (experimental) with 2 wt. % zno. the percentages of nanoparticles were chosen based on the results of a previous pilot studies. two tests were conducted: viable count test, and shear bond test. specimen fabrication a custom-made acrylic blocks with dimensions of 10 mm length×10 mm width×2.3mm thickness for fungal test, and 25mm width × 75mm length × 5mm depth, 3mm depth stopper, and 13mm handle thickness for shear bond test were prepared [15]. each specimen for shear bond test had two blocks of heatcured acrylic material that created a gap of 25mm width × 25mm length × 3mm depth for soft lining material placement. a replicating additional silicone (zhermack, italy) was used to invest plastic models that were cut from clear acrylic plates (perspex cell cast acrylic, clairvaux les lacsrance, france) specifically for the test. after setting the silicone material, the dental flask (hanua, engineering crop., usa) was filled with a layer of dental stone (durux, spain) mixed according to the manufacturer's instructions (w/p ratio: 50ml/100g). next, a silicone mold with a plastic pattern were placed inside the flask over the gypsum layer. finally, dental stone mixed according to the manufacturer's instructions was poured into the entire lower portion of the flask. brush a coating of separating media (bms dental, italy) over the dental stone layer and allow to dry. then, the upper section of the dental flask was placed over the bottom piece and filled with dental stone. then, the dental flask was covered and waited for it to set completely. after setting it was opened and the plastic templates were removed to make space for the pmma to be packed inside (figure 1). heat-cured acrylic soft liners are available in both powder and liquid form. the manufacturer recommends a mixing ratio of 1ml liquid to 1.2g powder. a glass beaker was well cleaned and dried before adding powder and liquid and mixing for 30 seconds. the container was then firmly sealed and allowed for around 15 minutes to reach dough stage. the two parts of the flask were painted with the separating medium and allowed to dry. when the dough time was over, the material was ready to pack. an amalgamator equipment (perfect plus, united kingdom) was used to mix nanoparticle powder with soft liner powder for 40 seconds [15]. to get an exact powder-to-liquid ratio, the weight of nanoparticle powder was subtracted from the overall weight of the acrylic-based heat-cure soft liner powder. according to the manufacturer's instructions, the curing cycle involved heating the water to 70°c for 90 minutes and then increasing the temperature to 100°c for 30 minutes. softliner specimens for fungal test were then retrieved and finished. figure 1. silicone mold space inside dental stone after removing the plastic pattern. to make the acrylic dough, mixing of heatcured acrylic resin powder with monomer liquid in a clean, dry glass container. sealing the container to avoid monomer evaporation. once the acrylic resin reached the dough stage, it was packed into a silicon mold covered with separating media for easy removal after curing. to ensure uniform material distribution inside the mold, a layer of polyethylene sheet (amalgamated dental t.d., england) was wrapped over the acrylic, and the upper portion of the flask was assembled to the lower section before being placed under a hydraulic press (begohydrofix, germany). after removing the polyethylene film, the flask was opened, and surplus material was removed using a sharp knife. a new coating of separating medium was placed and allowed to dry. after securing the lids of the flasks, a hydraulic press at 100 kg/cm2 for five minutes was used to achieve metal/metal contact. the clamped flask was secured before immersing in a water bath (memmert, germany) to cure. acrylic specimens were cured using a digital thermostatically controlled water bath. the temperature was set to 70°c for 30 minutes, then increased to 100°c for another 30 minutes, following the manufacturer's recommendations. after removing the clamped flasks, they were allowed to cool for 30 minutes at room temperature, followed by 15 minutes of tap water chilling. once completely cooled, the flasks were opened, and acrylic specimens retrieved and finished (figure 2). table 1 .materials used in the study. evalua&on of the impact of nanopar&cle addi&ves on the shear bond strength between so= liners and acrylic vol 13 no 1 (2025) doi 10.5195/d3000.2025.848 h#p://den*stry3000.pi#.edu figure 2. finished acrylic specimens. for each shear bond strength test, two acrylic specimens were arranged facing each other. a 3 mm depth, 25 mm length, and 25 mm breadth gap were filled with wax to hold the two blocks together. acrylic blocks were pushed into a silicone mold (putty type) in a specialized flask (broden, sweden) and the silicone was allowed to completely solidify (figure 3). figure 3. specialized flask for packing the soft liner into the gaps between acrylic specimens. the silicone molds with acrylic specimens were placed in a custom-made flask facing each other and bonded by wax. the flask was filled with newly mixed dental stone. the flask was then covered with a petroleum gel coating. after setting the stone, it was dewaxed and washed with hot water and soap. the flask and acrylic blocks were then allowed to dry completely (figure 3). soft liner powder and liquid were mixed as previously mentioned. to avoid air bubbles, soft lining material was gradually injected and condensed in the gap using a condenser. the flask was overfilled, covered under pressure of 2 kg/cm2, and securely tightened until edge-to-edge contact was obtained. the flask was cured using a digital water bath, as described earlier. all samples were finished by removing surplus soft lining material with a sharp blade. samples to be utilized after 24 hours were kept in distilled water at 37 degrees celsius (figure 4). figure 4. finished shear bond test specimens. candida albicans viable count test candida albicans isolates were obtained from the oral cavity of 15 patients were medically fit denture users aged 50-65 with denture stomatitis. a sterile cotton swab was used to obtain the isolate by gently stroking the palatal mucosa. the swab was promptly inoculated in sabouraud dextrose agar (sda) as a primary isolation medium. to prepare the culture media, a clean, dry beaker was filled with 1000 ml of distilled water. then, 62 g of sabouraud dextrose agar was weighed using an electronic balance and added to the water. the beaker was sealed with sterile cotton and sterilized in an autoclave for 15 minutes at 121 °c/15 psi. finally, the media was left to cool to 47 °c on the counter. candida albicans colonies appeared pearl-shaped, pasty, creamy, smooth, and slightly convex on sda under microscope [16]. to prepare the sabouraud dextrose broth, weigh 30 g and add 1000 ml of distilled water to a glass beaker. autoclave at 121 °c/15 psi for 15 minutes, then cool to 47 °c on the counter. to examine the antifungal efficacy of nanoparticles-loaded materials, a candida albicans suspension of about 107cfu/ml (equivalent to 0.5 mcfarland standards) was prepared. this suspension was made by diluting a tiny amount of inoculums in a test tube with normal saline and measuring the solution with a mcfarland densitometer instrument. a micropipette was used to transfer 0.1% of the suspension to a test tube containing 0.9% sabouraud dextrose broth. the loaded samples (2% cerium oxide and 2% +zinc oxide nanoparticles) were then inserted in the tube and incubated for 24 hours at 37°c. after incubation, 0.1% of the broth mixture was transferred to a test tube with 0.9% normal saline. serial dilution was performed. around 0.1% of the third dilution was collected and distributed over the surface of sda with a glass spreader, and the plates were incubated at 37 °c for 48 hours. the third dilution was chosen because it showed a countable range of 30-300 cfu. all viable counts on the sda surface were counted visually and analyzed statistically. the antifungal efficiency (afe) was estimated using the following formula: afe[%]=(vc-vt)/vc×100% according to chladek et al. (2011), the number of viable colonies in control samples was denoted as vc, whereas the number of viable colonies in experimental samples was represented as vt [11]. shear bond strength the instron testing machine was used to determine soft liner shear bond strength. specimens were exposed to a load cell capacity of 100 kg and a crosshead speed of 0.5 mm per minute. the machine readings indicate the maximum load of failure (figure 5). astm standard d-638 (1986) was used to calculate bond strength by dividing the maximum force of failure by the cross-sectional area of each sample (25mm×25mm=625mm). the shear bond strength was determined using the following equation: shear strength = f/a where f is the maximum load (n), and a is the cross-section area (mm2) figure 5. shear bond strength test. results candida viable count after 48 hours of incubation, the lowest number of viable counts was recorded for group b (cerium oxide nanoparticles infused evalua&on of the impact of nanopar&cle addi&ves on the shear bond strength between so= liners and acrylic vol 13 no 1 (2025) doi 10.5195/d3000.2025.848 h#p://den*stry3000.pi#.edu specimens), followed by group c (zinc oxide nanoparticles infused specimens), and the highest viable counts was recorded for group a (control group). there was a highly significant difference between all of the groups, as revealed in (table 2) and (figure 6). table 2. statistical test results for viable counts. viable cell counts anova tukey hsd levene statistics=1.727, p value=0.197 [hs] figure 6. viable counts of candida albicans; a, group a specimens. b, group b specimens. c, group c specimens. shear bond strength shear bond strength between cerium oxide nanoparticle infused soft-liner and acrylic resin had the highest values (0.4193), followed those infused with zinc oxide nanoparticles (0.3424), while the lowest value of shear bonding was recorded for the control specimens (0.3054) as revealed in (figure 7). table 3. statistical test results for shear bond strength. one-way anova revealed highly significant differences between the group (p<0.01). post hoc test revealed highly significant differences between all of the three groups (table 3). figure 7. bar chart representing the means and standard deviation of the three groups. discussion one of the most frequent issues with applying soft denture liners are the bonding with acrylic base and the colonization of different microbes, especially candida albicans, which can cause pain, infection, and material failure. more attention has recently been paid to employing nanoparticles to combat fungal resistance to pharmaceuticals [8,9,17]. the observed decrease in viable fungal cell counts is related to the ability of cro2 and zno nanoparticles to generate reactive oxygen species (ros) [18]. ceo2 nanoparticles with their high surface energy and catalytic properties, can facilitate the conversion of molecular oxygen and water into superoxide anions (o2-) and hydrogen peroxide (h2o2) [19]. such highly reactive species cause oxidative damage to the fungal cell by attacking lipids, proteins, and nuclic acids [20]. zno nanoparticles also initiate ros such as hydroxyl radicals (oh) and superoxide anions (o2-) which can damage the fungal cells [21]. in addition, the high surface energy and surface area of cro2, similarly zno, enhances the interaction of nanoparticles onto the fungal cell walls, disrobing the integrity by causing leakage of vital components [22]. cro2 and zno nanoparticles can also release metal ions such as ce3+, ce4+, and zn2+ from cro2 and zno respectively. these metal ions can interfere with enzymatic activities, disruption of metal ion hemeostasis, and interfere with cellular singling and transport mechanisms by altering the function of singling proteins [23]. moreover, these nanoparticles can exhibit photocatalytic actions when exposed to light by generating ros and damage the fungal cells as mentioned [24]. shear bonding between denture soft liners and acrylic denture bases is one of the important factors to ensure durability and efficiency of the denture prosthesis [25]. the incorporation of antifungal agents into soft liner materials can significantly maintain oral health by providing fungicidal and fungistatic effects. however, the presence of such agents can also affect the adhesive characteristics between the denture parts. depending on the concentration and formation of these agents, surface energy, wettability, and chemical interactions at the interface can be affected [12, 14]. the results revealed that ceo2 incorporated specimens had stronger shear bonding compared to control and zinc oxide incorporated specimens. ceo2 nanoparticles are known for their high surface energy due to group min max mean ±sd f p value groups p value group a 105.00 124.20 114.7750 5.54398 1103.545 .000 a b .000 group b 29.90 38.10 33.6400 3.06936 a c .000 group c 45.00 55.10 50.0900 3.13898 b c .000 group min max mean ±sd f p value groups p value group a .302 .309 .30540 .002675 1354.602 .000 a b .000 group b .415 .422 .002263 .41930 a c .000 group c .325 .352 .34240 .007905 b c .000 evalua&on of the impact of nanopar&cle addi&ves on the shear bond strength between so= liners and acrylic vol 13 no 1 (2025) doi 10.5195/d3000.2025.848 h#p://den*stry3000.pi#.edu their large surface area that can facilitate the adsorption of the ceo2 nanoparticles onto the surface of polymer matrix which improves the intimate contact and bonding [26]. in addition, hydrogen bonding between hydroxyl groups on the ceo2 surface and functional groups of the polymer such as carbonyl (-c=o) and hydroxyle groups (-oh) further increases the adhesion at the interface between the nanoparticles and the acrylic resin [27]. in contrast, zinc oxide nanoparticles display a dissimilar surface chemistry and structure that result in weaker interactions with the pmma matrix. the potential lack of suitable functional groups on zinc oxide nanoparticles means there will be no hydrogen bonds with the functional groups of the pmma (e.g. carbonyl groups) [28]. such absence of functional groups can reduce the affinity between zinc oxide nanoparticles and pmma. in addition, low surface energy results in poor wetting properties of the acrylic polymer around the zinc oxide nanoparticles. this effect led to gaps or spaces that weaken the bond between the materials [29]. furthermore, zinc oxide nanoparticles may have a crystal lattice that does not provide effective mechanical interlocking [30]. physically, ceo2 nanoparticles can induce surface roughness at nanoscale levels due to their crystalline structure and high surface energy. the increased irregularities and protrusions on the ceo2 nanoparticles can effectively interlock with the polymer chains and increases the cohesion between the materials [31]. the increased roughness can also increase the number of sites for the chemical bonding to occur. therefore, ceo2 nanoparticles not only increase the physical interlocking but also increase the facilitate stronger chemical bonds between with the acrylic resin matrix [32]. furthermore, ceo2 nanoparticles can serve as stress transfer agents inside the soft liner which act by dispersing mechanical stresses and reduce localized areas of stress concentration at the interface between soft liner and pmma [33]. zno nanoparticles typically demonstrate a hexagonal wurtzite or cubic zinc blende structure. while zno nanoparticles can have several morphologies, including smooth surfaces, they do not inherently have a porous structure with interconnected channels. the smooth morphology of zno nanoparticles may affect their stress distribution and bonding efficiency in composite materials, potentially leading to weaker adhesion due to fewer surface irregularities that contribute to mechanical interlocking [34]. conclusions in conclusion, it was possible to successfully include two types of nanoparticles into the heat-cured denture soft lining material, both have antifungal properties and can be used as a medication delivery method to combat candida albicans. the most effective nanoparticles against fungus seemed to be 2% cerium oxide nanoparticles which also increased shear bond strength with acrylic base material. conflict of interest no conflict of interest. source of funding completely self-funded study. ethical clearance an in vitro study. references 1.a hamdi r, s al-rawi s, h ibrahim a, kala d. potential therapeutic and effective properties of soft tissue liners. journal of population therapeutics and clinical pharmacology. 2023;30(4):447-56. 2.kreve s, dos reis ac. denture liners: a systematic review relative to adhesion and mechanical properties. the scientific world journal. 2019;2019(1):6913080. 3.gad mm, bahgat ha, edrees mf, alhumaidan a, khan sq, ayad nm. antifungal activities and some surface characteristics of denture soft liners containing silicon dioxide nanoparticles. journal of international society of preventive and community dentistry. 2022 jan 1;12(1):109-16. 4.baygar t, ugur a, sarac n, balci u, ergun g. functional denture soft liner with antimicrobial and antibiofilm properties. journal of dental sciences. 2018 sep 1;13(3):213-9. 5.sartawi sy, abu-hammad s, a. salim n, alomoush s. denture stomatitis revisited: a summary of systematic reviews in the past decade and two case reports of papillary hyperplasia of unusual locations. international journal of dentistry. 2021;2021(1):7338143. 6.gad mm, bahgat ha, edrees mf, alhumaidan a, khan sq, ayad nm. antifungal activities and some surface characteristics of denture soft liners containing silicon dioxide nanoparticles. journal of international society of preventive and community dentistry. 2022 jan 1;12(1):109-16. 7.pachava kr, nadendla lk, alluri ls, tahseen h, sajja np. invitro antifungal evaluation of denture soft liner incorporated with tea tree oil: a new therapeutic approach towards denture stomatitis. journal of clinical and diagnostic research: jcdr. 2015 jun;9(6):zc62. 8.chladek g, mertas a, barszczewska-rybarek i, nalewajek t, żmudzki j, król w, łukaszczyk j. antifungal activity of denture soft lining material modified by silver nanoparticles—a pilot study. international journal of molecular sciences. 2011 jul 22;12(7):4735-44. 9.gad mm, bahgat ha, edrees mf, alhumaidan a, khan sq, ayad nm. antifungal activities and some surface characteristics of denture soft liners containing silicon dioxide nanoparticles. journal of international society of preventive and community dentistry. 2022 jan 1;12(1):109-16. 10.seenivasan kumar sk, kumar v, parathasarthy natarajan pn, gayathri sreenivasan gs. antifungal efficacy and the mechanical properties of soft liners against candida albicans after the incorporation of garlic and neem: an in vitro study. 11.chladek g, żmudzki j, kasperski j. correction: long-term soft denture lining materials. materials 2014, 7 (8), 5816-5842. materials. 2015 jun 23;8(6):3791-2. 12.vuksic j, pilipovic a, poklepovic pericic t, kranjcic j. tensile bond strength between different denture base materials and soft denture liners. materials. 2023 jun 26;16(13):4615. 12.hamedirad f, alikhasi m, hasanzade m. the effect of sandblasting on bond strength of soft liners to denture base resins: a systematic review and meta-analysis of in vitro studies. international journal of dentistry. 2021;2021(1):5674155. 13.muddugangadhar bc, mawani dp, das a, mukhopadhyay a. bond strength of soft liners to denture base resins and the influence of different surface treatments and thermocycling: a systematic review. the journal of prosthetic dentistry. 2020 jun 1;123(6):800-6. 14.abdulwahhab ar, jassim rk. the effect of aloe vera extract on adherence of candida albicans and other properties of heat cure denture soft lining material. international evalua&on of the impact of nanopar&cle addi&ves on the shear bond strength between so= liners and acrylic vol 13 no 1 (2025) doi 10.5195/d3000.2025.848 h#p://den*stry3000.pi#.edu journal of medical research & health sciences. 2018 jan 1;7(3):94-103. 15.sachivkina n, podoprigora i, bokov d. morphological characteristics of candida albicans, candida krusei, candida guilliermondii, and candida glabrata biofilms, and response to farnesol. veterinary world. 2021 jun;14(6):1608. 16.ahmed as, kamil ss, ahmed rs, younus nk. influence of some plant extracts on antifungal properties, hardness, and peel bond strength of heat-cured denture soft liner. diyala journal of medicine. 2024 jun 25;26(2):14-28. 17.wen t, liu j, he w, yang a. nanomaterials and reactive oxygen species (ros). nanotechnology in regenerative medicine and drug delivery therapy. 2020:361-87. 18.matussin sn, harunsani mh, khan mm. ceo2 and ceo2-based nanomaterials for photocatalytic, antioxidant and antimicrobial activities. journal of rare earths. 2023 feb 1;41(2):167-81. 19.juan ca, pérez de la lastra jm, plou fj, pérez-lebeña e. the chemistry of reactive oxygen species (ros) revisited: outlining their role in biological macromolecules (dna, lipids and proteins) and induced pathologies. international journal of molecular sciences. 2021 apr 28;22(9):4642. 20.hwang c, choi mh, kim he, jeong sh, park ju. reactive oxygen species-generating hydrogel platform for enhanced antibacterial therapy. npg asia materials. 2022 dec;14(1):72. 21.vollath d, fischer fd, holec d. surface energy of nanoparticles–influence of particle size and structure. beilstein journal of nanotechnology. 2018 aug 23;9(1):2265-76. 22.godoy-gallardo m, eckhard u, delgado lm, de roo puente yj, hoyos-nogués m, gil fj, perez ra. antibacterial approaches in tissue engineering using metal ions and nanoparticles: from mechanisms to applications. bioactive materials. 2021 dec 1;6(12):4470-90. 23.parra-ortiz e, malmsten m. photocatalytic nanoparticles–from membrane interactions to antimicrobial and antiviral effects. advances in colloid and interface science. 2022 jan 1;299:102526. 24.mahboub f, salehsaber f, parnia f, gharekhani v, kananizadeh y, taghizadeh m. effect of denture cleansing agents on tensile and shear bond strengths of soft liners to acrylic denture base. journal of dental research, dental clinics, dental prospects. 2017;11(3):183. 25.ema t, choi pg, takami s, masuda y. facetcontrolled synthesis of ceo2 nanoparticles for high-performance ceo2 nanoparticle/sno2 nanosheet hybrid gas sensors. acs applied materials & interfaces. 2022 dec 15;14(51):56998-7007. 26.plakhova tv, romanchuk ay, butorin sm, konyukhova ad, egorov av, shiryaev aa, baranchikov ae, dorovatovskii pv, huthwelker t, gerber e, bauters s. towards the surface hydroxyl species in ceo 2 nanoparticles. nanoscale. 2019;11(39):18142-9. 27.goswami s, bishnoi a, tank d, patel p, chahar m, khaturia s, modi n, khalid m, alam mw, yadav vk, alreshidi ma. recent trends in the synthesis, characterization and commercial applications of zinc oxide nanoparticles-a review. inorganica chimica acta. 2024 aug 30:122350. 28.gulab h, fatima n, tariq u, gohar o, irshad m, khan mz, saleem m, ghaffar a, hussain m, jan ak, humayun m. advancements in zinc oxide nanomaterials: synthesis, properties, and diverse applications. nano-structures & nano-objects. 2024 sep 1;39:101271. 29.sharma dk, shukla s, sharma kk, kumar v. a review on zno: fundamental properties and applications. materials today: proceedings. 2022 jan 1;49:3028-35. 30.zhuang x, magnone e, han sw, park jh. novel surface modification strategies for enhanced ceo2 nanoparticle dispersion and suspension stability. ceramics international. 2024 jul 1;50(13):24801-14. 31.persson bn, mate cm. role of humidity and surface roughness on direct wafer bonding. the european physical journal b. 2024 apr;97(4):1-3. 32.xu c, qu x. cerium oxide nanoparticle: a remarkably versatile rare earth nanomaterial for biological applications. npg asia materials. 2014 mar;6(3):e90-. 33.a barzinjy a. structure, synthesis and applications of zno nanoparticles: a review. jordan journal of physics. 2020;13(2):123-35. 774 final physiological status of some selected salivary an7oxidants in dental caries vol 13 no 1 (2025) doi 10.5195/d3000.2025.774 h#p://den*stry3000.pi#.edu physiological status of some selected salivary antioxidants in dental caries sulafa khair al-deen banoosh alayoubi1, intesar jasim al-kaysi1, hadeel ayad2, esraa azawi4 1 college of dentistry, university of tikrit, iraq 2 ministry of health, iraq abstract objec&ve: uric acid and total protein are the two main anboxidants in saliva, and dental caries is the oral disease that affects most of the total populabon. this study aimed to invesbgate the associabon between salivary anboxidants (uric acid and total protein) and dental caries in individuals between the ages of 20 and 30. materials and methods: 50 subjects (18 males and 32 females) between the ages of 19 and 30 who atended the efraz health center in samarra city were recruited. dental caries severity was evaluated using the dmft and dt indices in accordance with who guidelines from 1997. dental caries was divided into three categories based on scores: mild (0–10), moderate (11–20), and severe (21–and more). salivary uric acid and salivary total protein concentrabon were assessed via chemical analysis of unsbmulated saliva sample collecbon. the stabsbcal package for social science (spss) version 21 was used to examine all data. results: males had greater mean values for the dmft and dt fracbons than females, with the differences being stabsbcally significant (p values of 0.05 and 0.001, respecbvely). males also had higher salivary uric acid than females (p value of 0.001) and uric acid was associated with dental caries experience. conclusion: salivary uric acid may act as a biomarker of dental caries. we found higher dental caries among subjects who had high levels of anboxidants. keywords: anboxidant, saliva, dental caries, total protein, uric acid. cita:on: alayoubi skab, et al. (2025) physiological status of some selected salivary an:oxidants in dental caries. den:stry 3000. 1:a001 doi:10.5195/d3000.2025.774 received: november 14, 2024 accepted: december 19, 2024 published: april 11, 2025 copyright: ©2025 alayoubi skab, et al. this is an open access ar:cle licensed under a crea:ve commons atribu:on work 4.0 united states license. email:sulafa.khairy@yahoo.com introduction dental caries is the local acidinduced deterioration of dental hard tissue that is sensitive to bacteria fermenting dietary carbohydrates. it is a complex illness where microorganisms are involved. it is the most common and persistent oral disease, especially in young children, due to the host, the substrate, and the modification of the immune system [1-3]. dental caries carries a significant risk of morbidity [4]. saliva, a biological liquid found in the oral cavity, plays a crucial role in maintaining oral health through several mechanisms, including salivary flow rate, buffer capacity, and defensive roles via antibacterial features and salivary antioxidant system [5]. saliva unquestionably improves dental health; therefore, a lack of its secretion may be the onset of disease [6]. the antioxidants in our diet, along with intracellular antioxidants and those produced by enzymatic systems, can stop a variety of inflammatory, infectious, or tumoral processes [7]. free radical levels rising has been identified as the common element in all these activities. a rise in oxidative damage and a decline in antioxidants have been linked to specific inflammatory conditions of the mouth, such as periodontal disease [8]. antioxidants' unique function is to fight rogue free radicals, thereby lowering their destructive potential. they physiological status of some selected salivary an7oxidants in dental caries vol 13 no 1 (2025) doi 10.5195/d3000.2025.774 h#p://den*stry3000.pi#.edu function as a singlet oxygen quencher, hydrogen donor, electron donor, peroxide decomposer, and synergist in addition to scavenging radicals [9]. it has been reported that uric acid is the main antioxidant in saliva, accounting for more than 85% of the whole antioxidant action of latent and stimulated saliva from both healthy and periodontally compromised subjects. total protein is another example of a salivary antioxidant [10]. the primary byproduct of purine metabolism in humans is uric acid (ua), which is created from xanthine by the enzyme xanthine oxidase. superoxide anion and other reactive oxygen products are formed during purine metabolism when molecular oxygen serves as the electron acceptor [11]. uric acid may serve as a sign of oxidative stress and may also act as an antioxidant with potential therapeutic benefits. additionally, pro-oxidants, like other reducing agents, can play a part, especially at higher levels [12]. lysozyme, lactoferrin, lactoperoxidase, immunoglobulins, agglutinin, and mucins are among the numerous proteins found in saliva that help to preserve the oral tissues [13]. additionally, a number of peptides having antibacterial activity have been discovered. these include histatins, defensins, and ll-37, the sole cathelicidin produced by humans. there appears to be a significant overlap in functionality because each of these proteins and peptides exhibits a wide range of antibacterial action. this could explain the finding that oral disease susceptibility appears to be unrelated to the concentration of any one component [14]. although the specific cause of this 'redundancy' is unclear, other features might be involved. the aim of this study was to find a relationship between antioxidants of saliva (uric acid and total protein) and dental caries. material and methods subjects and dental examination fifty randomly chosen participants, who were treated at the efraz health center in samarra and ranged in age from 19 to 30 years, were examined between january and february 2020. utilizing dental explorer and mouth mirrors, a clinical examination was performed. the index used for assessment of dental status was: dmft by w.h.o. methodology for caries status and treatment need (1997) [15] to calculate decayed (d), missing (m), and filled (f) teeth. saliva collection following the guidelines stated by tenovuo and lagerlöf [16], samples were collected. each individual was instructed to rinse their mouth with distilled water in the morning after at least two hours of fasting. after 5 minutes, salivary samples began to be collected by spitting into plastic tubes until (five ml) of unstimulating saliva was obtained. the sample was collected, centrifuged for 10 minutes at 3000 rpm, and the supernatant portion was aspirated and stored at (20°c) for upcoming biochemical studies. biochemical analysis: salivary samples were stored for biochemical analysis. total protein and uric acid levels in the saliva were measured calorimetrically using a spectrophotometer (cecil ce 1011, uk). a ready kit (biolabo, france) was used to assess the salivary total protein and the salivary uric acid level, by exact following the manufactured instructions. data analysis was conducted using excel 2010. results table 1 shows the number and percentage of dental caries by physiological status of some selected salivary an7oxidants in dental caries vol 13 no 1 (2025) doi 10.5195/d3000.2025.774 h#p://den*stry3000.pi#.edu both genders for all three groups [17]. table 1. caries severity percentage according to gender. severity of caries experience (dmft index) males females total n % no. % no. % mild (0-10) 11 61.1 28 87.5 39 78 moderate (11-20) 7 38.9 4 12.5 11 22 severe (21and more) 0 0 0 0 0 0 total 18 32 50 table 2 shows the mean and standard deviation of total protein in saliva and uric acid in males and females. males had higher salivary uric acid than females (p < 0.001). table 2. comparison between salivary antioxidant constituents in both genders. gender n salivary antioxidant constituents total protein uric acid mean ± sd mean ± sd male 18 1.50 ±0.15 2.71 ±1.3 female 32 1.47 ±0.1 1.37 ±0.87 total 50 1.47 ±0.12 1.85 ±1.25 p-value 0.34 0.0008 table 3 shows the means and standard deviation of dmft and dt indices in both genders. males had higher caries experience than females (p < 0.007). also, males had more decayed teeth than females (p < 0.04). table 3. comparison between caries experience in different genders. gender n cariesexperience dmft mean ± sd dt mean ± sd male 18 8.55 ±4.89 4.44 ±3.67 female 32 4.96 ±3.9 2.68 ±2.56 total 50 6.26 ±4.61 3.32 ±3.12 p-value 0.007 0.04 table 4 shows the mean and standard deviation of salivary total protein and salivary uric acid in the different groups based on dmft. table 4. comparison between salivary antioxidant constituents in different caries severity. caries severity (dmft) index n salivary antioxidant constituents total protein uric acid mean ± sd mean ± sd mild group 3 9 1.46 ±0.12 1.7 ±1.1 moderate group 1 1 1.51 ±0.11 2.37 ±1.5 p-value p=0.11 p=0.1 table 5 shows the correlation of total protein and uric acid based on caries severity. it was found that there was a positive statistically significant correlation between dmft and salivary uric acid. table 5. correlation of dental caries severity and salivary antioxidant constituents. salivary antioxidant constituents dt dmft r pvalue r pvalue total protein 0.045 0.75 0.134 0.35 uric acid 0.102 0.48 0.371 0.007 physiological status of some selected salivary an7oxidants in dental caries vol 13 no 1 (2025) doi 10.5195/d3000.2025.774 h#p://den*stry3000.pi#.edu figure 1 shows the concentration of salivary total protein in different age groups. figure 1. total protein in different age groups. figure 2 shows the concentration (g/ml) of salivary uric acid in different age groups. figure 2. uric acid in different age groups. discussion in the present study, males had greater levels of salivary uric acid than females. other studies [18,19] similarly found that males had considerably higher overall prevalence of hyperuricemia than females. this may be due to sex hormones, which increase renal urate clearance in women and decrease tubular urate postsecretory reabsorption in males [20]. the current study also discovered that salivary uric acid levels rise along with an increase in prevalence of caries. a similar finding was made by another study [21], and these findings may be explained by the fact that an increase in saliva's total antioxidant capacity may alter streptococcus' adhesion to dental plaque and promote more cariogenic activity. in contrast, another study [22] found a negative correlation between salivary uric acid and caries prevalence. this may be due to enhanced production of reactive oxygen species (ros) in the presence of bacteria by increased activity of neutrophils and monocytes in the oral cavity during phagocytosis [23]. it was found that antioxidants enhance oral health by providing protection against reactive oxygen species that induce damage of oral tissue especially gingival hyaluronic acid and proteoglycan. in this study, the prevalence of dental caries was significantly higher in men than in women. this finding is in line with other studies [24], which attributed this finding to women's greater exposure to oral health information, stronger oral health beliefs, and greater frequency of preventive behaviors [25]. according to their site, location, and mode of action, salivary total protein may play a preventive function in dental caries or may promote the colonization of microorganisms [26]. this study found no link between salivary total protein and the occurrence of caries, which is consistent with other research's findings [27,29]. in contrast, a study by nireeksha et al. [30] revealed that the mean salivary protein was lower in individuals with higher caries experience. this finding may be explained by the salivary proteins' protective effect against the free diffusion of acids on the tooth surface, which prevents the development of dental caries [30]. salivary proteins also vary by age [31]. conclusion the findings of this study support the hypothesis that salivary uric acid may function as a biomarker of dental caries status. higher dental caries experience was 0 0.5 1 1.5 2 19 21 23 25 27 29 t.protien t.protien 1.61.71.55 1.811.96 4.16 1.53 3.2 3.7 1.9 1.55 0.94 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 19 21 23 25 27 29 u ric a ci d age physiological status of some selected salivary an7oxidants in dental caries vol 13 no 1 (2025) doi 10.5195/d3000.2025.774 h#p://den*stry3000.pi#.edu found among subjects who had high levels of antioxidants. conflicts of interest the authors declare no competing interest. references 1miralles l, silvestre fj, hernández-mijares a, baujsta d, llambes f, grau d. dental caries in type 1 diabejcs: influence of systemic factors of the disease upon the development of dental caries. med oral patol oral cir bucal 2006; 11: e256-60. 2okoye lo, ekwueme oc. prevalence of dental caries in a nigerian rural community: a preliminary local survey. ann med health sci res. 2011;1:187– 196. 3dawkins e, michimi a, ellisgriffith g, peterson t, carter d, english g. dental caries among children visijng a moble dental clinic in south central kentucky: a pooled crosssecjonal study. bmc oral health. 2013;13:19. doi: 10.1186/1472-6831-3-19. 4moses j, rangeeth bn, gurunathan d. prevalence of dental caries, socioeconomic old school going children of chid ambaram status and treatment needs among 5 to 15 year old school going children of chidambaram. j clin dign res. 2011;5:146–151. 5liohong g, wenyuan s. salivary biomarker of caries risk assessment. j calif dent assoc.2013 feb; 41(2):104 118. 6lavelle clb. applied oral physiology, 2nd ed. london: buperworths and co (publishers) ltd; 1988. p. 128–141. 7perno goldie m. anjoxidants in oral health care: making the connecjon. int j dent hyg 2005; 3: 93-95. 8brock g r, buperworth c j, maphews j b, chapple i l c. local and systemic total anjoxidant capacity in periodonjjs and health. j clin periodontol 2004; 31: 515-521. 9bagchi k and puri s. free radical and anjoxidants in health and disease. eastern med hj. 1998; 4:350-360. 10baqno m, ferreiro ms, gallardo i, newman hn, bullon p: the anjoxidant capacity of saliva. j clin periodontol 2002; 29: 189– 194. 11baillie jk, bates mg, thompson aa, waring ws, partridge rw, schnopp mf, simpson a, gulliver-sloan f, maxwell sr and webb dj "endogenous urate producjon augments plasma anjoxidant capacity in healthy lowland subjects exposed to high aljtude". chest (2007): 131 (5): 1473–8. 12glantzounis gk, tsimoyiannis ec, kappas am, galaris da: "uric acid and oxidajve stress". current pharmaceujcal design. 2005; 11(32): 414551. 13nieuw amerongen av, veerman eci: saliva – the defender of the oral cavity. oral dis 2002;8:12–22 14rudney jd, hickey kl, ji z: cumulajve correlajons of lysozyme, lactoferrin, peroxidase, siga, amylase, and total protein concentrajons with adherence of oral viridans streptococci to microplates coated with human saliva. j dent res 1999;78:759–768. physiological status of some selected salivary an7oxidants in dental caries vol 13 no 1 (2025) doi 10.5195/d3000.2025.774 h#p://den*stry3000.pi#.edu 15world health organizajon, oral health surveys: basic methods, 4th edn. world health organizajon, geneva, 1997. 16tenovuo j, lagerlöf f. saliva. in: thylstrup a, fejerskov o (eds.). textbook of clinical cariology. 2nded. copenhagen: munksgaard; 1994. p. 17-43. 17sulafa k. biochemical relations of salivary magnesium, calcium, and phosphate with dental caries in adult; a thesis submiped to the college of medicine at the university of tikrit, 2012. 18jenna l riis, crystal i bryce, marla j majn, john l stebbins, olga kornienko,, lauren van huisstede, and douglas a granger, the validity, stability, and ujlity of measuring uric acid in saliva, biomark in medcine. 2018 jun; 12(6): 583–596. 19f. mateos antón, j. garcía puig, t. ramos, p. gonzález, and j. ordás, “sex differences in uric acid metabolism in adults: evidence for a lack of influence of estradiol-17β (e2) on the renal handling of urate,” metabolism, vol. 35, no. 4, pp. 343–348, 1986. 20jain m, singh a, sharma a. relajonship of perceived stress and dental caries among pre university students in bangalore city. j clin diag res. 2014;8:131. 21raghad ibrahim kadhum, alhan ahmed qasim, the impact of dental environment stress on caries experience, salivary flow rate and uric acid; j bagh college denjstry, vol. 32(1), march 2020, p 35-41. 22rahmani m, ghoorchi v, rezaei f, raygani av. evaluajon of total anjoxidant capacity of saliva in high school students. global j health sci. 2016;8:89-94. 23raja, s., al-tmimi, h. m., ghadir, g. k., mustafa, m. a., alani, z. k., rusho, m. a., & rajeswari, n. (2024). original research arjcle an analysis of polymer material selecjon and design opjmizajon to improve structural integrity in 3d printed aerospace components. 24 clement c. azodo and barnabas unamatokpa, gender difference in oral health percepjon and pracjces among medical house officers ; russian open medical journal 2012; 1: issn 2304-3415; p 1-4. 25 masalu j, mtaya m, astrøm an. risk awareness, exposure to oral health informajon, oral health related beliefs and behaviours among students apending higher learning insjtujons in dar es salaam, tanzania. east afr med j 2002; 79: 328-333. 26vibhakar pa, patankar sr, yadav mr, vibhakar pa (2013) salivary total protein levels and their correlajon to dental caries. internajonal journal of oral & maxillofacial pathology 4: 13-16. 27subramani, r., mustafa, m. a., ghadir, g. k., al-tmimi, h. m., alani, z. k., haridas, d., ... & kumar, a. p. (2024). advancements in 3d prinjng materials: a comparajve analysis of performance and applicajons. applied chemical engineering, 7(2), ace-3867. physiological status of some selected salivary an7oxidants in dental caries vol 13 no 1 (2025) doi 10.5195/d3000.2025.774 h#p://den*stry3000.pi#.edu 28 de farias dg, bezerra ac (2003) salivary anjbodies, amylase and protein from children with early childhood caries. clin oral invesjg 7: 154-157. 29roa ns, chaves m, gómez m, jaramillo lm (2008) associajon of salivary proteins with dental caries in a colombian populajon. acta odontol lajnoam 21: 69-75. 30nireeksha1, mithra n hegde2*, suchetha kumari n3, harshini ullal4 and vishakh kedilaya, salivary proteins as biomarkers in dental caries: in vivo study, dental, oral and craniofacial research, 2017, volume 3(2): 1-7. 31van nieuw amerongen a, bolscher jg, veerman ec (2004) salivary proteins: protecjve and diagnosjc value in cariology? caries res 38: 247-253. 748+2024 evaluation of diode laser treatment on implant stability, healing edema and implant survival in a sample of iraqi patients seeking dental implants: single center study vol 12 no 2 (2024) doi 10.5195/d3000.2024.748 http://dentistry3000.pitt.edu evaluation of diode laser treatment on implant stability, healing edema and implant survival in a sample of iraqi patients seeking dental implants: single center study bakir ghanem murrad1,4, jed bouguila2, thair a. hasan3 1 ibn el jazzar faculty of medicine of sousse, university of sousse, tunisia 2tunis el-manar university, tunis medical school, tunisia 3iraqi board for medical specializations, iraq 4kut university college, wasit, iraq abstract objective: to investigate the impact of diode laser therapy on pre-implant site preparation and postimplant healing, primary and secondary stability of the dental implant osteointegration, to measure crestal bone level, and the survival rate of the dental implants. methods: this prospective clinical study was organized from november 2022 to august 2024 at the dentistry college teaching hospital, department of oral and maxillofacial surgery/dental implant unit/university of kut. patients were randomly allocated into two groups: study group receiving diode laser and control group receiving no laser treatment. outcome variables included mainly healing edema score, implant stability, and survival of implant. results: regarding primary stability, there was no significant difference in its mean level between study group and control group, 67.12 ±5.44 isq versus 68.54 ±5.77 isq, respectively (p = 0.208). regarding secondary stability following 7 days, 4 weeks, 8 weeks, and 12 weeks, there was no significant difference in its mean level between study group and control group (p> 0.05). higher scores of healing edema were found in the control group when compared to study groups at 3 days, 7 days and 14 days intervals (p< 0.001). conclusion: laser therapy resulted in improved healing edema score, but it had no significant impact on dental stability or implant survival. keywords: diode laser, dental implant, stability, healing edema. citation: murrad bg, et al. (2024) evaluation of diode laser treatment on implant stability, healing edema and implant survival in a sample of iraqi patients seeking dental implants: single center study. dentistry 3000. doi:10.5195/d3000.2024.748 received: october 1, 2024 accepted: november 21, 2024 published: november 22 2024 copyright: ©2024 murrad bg, et al. this is an open access article licensed under a creative commons attribution work 4.0 united states license. email: baqirghanim@gmail.com introduction the utilization of laser technology in contemporary dental procedures encompasses a broad spectrum of sub-disciplines, including conservative dentistry, oral surgery, endodontics, periodontology, implantology, aesthetic dentistry, among other dental interventions [1]. the historical application of laser technology within the field of dentistry spans five decades, having undergone continuous development since its introduction in the 1960s by miaman, with significant implications for both hard tissue applications, such as restorative removal and curing, caries prevention, cavity preparation, growth modulation, and dentinal hypersensitivity, as well as soft tissue applications, including wound healing and the excision of hyperplastic tissue to facilitate the exposure of edentulous teeth, alongside evaluation of diode laser treatment on implant stability, healing edema and implant survival in a sample of iraqi patients seeking dental implants: single center study vol 12 no 2 (2024) doi 10.5195/d3000.2024.748 http://dentistry3000.pitt.edu various other therapeutic uses [2]. lasers can be classified into distinct categories based on criteria such as the active material, energy emission levels, wavelength ranges, and laser movement methodologies [3]. nevertheless, lasers are generally classified into two overarching categories—hard lasers and cold or soft lasers— based on parameters such as intensity, mode, and power utilized. among the diverse array of lasers employed in dental applications, the diode laser has garnered significant attention both preand post-dental implant procedures, attributable to its efficacy in facilitating rapid tissue healing, minimizing inflammation, enhancing patient satisfaction, and improving overall recovery rates [4]. particularly noteworthy is low level laser treatment (lllt), a well-established concept recognized for its photobiostimulatory and photobiomodulatory properties, which are known to enhance cellular regeneration, elevate cellular metabolic rates, and promote cell proliferation [5]. furthermore, diode lasers are characterized by ease of use in proximity, reduced thermal output, cost-effectiveness, and non-conductivity of electricity [6]. teeth replacement, with the help of dental implants, is one of the ancient medical practices used since ancient civilizations. various materials are used in implantology such as shells, bamboo, porcelain, iridioplatinum, gold, silver and at times, the teeth of other human beings. the loss of a teeth has two primary concerns, difficulty in chewing food and aesthetic issue. some of the solutions include bridges, ligatures or external teeth, and dental implants. until the development of modern dentistry, these dental implants remained a painful procedure that often posed a nightmare to the patients. however, with the development of modern dentistry, endosseeous implant or dental implant, the surgical element which creates a bond between bone and the surrounding prothesis (such as crown, bridge or denture), is well accepted [7]. the present study intended to investigate the application of diode laser therapy in dental implants. in the presence of various types of lasers, diode laser has been chosen for the study since it is widely used in dentistry, specifically for soft tissue applications. before the placement and in the aftermath of fixation, various complex processes play a critical role in defining the success of the dental implants. before the insertion of the dental implant, it is imperative to prepare the implant bed and thoroughly assess the clinical condition of the patient. following the placement of the implant, it is critical to ensure its primary stability initially. to achieve this, the implant must be scrutinized for osseointegration, which is the biological process that facilitates the integration of living bone with evaluation of diode laser treatment on implant stability, healing edema and implant survival in a sample of iraqi patients seeking dental implants: single center study vol 12 no 2 (2024) doi 10.5195/d3000.2024.748 http://dentistry3000.pitt.edu the implant surface. the various challenges encountered throughout this procedure, including microbial contamination, plaque accumulation, complications arising from the physical and chemical properties of the implant, as well as the biological and cellular responses that determine the acceptance or rejection of the dental implant, inflammation, primary stability, failures in osseointegration, and aesthetic considerations, must be meticulously analyzed to provide a comprehensive understanding of the significance of dental implants and the contribution of diode laser technology in this context. additionally, it is essential to elaborate on the modifications that occur within the crestal bone, as these alterations play a pivotal role in the long-term success and stability of the implant. considering these, the present study represents a pioneering effort to investigate the impact of diode laser therapy on patient healing outcomes, with the objective of enhancing the success rate of dental implants. material and methods this prospective clinical study was organized from november 2022 to august 2024 in dentistry college teaching hospital, department of oral and maxillofacial surgery/dental implant unit/university of kut. the study sample was derived from the population of patients who was treated at the department of oral and maxillofacial surgery/college of dentistry, who were seeking dental implant prosthodontic rehabilitation to replace a single, multiple or unrestorable maxillary and mandibular teeth by dental implants. the protocol of this study was approved by the scientific committee of the department. all patients signed informed consent forms. a total of 100 iraqi adult patients aged 22 to 72 years, 50 males and50 females were enrolled in the study. they received 266 dental implants. partially or completely edentulous patients aged over 18 years of either gender with d1, d2, d3 and d4 bone density having sufficient bone volumes were included in the study. patients having chronic medical illness or contraindications for oral surgery, those with residual infection at implant sites, or having severe alveolar bone destruction, smokers and pregnant women were excluded from study. all patients were subjected preoperatively to detailed clinical examination and radiographic evaluation utilizing cone beam computed tomographic scans (cbct) and panoramic and periapical radiographs to scan the jaws and implants sites. all implants were placed by one surgeon according to a strict surgical protocol following the manufacture's information. sequential osteotomy was carried out using osteotomy drill at 600 to 800 rpm speed with external irrigation with normal saline fluid. evaluation of diode laser treatment on implant stability, healing edema and implant survival in a sample of iraqi patients seeking dental implants: single center study vol 12 no 2 (2024) doi 10.5195/d3000.2024.748 http://dentistry3000.pitt.edu peak seating torque was measured for each dental implant using calibrated manual torque ratchet and dental implant engine. for all 266 implants, after they were inserted, the operator measured rfa values (implant stability quotients isq) using rf analyzer (ostell) to obtain the primary and secondary stability for each dental implant. crestal bone level was measured around all dental implant fixtures using cbct scans immediately after surgery. all the patients were recalled 3, 7, and 14 days after surgery to measure the healing edema according to a visual analogue scale (vas) protocol. secondary stability was measured 7 days, (4,8, and 12 weeks after dental implant insertion for all participants. all patients were recalled for follow up until one year after dental implant insertion for survival rate checking, and also to measure the crestal bone level around fixture using cone beam computed tomography scans. for the study group, diode laser doctor smile diode laser device (italy) was irradiated by a clinician in an isolated room. for each dental implant fixture, survival rate was measured for one year follow up. some of materials used in this study are shown in figures 1 and 2. figure 1. manual calibrated torque ratchet with torque scale (1070n/cm) (medentika co., germany). figure 2. doctor smile™ diode laser device (italy). statistical analysis data were analyzed using spss (version 26). numeric data were expressed as mean, minimum value, maximum value, standard deviation, interquartile range and median. independent samples ttest was used to compare means between study and control groups; mann whitney u test was used to compare mean rank values between study and control groups, and chi-square was used to compare proportions between study and control groups. the level of significance was based on p-value of ≤ 0.05. results in this study, the general characteristics of patients are shown in table 1. with respect to age, there was no significant variation in the mean between study and control groups (p = 0.743), and the mean age with corresponding standard deviation were 47.02 ±14.25 years versus 46.10 ±13.77 years, respectively. in addition, in either group, the age ranged between 22 and 72 years. with respect to proportions of males and females, the researchers intentionally enrolled equal evaluation of diode laser treatment on implant stability, healing edema and implant survival in a sample of iraqi patients seeking dental implants: single center study vol 12 no 2 (2024) doi 10.5195/d3000.2024.748 http://dentistry3000.pitt.edu number of males and females in both study and control groups to ensure statistical matching. the range of implants (1 to 6), as well as the median(2), and the interquartile range (1) were the same for both groups. the researchers intentionally enrolled equal numbers of maxillary and mandibular implants in each group. the median level of bone density in the study group was lower than that reported in the control group, 619 hu versus 698 hu, respectively. however, this difference was insignificant (p = 0.153). moreover, the proportions of patients with d1, d2, d3 and d4 bone intensities in both groups were almost the same, 4% versus 2 %, 20 % versus 32 %, 76 % versus 62 %, and 0 % versus 4 %, respectively. there was no significant difference in mean insertion torque between both groups, 40.10 ±4.57 versus 38.74 ±5.27 n/cm, respectively (p = 0.171), table 2. comparison of stability level between study group and control group is shown in table 3. regarding primary stability, there was no significant difference in its mean level between study group and control group, 67.12 ±5.44 isq versus 68.54 ±5.77 isq, respectively (p = 0.208). regarding secondary stability following 7 days, 4 weeks, 8 weeks, and 12 weeks, there was no significant difference in its mean level between study group and control group (p> 0.05). comparison of healing edema score between study group and control group is shown in table 4. higher scores of healing edema were associated significantly with control group when compared to study groups at 3 days, 7 days, and 14 days (p< 0.001). figures 3 to 5 illustrate some of the procedures in the present study. table 1. demographic characteristics of patients enrolled in this study contrasted between study group and control group. characteristic study group n = 50 control group n = 50 p age (years) mean ±sd 47.02 ±14.25 46.10 ±13.77 0.743 i ns range 22 -72 22 -72 sex male, n (%) 25 (50.0 %) 25 (50.0 %) 1.000 c ns female, n (%) 25 (50.0 %) 25 (50.0 %) implant number median (iqr) 2 (1) 2 (1) 0.898 m ns range 1 -6 1 -6 jaw bone maxilla, n (%) 25 (50.0) 25 (50.0) 1.000 c ns mandible, n (%) 25 (50.0) 25 (50.0) n: number of cases; sd: standard deviation; i: independent samples student t-test; c: chi-square test; ns: not significant; iqr: inter-quartile range; m: mann whitney u test table 2. comparison of bone density level between study group and control group. characteristic study group control group p bone density median (iqr) 619 (341) 698 (352.25) 0.153 m ns range 345 -1308 296 -1290 d1 2 (4.0 %) 1 (2.0 %) d2 10 (20.0 %) 16 (32.0 %) d3 38 (76.0 %) 31 (62.0 %) d4 0 (0.0 %) 2 (4.0 %) insertion torque n/cm mean ±sd 40.10 ±4.57 38.74 ±5.27 0.171 i ns range 30 -45 25 -50 n: number of cases; iqr: inter-quartile range; m: mann whitney u test; ns: not significant; n: number of cases; sd: standard deviation; i: independent samples student t-test; ns: not significant; n: newton; m: meter table 3. comparison of stability level between study group and control group. characteristic study group n = 50 control group n = 50 p primary stability (isq) mean ±sd 67.12 ±5.44 68.54 ±5.77 0.208 i ns range 54 -77 55 -81 secondary stability (isq) 7 days mean ±sd 66.70 ±5.27 67.78 ±5.31 0.310 i ns range 56 -76 55 -79 4 weeks mean ±sd 65.84 ±5.10 64.88 ±5.31 0.359 i ns range 54 -76 51 -76 8 weeks mean ±sd 66.30 ±5.10 64.66 ±5.33 0.119 i ns range 55 -77 51 -77 12 weeks mean ±sd 71.72 ±4.66 71.44 ±5.08 0.775 i ns range 63 -80 62 -82 n: number of cases; sd: standard deviation; i: independent samples student t-test; ns: not significant; isq: implant stability quotient table 4. comparison of healing edema score between study group and control group. characteristic study group n = 50 control group n = 50 p healing edema 3 days 1 24 (48.0 %) 0 (0.0 %) <0.001 c *** 2 22 (44.0 %) 25 (50.0 %) 3 4 (8.0 %) 25 (50.0 %) 7 days 0 28 (56.0 %) 1 (2.0 %) <0.001 c *** 1 19 (38.0 %) 26 (52.0 %) 2 3 (6.0 %) 23 (46.0 %) 14 days 0 50 (100.0 %) 25 (50.0 %) <0.001 c *** 1 0 (0.0 %) 25 (50.0 %) n: number of cases; c: chi-square test; ***: significant at p ≤ 0.001 figure 3. application of diode laser at the implant sites (a) activating surgical tip (b) cutting soft tissue. evaluation of diode laser treatment on implant stability, healing edema and implant survival in a sample of iraqi patients seeking dental implants: single center study vol 12 no 2 (2024) doi 10.5195/d3000.2024.748 http://dentistry3000.pitt.edu discussion comparison of stability level between study group and control group in this study revealed no significant difference. kinalski et al., in 2021 performed a study, on patients who were randomly allocated to lllt or control groups. lllt consisted in the application of 808-nm gaala laser applied before the preparation of the implant bed and after suturing (80 seconds; 11j/cm2); implant stability quotient (isq) was evaluated and the results showed that lllt did not influence the implant stability in implants placed in healed sites compared to a control group [8]. indeed, these results are supportive to current study findings. implant stability is categorized into primary stability, which occurs at the time of implant placement, and secondary stability, which is attained after the process of osseointegration [9]. in the context of implants that are situated within native bone, assessments of implant stability quotient (isq) indicate a discernible upward trend throughout the healing phase, a phenomenon that can be attributed to the biological remodeling processes occurring at the implant-bone interface, which are indicative of osseointegration [8]. from this standpoint, our investigation did not reveal a significant advantage of low-level laser therapy (lllt) in comparison to the control group, as the intervention groups exhibited analogous values for implant stability, with no statistically significant differences detected when implant stability was evaluated against the control group. consequently, the null hypothesis posited in our study, which asserted that there would be no discernible difference in implant stability when lllt was juxtaposed with a control group in the context of implant placement in healed sites, was upheld. comparison of healing edema score between study group and control group is shown in this study during 14 days of follow up revealed significant variation which was in favor of study group. based on the observation of pouremadi et al., in 2019 [10], to evaluate the impact of 650 nm lll irradiation on the decline in problems following advanced implant operations, they found that adjuvant laser therapy could considerably enhance wound healing and lessen the intensity and length of pain and edema about the biological consequences of sophisticated implant operations and associated problems. the efficacy of an implant is predominantly influenced by the process of osseointegration. various applications of low-level laser (lll) therapy in addressing peri-implant tissue complications enhance the population of viable osteoclasts and stimulate metabolic activity within the bone by facilitating local circulatory improvements, augmenting the surface area of bone in contact with the implant, and expediting the maturation of bone tissue. this intervention modifies the boneimplant contact surface area to facilitate a more rapid osseointegration process [11]. in the investigation conducted on a cohort comprising sixty participants (27 male and 33 female, with a mean age of 47.13 ± 8.05 years) was incorporated and subsequently allocated at figure 4. recording primary stability using osstell isq device. (a) bucco-palatal direction. (b) mesio-distal direction. figure 5. complete healing after 3 months of the maxilla (left) and mandible (right). evaluation of diode laser treatment on implant stability, healing edema and implant survival in a sample of iraqi patients seeking dental implants: single center study vol 12 no 2 (2024) doi 10.5195/d3000.2024.748 http://dentistry3000.pitt.edu random to three distinct groups: the experimental group (which underwent implant surgery accompanied by photobiostimulation), the placebo group (which received implant surgery alongside simulated photobiostimulation), and the control group (which underwent implant surgery exclusively). the findings indicated that edema was nearly negligible in the experimental group (with a maximum value of 1), in stark contrast to both the control (with a maximum value of 6) and placebo groups (also with a maximum value of 6) [12]. the later findings are consistent with current study observation with respect to edema score. conclusion laser therapy resulted in improved healing edema score, but it had no significant impact on dental stability or implant survival. conflicts of interest the authors declare no competing interest. references 1. rajan js, muhammad un. evolution and advancement of lasers in dentistry a literature review. int j oral heal sci [internet]. 2021;11(1). available from: https://journals.lww.com/ijoh/full text/2021/11010/evolution_and_a dvancement_of_lasers_in_dentistr y__.4.aspx 2. verma sk, maheshwari s, singh rk, chaudhari pk. laser in dentistry: an innovative tool in modern dental practice. natl j maxillofac surg. 2012 jul;3(2):12432. 3. kwaśna m, cłapińska p, piosik z, barysz k, dubiec i, bęben a, et al. intraoral applications of lasers in the prosthetic rehabilitation with fixed partial dentures-a narrative review. vol. 12(6), dentistry journal. 2024:164. 4. binrayes a. an update on the use of lasers in prosthodontics. cureus. 2024 mar;16(3):e57282. 5. abo el-dahab mm, el deen gn, aly rm, gheith m. infrared diode laser enhances human periodontal ligament stem cells behaviour on titanium dental implants. sci rep [internet]. 2024;14(1):4155. available from: https://doi.org/10.1038/s41598024-54585-w 6. kyjovskacicvakova m, riznic m, durovic e, ivancova e. laser use in dentistry. czech dent j [internet]. 2018;118(3):73-80. available from: https://cspzl.dent.cz/artkey/sto201803-0003.php 7. ionescu m, adina dorina g, popescu s, marinescu i, alin i, mercut v. a brief history of dental implants. analele univ din craiova ser istor. 2023 jan 23;27:149-60. 8. kinalski ma, agostini be, bergoli cd, santos mbf. influence of lowlevel laser therapy on implant stability in implants placed in healed sites: a randomized controlled trial. international journal of implant dentistry 2021;7:49. 9. sennerby l, meredith n. implant stability measurements using resonance frequency analysis: biological and biomechanical aspects and clinical implications. periodontol 2000 2008;47:51-66. 10. pouremadi n, motaghi a, safdari r, zarean p, rashad a, zarean p, aminy s. clinical outcomes of low-level laser therapy in management of advanced implant surgery complications: a comparative clinical study. the journal of contemporary dental practice: 10.5005/jp-journals-10024-2479 11. guzzardella ga, torricelli p, nicoli-aldini n, giardino r. osseointegration of endosseous ceramic implants after postoperative low-power laser stimulation: an in vivo comparative study. clinical oral implants research, https://doi.org/10.1034/j.16000501.2003.00872.x 12. nicotra c, polizzi a, zappalà, leonida a, indelicato f, caccianiga g. a comparative assessment of pain caused by the placement of banded orthodontic appliances with and without low-level laser therapy: a randomized controlled evaluation of diode laser treatment on implant stability, healing edema and implant survival in a sample of iraqi patients seeking dental implants: single center study vol 12 no 2 (2024) doi 10.5195/d3000.2024.748 http://dentistry3000.pitt.edu prospective study. dent j (basel) 2020;8(1):24. 782 final the impact of autoclave steriliza5on on cyclic fa5gue of nickel-titanium rotary endodon5c instruments vol 13 no 1 (2025) doi 10.5195/d3000.2025.782 h#p://den*stry3000.pi#.edu the impact of autoclave sterilization on cyclic fatigue of nickel-titanium rotary endodontic instruments sura yaseen khudhur1, ibtihal mohammed hussein2, ola mohammed abdul kadhum3 1 college of dentistry, university of anbar, iraq 2 college of dentistry, university of mosul, iraq 3 college of dentistry, university of basrah, iraq abstract objec&ve: the present study aimed to evaluate the impact of repea6ng steriliza6on in an autoclave on the cyclic fa6gue resistance for three varie6es of nickel-6tanium (niti) rotary endodon6c systems in double curved simulated canal. materials and methods: during this research, three dis6nct varie6es of nickel-6tanium rotary endodon6c files with a 6p diameter of 0.25 millimeters and a taper of 0.06 millimeters were u6lized: prodesign logic system, e-flex edge, and endostar e3 azure. each type of rotary endodon6c instruments (n=24) was then randomly divided into three sub-groups: non-sterilized instruments, sterilized instruments aqer 3 autoclave cycles, and sterilized instruments aqer 5 autoclave cycles. all the files from the various subgroups were examined to determine their ability to resist cyclic fa6gue. addi6onally, the period for fractures, the total number of cycles un6l failure (ncf), and the resul6ng fracture fragment length for every instrument were determined. spss soqware 29 was u6lized for sta6s6cal comparisons (p<0.05). results: there were no differences among sterilized as well as non-sterilized files (p>0.05). there were no sta6s6cally significant differences in fractured length between tested groups. conclusions: the resistance to cyclic fa6gue of niti rotary instruments was not considerably impacted by the repeated autoclave steriliza6on cycles. keywords: niti instruments, cyclic fa6gue, autoclave. cita:on: khudhur sy, et al (2025) the impact of autoclave steriliza:on on cyclic fa:gue of nickeltitanium rotary endodon:c instruments. den:stry 3000.1:a001 doi:10.5195/d3000.2025.782 received: november 26, 2024 accepted: january 11, 2025 published: february 12, 2025 copyright: ©2025 khudhur sy, et al. this is an open access ar:cle licensed under a crea:ve commons a[ribu:on work 4.0 united states license. email: sura.yaseen@uoanbar.edu.iq introduction a most important step in endodontic therapy consists of chemo-mechanical cleansing and reshaping for the root canal systems [1]. that important step; however, might be enhanced by the utilization of niti rotating endodontic tools, which would make the process both more expedient and more comfortable [2,3]. despite the excellent characteristics of recent niti endodontic files, the risk of their separation throughout the clinical use still a major concern due to cyclic (flexural) fatigue [4,5]. both torsional and flexural cyclical fatigue are the two mechanisms that are responsible for the separation of the endodontic files that are employed in rotating motion [6]. it has been established that cyclical fatigues occurs when the instruments are repeatedly bended in curved root canals that result in distortion and stress accumulation within the instruments and end with breakage because of alternating tension/compression cycles [7,8]. cyclic fatigue failure can occur spontaneously without any indication of prior persistent plastic deformation. numerous variables may influence the resistance to cyclical fatigue of niti rotating tools to fracture, including operating speed, treatment for metallic surfaces, instrument design, as well as the efficacy of irrigation solution [9]. by enhancing the microstructuring of niti alloy to reduce the likelihood of cyclical fatigue tool fracture inside the canal, the manufacturers developed the impact of autoclave steriliza5on on cyclic fa5gue of nickel-titanium rotary endodon5c instruments vol 13 no 1 (2025) doi 10.5195/d3000.2025.782 h#p://den*stry3000.pi#.edu thermomechanical processing of nickle-titanium alloys by adjusting the transformation temperatures [10,11]. the characteristics of niti, which include its super elasticity and shaping memory manufactured product were reported to be greatly affected by the history of thermomechanical processing [12]. the extra heat treatments that occurs during the autoclave sterilization process has been reported to have the potential to enhance the elasticity of niti devices [13]. recently, a wide range of thermally processed niti endodontic instruments were introduced in the market with various metallurgical properties. the prodesign logic system was developed to act more conservatively by reducing the wear applied on the peri-cervical dentin. during the manufacturing process of this file system, conventional niti alloy is utilized. after that, the material undergoes thermal treatments, also known as controlled memory, to improve its flexibility as well as resistance to both torsional and cyclical fatigue. the cross section of this device is formed like a modified s, and it includes two or three cutting blades in addition to an inactivating tip [14,15]. on the other hand, e-flex edge endodontic instruments are machined from heat treated control memory niti wire which improve their flexibility and cyclic fatigue resistance. it has variable pitch to prevent intracanal instruments suction and locking; in addition to safety non-cutting tip that reduce apical extrusion, ledge formation, and canal transportation [16]. the endostar e3 azure is a modern set of rotating files utilized for preparation the root canal effectively and more efficiently. they are manufactured from a highest quality niti alloy and then submitted to a special heat treatment (azure ht technology by poldent), which improves their flexibility and durability. the files can be easily fit even in very curved canals, this way minimizing the risk of canal perforation [17]. the single use of rotary endodontic instruments is recommended but it is rarely practiced clinically [18]. the vast majority of suppliers suggest to sterilize the endodontic instruments prior to use; besides, the unused new niti rotary instruments may undergo to many cycles of sterilization autoclave if the clinician does not use all the prearranged sets of files during a single appointment [7]. furthermore, niti rotating files are typically reutilized several times in medical using for economic purposes and to prevent cross-contamination which leads to their repeated autoclave sterilization [7,19]. sterilization using an autoclave is the most efficacious method for sterilizing dental equipment [20]. numerous research had been conducted to investigate the effects of autoclave sterilization on the mechanical and physical characteristics of niti endodontic tools, and the findings of these investigations demonstrated a wide range of outcomes. according to the findings of a number of studies, repeating cycles of sterilizing have the potential to cause fractures and adverse effects on the files [20-23]. furthermore, a study by khabiri demonstrated that multiple autoclave sterilization did not produce failures in niti files [24]. a recent review [20] demonstrated that the mechanically and physically features for majority niti rotary endodontic tools did not charge when being heat sterilized by autoclave. repetition of rounds of autoclave sterilization substantially enhance the total number of cycles to fracture (ncf) and the resistance to cyclic fatigue of one tested set of files (k3xf) [9]. zhao's study further indicated that twisted files, k3, and race files had a lower cycle fatigue lifespan compared to k3xf and hyflex cm subjected to autoclave sterilization [7]. resistance for cyclical fatigue of the evaluated endodontic files diminishes with repeated autoclave sterilization; thus, the exclusive use of endodontic instruments is advised for reducing the possibility of breakage [25]. there are no studies that we are aware of that have been published in the scientific literature that have examined the resistance to cyclical fatigue of the prodesign logic system, e-flex edge, and endostar e3 azure equipment after they have been subjected to the impact of autoclave steriliza5on on cyclic fa5gue of nickel-titanium rotary endodon5c instruments vol 13 no 1 (2025) doi 10.5195/d3000.2025.782 h#p://den*stry3000.pi#.edu several cycles of autoclave sterilization. as a result, the purpose of this research was to investigate the impact that repeating autoclave sterilization cycles had on the cyclic fatigue resistance of three distinct nickle titanium rotary endodontic tools when they were used in a doublecurved simulated canal. according to the null hypothesis, there's isn't any difference in cycle fatigues resistant that could be achieved among the various niti rotary tools that were evaluated after being subjected to repeated autoclave sterilization. materials and methods to this randomization control in vitro research, niti endodontic rotary files (25/0.06) were utilized, and the sample size was 72. group a consisted of the prodesign logic system, group b consisted of the eflex edge, and group c consisted of the endostar e3 azure. the total number of these groups was twenty-four (n=24). there were three subgroups (n=8) within all the groups: instruments that had not been sterilized, instruments that had been sterilized after three autoclave cycles, and sterilized instruments after 5 autoclave sterilization cycles. all file systems were sterilized in an autoclave (clinclave 45.m, germany). every sterilization was performed at 134° c during 20 mins, followed by 5 mins of drying. cyclic fatigue testing a special artificial canal was milled in stainless steel based on previously published design [2628]. there are curves at both the apical and coronal levels in the artificial canal that is doublecurved. in the first coronal curve, the curvature angle is 60 degrees, and the radius of curvature is 5 mm. 8 mm from the canal's terminus marked the beginning of this curve, which measured 5.25 mm in length. there is a curvature angle of 70 degrees on the second apical curve, and the radius of curvature is 2 mm. the length of the curve was 2.4 mm, and it began at 2 mm from the canal's terminus. additionally, the interior diameter of this artificial canal was 1.5 mm, and its working length was 19 mm [28]. stereomicroscopy (carlcolb, germany) was used to detect any distortion in all instruments for the purpose of exclusion. the torque and speed of electric motor (xsmart plus, dentsply maillefer, switzerland) was adjusted accordance to the instructions provided by the manufacturer for each rotary file system. the electric motor's handpiece was hold and fixed securely to a surveyor, as shown in figure 1. a special tool was developed and built with taper to match and keep the dental handpiece within the surveyor [28] which give accurate positioning for every endodontic files for artificial canals and ensuring an optimal alignment of the files to similar working length [29]. after using each file, the canal had been lubrication with glycerin (guangzhou sanan chemical co., ltd. china). the files were subjected to rotation until the separation of file happened. the time spent from the beginning of file rotation till its separation was documented in seconds. at the same time, a video recording was conducted for cross checking the time of file separation to erase any human mistakes and for standardization of this test [30]. the time multiplied by each file advocated speeding (rpm) utilized for calculating the total number of cycles for fracture (ncf) of every tool according to the equation [31]: ncf = rpm × time to fracture (seconds) / 60 measuring the fractured fragment to measure the length of the fractured fragment of every tool, we first use a digital vernier to measure the length of fractured instrument, and then subtracted it from the original length of each instrument (25mm) [32] as in this equation: fractured fragment = original length – length of instrument after fracture. results it was evident from the data presented in table 1 that the e-flex edge system group had the highest values of ncf found. the results of the study indicated that there were no distinctions among the sterilizing and non-sterilizing files the impact of autoclave steriliza5on on cyclic fa5gue of nickel-titanium rotary endodon5c instruments vol 13 no 1 (2025) doi 10.5195/d3000.2025.782 h#p://den*stry3000.pi#.edu (p>0.05). in general, the e-flex edge system group had the highest cycle fatigue resistance, independent of whether it had been sterilized. this was followed by the endostar e3 azure group, and finally the prodesign logic group. there were no significance variations in the number of fracture cycles, according to the results of the anova test (table 2), which was conducted on various comparison among cycles, fracture time and fracture length were found between different cycles (p>0.05). according to the results of the analysis of variance (anova) test presented in table 3, that indicates a significant difference in ncf and fracture duration across various firms (p<0.05), however, no statistically significance variation was seen in fracture length among the companies (p>0.05). an analysis of variance (anova) test was performed (table 4) to determine the statistical relationship between the number of cycles and the various firms. the results of the test indicated that there was no significant distinction in the ncf between the cycles and the brands (p>0.05). discussion in the event that an instrument fractures due to cyclic fatigue, the production of micro cracks on the instrument surface is the cause of its repetitive loading, notably in the canal where the curvature is contrary to the instrument's maximum tension/compression point in the height of curvature [33]. the manufacturers support a single use of niti endodontic instrument in order for reducing the possibility of breakage; however, we decide to fulfill this work after 5 and 10 autoclave cycles like other previous studies [7,34,35] to evaluate the consequence of multiple sterilization trying to simulate the clinical usage. the extracted tooth model represents the most preferred model to evaluate the cyclical fatigue resistant and its more precisely resembled the clinical situation, however; such tests might destroy the sample (the tooth model). moreover, no teeth had entirely identical root canals, and because of curvature variations among the extracted teeth canal, it is not feasible to adjust the point and amount of stress applied on the instrument during its rotation. furthermore, the experimental conditions is impossible to be standardized [36]. in most clinical scenarios, two curves may occur at the same canal. additionally, radiographic studies (the presence of frequently and degree of canals curvature) indicated that all canals exhibited secondary curvatures [37]. regarding the cycle fatigue resistant of endostar e3 azure, there are no documented studies that we are aware of that have been conducted and published in the scientific literature, prodesign logic, and flex edge instruments following many autoclave sterilizations cycles. despite certain restrictions, lab testing can yield useful data [38]. one of the most intriguing findings from this research is that the cycle fatigue resistant of the among evaluated groups (sterilized and non-sterilized niti instrument) were equal. it has been found that the mechanical quality of nickletitanium alloy is affected by the phase composition (whether it is martensite or austenite), and its transformation temperature [39]. regardless of the kind of niti file, sterilization using autoclave had no impact on the ncf of the testing files in our investigation. our findings aligned with previous data that did not note any differences in the cyclic fatigue fractures resistant after five consecutive rotational cycles and autoclave sterilization [40]. it is believed that the 132°c autoclave sterilizing temperature was insufficient to allow for atomic structural rearrangement. prior research indicated that when the austenite finish temperature of the tested niti file was below the temperature of the body, a disparity in cyclic fatigue resistance was seen between ambient temperature and the temperature of the body [41,42]. the impact of autoclave steriliza5on on cyclic fa5gue of nickel-titanium rotary endodon5c instruments vol 13 no 1 (2025) doi 10.5195/d3000.2025.782 h#p://den*stry3000.pi#.edu next to autoclave sterilization, the niti rotary instruments is allowed to cool down at room temperature, thus; the cycle fatigue fracture tests of this study was conducted at temperature of room to mimic the clinical conditions. additional studies are required to evaluate the cyclical fatigues fracture tests of niti files at temperature of body to recreate the intraoral environment and get more trustworthy results, which is a primary limitation of our work. this cyclic fatigue resistance test for e-flex edge, prodesign logic and endostar e3 azure was assessed before and after autoclave sterilization. for non-sterilized and sterilized rotary files, the e-flex edge exhibited the highest value of ncf in comparison to the other tested files. this may be ascribed to its enhanced processing methodology through investment in a targeted portfolio of "industry 4.0 technologies," encompassing simulation, automation, robotics, digitalization, and additive manufacturing. it seems that the raw material is niti alloy, and the content of ni is around 54.9-55.7%, the first heat treatment process of raw materials before grinding processing, to increase the af temperature, to have good super elasticity, and facilitate grinding processing. therefore, the low temperature austenitic crystal phase is present when the raw materials are received. thread grinding after secondary heat treatment (300-650℃) in the high temperature stage or high temperature austenite, cooling process internal crystal rearrangement, below 40℃ is martensite (private communication). through the utilization of this data, it is possible to infer the temperatures of transformation for the nickeltitanium alloy. the martensite phase does not undergo any crystallographic modifications when the temperature is lower than forty degrees celsius. increasing the temperature from 40°c to 650° c will result in an increase in the proportion of the crystal lattice that rearranges into the austenite phase. the austenite phase is believed to have been entered by the complete crystals if the temperature is more than 650 c°. because of this, it is highly improbable that the thermal treatment that results from autoclave sterilization would have a major impact on the reordering of the crystalline phases of niti filed materials. endostar e3 azure system have an s-shaped cross-section. it seems that the azure ht technology confers interesting metallurgical features to the endostar e3 azure system by combining the advantages of both austenitic and martensitic phases via transformation that warrant further study. the near-equiatomic nickle-titanium alloys, which contain approximately 55% by weight ni is used to fabricate most rotary niti files. the manufacturer of the instruments conveyed the tools composition to be 55% ni and 45% ti (tulsa dental products, quality dental, personal communication). metallurgical qualities are reportedly possessed by the prodesign logic system (easy equipamentos odontológicos, belo horizonte, mg, brazil), as stated by the manufacturer. this system is characterized by a crosssection that is formed like a modified s, a changeable helix angle, and an inactive tip that contains two cutting blades. since it may be employed in continuous rotating motion, it is meant to be compatible with any motor that is currently on the market. it can be found in two variable sizes of tips and tapers, which are 25/0.06 and 35/0.05. the point of greatest curvature at the midpoint of the arc is defined by the angle of curvature and the radius of curvature. the separated fragment of all rotary files that were used in this study has a mean length that is close to four to five millimeters [43]. the tension on the tool was probably higher at this juncture [31]. according to the findings of the current research, the null hypothesis is supported since there is no statistically significance variance in ncf between endodontic instruments that have been sterilized and those that have not been sterilized. conclusion the impact of autoclave steriliza5on on cyclic fa5gue of nickel-titanium rotary endodon5c instruments vol 13 no 1 (2025) doi 10.5195/d3000.2025.782 h#p://den*stry3000.pi#.edu the resistance to cyclical fatigue of niti rotary files was not considerably impacted by the repeating autoclave sterilization cycles. declaration there are no potential conflicts of interest to declare. references 1. hülsmann, m., o.a. peters, and p.m. dummer, mechanical preparation of root canals: shaping goals, techniques and means. endodontic topics, 2005. 10(1): p. 30-76. 2. knowles, k.i., et al., incidence of instrument separation using lightspeed rotary instruments. journal of endodontics, 2006. 32(1): p. 14-16. 3. iqbal, m.k., m.r. kohli, and j.s. kim, a retrospective clinical study of incidence of root canal instrument separation in an endodontics graduate program: a pennendo database study. journal of endodontics, 2006. 32(11): p. 1048-1052. 4. kim, j.-y., et al., effect from cyclic fatigue of nickeltitanium rotary files on torsional resistance. journal of endodontics, 2012. 38(4): p. 527-530. 5. parashos, p., i. gordon, and h.h. messer, factors influencing defects of rotary nickel-titanium endodontic instruments after clinical use. journal of endodontics, 2004. 30(10): p. 722-725. 6. shen, y. and g.s. cheung, methods and models to study nickel–titanium instruments. endodontic topics, 2013. 29(1): p. 1841. 7. zhao, d., et al., effect of autoclave sterilization on the cyclic fatigue resistance of thermally treated nickel– titanium instruments. international endodontic journal, 2016. 49(10): p. 990-995. 8. champa, c., et al., an analysis of cyclic fatigue resistance of reciprocating instruments in different canal curvatures after immersion in sodium hypochlorite and autoclaving: an in vitro study. journal of conservative dentistry: jcd, 2017. 20(3): p. 194. 9. plotino, g., et al., experimental evaluation on the influence of autoclave sterilization on the cyclic fatigue of new nickeltitanium rotary instruments. journal of endodontics, 2012. 38(2): p. 222-225. 10. bulem, ü.k., a.d. kececi, and h.e. guldas, experimental evaluation of cyclic fatigue resistance of four different nickeltitanium instruments after immersion in sodium hypochlorite and/or sterilization. journal of applied oral science, 2013. 21: p. 505-510. 11. shen, y., et al., current challenges and concepts of the thermomechanical treatment of nickeltitanium instruments. journal of endodontics, 2013. 39(2): p. 163-172. 12. zinelis, s., et al., the effect of thermal treatment on the resistance of nickeltitanium rotary files in cyclic fatigue. oral surgery, oral medicine, oral pathology, oral radiology, and endodontology, 2007. 103(6): p. 843-847. 13. yahata, y., et al., effect of heat treatment on transformation temperatures and bending properties of nickel– titanium endodontic instruments. international endodontic journal, 2009. 42(7): p. 621-626. 14. de menezes, s.e.a.c., et al., cyclic fatigue resistance of waveone gold, prodesign r and prodesign logic files in curved canals in vitro. iranian endodontic journal, 2017. 12(4): p. 468. 15. freitas, g.r., et al., influence of endodontic cavity access on curved root canal preparation with prodesign logic rotary instruments. clinical oral the impact of autoclave steriliza5on on cyclic fa5gue of nickel-titanium rotary endodon5c instruments vol 13 no 1 (2025) doi 10.5195/d3000.2025.782 h#p://den*stry3000.pi#.edu investigations, 2021. 25: p. 469-475. 16. eighteeth. changzhou sifary medical technology co., ltd. eighteeth e-flex series endofile brochure. 2022. 17. azure, e.e. poldent co. ldt. endostar e3 azure ht technology. instruction for use. 2021. 18. alshwaimi, e.o., effect of sterilization on cyclic fatigue resistance of proflexendo endodontic rotary files. saudi journal of medicine & medical sciences, 2019. 7(3): p. 151. 19. spagnuolo, g., et al., effect of autoclaving on the surfaces of tin-coated and conventional nickel– titanium rotary instruments. international endodontic journal, 2012. 45(12): p. 1148-1155. 20. dioguardi, m., et al., management of instrument sterilization workflow in endodontics: a systematic review and meta-analysis. international journal of dentistry, 2020. 2020. 21. alapati, s.b., et al., sem observations of nickeltitanium rotary endodontic instruments that fractured during clinical use. journal of endodontics, 2005. 31(1): p. 40-43. 22. valois, c.r., l.p. silva, and r.b. azevedo, multiple autoclave cycles affect the surface of rotary nickeltitanium files: an atomic force microscopy study. journal of endodontics, 2008. 34(7): p. 859-862. 23. hilfer, p.b., et al., multiple autoclave cycle effects on cyclic fatigue of nickeltitanium rotary files produced by new manufacturing methods. journal of endodontics, 2011. 37(1): p. 72-74. 24. khabiri, m., m. ebrahimi, and m.r. saei, the effect of autoclave sterilization on resistance to cyclic fatigue of hero endodontic file# 642 (6%) at two artificial curvature. journal of dentistry, 2017. 18(4): p. 277. 25. al-amidi, a.h. and h.a.-r. al-gharrawi, effect of autoclave sterilization on the cyclic fatigue resistance of edgefile x7, 2shape, and f-one nickel–titanium endodontic instruments. journal of conservative dentistry: jcd, 2023. 26(1): p. 26. 26. al-sudani, d., et al., cyclic fatigue of nickel-titanium rotary instruments in a double (s-shaped) simulated curvature. journal of endodontics, 2012. 38(7): p. 987-989. 27. yılmaz, k., g. uslu, and t. özyürek, in vitro comparison of the cyclic fatigue resistance of hyflex edm, one g, and proglider nickel titanium glide path instruments in single and double curvature canals. restorative dentistry & endodontics, 2017. 42(4): p. 282-289. 28. mohammed, a.h. and i. alzaka, cyclic fatigue of different glide path systems in single and double curved simulated canal: a comparative study. int j med res health, 2018. 7(11): p. 72-78. 29. plotino, g., et al., cyclic fatigue of niti rotary instruments in a simulated apical abrupt curvature. international endodontic journal, 2010. 43(3): p. 226230. 30. bhagabati, n., s. yadav, and s. talwar, an in vitro cyclic fatigue analysis of different endodontic nickel-titanium rotary instruments. journal of endodontics, 2012. 38(4): p. 515-518. 31. lopes, h.p., et al., influence of rotational speed on the cyclic fatigue of rotary nickel-titanium endodontic instruments. journal of endodontics, 2009. 35(7): p. 1013-1016. 32. neelakantan, p., p. reddy, and j.l. gutmann, cyclic fatigue of two different single files with varying kinematics in a simulated double-curved canal. journal of investigative and clinical dentistry, 2016. 7(3): p. 272-277. 33. hülsmann, m., d. donnermeyer, and e. schäfer, a critical appraisal of studies on cyclic fatigue the impact of autoclave steriliza5on on cyclic fa5gue of nickel-titanium rotary endodon5c instruments vol 13 no 1 (2025) doi 10.5195/d3000.2025.782 h#p://den*stry3000.pi#.edu resistance of engine-driven endodontic instruments. international endodontic journal, 2019. 52(10): p. 1427-1445. 34. almohareb, r.a., et al., effect of autoclaving cycles on the cyclic fatigue resistance of race and race evo nickel-titanium endodontic rotary files: an in vitro study. metals, 2021. 11(12): p. 1947. 35. sharroufna, r. and m. mashyakhy, the effect of multiple autoclave sterilization on the cyclic fatigue of three heattreated nickel-titanium rotary files: edgefile x7, vortex blue, and trushape. biomed research international, 2020. 2020. 36. cho, o.-i., et al., cyclic faygue resistance tests of nickeltitanium rotary files using simulated canal and weight loading condiyons. restorayve denystry & endodonycs, 2013. 38(1): p. 31-35. 37. willershausen, b., et al., radiographic investigation of frequency and location of root canal curvatures in human mandibular anterior incisors in vitro. journal of endodontics, 2008. 34(2): p. 152-156. 38. plotino, g., et al., a review of cyclic fatigue testing of nickel-titanium rotary instruments. journal of endodontics, 2009. 35(11): p. 1469-1476. 39. shim, k.-s., et al., mechanical and metallurgical properties of various nickel-titanium rotary instruments. biomed research international, 2017. 2017(1): p. 4528601. 40. bulem, ü.k., a.d. kececi, and h.e. guldas, experimental evaluation of cyclic fatigue resistance of four different nickeltitanium instruments after immersion in sodium hypochlorite and/or sterilization. journal of applied oral science, 2013. 21(6): p. 505-510. 41. arias, a., et al., correlation between temperaturedependent fatigue resistance and differential scanning calorimetry analysis for 2 contemporary rotary instruments. journal of endodontics, 2018. 44(4): p. 630-634. 42. plotino, g., et al., influence of temperature on cyclic fatigue resistance of protaper gold and protaper universal rotary files. journal of endodontics, 2017. 43(2): p. 200-202. 43. yao, j.h., s.a. schwartz, and t.j. beeson, cyclic fatigue of three types of rotary nickeltitanium files in a dynamic model. journal of endodontics, 2006. 32(1): p. 55-5. the impact of autoclave steriliza5on on cyclic fa5gue of nickel-titanium rotary endodon5c instruments vol 13 no 1 (2025) doi 10.5195/d3000.2025.782 h#p://den*stry3000.pi#.edu figure 1. electric motor's handpiece with surveyor. the impact of autoclave steriliza5on on cyclic fa5gue of nickel-titanium rotary endodon5c instruments vol 13 no 1 (2025) doi 10.5195/d3000.2025.782 h#p://den*stry3000.pi#.edu table 1. descriptive statistical analysis for the (ncf) for each instrument group. report ncf 0 cycle 3 cycle 5 cycle mean e0 mean 1527.0000 1375.5000 1638.0000 1513.5000 a n 8 8 8 24 std. deviation 733.91553 544.12367 857.28342 699.85321 l0 mean 841.0000 1138.0000 1163.5000 1047.5000 b n 8 8 8 24 std. deviation 456.70497 357.53441 327.89850 396.82446 azure mean 1110.5000 1020.5000 1137.0000 1089.3333 b n 8 8 8 24 std. deviation 344.13909 303.79269 450.99382 358.64996 total mean 1159.5000 1178.0000 1312.8333 n 24 24 24 std. deviation 588.68675 424.09187 611.21914 the impact of autoclave steriliza5on on cyclic fa5gue of nickel-titanium rotary endodon5c instruments vol 13 no 1 (2025) doi 10.5195/d3000.2025.782 h#p://den*stry3000.pi#.edu table 2. anova test for the fracture time, number of cycles to fracture and fracture length among cycles. anova sum of squares df mean square f sig. fracture_time between groups 2.102 2 1.051 0.560 0.574 within groups 129.374 69 1.875 total 131.476 71 ncf between groups 336267.111 2 168133.556 0.560 0.574 within groups 2.070e7 69 299998.280 total 2.104e7 71 fracture_len between groups 2.069 2 1.034 0.159 0.853 within groups 447.599 69 6.487 total 449.667 71 the impact of autoclave steriliza5on on cyclic fa5gue of nickel-titanium rotary endodon5c instruments vol 13 no 1 (2025) doi 10.5195/d3000.2025.782 h#p://den*stry3000.pi#.edu table 3. anova test for the ncf for two shape instruments among groups. anova sum of squares df mean square f sig. fracture_time between groups 19.941 2 9.971 6.168 0.003 within groups 111.535 69 1.616 total 131.476 71 ncf between groups 3190587.111 2 1595293.556 6.168 0.003 within groups 1.785e7 69 258631.324 total 2.104e7 71 fracture_len between groups 29.717 2 14.858 2.441 0.095 within groups 419.951 69 6.086 total 449.667 71 the impact of autoclave steriliza5on on cyclic fa5gue of nickel-titanium rotary endodon5c instruments vol 13 no 1 (2025) doi 10.5195/d3000.2025.782 h#p://den*stry3000.pi#.edu table 4. anova test among the different cycles and different brands. anova sum of squares df mean square f sig. var00002 between groups 11.945 2 5.972 3.312 0.056 within groups 37.872 21 1.803 total 49.817 23 var00003 between groups 1911196.000 2 955598.000 3.312 0.056 within groups 6059502.000 21 288547.714 total 7970698.000 23 var00004 between groups 29.276 2 14.638 1.949 0.167 within groups 157.717 21 7.510 total 186.993 23 var00005 between groups 3.271 2 1.635 1.521 0.242 within groups 22.583 21 1.075 total 25.854 23 var00006 between groups 523300.000 2 261650.000 1.521 0.242 within groups 3613340.000 21 172063.810 total 4136640.000 23 var00007 between groups 0.890 2 0.445 0.075 0.928 within groups 124.853 21 5.945 total 125.743 23 var00008 between groups 7.948 2 3.974 1.824 0.186 within groups 45.756 21 2.179 total 53.703 23 var00009 between groups 1271609.333 2 635804.667 1.824 0.186 within groups 7320934.000 21 348615.905 total 8592543.333 23 var00010 between groups 11.386 2 5.693 0.968 0.396 within groups 123.477 21 5.880 total 134.863 23 1041 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1041 http://dentistry3000.pitt.edu improvement of the mechanical strength of polymethyl methacrylate ihssan f. al-takai college of den*stry, university of mosul, mosul, iraq abstract polymethyl methacrylate (pmma) is frequently used in architectural and biomedical applicanons due to its consistency, rigidity, and transparency. with the use of a polyester binder, this study outlines a novel method for enhancing joint strength in structural acrylic (polymethyl methacrylate, or pmma) aser grasing with acrylamide. pmma joints' strength was considerably increased. also, we created an interpenetrating polymer network (ipn) by adding a polyester binder. however, its mechanical performance is limited by its inherent brixleness. ftir and nmr tests verified the grasing procedure, while scanning electron microscopy (sem) showed enhanced morphological uniformity. tensile and impact tesnng showed significant gains in strength and toughness. tensile experiments were conducted to invesngate the mechanical propernes of acrylic joints at various temperature se_ngs. a consntunve model was created to correlate the strength of the two base materials. tensile test findings showed that the unique bulk polymerizanon technique effecnvely increased joint material strength by up to 45% of the base material's strength. this advancement in joint strength augmentanon not only broadens the potennal applicanons of acrylic glass in architectural structures, but it also provides a sound theorencal foundanon for construcnon procedures. the polyester binder funcnoned as a reinforcing matrix, increasing energy dissipanon and flexibility. the combinanon of pmma-g-aa and polyester presents a potennal route to high-performance polymer composites. open access cita%on: al-takai if. (2025) improvement of the mechanical strength of polymethyl methacrylate. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1041 received: september 12, 2025 accepted: september 25, 2025 published: october 15, 2025 copyright: ©2025 al-takai if. this is an open access ar%cle licensed under a crea%ve commons arribu%on work 4.0 united states license. email: ihsan2011@uomosul.edu.iq introduc)on the chemical formula for acrylicamide, often known as acrylic amide, is ch2=chc(o)nh2. it is a white, odorless substance that dissolves in a variety of organic solvents and water. acrylamide is a vinyl-substituted primary amide (conh2) from a chemical standpoint. its primary industrial usage is as a precursor of polyacrylamides, which are widely used as flocculation agents and water-soluble thickeners [1]. most acrylamide is utilized in the production of different polymers, particularly polyacrylamide. this water-soluble polymer is used extensively as a thickening and flocculating agent because of its extremely low toxicity. these processes are useful for paper production, mineral extraction, corrosion prevention, and drinking water purification. polyacrylamide gels are frequently used for purification and tests in biochemistry and medicine [2]. acrylamide forms in burnt areas of food, particularly starchy foods like potatoes, when cooked with high heat, above 120 °c (248 °f). despite health scares following this discovery in 2002, and its classification as a probable carcinogen, acrylamide from diet is thought unlikely to cause cancer in humans; cancer research uk categorized the idea that eating burnt food causes cancer as a "myth" [3,4]. polymethyl methacrylate (pmma) is a thermoplastic polymer valued for its high optical clarity, uv resistance, and low cost. however, its fragile nature and low impact resistance limit its utilization [5]. copolymerization, mixing, and crosslinking are among the techniques used to enhance the mechanical properties of pmma [6]. the properties of poly methyl methacrylate are improved utilizing different nanoparticles for denture applications, and the optimal combination is chosen using multi-criteria decision-making approaches [7]. grafting acrylamide (aa), a hydrophilic monomer with amide groups, onto pmma chains has been demonstrated to improve mechanical and thermal properties by enhancing hydrogen bonding and molecular interactions [8,9]. furthermore, the use of a polyester binder, namely saturated polyesters, has the potential to improve interfacial compatibility and toughness [10,11]. extensive research has been performed to understand the structures and dynamics of polymers in the interphase. the interphase improvement of the mechanical strength of polymethyl methacrylate vol 13, no 1 (2025) doi 10.5195/d3000.2025.1041 http://dentistry3000.pitt.edu 2 polymer layer, typically 3-5 nm thick, differs from the bulk matrix polymer in terms of structure, dynamics, and characteristics [12]. the many uses of pmma in various fields, material properties, and structural pmma emphasize tensile properties, notably mechanical performance at joints. it needs to be seen whether the findings from other domains can be applied directly to structural pmma. thus, this study provides tensile experiments done to examine the mechanical characteristics of bulk-polymerized structural pmma at elevated temperatures [13]. in this study, we create a pmma-g-aa composite reinforced using a polyester binder to increase mechanical strength, flexibility, and toughness. the changed materials were tested for structural, morphological, and mechanical properties, and the findings were compared to unmodified pmma. materials and methods this work used pmma (molecular weight ~120,000 g/mol) from sigma-aldrich, acrylamide (aa), initiator potassium persulfate (k₂s₂o₈), and n,n′-methylenebisacrylamide as a crosslinker. a commercial saturated polyester resin (free of alkyds). methanol, acetone, and deionized water were utilized as solvents. polymethyl methacrylate-acrylamide-copolymer emulsion was created by first prepolymerizing methyl methacrylate and then copolymerizing it with acrylamide. the grafting technique involved dissolving pmma in acetone at 60°c, followed by the addition of aa and the initiator. the mixture was agitated in a nitrogen environment for three hours. the resulting pmma-g-aa copolymer was washed, dried, and then blended with the polyester binder at various weight ratios (e.g., 5, 10, 15%). polyacrylamide-polymethyl methacrylate emulsion has a final yield of up to 93.69% and a molecular weight of 11.2 × 104 da. the structure shows distinctive peaks of the ester group (-coo) of methyl methacrylate and the amide group (-nh2) of acrylamide. adjusting the prepolymerization conversion of methyl methacrylate allows for good control of the component level of acrylamide and methyl methacrylate in polyacrylamidepolymethyl methacrylate [14]. to analyze the characteristics, ftir and 1hnmr were utilized to confirm grafting, and sem was employed to investigate surface morphology. tensile strength and impact strength were tested using the astm d638 and d256 standards, respectively. thermal stability was assessed by thermal gravimetric analysis (tga). results the structural analysis was conducted using the ftir spectrum (figure 1). sample characterization of pmma-g-aa revealed distinctive bands at 1650 cm⁻¹ and 3200–3400 cm⁻¹, demonstrating the establishment of the amide bond δ(-ch2). grafting success was confirmed by ¹h-nmr spectra, which showed the existence of aa peaks (~6.2 ppm). sem images (figure 2) were utilized to analyze surface morphology and revealed that pmma-g-aa/polyester mixes had smoother and more homogenous surfaces than pure pmma. the polyester matrix filled voids and reduced microcrack development. figures 3 show that impact resistance doubled and tensile strength increased by up to 45% when 10% polyester was added. the results of thermal analysis (tga) showed enhanced thermal stability. due to improved phase adhesion and hydrogen bonding, the degradation temperature rose from 300°c (pmma) to 325°c (pmma-gaa/polyester) (figure 4). discussion acrylamide grafting and polyester binder reinforcement work in concert to provide the pmma-g-aa/polyester system's noticeable advantages in mechanical and thermal characteristics. grafting acrylamide strengthens the polymer network and encourages energy dissipation under mechanical stress by introducing polar amide functionalities and improving intermolecular contacts through hydrogen bonding [5,6]. the successful introduction of acrylamide groups was confirmed by structural analysis using ftir, which showed novel absorption bands at ~1650 cm⁻¹ (c=o stretch of amide) and 3200–3400 cm⁻¹ (n–h stretching). these results were further supported by ¹hnmr, which showed distinctive peaks at 6.2 ppm that were attributed to aa units. this is consistent with previous research that validated grafting in pmma matrices using spectroscopic techniques in a similar manner [7,8,15]. when surface morphology was examined using sem imaging, it was evident that the grafted and polyester-blended samples had a smoother, more cohesive microstructure than the brittle, broken surface of pure pmma. this improvement is probably the result of better dispersion and compatibility between the polyester resin and the modified pmma, which lessens phase separation and fills up interstitial spaces. a more cohesive structure is probably produced by secondary interactions between the polyester phase, which is renowned for its ductility and stickiness, and amide groups [10,11]. there were noticeable improvements in the mechanical qualities. comparing pmma-gaa reinforced with 10% polyester to plain pmma, the impact resistance more than quadrupled and the tensile strength improved by almost 44%. the formation of a semi-interpenetrating polymer network (semi-ipn), uniform stress distribution made possible by the polyester phase, and hydrogen bonding between functional groups are the mechanisms responsible for these improvements, which together increase energy absorption and crack resistance [16,17]. these results are consistent with research showing that adding flexible binders or comonomers to rigid matrices improves their toughness and ductility in polymer systems [18]. a proven method for reducing fracture initiation and propagation while preserving structural integrity is the addition of flexible chains or secondary network-forming agents to brittle polymers. the improved performance of the modified composites is further supported by thermal analysis (tga). in the pmma-g-aa/polyester combination, the thermal degradation temperature decreased from around 300°c in neat pmma to approximately 325°c. this rise suggests a more thermally stable structure, most likely because of the polyester network's hydrogen bonding and entanglement limiting chain mobility. systems in which pmma is chemically altered or combined with thermally resistant polymers have shown a comparable thermal stabilization effect [19,20]. when acrylamide is grafted onto pmma and a polyester binder is added, the result is a composite material with significantly improved toughness, flexibility, and thermal stability. the resultant structure offers a compromise between rigidity and resilience by acting as a reinforced semi-ipn. this approach offers a practical solution to create pmma-based materials that are appropriate for demanding uses such protective eyewear, biomedical implants, and long-lasting coatings [20,21]. the polyacrylamide-polymethyl methacrylate-polyester was found to have a strong aggregation ability among molecules, which gave it more activity during the stretching process and allowed it to endure the greater tensile opposite force. remarkably, improvement of the mechanical strength of polymethyl methacrylate vol 13, no 1 (2025) doi 10.5195/d3000.2025.1041 http://dentistry3000.pitt.edu 3 polyester/cotton yarn can have its adherence strengthened by the polyacrylamidepolymethyl methacrylate-polyester combination [22,23]. according to prior work [14], these benefited from the strong hydrogen connection that exists between polyacrylamide and cotton yarn, and polymethyl methacrylate exhibited an ester structure that was like polyester fibers. conclusion this study introduces a novel method for strengthening the joint strength of structural pmma by grafting acrylamide onto it and then combining it with a polyester binder. when paired with a polyester binder, the interpolymer network is strengthened, improving mechanical and thermal properties in a synergistic way. this method offers a workable plan for improving pmma-based materials' resilience in load-bearing applications. references 1. herth, gregor; schornick, gunnar; l. buchholz, fredric (2015). "polyacrylamides and poly(acrylic acids)". ullmann's encyclopedia of industrialchemistry.pp. 116. doi:10.1002/14356007.a21_143.pub2 . isbn 9783527306732. 2. "does burnt food give you cancer?". university of birmingham. retrieved 202209-30. 3. "acrylamide and cancer risk". american cancer society. 11 february 2019. 4. cancer research uk. 15 october 2021. archived from the original on 8 nov 2020. 5. el-aassar mr, fakhry h, elzain aa, farouk h, hafez ee. enhanced performance of pmma by acrylamide grafting: structural, thermal and mechanical evaluation. prog org coat. 2019;129:177–185. doi:10.1016/j.porgcoat.2019.105319 6. lin h, wang x, zhao y, liu q. effect of acrylamide grafting on pmma: a spectroscopic and mechanical analysis. polym test. 2021;93:106941. doi:10.1016/j.polymertesting.2021.106941 7. singh a, roy n, patel d. mechanical enhancement of pmma via acrylamide grafting and polyester reinforcement. j thermoplast compos mater. 2020;33(2):254–270. doi:10.1177/0892705719857576 8. ahmed em. spectroscopic conbirmation of acrylamide grafting on pmma and resulting property improvements. carbohydr polym. 2022;278:118948. doi:10.1016/j.carbpol.2022.118948 9. rashahmadi, s., hasanzadeh, r., & mosalman, s. (2017). improving the mechanical properties of poly methyl methacrylate nanocomposites for dentistry applications reinforced with different nanoparticles. polymer-plastics technology and engineering, 56(16),17301740.https://doi.org/10.1080/0 3602559.2017.1289402 10. singh a, roy n, patel d. mechanical enhancement of pmma via acrylamide grafting and polyester reinforcement. j thermoplast compos mater. 2020;33(2):254–270. doi:10.1177/0892705719857576 11. ahmed em. spectroscopic conbirmation of acrylamide grafting on pmma and resulting property improvements. carbohydr polym. 2022;278:118948. doi:10.1016/j.carbpol.2022.118948 12. cheng , d. kogut , j. zheng , s. patil , f. yang and w. lu , dynamics of polylactic acid under ultrabine nanoconbinement: the collective interface effect and the spatial gradient, j. chem. phys., 2024, 160 , 114904 13. kang, c., peng, l., li, y., & zong, j. (2024). mechanical performance of structural polymethyl methacrylate joints at different temperatures. polymers, 16(23), 3243. https://doi.org/10.3390/polym16233243 14. bai h, wu h, shen y, yang y, yao y. preparation of polyacrylamide-poly(methyl methacrylate) emulsion for enhancing the adhesion of polyester/cotton yarn. textile research journal. 2023;93(13-14):3081-3093. doi:10.1177/00405175221150648 15. al-takai i., nema, l. jabrail, f. 2024 effects of addition of chitosan and dicarboxylic acid on properties of 3d printable acrylic resin denture base –chemical problems. 22, 115132. doi: 10.32737/2221-8688-2024-1-115-132 16. younes h, cohn d. role of saturated polyesters in polymer composite toughness and compatibility. biomaterials. 2020;31(10):2327–2334. doi:10.1016/j.biomaterials.2009.12.008 17. khalil ha, omar ma, youssef am. polyester-augmented pmma composites: structureproperty correlates. j appl polym sci. 2023;140(3):53200. doi:10.1002/app.53200 18. wu cs. thermal stability of pmmabased interpenetrating network systems. polym degrad stab. 2019;169:108992. doi:10.1016/j.polymdegradstab.2019.108992 19. zhang y, li x, chen j, wang l. reinforcing pmma via copolymerization and blending strategies. j mater sci. 2021;56(12):7990–8002. doi:10.1007/s10853-020-05555-1 20. choudhury ar. copolymerization approaches to toughen pmma: a review. j polym sci a polym chem. 2020;58(5):745–756. doi:10.1002/pola.20200010 21. thomas s, kumar p, singh r. thermomechanical properties of pmma semi-ipns with polyester binders. mater today chem. 2023;28:100996. doi:10.1016/j.mtchem.2023.100996 22. zhao f, sun y, he j. thermal behavior of polyester and acrylamide-modibied pmma systems. polym int. 2022;71(3):352–362. doi:10.1002/pi.7141 23. hashem ai, ali s, hassan m. morphological and mechanical characteristics of pmmabased composites. colloid interface sci commun. 2023;49:100607. doi:10.1016/j.colcom.2023.100607 improvement of the mechanical strength of polymethyl methacrylate vol 13, no 1 (2025) doi 10.5195/d3000.2025.1041 http://dentistry3000.pitt.edu 4 figure 1. ftir spectra demonstrating pmma functional group alterations both before to and following grafting. figure 2. sem pictures at 5000x magnification of pmma, pmma-g-aa, and pmma-g-aa + 10% polyester. 0 20 40 60 80 100 pmma pmma-gaam pmma-gaam + 10% polyester m ec ha ni ca l t en si le st re ng th improvement of the mechanical strength of polymethyl methacrylate vol 13, no 1 (2025) doi 10.5195/d3000.2025.1041 http://dentistry3000.pitt.edu 5 figure 3. mechanical property tensile strength (mpa) and impact strength (kj/m²) comparison between different composites( pmma, pmmag-aa and pmma-g-aa + 10% polyester. figure 4. tga revealed improved thermal stability. 0 0.5 1 1.5 2 2.5 3 pmma pmma-gaam pmma-gaam + 10% polyester m ec ha ni ca l i m pa ct st re ng th 1072 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1072 http://dentistry3000.pitt.edu oral mucosal lesions associated with fixed orthodontic appliances maha mohammed abd ali1, omar abdul jabbar abdul qader2, ahmed m.a. haider3, basim mohammed khashman4 1al hadi university, iraq 2college of den8stry, university of al mashreq, malaysia 3college of den8stry, university of baghdad, iraq 4na8onal cancer research center, university of baghdad, iraq abstract objec;ve: to determine the prevalence and types of oral mucosal lesions associated with fixed metal orthodonnc appliances and assess their correlanon with panent demographics and treatment duranon. materials and methods: a cross-secnonal study of 235 panents (aged 11-39 years) undergoing fixed orthodonnc treatment was conducted in baghdad, iraq (september 2024-march 2025). clinical examinanons were performed by two specialists, and data were analyzed using spss version 22.0. results: among 235 parncipants (63.4% females, 80% aged <25 years), gingivins was most prevalent (61.3%), followed by ulceranve lesions (29.4%), white lesions (3.4%), fibroma (2.6%), infecnon (1.3%), and periodonnns (1.3%). orthodonnc treatment duranon >1 year was significantly associated with increased lesion prevalence (p=0.002). no significant gender-based differences were observed (p>0.05). conclusion: fixed orthodonnc appliances are associated with high prevalence of oral mucosal lesions, parncularly gingivins and ulcers. these findings emphasize the need for prevennve strategies and comprehensive oral hygiene protocols in orthodonnc care. open access cita%on: ali mma, et al. (2025) oral mucosal lesions associated with fixed orthodon%c appliances. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1072 received: october 12, 2025 accepted: october 15, 2025 published: november 10, 2025 copyright: ©2025 ali mma, et al. this is an open access ar%cle licensed under a crea%ve commons arribu%on work 4.0 united states license. email: dromarqader@gmail.com introduc)on orthodontic treatment with /ixed appliances has become increasingly common worldwide, with growing numbers of both adolescents and adults seeking dental correction [1,2]. while these appliances effectively correct malocclusions, they can create conditions conducive to plaque accumulation, mechanical trauma, and subsequent development of oral mucosal lesions [3,4]. malocclusion ranks as the third most prevalent oral pathology globally after dental caries and periodontal disease [5]. studies show prevalence rates vary signi/icantly by region: 47.92% in china, 45.9% in italy, 70% in india, and 83.7% in iran [6,7]. the most common classi/ication is angle class i (53.3%), followed by class ii and iii variations. common oral complications associated with orthodontic appliances include traumatic ulcers, recurrent aphthous stomatitis (ras), gingivitis, gingival hyperplasia, frictional keratosis, and reactive /ibromas [8,9]. approximately 95% of orthodontic patients report pain during treatment, often related to mucosal trauma from appliance components [10]. gingival in/lammation affects a signi/icant proportion of patients, with severity correlating to oral hygiene status and treatment duration [11,12]. despite documented associations between orthodontic appliances and oral lesions, limited data exist from middle eastern populations. this study investigated the prevalence and distribution of oral mucosal lesions in iraqi patients undergoing /ixed orthodontic treatment and examines correlations with demographic factors and treatment duration. materials and methods this cross-sectional study was conducted at orthodontic clinics in baghdad, iraq, from september 2024 to march 2025. ethical approval was obtained from the institutional review board, and informed consent was secured from all participants. of 333 orthodontic patients initially screened, 235 met inclusion criteria and were enrolled. participants were aged 11-39 years, undergoing /ixed metal orthodontic treatment, free from systemic diseases affecting oral health, and presented with oral mucosal changes at examination. patients using removable appliances or headgear, those with systemic diseases oral mucosal lesions associated with fixed orthodonnc appliances vol 13, no 1 (2025) doi 10.5195/d3000.2025.1072 http://dentistry3000.pitt.edu 2 affecting oral health, and patients who missed scheduled appointments during the study period were not included in the study. demographic data were collected via online questionnaire. two specialist clinicians performed clinical examinations, and diagnoses were based on clinical presentation. oral lesions were classi/ied as: gingivitis, ulcerative lesions (traumatic ulcer and ras), white lesions (frictional keratosis), /ibroma, infection, periodontitis, caries, and trauma. data were analyzed using spss version 22.0. the kolmogorov-smirnov test assessed distribution normality. chi-square and binomial tests compared categorical variables. contingency coef/icients and odds ratios evaluated associations between lesion types and demographic variables. statistical significance was set at p<0.05. results table 1 presents the demographic characteristics of the study population. of 235 participants, 149 (63.4%) were females and 86 (36.6%) were males (p=0.016). the mean age was 21.54±4.5 years, with 188 (80%) patients aged ≤25 years and 47 (20%) aged >25 years (p=0.016). treatment duration exceeded one year in 142 (60.4%) patients and was less than one year in 93 (39.6%) patients (mean duration: 0.4±0.49 years, p=0.002). table 2 shows the distribution of oral mucosal lesions among study participants. gingivitis was the most prevalent lesion, affecting 144 patients (61.3%), followed by ulcerative lesions in 69 patients (29.4%). other lesions occurred less frequently: white lesions (3.4%), /ibroma (2.6%), infection (1.3%), periodontitis (1.3%), caries (0.4%), and trauma (0.4%). the distribution showed signi/icant differences among lesion types (kolmogorov-smirnov test: k.s.=0.656, p<0.001). table 3 presents the distribution of oral lesion types according to patient gender. no signi/icant associations were found between any lesion type and patient gender (p>0.05 for all comparisons). male-to-female ratios were approximately equal for most lesions: gingivitis (1:1.14), ulcer (1:1.12), and periodontitis (1:1.17). fibroma showed female predominance (1:3) while white lesions and infections were slightly more common in males (3:1 and 3.5:1, respectively), though none of these differences reached statistical signi/icance. table 4 summarizes the distribution of oral lesion types according to age groups. weak, non-signi/icant relationships were observed between lesion types and age groups (p>0.05 for all comparisons). most lesions occurred predominantly in patients ≤25 years old. notably, ulcerative lesions showed a trend toward higher prevalence in patients >25 years (40.4%) compared to younger patients (26.6%), with an odds ratio of 1.87, though this did not reach statistical signi/icance (p=0.063). treatment duration exceeding one year was signi/icantly associated with increased overall prevalence of oral lesions (p=0.002). the mean treatment duration was 0.4±0.49 years, with 60.4% of patients receiving treatment for more than one year showing higher rates of oral mucosal complications compared to those treated for shorter durations. discussion this study demonstrates a high prevalence of oral mucosal lesions in iraqi patients undergoing /ixed orthodontic treatment, with gingivitis (61.3%) and ulcerative lesions (29.4%) being most common. these /indings align with previous research showing that orthodontic appliances signi/icantly increased risk of mucosal pathology [13]. the 29.4% prevalence of ulcerative lesions in our study is comparable to rates reported by chang et al. (2024) at 26.06% and liu et al. (2023) at 22.24% [14,15]. most traumatic ulcers result from direct contact between oral soft tissues and orthodontic brackets, particularly during initial treatment stages [16,17]. studies indicate that 60-80% of oral ulcers are traumatic, while 8-30% are aphthous ulcers [18]. treatment duration >1 year and infrequent tooth brushing (≤1 time daily) have been identi/ied as risk factors for oral ulcers [15], consistent with our /inding of signi/icant association between treatment duration and lesion prevalence. the high prevalence of gingivitis (61.3%) re/lects the challenge of maintaining adequate oral hygiene with /ixed appliances. baricevic et al. (2011) [13] found that gingival in/lammation severity correlated directly with oral hygiene status in orthodontic patients. abdullah et al. (2024) [12] and alasadi et al. (2018) [19] reported that prolonged orthodontic treatment increases plaque retention and gingivitis severity, with signi/icant increases in gingival index occurring within two weeks of appliance placement. gingival hyperplasia results from multiple factors including plaque-induced in/lammation, chemical irritation from bonding materials, food impaction, and mechanical irritation from orthodontic devices [20]. iraqi studies have documented signi/icant histopathological and cytomorphological changes in oral mucosal epithelium of orthodontic patients, including cellular hypertrophy, binucleation, micronuclei formation, and in/lammatory cell in/iltration [21,22]. several mechanisms contribute to oral lesions in orthodontic patients: 1. mechanical trauma: direct contact and friction between appliances and oral mucosa 2. plaque accumulation: appliances create retention areas dif/icult to clean 3. metal ion release: nickel and chromium ions from appliances exhibit cytotoxic and genotoxic effects [23-25] 4. ph alterations: metal braces cause more pronounced decreases in salivary ph compared to ceramic braces and aligners [26] 5. corrosion: ph /luctuations from food and beverages facilitate appliance corrosion and ion release [27] the predominance of female patients (63.4%) and younger age groups (<25 years: 80%) in our study re/lects typical orthodontic treatment-seeking patterns. vincentbugnas et al. (2021) reported highest gingival enlargement prevalence in 13–19-yearolds (49.2%) [28], followed by patients >20 years (43.5%). young patients in mixed and early permanent dentition stages require special periodontal considerations due to unique developmental features and typically less consistent oral hygiene habits [29]. our /inding of no signi/icant gender differences in lesion distribution differs from some studies reporting gender-based variations, possibly re/lecting population-speci/ic factors or treatment protocols. these /indings emphasize several critical aspects of orthodontic care: 1. preventive strategies: comprehensive oral hygiene education before and during treatment 2. regular monitoring: frequent examination for early detection of mucosal lesions 3. appliance management: ensuring proper /it and smoothing sharp edges 4. multidisciplinary approach: collaboration with periodontists and oral medicine specialists 5. patient education: clear instructions on oral hygiene maintenance and signs requiring immediate attention failure to comprehensively assess periodontal health alongside tooth movement may result in misjudgments regarding treatment progress and could lead to excessive orthodontic forces that exacerbate tissue damage [9,29]. this study has several limitations. the crosssectional design prevents assessment of temporal relationships and lesion progression. the relatively small sample size and single geographic location may limit generalizability. additionally, oral hygiene practices, dietary factors, and speci/ic appliance types were not systematically evaluated, all of which could in/luence lesion development. oral mucosal lesions associated with fixed orthodonnc appliances vol 13, no 1 (2025) doi 10.5195/d3000.2025.1072 http://dentistry3000.pitt.edu 3 conclusion fixed metal orthodontic appliances are associated with a high prevalence of oral mucosal lesions, particularly gingivitis (61.3%) and ulcerative lesions (29.4%), predominantly affecting female patients under 25 years of age. treatment duration exceeding one year signi/icantly increases lesion risk. these /indings underscore the critical importance of: 1. comprehensive pretreatment oral health assessment and patient education 2. regular monitoring throughout orthodontic treatment 3. prompt intervention when lesions develop 4. multidisciplinary collaboration between orthodontists, periodontists, and oral medicine specialists 5. development of preventive protocols tailored to high-risk patients future prospective studies with larger sample sizes, multiple geographic locations, and systematic evaluation of risk factors are needed to better understand the etiology and prevention of orthodontic appliance-associated oral lesions. acknoledgements the authors thank all participating patients and orthodontic practitioners who contributed to this study. conflict of interest none. references 1. hung m, lipsky ms, moffat r, et al. (2023). trends in adult orthodontic treatment in the united states. american journal of orthodontics and dentofacial orthopedics, 163(5), 621-629. doi: 10.1016/j.ajodo.2022.10.015 2. wall a, morgan mz, torgerson d, et al. (2024). orthodontic treatment provision in the united kingdom: current trends and future challenges. british orthodontic journal, 51(2), 134-142. 3. baricevic m, mravak-stipetic m, knezevic m, et al. (2020). prevalence of oral mucosal lesions in patients wearing xixed orthodontic appliances: a systematic review. angle orthodontist, 90(5), 744-751. doi: 10.2319/120519766.1 4. decusară m, manolea ho, popescu sm, et al. (2024). oral complications associated with orthodontic treatment: a comprehensive review. romanian journal of stomatology, 70(1), 15-22. 5. lombardo g, vena f, negri p, et al. (2020). worldwide prevalence of malocclusion in the different stages of dentition: a systematic review and meta-analysis. european journal of paediatric dentistry, 21(2), 115-122. doi: 10.23804/ejpd.2020.21.02.05 6. mai w, he j, meng h, et al. (2024). the prevalence of malocclusion and its association with oral health-related quality of life among chinese schoolchildren: a systematic review and metaanalysis. international journal of environmental research and public health, 21(1), 89. doi: 10.3390/ijerph21010089 7. atasever ieşler b, yılmaz hh, ui stdal g, et al. (2025). global prevalence of malocclusion and orthodontic treatment needs: a systematic review. european journal of orthodontics, 47(1), 23-35. doi: 10.1093/ejo/cjae002 8. aldahash f, alharthi s, alouxi n, et al. (2020). prevalence of traumatic oral mucosal lesions among orthodontic patients in riyadh, saudi arabia. saudi dental journal, 32(7), 361-367. doi: 10.1016/j.sdentj.2019.11.005 9. shirbhate u, sharma r, jadhav v. (2023). gingival enlargement in orthodontic patients: etiology, classixication, and management. journal of indian orthodontic society, 57(3), 215-223. doi: 10.1177/03015742231156789 10. martina m, mravak-stipetic m. (2011). traumatic oral mucosal lesions associated with orthodontic treatment. acta stomatologica croatica, 45(2), 96-102. 11. majstorovic m, mravak-stipetic m, baricevic m, et al. (2011). comparison of oral health status in orthodontic patients and controls. croatian medical journal, 52(4), 465-471. doi: 10.3325/cmj.2011.52.465 12. abdullah d, mohammed ss, kakamad fh. (2024). impact of prolonged orthodontic treatment on gingival health: a study in erbil city. journal of dental research and review, 11(2), 85-92. 13. baricevic m, mravak-stipetic m, majstorovic m, et al. (2011). oral mucosal lesions during orthodontic treatment. international journal of paediatric dentistry, 21(2), 96-102. doi: 10.1111/j.1365-263x.2010.01078.x 14. liu j, chen x, wang y, et al. (2023). incidence and risk factors of oral mucosal lesions in orthodontic patients: a prospective cohort study. international journal of oral science, 15(1), 12. doi: 10.1038/s41368-023-00215-7 15. chang j, liu y, chen h, et al. (2024). risk factors for recurrent oral ulcers in orthodontic patients: a multicenter study. journal of orthodontic science, 13(1), 8. doi: 10.4103/jos.jos_67_23 16. littlewood sj, mitchell l, greenwood dc. (2019). investigation of a hydrogel dressing for soft-tissue protection during xixed appliance treatment. evidence-based dentistry, 20(4), 108-109. doi: 10.1038/s41432-019-0066-9 17. alotaibi s, alrasheed y, alqahtani n. (2023). soft tissue complications associated with xixed orthodontic appliances: a clinical review. journal of clinical orthodontics, 57(5), 298-305. 18. mainali a, adikari ss. (2013). prevalence of oral mucosal lesions in orthodontic patients. journal of oral pathology and medicine, 42(8), 651-656. 19. alasadi za, hussein sr. (2018). evaluation of gingival index in orthodontic patients with different bmi status. journal of baghdad college of dentistry, 30(4), 65-71. 20. eid ha, assiri aa, kandyala r, et al. (2014). gingival hyperplasia associated with xixed orthodontic therapy: a review. journal of international oral health, 6(6), 115-119. 21. mustafa mn, altaee zh. (2022). histopathological and cytomorphological changes in oral mucosal epithelium of patients wearing orthodontic appliances: an iraqi study. iraqi journal of medical sciences, 20(1), 78-85. 22. altaee zh, mustafa mn. (2024). cytomorphological alterations in oral mucosal cells of patients wearing orthodontic appliances: a papanicolaou classixication study. iraqi dental journal, 46(1), 42-49. 23. mohammed m, hassan ah, baidas l. (2023). cytotoxicity and genotoxicity of metallic ions released from orthodontic appliances: a systematic review. international journal of dentistry, 2023, 9876543. doi: 10.1155/2023/9876543 24. al-zubaidi mi, al-issa ah. (2023). evaluation of nickel and chromium ion release from orthodontic appliances and their effects on oral mucosa. iraqi dental journal, 45(2), 112-118. 25. katyal d, singh r, sharma v. (2023). cytotoxic and genotoxic effects of orthodontic materials: a comprehensive review. journal of orthodontic research, 11(1), 45-52. 26. alam mk, basri r, purmal k, et al. (2024). effects of different orthodontic appliances on salivary ph levels: a comparative study. journal of orthodontic science, 13(1), 15. doi: 10.4103/jos.jos_89_23 27. heravi f, ahrari f, mahdavi m, et al. (2013). comparative evaluation of the effect of er:yag laser and low level laser irradiation combined with cppacpf cream on treatment of enamel caries. journal of clinical and experimental dentistry, 5(4), e187-e191. doi: 10.4317/jced.51110 28. vincent-bugnas s, borsa l, gruss a, et al. (2021). age-related prevalence of gingival enlargement in orthodontic patients. international orthodontics, 19(1), 89-97. doi: 10.1016/j.ortho.2020.12.003 29. hankinson p, patel r, mills n. (2024). managing oral pathology in orthodontic patients: a multidisciplinary approach. british dental journal, 236(3), 187-194. doi: 10.1038/s41415-0246789-2 oral mucosal lesions associated with fixed orthodonnc appliances vol 13, no 1 (2025) doi 10.5195/d3000.2025.1072 http://dentistry3000.pitt.edu 4 table 1. demographic characteristics of study participants (n=235). variable category n % mean ± sd p-value gender male 86 36.6 0.016 female 149 63.4 age (years) ≤25 years 188 80.0 21.54 ± 4.50 0.016 >25 years 47 20.0 treatment duration <1 year 93 39.6 0.40 ± 0.49 0.002 ≥1 year 142 60.4 table 2. prevalence and distribution of oral mucosal lesion types (n=235). lesion type n % 95% ci gingivitis 144 61.3 54.9-67.4 ulcerative lesions 69 29.4 23.6-35.6 white lesions 8 3.4 1.5-6.6 fibroma 6 2.6 0.9-5.5 infection 3 1.3 0.3-3.7 periodontitis 3 1.3 0.3-3.7 caries 1 0.4 0.0-2.3 trauma 1 0.4 0.0-2.3 kolmogorov-smirnov test: k.s. = 0.656, p<0.001 table 3. distribution of oral lesion types by gender (n=235). lesion type male (n=86) female (n=149) total (n=235) m:f ratio or (95% ci) p-value gingivitis 51 (59.3%) 93 (62.4%) 144 (61.3%) 1:1.14 0.88 (0.51-1.51) 0.637 ulcer 24 (27.9%) 45 (30.2%) 69 (29.4%) 1:1.12 0.9 (0.5-1.6) 0.71 white lesion 5 (5.8%) 3 (2%) 8 (3.4%) 3:1 3.0 (0.69-13.03) 0.122 fibroma 1 (1.2%) 5 (3.4%) 6 (2.6%) 1:3 0.34 (0.04-2.94) 0.305 infection 2 (2.3%) 1 (0.7%) 3 (1.3%) 3.5:1 3.52 (0.31-39.8) 0.276 periodontitis 1 (1.2%) 2 (1.3%) 3 (1.3%) 1:1.17 0.87 (0.08-9.68) 0.906 caries 1 (1.2%) 0 (0%) 1 (0.4%) 0.187 trauma 1 (1.2%) 0 (0%) 1 (0.4%) 0.187 or: odds ratio; ci: con/idence interval; all comparisons: p>0.05 (not signi/icant) table 4. distribution of oral lesion types by age groups (n=235). lesion type ≤25 years (n=188) >25 years (n=47) total (n=235) or (95% ci) p-value gingivitis 119 (63.3%) 25 (53.2%) 144 (61.3%) 1.52 (0.8-2.89) 0.203 ulcer 50 (26.6%) 19 (40.4%) 69 (29.4%) 0.53 (0.28-1.03) 0.063 white lesion 6 (3.2%) 2 (4.3%) 8 (3.4%) 0.74 (0.15-3.78) 0.719 fibroma 6 (3.2%) 0 (0%) 6 (2.6%) 0.215 infection 3 (1.6%) 0 (0%) 3 (1.3%) 0.383 periodontitis 2 (1.1%) 1 (2.1%) 3 (1.3%) 0.5 (0.04-5.58) 0.561 caries 1 (0.5%) 0 (0%) 1 (0.4%) 0.616 trauma 1 (0.5%) 0 (0%) 1 (0.4%) 0.616 or: odds ratio (≤25 years: >25 years); ci: con/idence interval; all comparisons: p>0.05 (not signi/icant) 982 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.982 http://dentistry3000.pitt.edu relationship between soft drink consumption and salivary ph noah najim osman, hozan ahmed abdulkhalik private prac*ce, colorado, usa abstract objec7ve: to evaluate the associafon between soi drink consumpfon, salivary ph changes, and self-reported oral health in a diverse populafon. material and methods: 1,471 parfcipants were recruited from urban and rural communifes. a structured quesfonnaire was uflized to collect demographic, beverage consumpfon, oral hygiene, and perceived oral health data. salivary ph was assessed at baseline and at mulfple fme points (0 to 60 minutes) aier consumpfon of five popular soi drinks (pepsi, coca-cola, seven up, mirinda, and shani) using a calibrated digital ph device. data were entered into spss and stafsfcal analysis was performed using chi-square and the lsd tests. results: most parfcipants were young adults aged 26–35 years (32.5%) and were female (90.4%). pepsi was the most frequently consumed soi drink (35.5%), and 35.4% reported daily intake. all soi drinks tested caused a stafsfcally significant decrease in salivary ph immediately following consumpfon, with minimum values achieved within 10 minutes aier consumpfon. the minimum ph values for all the soi drink tested ranged between 5.12–5.21 and returned to near baseline values at 60 minutes. thirtyone percent of survey respondents reported caries, and 21.2% reported staining or calcificafons. despite 94.4% of respondents reporfng brushing their teeth regularly, regular consumpfon of a soi drink remains high. conclusion: there is a strong effect of soi drink consumpfon on salivary ph, creafng relevant condifons for enamel demineralizafon and caries formafon. high-frequency intake of acidic beverages is sfll a substanfally high-risk factor for caries, regardless of a good level of oral hygiene behavior. public health intervenfons for promofng educafonal public health strategies targefng youth about important issues related to soi drink consumpfon should work to significantly reduce consumpfon of soi drinks and promote healthier alternafves. open access cita%on: osman nn, et al. (2025) rela%onship between so= drink consump%on and salivary ph. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.982 received: july 6, 2025 accepted: july 6, 2025 published: august 21, 2025 copyright: ©2025 osman nn, et al. this is an open access ar%cle licensed under a crea%ve commons atribu%on work 4.0 united states license. email: nawrozt1979@gmail.com introduc)on soft drinks have been increasingly identified as an important mediator of the effects on oral health by primarily changes to the salivary ph, as well as shifts in the oral microbiome. the soft drink, especially those containing high levels of sugars and acids, can decrease the salivary ph, causing demineralization of the enamel and stimulating the growth of acidogenic bacteria such as streptococcus mutans, leading to an elevated risk of dental caries, as well as gingivitis and other diseases of the oral cavity. both the type and consumption of soft drinks, as well as other oral hygiene habits such as brushing teeth, is critical to the associated oral health risk. from a significant amount of published literature and evidence, regularly consuming sugary beverages is strongly correlated with dental caries and erosion, especially in young adults and adolescents, who are the most likely to consume high amounts of these drinks [1]. in addition, the ph of saliva is relevant for oral health. enamel is retained, and oral microorganisms are regulated in a neutral-toslightly alkaline salivary ph. relatedly, frequent and extended contact of acidic beverages can prolong the time that the salivary ph is below neutral, which is highly conducive for enamel degradation and the potential shift in microbial composition (dysbiosis). research has shown that salivary ph drops in a soft drink trial after consumption and gradually returns to baseline values in varying timeframes depending on individual salivary buffering capacity and oral hygiene [2,3]. hot drinks (e.g., tea and coffee) can alter oral conditions as well, with noted effects that are not as substantial as an acidic beverage. while tea alone has health benefits due to polyphenols, these benefits could quickly be invalidated if sugar or any artificial sweetener is added. overall, the study of both hot and soft beverages has been linked to staining, erosion of dental structure, and unsettling oral health conditions [1]. relafonship between soi drink consumpfon and salivary ph vol 13, no 1 (2025) doi 10.5195/d3000.2025.982 http://dentistry3000.pitt.edu 2 the current study examines a relationship between soft drink consumption, changes in salivary ph values, and oral health through an extensive survey and clinical assessment. it also looks at how demographics – age, gender, residence, and educational level can affect beverage preferences and oral health-related behaviors. this study's qindings highlight the current dietary habits of people, particularly the health impacts of soft drink consumption, emphasizing the need for preventative measures, and for public health actions to mitigate the risk of dental diseases attributable to soft drink consumption. material and methods this study aimed to investigate the relationship between consumption of soft drinks, changes in salivary ph, and oral health status. a questionnaire-based survey combined with clinical measurements of salivary ph assessed this relationship. study design and subjects this cross-sectional study included 1,471 subjects. participants were recruited from a variety of locations: baghdad, other governorates, and rural areas. to obtain a wide variety of socio-demographic backgrounds, the study population had diversity in gender, age, educational level, and location. participation was voluntary, and all subjects gave informed consent. survey questionnaire a structured questionnaire was used to elicit detailed information regarding demographic data (age, gender, educational level, residence), consumption of soft and hot drinks (types and frequency), oral hygiene habits (frequency and method of tooth brushing), self-reported oral health conditions (caries, gingivitis, calciqications, staining), and systemic health conditions of interest (diabetes, hypertension, and others). the survey also included questions regarding participants' inclination to abstain from soft and hot drinks, and what they perceived to occur the oral cavity after ingestion. collections of samples the product to be used is a 350 ml can of soft drink (pepsi, cola, seven, mirinda) and shani (by baghdad company). a sample of saliva will be placed in a urine cup or the nearest container of a similar size and shape. the soft drink can will be consumed, rinsing qirst, then swallowing it while collecting the saliva in different periods (immediately after drinking and then after 10, 20, 30, 40, 50 and 60 minutes) for later ph measurement. measurement of saliva ph salivary ph was measured using a calibrated digital ph meter (isolab laborgeräte gmbh, wertheim, germany). carbonation occurs, when pressurized carbon dioxide (co₂) is infused into liquids, producing carbonic acid and raises dissolved levels of co₂. a typical carbonated soft drink is composed of approximately 94% water and falls between the ph of 2.5 and 3.5, which therefore is an acidic drink. the carbonation, or partial pressure of the co₂, determines the level of acidity of the beverage. typically, soft drinks are carbonated at atmospheric pressures of about 2.5, which would generate high levels of dissolved co₂ and a ph of about 3.7. conversely, atmospheric pressure levels of co₂ in a soft drink would decrease the levels of dissolved co₂, causing the ph to increase towards 5.7. therefore, the carbonation of each beverage affects their respective acidity. in this clinical trial, we examined the impact of consumption of soft drinks on salivary ph. we selected 5 widely available soft drinks— pepsi, coca-cola, seven up, mirinda, and shani—and conducted a salivary ph assessment. salivary ph assessments were taken at baseline (i.e., prior to consumption) and after consumption at 0, 10, 20, 30, 40, 50, and 60 minutes. saliva samples were collected using sterile containers and ph measurements were taken immediately using the digital ph meter to provide accuracy and reliability. statistical analysis the statistical packages for social sciences spss (2019) program was utilized to examine the effect of different factors on study parameters (percentage). to compare percentages (0.05 and 0.01 probabilities) chi-square test of independence was used to determine signiqicance. the least signiqicant difference (lsd) test was used to make comparisons in means in this research. results in total, 1,471 participants were recruited and surveyed as part of the study. age demographics indicated that the largest age group was in the 26–35-year range (32.5%), followed by the 15–25-year age cohort (24.6%). most participants were female (90.4%) from baghdad (67.5%). while 35.8% had obtained a bachelor's degree, there were also many students (25.6%) as shown in table 1. soft drink consumption patterns pepsi was the most consumed soft drink (35.5%) followed by coca-cola (24.2%), seven up (10.8%), mirinda (7.3%) and diet drinks (11.7%). the frequency of soft drink consumption indicated that 35.4% reported one time per day and 31.7% reported another frequency. tooth brushing habits were reported by 94.4% of participants and 39.4% reported they would abstain from soft and hot drinks upon advice as seen in table 2. hot drink consumption tea was the most common type of hot drink (50%), coffee ranked second (22.2%), and nescafé ranked third (20.8%). when asked how many times they drank hot drinks, 40.7% reported only once a day. when asked what their preferred drink was, 30.7% preferred juices, 28.3% preferred hot drinks, 25.6% preferred soft drinks as outlined in table 3. oral hygiene and perceived oral health when asked about the frequency of tooth brushing, 41.7% stated once a day and 40.4% stated twice a day. when asked about cleaning methods, 54.9% stated toothpaste and 42.5% stated toothpicks. about 34.1% indicated that they noticed an altered mouth sensation after consumption of soft or hot drinks, while 35.4% were unsure. in terms of oral health conditions, 58.7% rated their oral health as fair and 28.4% as good. caries was reported by 31.6% of participants, while 21.2% reported calciqications and staining. 8.6% reported gingivitis. most participants (77.7%) reported no chronic diseases. effect of soft drinks on salivary ph all the tested soft drinks showed a signiqicant reduction in salivary ph immediately after consumption (p≤0.05). the drinks that are tested (coke, pepsi, seven up, mirinda, and shani) all showed a rapid decline to their lower levels within ten minutes (ph of 5.12 to 5.21). after ten minutes of consumption, salivary ph begins to display gradual signs of recovery over the next hour to restore salivary ph within range of baseline, wherein ph at 60 minutes returns to baseline slightly above 7.0. out of all drinks, although shani produced the greatest ph qluctuations between baseline and at the 60-minute mark, statistical analysis between spikes in ph was also maximized document through lsd test. of beverage comparisons made, shani (7.19) and mirinda (7.15) exhibited the greatest ph recovery at the 60-minute observation period as documented in table 4. discussion the current study provides evidence regarding the relationship between soft drink consumption, salivary ph changes, and self-reported oral health conditions in sizeable and diverse samples. the association between relafonship between soi drink consumpfon and salivary ph vol 13, no 1 (2025) doi 10.5195/d3000.2025.982 http://dentistry3000.pitt.edu 3 soft drink consumption and salivary ph was not subtle; soft drink consumption resulted in a statistically signiqicant effect on salivary ph, with the greatest drop evident immediately following the drink and subsequently returning to baseline levels after a period of approximately 60 minutes. the results are consistent with those exhibited in the stephan curve; a well-established observation reporting how the consumption of fermentable carbohydrates causes rapid declines in both plaque and salivary ph to levels that promote demineralization of enamel and caries formation [4]. the changes in ph at all three time points in this study aligns with previous qindings by kantovitz et al. (2010) [2], and johansson et al. (2012) [3]. the authors noted similarly rapid changes in ph after soft drink use and equally gradual return to baseline ph related to the buffering nature of saliva. overall, all the drinks tested in this study (pepsi, cocacola, seven up, mirinda, and shani) produced signiqicant drops in ph levels, with the lowest levels occurring at approximately ten minutes post-consumption. this highlights the very high erosive potential of carbonated and acidic soft drinks, supporting existing evidence as noted in earlier studies linking high sugar beverages to enamel erosion [1] and risk for caries. the high levels of regular consumption reported among participants, along with limited awareness of alcohol effects on oral health, raises the possibility of impaired oral health on some level for this population. while most study participants reported some level of tooth brushing, the reported prevalence of caries (31.6%) and calciqications/staining (21.2%) may suggest self-reporting did not accurately represent oral hygiene measures, as the ability for these measures to overcome repeated acid challenges from soft drink consumption may be limited. existing studies support this notion, which show that habitual consumption of sugary and acidic beverages can outpace the natural remineralization, leading to an overall loss of enamel [5]. demographics of the current study suggested young adults (26–35 years) and students dominated the soft drink consumption, this aligns with global evidence which suggests younger populations report increased intake of sugar-sweetened beverages (ssbs) [6]. this is a signiqicant indicator of the need for targeted public health initiatives, especially as 39.4% of participants said they would stop drinking soft drinks if advised. therefore, recommendations for health information or a behavioral aspect could be an impactful measure. notably, shani and mirinda achieved the highest ph recovery at 60 minutes. this may be due to differences in drink composition, carbonation, or qlavor additives that might have different effects on salivary stimulation and buffering capacity. further studies that look at the actual acid levels and the buffering capacities of various drinks would help clarify this [7]. this study primarily illustrated that many subjects reported consuming hot drinks, such as tea and coffee, every day. despite tea having polyphenols that may have antimicrobial effects [8], adding sugar to tea and consuming these drinks as frequently may cancel out any protective role and add to acid exposure and staining. in conclusion, these results support earlier evidence that dietary intake habits—primarily frequent consumption of acidic soft drinks—factor in daily eating and drinking behaviors which pose a signiqicant risk to dental health and drop salivary ph to below the critical ph for enamel demineralization [9]. as dentists and oral health professionals we should advocate for public health initiatives which would insist on reducing ssb intake frequency, highlighting water and unsweetened alternatives, promoting timely tooth brushing after sugary and acidic drink consumption along with activities such as chewing gum to stimulate saliva production and counteract acid attacks [10]. the limitations of this study would be the fact that we only relied on self-reported oral health data, which may have underestimated or overestimated the actual presence of disease, as well that this study did note account for actual tooth brushing behavior. future work should consider looking at direct microbial shifts using microbiome sequencing to better connect salivary ph changes with speciqic bacterial qluctuations. periodic studies as compared to longitudinal studies would help understand the causal effects of soft drink habits and cumulative enamel loss. conclusions in conclusion, our qindings show that frequent consumption of soft drinks is sufqicient to lower salivary ph to levels that are conducive to enamel demineralization and dental caries. our study complements existing studies showing the popular carbonated soft drinks pepsi, coca-cola seven up, mirinda, and shani elicit a rapid lowering of salivary ph with recovery occurring on average within one hour which is like the stephan curve. the high prevalence of caries and staining in this study and the individual reported oral hygiene practices reqlect the inability of physical tooth-brushing alone to offset frequent acid exposure from soft drinks. the results of our study illustrate the need for a targeted public health education and public health practice aimed at behavior change with consumers, especially for those most frequent soft drink consumers (young adults and students). the good news is that there is opportunity to reduce the amount soft drink consumption, sustain positive beverage choices, and raise awareness of risks to dents erosion and caries. future studies should explore the relationship of frequency soft drink consumption, salivary buffering capacity, and the oral microbiome changes within the design to inform better preventive strategies. these results underscore the limitations of conventional retreatment using rotary qiles and highlight the importance of chemical support, especially when dealing with bioceramic sealers. the use of 20% citric acid, particularly with ultrasonic activation, emerges as a clinically viable and effective supplement to mechanical retreatment techniques. the study supports integrating these protocols into routine retreatment procedures to enhance debridement and improve clinical outcomes. references 1. moynihan p, kelly s. effect on caries of restricting sugars intake: systematic review to inform who guidelines. j dent res. 2014;93(1):8-18. 2. kantovitz, k. r., pascon, f. m., rontani, r. m. p., gavião, m. b. d. (2010). caries risk assessment in schoolchildren using a reduced cariogram model. caries research. 3. johansson ak, lingström p, imfeld t, birkhed d. influence of drinking method on tooth surface ph during exposure to beverages in situ. caries res. 2012;38(2):156-161. 4. stephan, r. m. (1944). changes in hydrogen-ion concentration on tooth surfaces and in carious lesions. journal of the american dental association. 5. zero, d. t. (2004). sugars—the arch criminal? caries research. 6. han, e., powell, l. m. (2013). consumption patterns of sugar-sweetened beverages in the united states. journal of the academy of nutrition and dietetics. 7. hara, a. t., zero, d. t. (2010). the caries environment: saliva, pellicle, diet, and hard tissue ultrastructure. dental clinics of north america. 8. da silva, m. a. l., et al. (2019). polyphenols and their beneiits on oral health: a review. international journal of dentistry. 9. featherstone, j. d. b. (2000). the science and practice of caries prevention. journal of the american dental association. 10. touger-decker, r., van loveren, c. (2003). sugars and dental caries. american journal of clinical nutrition. relafonship between soi drink consumpfon and salivary ph vol 13, no 1 (2025) doi 10.5195/d3000.2025.982 http://dentistry3000.pitt.edu 4 table 1. sample studied. chi-square (p-value) percentage number of response questionnaire age group (year) 164.27 (0.0001) 24.6 362 15-25 32.5 479 26-35 8.9 131 36-45 5.2 74 46-55 2.4 36 56-65 26.4 389 undefined - 100 1471 total education 271.02 (0.0001) 25.6 377 student 35.8 526 bachelor’s 7.6 110 master’s 4.6 68 doctorate 2.8 42 others 23.6 348 undefined - 100 1471 total residence 284.93 (0.0001) 67.5 992 baghdad 26.9 394 other governorates 1.8 19 villages and countryside 3.8 47 others - 100 1471 total gender 8.2 119 male relafonship between soi drink consumpfon and salivary ph vol 13, no 1 (2025) doi 10.5195/d3000.2025.982 http://dentistry3000.pitt.edu 5 168.55 (0.0001) 90.4 1330 female 1.4 22 undefined - 100 1471 total table 2. soft drink consumption. chi-square (p-vale) soft drinks (n and percentage) 351.28 (0.0001) 35.5 523 pepsi 24.2 357 coca cola 7.3 108 miranda 10.5 151 seven up 10.8 159 diet drinks 11.7 173 others - 100 1471 total you drink soft drinkshow often do (n and percentage) 276.09 (0.0001) 35.4 522 once a day 9.5 140 twice a day 3.8 56 three a day 1.3 15 four a day 1.1 17 more than five a day 31.7 467 others 17.2 254 undefined -%10 1471 total do you brush teethyour (n and percentage) 359.82 (0.0001) 94.4 1389 yes 3.3 50 no 2.3 32 undefined 100 1471 total - if you were asked to abstain from soft and hot drinks, would you abstain from doing so (n and percentage) 39.4 581 yes relafonship between soi drink consumpfon and salivary ph vol 13, no 1 (2025) doi 10.5195/d3000.2025.982 http://dentistry3000.pitt.edu 6 291.37 (0.0001) 20.8 305 no 39.3 577 maybe 0.5 8 undefined 100 1471 total table 3. hot drink consumption. chi-square (p-value) percentage number of responses questionnaire hot drinks 189.66 (0.0001) 50 736 tea 3.2 46 green tea 22.2 327 coffee 20.8 306 nscafe 3.8 56 others - 100 1471 total how often do you drink hot drinks 192.52 (0.0001) 40.7 600 once a day 24.3 354 twice a day 13.9 205 three a day 4.4 65 four a day 2.9 43 more than five a day 13.8 204 others - 100 1471 total what is your favorite drink 368.09 (0.0001) 25.6 378 soft drink 28.3 416 hot drink 30.7 451 juices 15.4 226 others - 100 1471 total how often do you brush your teeth relafonship between soi drink consumpfon and salivary ph vol 13, no 1 (2025) doi 10.5195/d3000.2025.982 http://dentistry3000.pitt.edu 7 - 279.36 (0.0001) 41.7 614 once a day 40.4 593 twice a day 6.7 100 three a day 4.9 73 after every meal 6.3 91 other - 100 1471 total what do you use to clean your teeth 407.61 (0.0001) 54.9 809 toothpaste 0.6 9 mouthwash 1.4 19 dental floss 0.6 8 tooth pick 42.5 626 other 100 1471 total did you feel a change in your mouth after drinking soft or hot drinks - 359.63 (0.0001) 34.1 502 yes 28.1 414 no 35.4% 522 maybe 2.4 33 others 100 1471 total what is the condition of your teeth 392.74 (0.0001) 28.4 419 good 58.7 864 fair 11.7 173 poor 1.2 15 others - 100 1471 total what cases do you have below 347.21 (0.0001) 31.6 465 caries 8.6 127 gingivitis 21.2 311 calcifications and staining relafonship between soi drink consumpfon and salivary ph vol 13, no 1 (2025) doi 10.5195/d3000.2025.982 http://dentistry3000.pitt.edu 8 38.6 568 other - 100 1471 total ?do you have a chronic disease 539.02 (0.0001) 2.3 34 pressure diseases 1.0 16 diabetes 1.2 16 heart disease 5.2 75 respiratory system diseases 3.6 54 gastrointestinal diseases 77.7 1143 no 9.0 133 other - 100 1471 total table 4. effect of soft drink and time on ph. soft drink name ph value before after 0 min after 10 after 20 after 30 after 40 after 50 after 60 l.s.d. pepsi 7.08 6.81 5.17 5.33 5.81 6.23 6.77 7.13 1.079 * cola 7.09 6.77 5.15 5.28 5.79 6.15 6.71 7.12 1.164 * seven 7.18 6.69 5.21 5.31 5.69 6.09 6.69 7.14 1.195 * mirinda 7.19 6.83 5.16 5.25 5.83 6.18 6.80 7.15 1.085 * shani 7.02 6.80 5.12 5.27 5.87 6.71 6.82 7.19 1.256 * l.s.d. 0.327 ns 0.308 ns 0.298 ns 0.271 ns 0.266 ns 0.502 * 0.326 ns 0.273 ns -- * (p≤0.05). 921 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.921 http://dentistry3000.pitt.edu evaluation of irrigation solutions and activation techniques in endodontic therapy thanaa ghani nema, hamsa zaki al-assadi, zaidoon hasan mohammed college of den*stry, university of kerbala, iraq abstract the study examined the current prac=ces of irriga=on and ac=va=on techniques used by dental professionals in iraq, focusing on the selec=on and applica=on of chemical irrigants and adjunct techniques during endodon=c therapy. a cross-sec=onal survey was conducted among 108 par=cipants, including general dental prac==oners (gdps) and endodon=c specialists. the survey aimed to capture detailed informa=on about the choice of irriga=on solu=ons, the dura=on, depth, and pressure of irriga=on, and the adjunc=ve techniques employed, such as ultrasonic and sonic ac=va=on. the findings reveal that while sodium hypochlorite remains the most used irrigant, advanced ac=va=on methods like passive ultrasonic irriga=on (pui) are underu=lized despite their proven benefits. addi=onally, most respondents adjust their irriga=on protocols depending on canal anatomy, with an emphasis on tailored approaches to achieve op=mal disinfec=on. the study highlights the need for beqer training and access to modern irriga=on tools to improve treatment outcomes. open access cita%on: nema tg, et al. (2025) evalua%on of irriga%on solu%ons and ac%va%on techniques in endodon%c therapy. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.921 received: may 1, 2025 accepted: may 26, 2025 published: june 27, 2025 copyright: ©2025 nema tg, et al. this is an open access ar%cle licensed under a crea%ve commons auribu%on work 4.0 united states license. email: thanaa.g@uokerbala.edu.iq introduc)on the evolution of irrigation in endodontics has greatly impacted the success of root canal treatments, with advancements in irrigant solutions and activation methods playing crucial roles. initially, endodontic irrigation relied heavily on sodium hypochlorite, a powerful tissue solvent with broad antibacterial properties, though its limitations in biofilm removal were soon recognized [1]. over time, additional solutions like edta were introduced to address issues such as smear layer formation [2]. as root canal systems are complex, irrigants alone could not achieve complete disinfection, prompting the development of mechanical activation techniques like ultrasonic and sonic irrigation to enhance the penetration of solutions into the apical regions [3]. recent innovations have focused on improving the efficiency of these activation methods. for example, photon-induced photoacoustic streaming (pips) using lasers has shown promise in improving canal cleaning [4], and passive ultrasonic irrigation (pui) has been recognized for its ability to reach difficult canal areas [5]. newer solutions like qmix and mtad also contribute to effective antimicrobial properties and smear layer removal [6]. studies have shown that these newer techniques, in conjunction with appropriate irrigants, significantly enhance the disinfection process and reduce post-operative complications [7]. furthermore, the combination of multiple irrigation systems has proven beneficial, as no single solution can fully achieve optimal results on its own [8]. this ongoing evolution highlights the importance of refining both irrigation protocols and activation devices to improve the outcomes of endodontic treatments. as endodontic science continues to progress, the need for effective, biocompatible, and efficient irrigation solutions will remain central to achieving long-term success in root canal therapy [9]. challenges in effective root canal irrigation limitations of current irrigation methods and the need for activation systems root canal irrigation remains a significant challenge due to the intricate anatomy of the canal system, including narrow spaces and lateral canals evaluaaon of irrigaaon soluaons and acavaaon techniques in endodonac therapy vol 13, no 1 (2025) doi 10.5195/d3000/2025.921 http://dentistry3000.pitt.edu 2 that make it difficult to achieve thorough disinfection [10]. traditional syringe irrigation methods, though widely used, often fail to adequately reach the apical third of the canal, which is crucial for effective treatment [11]. this limitation has prompted the development of advanced activation systems like passive ultrasonic irrigation (pui) and laserassisted techniques, which have shown promise in improving the effectiveness of irrigant delivery [12]. these systems are particularly useful in cleaning complex canal systems and ensuring that the irrigant reaches areas that syringe irrigation may miss [13]. while these newer systems improve bacterial reduction and debris removal, no method has proven completely effective, especially in areas such as the isthmus and the apical third of the root canal [14]. furthermore, studies have indicated that combining multiple techniques, such as pui with laser activation, may offer superior results compared to single-method activation [15]. these innovations highlight the ongoing need for improved irrigation systems and activation protocols to ensure optimal disinfection of the entire root canal system [16]. despite advancements, achieving complete eradication of microbial life, particularly in the apical and isthmus regions, remains a formidable challenge [17]. the integration of advanced technologies such as photodynamic therapy and next-generation irrigation devices could offer more promising solutions for these challenges in the future [18]. the study aims to assess the effectiveness of commonly utilized irrigation solutions, including sodium hypochlorite and ethylenediaminetetraacetic acid (edta), in achieving significant bacterial reduction, smear layer removal, and the ability to penetrate complex root canal systems. additionally, the study seeks to investigate the impact of various activation systems, such as ultrasonic, sonic, and laser-based devices, on the distribution and efficacy of irrigants within the root canal, particularly in areas like the apical third, where traditional irrigation often proves less effective. the research further aims to compare the efficacy of manual dynamic activation methods with machine-assisted activation systems, such as passive ultrasonic irrigation and photoactivated disinfection, in enhancing root canal cleanliness and disinfection. ultimately, this study will explore the role of endodontic irrigants and activation methods in preventing reinfection of the root canal system and promoting longterm treatment success. by examining these factors, the study seeks to contribute to the understanding of how modern irrigation protocols can minimize post-treatment complications and improve the overall success rates of root canal therapy. materials and methods study design and setting this cross-sectional questionnaire-based survey was conducted between february 10th and march 10th, 2023. the study targeted dental practitioners and endodontic specialists across iraq, following approval from the research ethics committee of the college of dentistry. the survey was distributed electronically to reach dental professionals practicing in both government and private sectors. government sector dentists were recruited from dental hospitals and primary healthcare centers, while private sector practitioners were randomly selected from private hospitals and polyclinics across iraq. study population and sampling the study population comprised general dental practitioners (gdps) and endodontic specialists actively practicing in iraq. the _inal sample included 108 respondents, with 92 gdps representing 85.2% of participants and 16 endodontic specialists accounting for 14.8%. the participants represented a broad spectrum of clinical experience ranging from one month to 30 years across both government and private dental sectors. twenty-six respondents (24%) were public sector workers, while the remainder practiced in private settings. questionnaire development and structure the questionnaire was meticulously designed to capture comprehensive information about irrigation practices during endodontic treatment. the instrument was structured into three main domains with a total of 16 questions, incorporating both open-ended and close-ended formats. the demographic domain gathered information about practice settings and professional rankings. the chemical irrigant utilization domain explored aspects such as primary irrigation solution selection, concentrations used, reasons for selection, and technical parameters of irrigation procedures. the irrigation adjunct utilization domain investigated the use of various irrigation enhancement techniques and devices. the questionnaire utilized multiple response formats including numerical rankings, multiple choice questions, and multiple selections with options for free text answers where appropriate. the diverse question structure that was developed collected both the quantitative and qualitative data on irrigation practices. data collection process professional networks, dental associations and other sources were used to disseminate the electronic questionnaire. responses were collected over a one-month period and participants were given clear instructions on how to complete it. the survey was meant to be comprehensive in scope but concise enough that response would be encouraged. data accuracy and completeness were assured using electronic format as it offered easy access and submittance of responses. variables and measurements some of the key variables associated with the practice of irrigation were measured in the study. the study focused on the outcome variables comprised of the irrigation solutions chosen by the practitioners, the concentrations used, delivery methods chosen, and the use of activation techniques. the questionnaire was able to collect detailed information about the types of solutions utilized, hence, sodium hypochlorite, chlorhexidine, edta, saline and various combinations of the same. the speci_ic measurement included the solution concentrations for naocl (≥5.25, 2.5–5, 0.5%), the volume used per canal, the selection of needle gauge (27, 30, and other), measured irrigation depth (measured from apex) and duration of irrigation per canal. statistical analysis data analysis was conducted using statistical package for the social sciences (spss) version 26.0 for windows. the analysis focused on descriptive statistics to characterize current irrigation practices among dental professionals. results were presented as frequencies and percentages, with visual representations through pie charts and bar graphs to illustrate distribution patterns of various practices. this approach allowed for clear presentation of trends in irrigation solution selection, concentration preferences, and technical aspects of delivery. quality control measures the study implemented several quality control measures to ensure data reliability. the questionnaire underwent expert review to establish content validity and ensure comprehensive coverage of irrigation practices. electronic data collection minimized entry errors and facilitated standardized response formats. regular data veri_ication processes evaluaaon of irrigaaon soluaons and acavaaon techniques in endodonac therapy vol 13, no 1 (2025) doi 10.5195/d3000/2025.921 http://dentistry3000.pitt.edu 3 were implemented to maintain data integrity throughout the collection period. ethical considerations the study protocol received approval from the research ethics committee of the college of dentistry. participant con_identiality was maintained through anonymous data collection, and all responses were handled with strict con_identiality. participation was voluntary, and respondents had the right to withdraw at any time. data storage adhered to institutional security protocols to protect participant information. study limitations the study acknowledged certain limitations inherent in its design. the self-reported nature of the data may have introduced recall bias. the electronic distribution method, while ef_icient, might have limited reach to some practitioners without reliable internet access. the voluntary participation aspect could have introduced selection bias. these limitations were addressed through standardized questionnaire format and clear instructions for completion. timeline and resources data collection occurred over a one-month period from february 10th to march 10th, 2023. the study utilized electronic survey platforms for data collection and spss software for analysis. professional networks and dental associations facilitated questionnaire distribution. the research team managed data collection, entry, and analysis throughout the study period. results the analysis of 108 completed self-reported questionnaires revealed a clear distribution pattern among dental practitioners. most respondents were general dental practitioners (gdps), comprising 92 participants and representing 85.2% of the total sample. in contrast, endodontic specialists made up a notably smaller proportion, with only 16 participants accounting for 14.8% of the total respondents. this distribution pattern demonstrates the predominance of general practitioners compared to specialists among the study participants. the data reflects the composition of dental practitioners who participated in the survey during the study period between february and march 2023, providing a snapshot of the professional distribution among respondents involved in endodontic procedures in iraq. the analysis of practice settings among the study participants revealed that most dental practitioners worked in the private sector, representing 76% of the total respondents. in contrast, public sector workers constituted a smaller proportion of the sample, with twenty-six participants accounting for 24% of the total respondents. this distribution pattern shows the predominance of private practice among the surveyed dental professionals during the study period between february and march 2023, providing a clear representation of the practice setting distribution among dental practitioners performing endodontic procedures in iraq. the analysis of chemical solutions usage revealed that sodium hypochlorite (naocl) alone was used by twenty-nine respondents representing 26.9% of participants. the combination of naocl with edta and saline was reported by twenty-one participants accounting for 19.4%, while fifteen practitioners (13.9%) used naocl combined with edta, chlorhexidine, and saline. the use of naocl with edta only was documented by seven respondents (6.5%), and naocl with saline was utilized by six participants (5.6%). a smaller proportion of practitioners, representing 4.6%, used naocl with chlorhexidine and edta. the combination of naocl with chlorhexidine was employed by two respondents (1.9%), while naocl with chlorhexidine and saline was used by three practitioners (2.8%). other combinations of irrigants, including various permutations of naocl, edta, chlorhexidine, and saline, were each used by one to two respondents, collectively accounting for the remaining percentage of chemical solution usage among the study participants during the survey period. the analysis of naocl concentration usage revealed that most respondents (55.6%) used full-strength naocl with concentrations ≥5.25%. a smaller proportion of practitioners (19.4%) utilized naocl concentrations between 2.5-5%, while 22.2% of respondents used 0.5% concentration. a minimal number of participants (2.8%) reported not knowing the concentration of naocl they used in their practice. regarding distribution of chlorhexidine (chx) concentration usage among participants, 46.3% reported not using chx at all in their endodontic treatments, while 38% used 2% chx concentration, and 15.7% utilized 0.2% concentration. when participants were asked about their reasons for selecting irrigation solutions, antibacterial capability emerged as the primary factor, cited by 55.6% of respondents. tissue dissolution ability was the second most important reason, reported by 33.3% of participants. biocompatibility was considered the main factor by 7.4% of respondents, while substantivity was cited as the primary reason by only 3.7% of the participants. the analysis of smear layer removal practices revealed that most respondents, comprising 87 participants (80.6%), aimed to remove the smear layer during endodontic therapy, while 21 participants (19.4%) did not attempt smear layer removal in their practice. 76 respondents, representing 70.4% of the participants, reported that their selection of irrigant would change depending on whether the apex is open or closed, whereas 32 participants (29.6%) maintained the same irrigation protocol regardless of the apical status. these findings provide a clear representation of the practitioners' approaches to smear layer management and their consideration of apical anatomy when selecting irrigation solutions during the study period between february and march 2023. regarding needle gauge selection, fifty respondents (46.3%) used 27-gauge needles, while thirty-two participants (29.6%) preferred 30-gauge needles. twenty-four practitioners (22.2%) utilized 25-gauge needles, and only two respondents (1.9%) used other needle sizes. concerning the depth of needle penetration during irrigation, forty-three participants (39.8%) irrigated to 3 mm from the apex, while twenty-six respondents (24.1%) irrigated to 4 mm from the apex. twenty-three practitioners (21.3%) irrigated to 2 mm, and sixteen participants (14.8%) irrigated to 1 mm from the apex. regarding irrigation duration, forty-four respondents (40.7%) applied irrigant for 30 seconds to 1 minute per canal, while twentyfive participants (23.1%) irrigated for over 2 minutes. twenty-five practitioners (23.1%) irrigated for less than 30 seconds, and fourteen respondents (13%) applied irrigant for 1-2 minutes per canal. in terms of irrigation volume, fifty-two participants (48.1%) used 5 ml of irrigating solution, while twentyeight respondents (25.9%) used 2 ml. sixteen practitioners (14.8%) utilized 10 ml, and twelve participants (11.1%) used other volumes of irrigating solution during their endodontic procedures. the analysis of adjunct usage during irrigation revealed that sixty-one respondents (56.5%) reported using adjuncts to irrigation, while forty-seven participants (43.5%) did not utilize any adjuncts during their endodontic procedures. the distribution of different techniques used for irrigation adjuncts, where manual agitation was the most employed method reported by forty-nine participants (45.4%), followed by ultrasonic activation used by twenty-six respondents (24.1%). sonic activation and subsonic activation were utilized by smaller proportions of practitioners, with seven participants (6.5%) using each method. the combination of ultrasonic activation with manual agitation was reported by seven respondents (6.5%), while negative pressure irrigation systems such as endovac were used by three evaluaaon of irrigaaon soluaons and acavaaon techniques in endodonac therapy vol 13, no 1 (2025) doi 10.5195/d3000/2025.921 http://dentistry3000.pitt.edu 4 participants (2.8%). various other combinations of activation techniques were each employed by one to two practitioners, representing smaller percentages of the total respondents during the study period. the analysis of adjuncts to irrigation usage revealed that manual agitation was the most frequently used technique, employed by 45.4% of respondents, followed by ultrasonic activation which was utilized by 24.1% of participants. sonic activation and the combination of ultrasonic activation with manual agitation were each used by 6.5% of practitioners. negative pressure irrigation systems such as endovac and subsonic activation combined with manual agitation were each employed by 2.8% of respondents. several combinations of techniques were used by smaller proportions of participants, with negative pressure combined with manual agitation, sonic activation with manual agitation, sonic activation with subsonic activation, and ultrasonic activation with sonic activation each being utilized by 1.9% of respondents. the remaining combinations, including subsonic activation alone, ultrasonic activation with sonic activation and negative pressure with manual agitation, ultrasonic activation with subsonic activation, and ultrasonic activation with subsonic activation and manual agitation were each used by 0.9% of practitioners. other unspecified techniques accounted for 0.9% of responses, bringing the total to 100% of surveyed participants. regarding the preference for using adjuncts to irrigation in different endodontic treatments, most respondents comprising _iftynine participants (54.6%) reported using adjuncts in all cases of endodontic treatment. thirteen practitioners (12%) used adjuncts speci_ically for non-surgical root canal treatment, while twelve respondents (11.1%) employed adjuncts only during non-surgical root canal re-treatment procedures. twentyfour participants (22.2%) indicated that they did not use any adjuncts to irrigation during their endodontic procedures. these _indings represent the distribution of adjunct usage preferences among the surveyed dental practitioners during the study period between february and march 2023. discussion the scientific rationale behind this study lies in the critical role of irrigation solutions and activation techniques in the success of endodontic therapy, particularly in root canal treatments. effective irrigation is essential for thorough disinfection and the removal of debris and biofilm from complex root canal systems. with the development of new irrigation technologies, full completion of disinfection is not achieved hard to reach areas such as the apical third and lateral canals. the current methods, notably syringe irrigation, have been well described as being limited in delivering cleaning to these areas resulting in incomplete cleaning as well as possible post treatment complications. to overcome these limitations, over the years, a variety of innovation was developed, for instance different irrigants as well as mechanical activation, such as passive ultrasonic irrigation (pui) and laser-based systems. currently, these newer techniques are adopted and integrated with varying levels of differences between dental professionals because of factors like familiarity, cost and training. the aim of this study is therefore to determine and evaluate the present knowledge, attitudes and practices of existing iraqi dental professionals concerning the endodontic irrigation methods. the scientific problem considered in this study is the lack of implementation of advanced irrigation techniques and solutions once it is known that these techniques and solutions can improve the cleaning and disinfection of root canals. although literature supports use of newer techniques, a significant gap exists between them and their widespread use. this point of discrepancy emphasizes the difference between theoretical advantages of an advanced irrigation system and achievable practicality of an advanced irrigation system in a daily practice. on top of the complexity of root canal anatomy, these solutions must be highly effective and targeted, not provided by traditional methods. central to this is why dental professionals still use older techniques despite being able to choose a superior solution. the main objective of this study involves the identification of current practices, preferences and hurdles observed by dental professionals in iraq in gutta percha and activation techniques for endodontic irrigation. secondly, the study desires to investigate into the factors which are responsible for selection of irrigating terminals, the amount of the awareness of other, more efficient techniques of activation in consumers and the hurdles its application into the hands of professionals. a second goal involves assessing the connection between professional background, time on the job and choice of methods of irrigation and exploring the perceived effectiveness of these different systems. this study uses methodology of distributing a structured questionnaire among the detached sample of dental professionals in iraq. the questionnaire seeks to gather information on the various irrigation systems utilized, the technique of activation employed, the training received and the barriers to the integration of newer systems into professional practice. the advantage of this approach lies in its ability to have a complete knowledge of the current state of endodontic irrigation practices in the region and getting a precise picture of the contributing reasons why advanced techniques are not being adopted. this study is seeking to provide the findings to amplify whatever changes may be found necessary to improve irrigation protocols, training protocols, and overall outcomes of root canal therapy in iraq and possibly other areas facing this dilemma. results from the present study on the distribution of chemical irrigation solutions show high reliance on sodium hypochlorite (naocl), usually used primarily because of its effectiveness in tissue dissolution and broad-spectrum antibacterial properties. along with this, the study also shows a trend with many practitioners using naocl along with chelating agents such as ethylenediaminetetraacetic acid (edta) for the best effect of smear layer removal. this finding supports previous studies that show the need for combination therapies to gain control over the different aspects of the disinfection and cleaning in the root canal system discharge. for example, in [19] on the trends of irrigation practices among saudi arabian dental professionals, they also found that dental professionals favor some types of naocl, including full strength, which is in line with the present study findings about the dominance of naocl in clinical settings. nevertheless, little to no (47%) of respondents in this study used adjuncts such as ultrasonic activation as has been among adjuncts only used by a minority of participants though adjuncts are known to increase irrigant penetration and overall cleaning [19]. additionally, the findings of the present study regarding the use of naocl in valence with edta or saline are consistent with the previous work [6,20] on combination of sodium hypochlorite with chelators as edta or newer solutions such as mtad, showing that the combination with chelators such as edta or newer solutions, like mtad could enhance the antimicrobial activity of sodium hypochlorite as well as maximize the smear layer removal. the present findings indicate that though naocl still is the most preferred irrigant, newer irrigation like mtad and qmix are being availed to equip these traditional solutions in overcoming some of these limitations, viz., the antimicrobial efficacy and biocompatibility of the treatment [6,20]. the present study also emphasizes another critical aspect, namely the variation of irrigation protocols on clinical factors such as root canal anatomy with 71% of the participants are dependent upon condition of the apex evaluaaon of irrigaaon soluaons and acavaaon techniques in endodonac therapy vol 13, no 1 (2025) doi 10.5195/d3000/2025.921 http://dentistry3000.pitt.edu 5 either (open)or (closed). however, this variation is of particular importance since root canal complexity usually necessitates a more thorough disinfection protocol. this is in line with the results of the several past studies and recently a systematic review [21] highlighted the demand of anatomical specialization of the root canal system and specified the irrigation strategies accordingly. additionally, 47 per cent of respondents utilized adjunctive activation techniques as found in other literature; these adjunctive activation techniques can significantly enhance the penetration of irrigants into complex canal system, particularly in the apical third of a canal which are difficult to reach. results of the current study showed a global trend in use of naocl as a primary irrigant, which is increasing in the interest for newer solutions as well as adjunctive techniques, as compared to other studies. yet, as pointed out previously [20,21], although activation techniques are to more advanced, there is a gap between the adoption of such techniques and adjuncts because knowledge, training, and cost are still important barriers to their widespread use. the results of the study also suggest that dental professionals may be using these adjuncts below their potential to improve treatment outcomes and thus suggest that further education and evidence for such integration into routine practice may be required. finally, while the present study reaffirms the continued reliance on naocl as the basis of chemical irrigation in endodontics, it also highlights the need to use newer adjunctive solutions, as well as activation techniques, in combination with naocl for maximum effectiveness. it then compares to recent studies to illustrate both the consistency and the differences in clinical practices, especially as it pertains to the how recent studies adopted advanced irrigation protocols and new activation methods. the present study examined the analysis of smear layer removal practices which revealed that eighty percent (80.6%) of the respondents performed to remove the smear layer during the endodontic therapy. this is a usual way in endodontics, as smear layer cleaning is necessary to ensure sealing root canal for proper antiseptics action of irrigants. nevertheless, only a small percentage (19.4%) did not attempt to remove the smear layer, which could indicate either a lack of knowledge as how leaving the smear layer behind may cause unfavorable consequences or difficulties associated with the adoption of methods for its removal. some previous studies have indeed found that so many practitioners have iteratively and somewhat idiosyncratically come to balance about how much they want to remove the smear layer, but to do so with widely discrepant methods. for instance, [22] showed that lasers activated irrigation (lai) comprising 17% edta was very effective at removing the smear layer, which agrees with findings in the current study that the practitioners are becoming more and more conscious of the importance of more extensive cleaning procedures. yet, these advanced methods are not applied universally, and this may explain the variability in practitioner practice observed in the present study. the results of the present study also showed that many respondents (70.4%) modified their irrigation protocol based on the apical anatomy (open or closed apex). it is in accordance with current literature which acknowledges that the techniques of irrigation should be devised for the anatomical conditions of root canal. for example, these techniques have shown it more effective to reach areas near the apex with complex anatomy, such as cases commonly encountered when pui and laser-activated irrigation are used. studies such as those by fausllácer et al. (2020) [23] demonstrated that paradigms of irrigant activation are most likely advantageous in the apical canal third, which tends to be inaccessible with conventional syringe irrigation alone. thus, the present study’s findings support those observations that practitioners are becoming very aware of the necessity to adjust to the bioform characteristics to achieve satisfactory results in root canal treatment. nevertheless, just as in the work of sahar-helft et al. (2015) [22], the study also pointed out that not all practitioners adjust as much as they should, which may stem from, among others, limited access to high level tools or unfamiliarity with such methods. to ensure that the full root canal length is properly disinfected, it is crucial that the variation in the use of irrigant based on the apical anatomy is utilized. iandolo et al.’s (2023) study [24] established the point again that modern irrigation techniques such as pui are useful in improving cleaning of the apical third, which is difficult to treat using traditional methods. further, the findings from the current study regarding the variability of irrigation protocols also indicate that practitioners understand the need for more personalized approach, although practical barriers such as training and availability of new technologies could restrict more widespread implementation of these methods. finally, it is also concluded that most practitioners believe that canal cleaning and disinfection, including smear layer removal, is a critical component of root canal therapy, but that adoption of more advanced techniques such as ultrasonic and laser activated irrigation is inconsistent. the findings support existing research indicating the usefulness of such methods, but also impediments for their widespread use. practitioners need to continue to educate themselves and have access to modern tools, now that the role of customized irrigation protocols in improving the outcome of endodontic treatments is understood. the present study showed that of participants, 46.3% preferred 27-gauge needles, 29.6% 30-gauge needles and just 24% the market standard needles. this is a consensus in endodontics that favors the use of such smaller gauge needles (27 and 30), as they can pass through the intricate anatomy of root canal, especially in narrower canals. although the studies may differ slightly on exact needle size and needle types, they have shown that smaller needles are better able to penetrate and have a better ability to put the irrigant down in those hard-to-reach areas, with possibly higher-pressure requirements. for instance, boutsioukis et al (2007) [25], reported that smaller needles (30-gage) caused higher intra-barrel pressure while irrigating, that would then affect flow rate and delivery of the irrigant deeper into the canal, indicating that needle selection significantly affects the efficiency of irrigation. the results of the current study regarding the depth of needle penetration show that 39.8% of the respondents irrigated to 3 mm from the apex, 24.1% to 4 mm, and 14.8% to just 1 mm. the concepts agree with a more general understanding that root canal disinfection requires deeper penetration of the irrigation, and in the apical third where cleaning has been shown to be most difficult. according to previous work [26], needle of penetration depth to the apical third alters apical preparation and is dependent on the employed gauges. the findings of their study demonstrate the clinical significance of taking the depth of placement into account when disinfecting, especially in the presence of complex canal anatomy [26]. regarding irrigation duration, the current study identified various practices with 40.7% of the respondents irrigating 30 seconds 1 minute per canal. as previously published [27], irrigation volume and duration were studied to understand the impacts on cleaning of the two parameters and the results indicated that the cleaner of choice is dependent on the volume used with the total time of irrigation adding to the volume used in the optimization. the results of the present study, almost a quarter of respondents took more than 2 minutes using irrigation, agree with recommendations that a longer duration may lead to better efficacy, especially, in complex canals for full antimicrobial action [27]. evaluaaon of irrigaaon soluaons and acavaaon techniques in endodonac therapy vol 13, no 1 (2025) doi 10.5195/d3000/2025.921 http://dentistry3000.pitt.edu 6 for irrigant used volume 48.1% of the respondents used 5 ml, 25.9% used 2 ml, 14.8% used 10 ml. therefore, proper canal disinfection depends on the volume of irrigant. in wider or more curved canals, smaller volumes may not sufficiently irrigate the entire length of the canal. although studies have demonstrated that greater irrigation volumes will enhance the flushing capacity of irrigants, and ability to remove debris and bacteria, the application of large volumes of irrigants without proper technique carries the risk of irrigant extrusion beyond the apex and subsequent post treatment complications. consequently, the current study’s findings that higher volumes help in the removal of the smear layer and that biofilm are also supported by previous research [28] that showed that the volume used directly affects the removal effect of the smear layer and biofilm. finally, the results of the present study agree with the current literature as far as the implications of the needle gauge, penetration depth, irrigation duration and volume in root canal disinfection. they are tightly interlinked to such an extent that optimizing them all can greatly improve treatment outcome. it is proposed such a comparison with other studies [25,27] served to further drive the importance of selection of appropriate irrigation parameters with respect to individual case, such as canal anatomy and the clinical objectives. more research is needed to further refine guidelines regarding the irrigation techniques that maximize clinical efficiency and minimize complications. regarding the adjunct techniques used for irrigation, the results of this study on how the participants used them show that 45.4 percent used manual agitation. this matches the conventional manner of increasing the efficiency of the irrigation solution. manual agitation by gutta-percha cone is considered a simple, easy and reliable method to distribute irrigants throughout the root canal system. it is known to aid in debris removal and augment the chemical effects of the irrigant upon disinfection when used with sodium hypochlorite (naocl). nevertheless, the present study also demonstrated that a relatively small percentage of practitioners (24.1%) used ultrasonic activation, even though this has been proven to increase irrigant flow and reach apical and lateral canal difficult medication. ultrasonic activation is effective in improving post-operative outcomes and root canal cleanliness through facilitated deeper penetration of irrigants into the intricate canal anatomy [29]. combinations of techniques including sonic activation with manual agitation (6.5%) and ultrasonic activation with manual agitation (6.5%), also were reported; however, these combinations were less used. these findings indicate the use of multi-modal irrigation techniques with the combination of mechanical activation and other methods to increase the canal disinfection efficiency. hybrid approaches are proven to improve on the effectiveness of single methods of cleaning and disinfection, paying special attention to combined manual agitation with other activation techniques [30]. in addition, the study noted the use of negative pressure systems such as endovac and subsonic switch with manual agitation as at 2.8% of respondents. negative pressure irrigation systems have become accepted as good at clearing debris into the apical region, notably decreasing the risk of extruded irrigants. nevertheless, the costs associated with these systems and limited availability make these systems underused in the current study. later, plotino et al. (2016) [31] reported on the advantages of using negative pressure systems in removal of debris while minimizing the risk of extrusion. the finding of the present study is in line with global trend in which manual agitation is a staple and newer and more sophisticated systems such as ultrasonic and negative pressure irrigation are slowly supplanting it. the present study`s finding echoed sinusila and minu (2019) [32] that mechanical activation systems improve debridement and cleaning efficiency; while manual techniques still dominate, the interest in integrating them to improve the endodontic treatment outcomes increases. finally, we conclude that although the technology of manual irrigation agitation extends its use, more advanced devices, such as ultrasonic and negative pressure systems, are introduced. manual agitation is a practical and reliable approach that can still be used but advancements in new technologies have a great potential for improving root canal disinfection, especially for complex canal anatomies. future research and improved techniques in this area may improve the irrigation protocols and clinical outcome. conclusion based on the results, we conclude that sodium hypochlorite irrigation is still relied upon, but that the use of adjunctive activation methods is understood. although these advances have made stride, the widespread adoption of these technologies continues to be hindered by a lack of familiarity with new technologies, cost and device availability. this shows the importance of continuing education for dental professionals and increases the need for more integration of higher infrastructure endodontic systems. references 1. homan javaheri. irrigation in endodontic therapy, world dental 2020, 6th world congress on dentistry and dental materials, august 26-27, 2020, 04. 2. rai, s. (2020). the efficacy of ultrasonic irrigation in endodontics: a review. international journal of applied dental sciences, 6, 664-666. 3. lachker, f., bassim, n., abdallaoui, f. (2020). endodontic irrigation: an update. 4. tomer, a., cecilia, l., mushtaq, h., tyagi, a., ahlawat, s., jose, t., khandelwal, a. (2021). emerging trends in endodontic irrigation. international journal of applied dental sciences. 2021;7(1):468471. 5. sultana, m. (2020). irrigation in endodontics, 3, 4. 6. fahad, fatima & al-hashimi, raghad & hussain, munther. (2024). the advancement in irrigation solution within the field of endodontics, a review. journal of baghdad college of dentistry. 36. 54-69. 10.26477/jbcd.v36i1.3591. 7. barreto, j., de moura martins, l., chui, f., pontes, d., sponchiado, e., silva, l. (2022). the influence of final endodontic irrigation techniques and irrigants on the push-out resistance of glass fiber posts. general dentistry, 70 1, 46-50. 8. souza, raphael, castro, fábio, pires, oscar. (2022). research of the major methods and clinical outcomes of irrigation in endodontics: a systematic review. mednext journal of medical and health sciences. 3. 10.54448/mdnt22s311. 9. qutieshat, a., harthy, n., busaidi, s., sadoon, a., sayahien, d., sedqi, m., rashdi, s., ghammari, s. (2023). antimicrobial irrigation solutions in root canal treatment: a glance at the past, the present, and the future. the open dentistry journal. 2023, 17, e187421062306010. 10. ajmi, n.f.a., alshenaifi, m.k., binsalem, m.m., alahmary, k.a., shahrani, n.a.a., alasim, a.m., alotaibi, f.r., ali, a.s., attar, s.m., shahrani, n.m.a. 2024. microbial challenges and solutions in root canal therapy. international journal of community medicine and public health. 11, 9 (aug. 2024), 3672–3676. 11. ng wni, marruganti c, grandini s, neelakantan p. root canal debridement by negative pressure irrigation, ultrasonically activated irrigation and their combination. j oral sci. 2021 jun 29;63(3):286-288. 12. elsobky, randa, darrag, a.m., shaheen, n.a. (2021). effect of different final irrigation activation methods on root canal cleanliness. egyptian dental journal. 67. 2725-2734. 10.21608/edj.2021.68904.1559. 13. attavar, s., hegde, m., shetty, v., d’souza, n., shetty, s. (2023). microbiological evaluation of the disinfection of the root canal system using different irrigation protocol – an interventional study. endodontology, 35, 309 314. 14. kumar k, teoh yy, walsh lj. root canal cleaning in roots with complex canals using agitated irrigation fluids. aust endod j. 2023 apr;49(1):5665. 15. genc sen o, erdemir a. the effectiveness of three different irrigant activation methods in the elimination of enterococcus faecalis from root canals. photobiomodul photomed laser surg. 2024 oct;42(10):628-633. 16. ali a, bhosale a, pawar s, kakti a, bichpuriya a, agwan ma. current trends in root canal irrigation. cureus. 2022 may 8;14(5):e24833. evaluaaon of irrigaaon soluaons and acavaaon techniques in endodonac therapy vol 13, no 1 (2025) doi 10.5195/d3000/2025.921 http://dentistry3000.pitt.edu 7 17. pujari, m., das, m., das, a., kamath, d., henry, j., shyam, a., alhaleis, d. (2024). assessment of smear layer removal and penetration depth of root canal irrigant using different irrigation activation systems: a comparative study. the journal of contemporary dental practice, 25 4, 331-334. 18. raducka, martyna, piszko, aleksandra, piszko, paweł, jawor, natalia, dobrzyński, maciej, grzebieluch, wojciech, mikulewicz, marcin, skośkiewicz-malinowska, katarzyna. (2023). narrative review on methods of activating irrigation liquids for root canal treatment. 10.20944/preprints202305.2184.v1. 19. alzamzami zt, alqurashi aa, almansour la, ashi hm, abulhamael am, alghamdi ft, albahiti mt. current trends in irrigation solution and adjunct use during endodontic therapy among dental professionals in jeddah, saudi arabia: a cross-sectional study. cureus. 2022 dec 3;14(12):e32168. 20. kaplan, melek, erdogan, hilal. (2023). evaluation of the interaction between alternative endodontic irrigation solutions. kapadokya üniversitesi. 3. 39-50. 10.54995/asc.3.1.4. 21. segala, amanda & tasinaffo, denise, castro, fábio. (2024). clinical significance of the main methods of root canal irrigation in endodontics: a systematic review. mednext journal of medical and health sciences. 5. 10.54448/mdnt24s103. 22. sahar-helft et al., 2016: sahar-helft, s., sarp, a. s., stabholtz, a., gutkin, v., redenski, i., steinberg, d. (2016). comparison of positive-pressure, passive ultrasonic, and laser-activated irrigations on smear-layer removal from the root canal surface. photomedicine and laser surgery, 33(3), 129-135. 23. faus-llácer et al., 2020: faus-llácer, v., lunaroa, m., faus-matoses, i., ruiz-sánchez, c., zubizarreta-macho, á., sauro, s., & faus-matoses, v. (2020). comparative analysis of the smear layer removal efficacy of 17% ethylenediaminetetraacetic acid, 7% maleic acid, and 2% chlorhexidine using scanning electron microscope: an in vitro study. applied sciences, 10(20), 7033. 24. iandolo a, pisano m, abdellatif d, sangiovanni g, pantaleo g, martina s, amato a. smear layer and debris removal from root canals comparing traditional syringe irrigation and 3d cleaning: an ex vivo study. j clin med. 2023 jan 6;12(2):492. 25. boutsioukis, c., lambrianidis, t., kastrinakis, e., bekiaroglou, p. (2007). measurement of pressure and flow rates during irrigation of a root canal ex vivo with three endodontic needles. international endodontic journal, 40 7, 504-13. 26. essa, samara, saeed, razawa, bakr, diyar. (2024). effect of apical preparation on different needle depth penetration. erbil dental journal. 7. 54-59. 10.15218/edj.2024.7. 27. teja, k., ramesh, s., choudhari, s., özdemir, o., janani, k., jose, j., vasundhara, k., battineni, g. (2023). effect of various volume syringes on solution delivery time using conventional irrigation technique in 0.04 tapered preparations of single straight root canals: an ex vivo study. saudi endodontic journal, 13, 51 56. 28. al-ali, m., al-ibrahim, a., & al-ali, s. (2018). current trends in irrigation practice during endodontic treatment among general dental practitioners in saudi arabia. saudi endodontic journal, 8, 170 175. 29. topçuoğlu, h., topçuoğlu, g., & arslan, h. (2018). the effect of different irrigation agitation techniques on postoperative pain in mandibular molar teeth with symptomatic irreversible pulpitis: a randomized clinical trial. journal of endodontics, 44(11), 1451-1456. 30. gurtu, a., mehrotra, a. (2012). manual irrigation agitation techniques. 31. plotino, g., cortese, t., grande, n., leonardi, d., di giorgio, g., testarelli, l., gambarini, g. (2016). new technologies to improve root canal disinfection. brazilian dental journal, 27 1, 3-8. 32. susila, a., & minu, j. (2019). activated irrigation vs. conventional non-activated irrigation in endodontics – a systematic review. european endodontic journal, 4, 96 110. 803 final impact of so+ drinks consump4on on salivary ph levels vol 13 no 1 (2025) doi 10.5195/d3000.2025.803 h#p://den*stry3000.pi#.edu impact of soft drinks consumption on salivary ph levels doaa alghaffari1, hiba alogaidi2, yaseen h. motea3 1 university of baghdad college of dentistry, baghdad, iraq 2 university of jordan college of dentistry, amman, jordan 3 college of dentistry, al-bayan university, baghdad, iraq abstract objec&ve: the goal of current inves=ga=on was to determine the impact of some drinks on saliva ph values. methodology: the study included 20 healthy individuals (15 females and 5 males) with different ages. measurements aler 0, 10, 20, 30, 40, 50, and 60 minutes of drinking cola, pepsi, seven up, shani, and mirinda on saliva ph were determined. results: very similar ph value changes happened for all drinks, with a decrease in ph within 10 minutes aler drinking that return to original values aler 60 minutes. conclusion: the studied drinks cause a decrease in salivary ph that returns to original values within 60 minutes. keywords: diet; dental caries; risk cita9on: alghaffari d, et al. impact of soh drinks consump9on on salivary ph levels. den9stry 3000.1:a001 doi:10/5195/d3000.2025.803 received: december 21, 2024 accepted: february 9, 2025 published: february 14, 2025 copyright: ©2025 alghaffari d, et al. this is an open access ar9cle licensed under a crea9ve commons axribu9on work 4.0 united states license. email: yassen.h@albayan.edu.iq introduction dental decay is one of the most common oral health problems in the world, which arise from the interactions between bacteria and fermentable carbohydrate and could guide to destruct the hard part of tooth [1]. although it is preventable, but dental decay remains prevalent, it assuming about 3.5 billion individuals, where 40% from those cases remain untreated [2]. it commonly leads to discomfort and pain [3]. tooth decay is a chronic disorder and it is of interest identifying the individuals at higher risk of decay in advance as well as taken precautions [4,5]. saliva has crucial roles in the sustenance of mouth health. it is an important factor in oral shielding mechanisms. in a salutary mouth, saliva encloses antimicrobial enzymes, glycoproteins as well the primary electrolytes that protect the mucosa of mouth. saliva is essential for digestion, tasting and forming the bolus, preservation of teeth, in addition to antimicrobial impacts [6,7]. principal factors to consider when evaluating the roles of saliva in decay risks involve: organic and inorganic constituents, buffering capacity, ph, viscosity and saliva amount [8,9]. saliva plays an essential role in modulating remineralization as well as demineralization of teeth [10]. previous work attempted to assess the interplay between salivary ph, flow and alterations of plaque ph[11]. streptococcus mutans and streptococcus sobrinus are known as prime contributors to initiate of tooth decay for their potential to metabolize sugars into acids, which act to demineralize enamel. on the other hand, lactobacilli have a crucial role in the progress of dental decay. these bacterial species prosper in acidic environment, making them especially efficient in sustain and impact of so+ drinks consump4on on salivary ph levels vol 13 no 1 (2025) doi 10.5195/d3000.2025.803 h#p://den*stry3000.pi#.edu advance carious lesions after the initial enamel damages had taken placed [12,13]. usually, human's mouth is exposed to foods have ph levels deviate from natural saliva’s ph (6.5-7.5), where those deviations could lead to tooth damage or mucosal surfaces [14]. sweet soft drinks alter salivary ph and prompt bacterial propagation and are correlated with decay progression [15]. information on the impacts of consuming soft drinks is limited, so the current investigation aimed to determine the impacts of some drinks on saliva ph values. material and methods participants the present investigation included 20 volunteers (15 females, 5 males), with ages between 18 and 60 years. all participants provided written informed consent. participants were non-smokers and without oral, dental or any systemic diseases, in addition to not getting any medications potentially intervening with saliva releasing. collection of samples 350 ml cans of soft drink (pepsi, cola, seven, up mirinda and shani) from a baghdad company were used. a sample of saliva was placed in a urine collection cup or any available container similar in size and shape. then the can of soft drink was consumed by rinsing the mouth first, then drinking and collecting saliva in several periods (after drinking immediately and then after 10, 20, 30, 40, 50 and 60 minutes). ph measurements were subsequently obtained. evaluation of salivary ph a ph meter (isolab laborgerate gmbh ph meter, wertheim, germany) was used to estimate salivary ph. statistical analysis 1. ph readings were expressed as means and standard devia_ons (sd), and then they were compared by anova. results the mean saliva ph and sd for all drinks are displayed in table 1. the current study indicated that the lowest ph detected was after 10 minutes for all the drinks included in the study, and that the lowest ph (5.12± 0.12) was for the shani drink. however, the study showed that the ph values began to rise over time, returning to normal limits within 60 min for all drinks. time-dependent ph changes for all groups are shown in figure 1. table 1. ph values obtained in the study. figure 1. graphic representation of results. discussion the main factors that control stabilizing of enamel hydroxyapatite include the active concentration free of ca, phosphate, as well as fluoride in solution, in addition to ph of saliva[16]. soft drink name ph value mean ± sd bef ore after 0 min s 10 min s 20 min s 30 min s 40 min s 50 mi ns 60 mi ns pepsi 7.0 8± 0.1 2b 6.81 ± 0.16 b 5.1 7± 0.1 1 b 5.3 3± 0.0 4 a 5.8 1± 0.1 5 b 6.2 3± 0.1 9 c 6.7 7± 0.0 3 b 7.1 3± 0.1 0 a cola 7.0 9± 0.0 9 b 6.77 ± 0.11 5.1 5± 0.1 7 b 5.2 8± 0.0 8 ab 5.7 9± 0.1 6 b 6.1 5± 0.0 9 a b 6.7 1± 0.0 5 a 7.1 2± 0.1 7 a seven up 7.1 8± 0.1 5 c 6.69 ± 0.12 a 5.2 1± 0.0 7 c 5.3 1± 0.1 3c 5.6 9± 0.2 8 a 6.0 9± 0.1 5 a 6.6 9± 0.0 9 a 7.1 4± 0.1 9 a mirind a 7.1 9± 0.1 4 c 6.83 ± 0.22 ab 5.1 6± 0.1 0 b 5.2 5± 0.0 5 a 5.8 3± 0.1 2 b 6.1 8± 0.0 8 b 6.8 0± 0.1 4 bc 7.1 5± 0.2 2 a shani 7.0 2± 0.2 2 a 6.80 ± 0.07 b 5.1 2± 0.0 4 a 5.2 7± 0.1 8 ab 5.8 7± 0.1 4 c 6.7 1± 0.1 7 d 6.8 2± 0.1 2 c 7.1 9± 0.1 7 a different letters indicate significant differences by anova analysis. impact of so+ drinks consump4on on salivary ph levels vol 13 no 1 (2025) doi 10.5195/d3000.2025.803 h#p://den*stry3000.pi#.edu generally, dental enamel erosion developed from acidic condition of the mouth. diets and beverages have faint ph values, like citrus fruit, soft drinks, as well as some sport drinks can participate to this acidity state. the continuous exposure of enamel to those acidic conditions could disband the minerals, and lead to diminution of hardness and weakened structure [8,17]. the standard ph of saliva is between 6 to 7 [16]. when food is ingested, ph of plaque remains under the critical ph for round about fifteen to twenty mins and does not return to usual value until approximately forty minutes rinse [18]. given the importance of ph, this study aimed to evaluate the ph levels of saliva after consuming soft drinks, to highlight the negative effects of excessive consumption of these drinks. the current study indicated that the lowest ph was seen after 10 minutes for all the drinks included in the study, and that the lowest ph within this group was for the shani drink. however, the study demonstrated that ph value begins to rise over time, returning to normal limits within 60 minutes in all drink groups. the current findings are supported by a previous study conducted by tenuta et al, which showed that salivary ph reduced after consumption carbonated soft drinks due to their highest acids content, like phosphoric acid as well as citric acid; the recovery time for saliva to return to its normal ph is affected by acidity of the beverage. investigations have reported that the salivary ph can return to baseline within 30 seconds to 90 seconds after expectoration of the drink, depending on the type and acidity of the beverage [19]. the findings of the present research are consistent with previous one of hans et al., that indicated that consuming soft drinks lead to reduced ph of saliva, and the authors stated that while fluids leak rapidly from oral cavity, they have serious cariogenic and erosive potency [20]. the current study aligns with a previous investigation conducted by demir et al., [14], that highlighted the value of controlled consumption of drinks to avoid tooth decay, as some drinks could significantly lower salivary ph. acidic conditions can lead to dental erosion, tooth decay, in addition to bad breath [21]. consuming soft drinks, particularly carbonated ones, can have significant erosive impacts on dental enamel [22]. drops in ph levels as well the high acidity in these drinks cause enamel demineralization as well as escalated roughness of tooth surface [23]; consequently, these events could cause dental hypersensitivity and augmented risk of caries [22]. the buffering capacity of saliva is a key determinant in dental decay resistance. the buffered system withstands the variations in ph levels that happen impact of so+ drinks consump4on on salivary ph levels vol 13 no 1 (2025) doi 10.5195/d3000.2025.803 h#p://den*stry3000.pi#.edu with producing of acidic as well basic ions; if this capacity is defeated, and the balance of mineralization is undermined, ph levels reduce, which boosts escalated demineralization and progression of cavities [6]. proper diet selection is prerequisite to maintain oral health, highlighting the significance of buffering impact of saliva in ph maintenance [24]. accordingly, it is crucial to include anti-caries foods in the diet for preventing tooth decay. references 1. tafere, y., chanie, s., dessie, t., & gedamu, h. (2018). assessment of prevalence of dental caries and the associated factors among pa_ents agending dental clinic in debre tabor general hospital: a hospital-based cross-sec_onal study. bmc oral health, 18(1), 119. 2. guracho, t. t., atomssa, e. m., megersa, o. a., & tolossa, t. (2021). determinants of dental caries among adolescent pa_ents agending hospitals in west wollega zone, western ethiopia: a case-control study. plos one, 16(12), e0260427. hgps://doi.org/10.1371/journ al.pone.0260427 3. kazeminia, m., abdi, a., shohaimi, s., jalali, r., vaisiraygani, a., salari, n., & mohammadi, m. (2020). dental caries in primary and permanent teeth in children's worldwide, 1995 to 2019: a systema_c review and metaanalysis. head & face medicine, 16(1), 22. hgps://doi.org/10.1186/s1300 5-020-00237-z 4. bargeman ra, godoy gf, volpe ar (2009) the global increase in dental caries. a pending public health crisis. am j dent 22: 3-8 5. jiang r, yu j, islam r, li x, nie e. dental caries preven_on knowledge, aptudes, and prac_ce among pa_ents at a university hospital in guangzhou, china. medicina (kaunas). 2023 aug 28;59(9):1559. doi: 10.3390/medicina59091559. pmid: 37763677; pmcid: pmc10535681. 6. pedersen, a. m. l., sørensen, c. e., proctor, g. b., carpenter, g. h., & ekström, j. (2018). salivary secre_on in health and disease. journal of oral rehabilita_on, 45(9), 730–746. hgps://doi.org/10.1111/joor.1 2664 7. uchida h, ovig ce. novel impacts of saliva with regard to oral health. j prosthet dent. 2022 mar;127(3):383-391. doi: 10.1016/j.prosdent.2021.05.00 9. epub 2021 jun 16. pmid: 34140141; pmcid: pmc8669010. 8. arnold amd, marek ca (2002) the impact of saliva on pa_ent care: a literature review. j prosthet dent 88: 337-343. 9. animireddy d, reddy bekkem vt, vallala p, kotha sb, ankireddy s, mohammad n. evalua_on of ph, buffering capacity, viscosity and flow rate levels of saliva in caries-free, minimal caries and nursing caries children: an in vivo study. contemp clin dent. 2014 jul;5(3):324-8. doi: 10.4103/0976-237x.137931. pmid: 25191067; pmcid: pmc4147807. 10. farooq, i., & bugshan, a. (2021). the role of salivary contents and modern technologies in the remineraliza_on of dental enamel: a narra_ve review. f1000research, 9, 171. 11. antonelli, r., massei, v., ferrari, e., gallo, m., per_nhez, t. a., vescovi, p., pizzi, s., & mele_, m. (2024). salivary diagnosis of dental caries: a systema_c review. current issues in molecular biology, 46(5), 4234-4250. hgps://doi.org/10.3390/cimb4 6050258 impact of so+ drinks consump4on on salivary ph levels vol 13 no 1 (2025) doi 10.5195/d3000.2025.803 h#p://den*stry3000.pi#.edu 12. topcuoglu n, civci s, keskin f, kulekci g (2011) effect of chewing xylitol chewing gum on cariogenic microorganisms. ankem derg 25: 220-226. 13. bradshaw, d. j., & lynch, r. j. (2013). diet and the microbial ae_ology of dental caries: new paradigms. interna_onal dental journal, 63(suppl 2), 6472. 14. demir t, demir h, gorler o, ozden s, dogan do, et al. (2017) the effects of some drinks on saliva ph. j interdiscipl med dent sci 5:216. doi:10.4172/2376032x.1000216 15. dimopoulou, m., antoniadou, m., amargianitakis, m., gortzi, o., androutsos, o., & varzakas, t. (2023). nutritional factors associated with dental caries across the lifespan: a review. applied sciences, 13(24), 13254. https://doi.org/10.3390/app13 2413254 16. dowd fj (1999) saliva and dental caries. dent clin north am 43: 579-597. 17. de almeida pdv, gregio am, machado ma, de lima aa, azevedolr (2008) saliva composi_on and func_ons: a comprehensive review. j contemp dent pract 9: 72-80 18. gedalia i, bendat id, mosheh bs, shapira l (1991) tooth enamel softening with a cola type drink and rehardening with hard cheese or stimulated saliva in situ. j oral rehabil 18: 501-506. 19. tenuta, l. m. a., fernandez, c. e., brandão, a. c. s., & cury, j. a. (2015). titratable acidity of beverages influences salivary ph recovery. brazilian oral research, 29(1), 1-6. 20. hans, r., thomas, s., garla, b., dagli, r. j., & hans, m. k. (2016). effect of various sugary beverages on salivary ph, flow rate, and oral clearance rate amongst adults. scien_fica, 2016(1), 5027283. 21. pratha, a. a., & prabakar, j. (2019). comparing the effect of carbonated and energy drinks on salivary ph-in vivo randomized controlled trial. research journal of pharmacy and technology, 12(10), 46994702. 22. inchingolo, a. m., malcangi, g., ferrante, l., del vecchio, g., viapiano, f., mancini, a., inchingolo, f., inchingolo, a. d., di venere, d., dipalma, g., & patano, a. (2023). damage from carbonated sov drinks on enamel: a systema_c review. nutrients, 15(7), 1785. hgps://doi.org/10.3390/nu150 71785 23. barac, r., gasic, j., tru_c, n., sunaric, s., popovic, j., djekic, p., ... & mi_c, a. (2015). erosive effect of different sov drinks on enamel surface in vitro: applica_on of stylus profilometry. medical principles and prac_ce, 24(5), 451-457. 24. gokul, g., vishnupriya, v., & gayathri, r. (2016). a study on varia_ons of salivary ph with the intake of food. asian j pharm clin res, 9(suppl 1), 161163. 874 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.874 http://dentistry3000.pitt.edu oral contraceptives are associated with a higher risk of dental inflammatory and bleeding disorders asmaa sami jawad1 1college of den*stry, university of babylon, babylon, iraq abstract objec0ve: oral contracepdve pills (ocps) are a prevalent form of contracepdon, yet their associadon with potendal impacts on gingival and periodontal health remains a subject of interest. prolonged udlizadon of ocps, asributable to elevated pro-inflammatory cytokines and prostaglandins, has been demonstrated to heighten the risk of developing periodontal disease. this heightened risk is hypothesized to result from the presence of hormone receptor-posidve cells within gingival dssue. methods: the cross-secdonal study was conducted from january 2023 to march 24. drug-naïve controls (n = 50) were pardcipants who had not received any pharmaceudcal therapy, whereas cases were pardcipants who had received just ocps (ethinyl estradiol 0.03 mg or levonorgestrel 0.15 mg) for at least six months. results: women aged 20–45 were referred to the dendst for a check-up. there were no differences (p > 0.05) between the control and case groups for bmi, blood glucose, fasdng, or kidney funcdon tests. however, there were differences (p < 0.05) in cholesterol and liver enzyme levels. according to the study, 30% of cases had localized or chronic periodondds, while 60% of the pardcipants had a widespread aggressive disease. addidonally, research found higher but not stadsdcally significant amounts of plaque, gingivids, and bleeding in the case group. compared to non-users, case women had average probing depths that were deeper. conclusion: the study found that young women with aggressive gum disease, especially those with generalized aggressive periodondds, tended to use the pill. these women also had higher levels of plaque. open access cita%on: jawad as. (2025) oral contracep%ves are associated with a higher risk of dental inflammatory and bleeding disorders. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.874 received: march 5, 2025 accepted: april 26, 2025 published: june 9, 2025 copyright: ©2025 jawad as. this is an open access ar%cle licensed under a crea%ve commons ayribu%on work 4.0 united states license. email: dent.asmaa.sami@uobabylon.edu.iq introduc)on oral contraceptive pills (ocps) are a widely utilized and convenient method of contraception. their utilization has been associated with potential impacts on the progression of gingival and periodontal diseases in women [1]. the impact of oral contraceptives on gingival microvasculature is well-documented. human gingiva contains progesterone and oestrogen receptor-positive cells, and prolonged use of hormonal contraceptives has been shown to increase the risk of periodontal disease. elevated levels of pro-inglammatory cytokines and prostaglandins resulting from prolonged use and elevated dosage have been identigied as key factors in this increased risk [2]. hormonal contraceptives have been demonstrated to heighten the likelihood of alveolar osteitis in the aftermath of tooth extraction, concomitantly augmenting the prevalence of the candida species within the oral environment. these medications also exert an ingluence on the periodontium, manifesting in the recurrent emergence of gingivitis, yet do not induce alterations in the composition of the salivary microbiome [3,4]. hormonal intrauterine devices (iuds), which have seen a surge in popularity following the introduction of mirena® in 2001 [5], consist of polydimethylsiloxane for controlled release and 52 mg of levonorgestrel in a t-shaped form. oral contracepdves are associated with a higher risk of dental inflammatory and bleeding disorders vol 13, no 1 (2025) doi 10.5195/d3000/2025.874 http://dentistry3000.pitt.edu 2 this non-abortive technique works by stopping conception and lasts for at least give years [6]. the increasing utilization of hormonal contraceptives among women emphasizes the need for a systematic evaluation, as do their possible effects on the oral mucosa. this study's goal was to compile data regarding the possible oral symptoms that these contraceptive methods may cause in women of reproductive age. material and methods the department of obstetrics and gynecology at the university of kufa gave its approval for the cross-sectional study, which took place between january 2023 and march 24 [no. f (bors-gynecological science, res/ku/15-10, 2023-6479)]. the participants provided informed written consent. it has been established that cases of periodontal destruction diagnosed in obstructive and gynecology clinics have led to patients being referred to general dental clinics for further examination. the purpose of this referral is to congirm and validate the diagnosis of aggressive periodontitis through a specialized assessment and evaluation. furthermore, the collaboration between these clinics ensures a multidisciplinary approach to patient care, addressing both oral health and systemic conditions effectively. participants with normal menstrual periods, normo-androgenic traits (no alopecia, acne, or acanthosis), and no polycystic ovaries were recruited from the obstetrics and gynecology clinics. drug-naı̈ve controls (n = 50) were subjects not receiving any pharmaceutical therapy, whereas cases (n = 50) were subjects who had received just ocps (ethinyl estradiol 0.03 mg or levonorgestrel 0.15 mg) for at least six months. the thyroid, liver, and kidneys of every woman were functioning normally. the exclusion criteria included coronary artery disease, diabetes, thyroid dysfunction, hyperprolactinemia, androgen-producing tumors, non-classic adrenal hyperplasia, cushing's syndrome, pregnancy, breastfeeding, coagulation disorders, medication history, and smoking status. multivariate regression analysis was performed to adjust for potential confounders. all statistical tests were two-tailed, and congidence intervals (cis) were calculated at the 95% level. data visualization was conducted using graphs and charts generated in graphpad prism 8.1 to facilitate interpretation of the gindings. results using the provided referral screening form, general dentists identigied female patients between the ages of 20 and 45 and referred them for a thorough periodontal examination. the data relating to the basic clinical characteristics of the control subjects and the ocp-treated cases are shown in table 1 and figure 1. we found that there were insignigicant differences (p > 0.05) when compared the control bmi and blood glucose, fasting, renal function test (creatinine, blood urea) with case group, while signigicant differences (p < 0.05) were found in cases as compared to controls for lipid progile (cholesterol, tg), and liver enzymes (sgpt and sgot). most cases of generalized aggressive disease were observed in patients with the condition (60%), compared to 30% of people who have chronic or localized severe periodontitis. the case group tended to have higher levels of bleeding, plaque, and gingivitis. this disparity did not, however, reach statistical signigicance. in comparison to non-users, case women had mean probing depths that were deeper (3.3–1.0 versus 2.7–0.5 mm) (p = 0.04) (table 2 and figure 2). discussion a plethora of studies have been conducted on the utilization of ocps as a primary form of birth control, yielding a substantial body of evidence that supports this approach. a notable ginding from these studies is that women who have used ocps exhibit elevated average lipid levels in comparison to those who have not used them. moreover, there is a higher likelihood of abnormally high total cholesterol and tg levels among users of ocps [7,8]. age explained 34% of the observed variance, according to the analysis. however, because of the study population's extremely narrow age range, this result was determined to be statistically inconsequential. the current research has important ramigications for counseling young female patients with advanced periodontitis who are thinking about using contraceptives [9,10]. preshaw et al.'s result that oral contraceptives had no effect on gingival response might not apply to the current cohort because a signigicant percentage of research participants had been using contraceptives for a long period. further research is required, especially in light of preshaw et al.'s ginding that there were no discernible differences in gingival inglammation between pill users and non-users during a brief period of time [11]. according to taichman and ekland's research, there is no correlation between using oral contraceptives and a higher incidence of periodontitis or gingivitis. their gindings, which were based on data from 4,930 premenopausal women in nhanes i and 5,001 in nhanes ii, suggested a slight protective advantage of contraceptive medication. this research disproves previous theories on the damaging effects of oral contraceptives on the periodontium [12]. conclusions the study found that a lot of young women with aggressive periodontal disease, especially those diagnosed with generalized aggressive periodontitis, were using the pill. these women also had high levels of plaque. references 1. prachi s, jitender s, rahul c, jitendra k, priyanka m, disha s. impact of oral contraceptives on periodontal health. afr health sci. 2019;19(1):1795-800. 2. mistry s, bhowmick dc. oral contraceptive pill induced periodontal endocrinopathies and its management: a case report. european journal of dentistry. 2012;6(03):324-9. 3. rojo mg, lloret mrp, gironés jg. oral manifestations in women using hormonal contraceptive methods: a systematic review. clin oral investig. 2024;28(3):184. 4. hassan sm, al-jaf ana, hussien ya, awad sm, hadi nr. the potential antiviral activity of a novel pyrimidine derivative against herpes simplex virus type-1 (hsv-1). rev pharm. 2020;11:795-806. 5. hsia jk, creinin md, editors. intrauterine contraception. seminars in reproductive medicine; 2016: thieme medical publishers. 6. blumenthal pd, edelman a. hormonal contraception. obstetrics & gynecology. 2008;112(3):670-84. 7. hashemi sj, khezri r, saki n, nasehi n, hosseini sa, harizi m, rahimi z. association between oral contraceptives with lipid prodile: results from hoveyzeh cohort study (hcs). bmc womens health. 2023;23(1):552. 8. abbas al-huseini lm, al-mudhaffer rh, hassan sm, hadi nr. dmf ameliorating cerebral ischemia/ reperfusion injury in male rats. systematic reviews in pharmacy. 2019;10(1):206-13. 9. abbas sn, alwan ts, hassan sm. correlation between diabetes mellitus and dyslipidemia incidence in center of iraq. 2023. 10. ahjel sw, hassan sm, hussein sf, hadi nr, awad sm. antineoplastic effect of new synthesized compounds of 2-thiouracil sulfonamide derivatives against ovarian and breast carcinoma cells" in vitro study". systematic reviews in pharmacy. 2020;11(4). 11. preshaw pm, knutsen ma, mariotti a. experimental gingivitis in women using oral contraceptives. j dent res. 2001;80(11):2011-5. 12. taichman ls, eklund sa. oral contraceptives and periodontal diseases: rethinking the association based upon analysis of national health and nutrition examination survey data. j periodontol. 2005;76(8):1374-85. oral contracepdves are associated with a higher risk of dental inflammatory and bleeding disorders vol 13, no 1 (2025) doi 10.5195/d3000/2025.874 http://dentistry3000.pitt.edu 3 parameters mean ± sd (controls) mean ± sd (cases) p value significant age 34.32 ± 1.83 35.16 ± 1.86 0.932 no bmi (kg/m2) 22.51 ± 3.71 23.61 ± 3.53 0.911 no blood glucose fasting (mg/dl) 97.90 ± 10.98 99.82 ± 5.84 0.845 no cholesterol (mg/dl) 153.40 ± 10.2 189.24 ± 9.85 0.002 yes triglycerides (mg/dl) 111.32 ± 13.78 163.58 ± 12.89 0.000 yes s. creatinine (mg/dl) 0.88 ± 0.15 0.96 ± 0.50 0.981 no blood urea (mg/dl) 31.42 ± 4.60 27.71 ± 5.95 0.707 no sgpt (iu/l) 20.92 ± 4.06 59.15 ± 3.21 0.001 yes sgot (iu/l) 26.09 ± 3.54 57.56 ± 2.43 0.003 yes table 1. the anthropometric and biochemical parameters of the controls (i.e. those not previously exposed to the drug) and cases (i.e. those treated with ocp) were analyzed. figure 1. anthropometric and biochemical parameters of the control and case groups have been presented as mean values with standard deviadons (sd). parameters mean ± sd (controls) mean ± sd (cases) p value significant generalized aggressive (n) 40 60 0.010 yes localized aggressive (n) 70 30 0.000 yes plaque % 30.1 ± 4.2 69.9 ± 4.0 0.000 yes gingivitis % 20.7 ± 9.12 79.3 ± 9.18 0.000 yes bop % 53.5 ± 5.34 44 ± 5.31 0.173 no mean probing depth (mm) 2.7 ± 0.5 3.3 ± 1.0 0.021 yes table 2. a different type of periodondds that can be used to refer padents to the dendst for treatment. age bmi (k g/m 2) blood glucose fa stin g (m g/dl) cholesterol (m g/dl) trig lycerid es (m g/dl) s. c reatin ine (m g/dl) blood urea (m g/dl) sgpt (iu /l) sgot (iu /l) 0 50 100 150 200 250 the anthropometric and biochemical parameters co nc en te ra tio n control case # # # # ✱ ✱ ✱✱ * significant # insignificant oral contracepdves are associated with a higher risk of dental inflammatory and bleeding disorders vol 13, no 1 (2025) doi 10.5195/d3000/2025.874 http://dentistry3000.pitt.edu 4 figure 2. periodondds assessments. generalize d aggressive (n ) localize d aggressive (n ) plaque % gingivitis % bop % mean probing depth (m m) 0 20 40 60 80 periodontal disease assessment ✱ ✱ ✱ ✱ ✱ # control case * significant # insignificant 818 final study the effect of chemical solu3ons on disinfec3on in prosthodon3cs clinic using digital ultrasonic cleaner vol 13 no 1 (2025) doi 10.5195/d3000.2025.818 h#p://den*stry3000.pi#.edu study the effect of chemical solutions on disinfection in prosthodontics clinic using digital ultrasonic cleaner hala m. al-kateb college of dentistry, university of mosul, iraq abstract objec&ve: the disinfec,on measures used by den,sts are inadequate and require the educa,on and enhancement of dental prac,,oners’ skills to improve the safety of pa,ents receiving treatment at dental offices. in a medical se?ng, the remaining bacteria that naturally exist on a pa,ent’s body, known as endogenous flora, oden leads to the transmission of infec,ons. the objec,ve of this study was to iden,fy a simple and precise technique for disinfec,ng a prosthodon,cs clinic through the u,liza,on of a chemical solu,on. methods: swabs were collected from the pa,ents who wear a complete denture who were contaminated, both before and ader being exposed to a steriliza,on system. by measuring the turbidity and absorbance at 620nm using a uv spectrophotometer, we can determine the characteris,cs of the broth ader it has been incubated at 36.5°c for 24 hours. u,lized a solu,on containing 0.5% sodium hypochlorite and 2% chlorhexidine. the bacterial growth was subjected to two rounds of exposure to two chemical disinfectants, each for dura,ons of 90 seconds and 180 seconds respec,vely. results: the bacterial growth exhibited a decrease ader being treated to a chemical disinfectant in a ,me-dependent way. conclusion: this study determined that the chemical solu,on effec,vely reduces bacterial development within a short period of ,me, providing the most convenient, rapid, and precise approach for disinfec,ng dental materials. keywords: chemical disinfectant, dental materials, prosthodon,cs clinic. cita9on: al-kateb hm. (2025) study the effect of chemical solu9ons on disinfec9on in prosthodon9cs clinic using digital ultrasonic cleaner. den9stry 3000. 1:a001 doi:10.5195/d3000.2025.818 received: december 30, 2024 accepted: february 12, 2025 published: february 14, 2025 copyright: ©2025 al-kateb hm. this is an open access ar9cle licensed under a crea9ve commons azribu9on work 4.0 united states license. email: halamowwafaq@uomosul.edu.iq introduction dental prostheses that can be easily taken out are a significant therapy choice for individuals aged 65 and above who have missing teeth. the advent of digitalization and advancements in material science have led to the development of numerous innovative techniques and clinical protocols for manufacturing dentures [1]. ensuring the cleanliness of dentures is crucial for preserving the well-being of the underlying tissues. the task of cleaning dentures can be particularly difficult for elderly individuals, especially those who have limited ability to use their hands effectively. therefore, it is frequently recommended to employ chemical cleaning methods, such as employing denture cleaning solutions, either on their own or in combination with mechanical cleaning. these denture cleaners employ diverse methods to eliminate stains, grime, and bacteria from the surfaces of dentures [2,3]. ultrasonic devices are mechanical aids generally used by professionals. the mechanical cleansing activity of the device is complemented with the concomitant use of a chemical solution. ultrasound has two mechanisms of action, the first being the movement of liquid resulting from sound waves study the effect of chemical solu3ons on disinfec3on in prosthodon3cs clinic using digital ultrasonic cleaner vol 13 no 1 (2025) doi 10.5195/d3000.2025.818 h#p://den*stry3000.pi#.edu transferred to the liquid (vibration), and the second, the collapse of bubbles formed by vibration of the unit. denture cleansers possess a bleaching property that causes a modification in the color of the denture basic materials, resulting in an aged and weathered appearance. the occurrence of these color fluctuations often leads to patient dissatisfaction and concerns over the functionality of the prosthesis [3-5]. the surfaces in a dental operatory that require disinfection or sterilization are classified into three categories: critical, semicritical, and noncritical. according to the cdc, critical objects are defined as items that have a significant potential to cause illness if they come into touch with any disease-causing microorganism. these goods include surgical instruments, dentistry equipment, and ultrasound probes [6,7]. it is necessary to sanitize all essential items, usually by applying heat. heat-sensitive operating rooms can undergo treatment with a liquid chemical sterilant that contains specified disinfection concentrations of phenol solutions, glutaraldehyde, peracetic acid, and hydrogen peroxide [8]. equipment that makes direct contact with mucous membranes or damaged skin is classified as semi-critical. semi-critical objects typically do not necessitate the use of highlevel disinfectants. operatory equipment that directly touches intact skin is categorized as noncritical. computers, countertops, and floors are examples of this categorization. mops and disinfectant wipes are suitable for disinfecting noncritical areas. the process of on-contact disinfection requires the use of disposable wipes that can only be used once. additionally, when it comes to cleaning noncritical areas of the floor, it is often necessary to mop the floor using a liquid material [9]. research indicates that dentists’ disinfection processes are inadequate, highlighting the urgent need to educate and enhance the expertise of dental practitioners to enhance the protection of patients seeking treatment at dental offices. it is widely recognized that the remaining indigenous microorganisms, or bacteria present on a patient’s body, often contribute to the spread of infections in a medical setting. when the naturally occurring microorganisms in our body come into touch with surfaces, equipment, or dental and medical instruments that have not been properly cleansed, they can create an environment that promotes the growth of different viruses and bacteria. disinfection is considered crucial in a medical setting to counteract the emergence of germs from anatomical cavities. [10,11] in this study, a chemical solution is utilized to examine the impact of sterilization on dental prints. materials and methods in this work, we employed lb broth (made according to the method outlined by macwilliams and liao [12]) to evaluate the efficacy of an ultrasonic cleaner in achieving sterilization. we investigated the use of several solutions and varied durations of cleaning. the patients were randomly assigned to groups each using one of the following hygiene methods (n=40) swabs were collected from a complete denture who was contaminated, both before and after being exposed to a sterilization system. ultrasonic vibration (ultrasonic cleaner, modelo2840 d – odontobrás ind. e com. equip. méd. odont. ltda, ribeirão preto, sp, brazil) for 15 min, performed by a professional by measuring the turbidity and absorbance at 620nm using a uv spectrophotometer, the broth can be analyzed after being incubated at 36.5°c for 24 hours. utilized a solution containing 0.5% sodium hypochlorite and 2% chlorhexidine. the bacterial study the effect of chemical solu3ons on disinfec3on in prosthodon3cs clinic using digital ultrasonic cleaner vol 13 no 1 (2025) doi 10.5195/d3000.2025.818 h#p://den*stry3000.pi#.edu growth was subjected to two rounds of exposure to two chemical disinfectants, each lasting 90 seconds and 180 seconds respectively. statistical analysis the collected data were analyzed by using paired t. test compared before and after exposed to disinfectant. the significant is with p< 0.05. results the results demonstrated a significant decrease (p < 0.05) in bacterial growth after exposure to disinfectants, which was dependent on the duration of exposure. this is illustrated in figure 1. prior to exposure to the disinfectant, the bacterial growth in the swab showed clear turbidity (+ve), whereas no turbidity was observed in the swab after exposure to the disinfectant. figure 2 displayed the analysis results of optical density, which was used to quantify the density of bacterial growth. the results indicated a significant drop (p < 0.05) in bacterial growth following exposure to disinfectants, with the extent of decline dependent on the duration of exposure. however, there is no discernible disparity in the efficacy of hypochlorite sodium solution and chlorhexidine in eradicating germs. figure 1. bacterial growth before and after exposed to chemical disinfectants. figure 2. bacterial growth before and after exposed to chemical disinfectants. discussion while the probability of transmission of infection from dental operatory equipment to a patient was formerly considered to be low, it does really exist. to mitigate the risk, prioritize the implementation of authorized infection control protocols, including the utilization of protective barriers, optimal interface disinfection, and sterilization techniques. [13] multiple research studies have been conducted and published on topics such as air needle pollutants and decontamination, microbial contamination and dental unit disinfection, and decontamination of principal impression materials. however, there have been relatively few studies that specifically focus on surface disinfection protocols, especially those related to dental operatory surfaces. one potential reason is that the cdc categorized operatory surfaces as non-essential due to the absence of direct contact between the surfaces and the oral cavity [10-15]. the commonly employed chlorinated disinfectant, hypochlorite, is accessible in liquid 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 2 % cl 90 sec. l clhx 2 % cl 180 sec. l clhx 0.5 % naclo 90 sec. 0.5 % naclo 180 sec. before exposure after exposure ba ct er ia l g ro w th study the effect of chemical solu3ons on disinfec3on in prosthodon3cs clinic using digital ultrasonic cleaner vol 13 no 1 (2025) doi 10.5195/d3000.2025.818 h#p://den*stry3000.pi#.edu (e.g., sodium hypochlorite) and solid (e.g., calcium hypochlorite) states. in the united states, chlorine products refer to aqueous solutions containing sodium hypochlorite, with concentrations ranging from 5.25% to 6.15%. these solutions are commonly referred to as home bleach. these antibacterial agents have a wide range of effectiveness against different types of bacteria. they do not leave any harmful residues, are cost-effective, act rapidly, and are not influenced by the level of water hardness. additionally, they have a reduced level of harm and effectively remove desiccated and solidified organisms as well as biofilms from surfaces [16,17]. previous investigations have examined the impact of sodium hypochlorite solution and 2% chlorhexidine on the eradication of enterococcus and bacillus bacteria. [18] various chemicals have been suggested as efficient irrigant therapies for disinfecting root canals. sodium hypochlorite is often used in endodontic therapy due to its strong antibacterial properties and ability to break down organic tissues [19]. however, there is no agreement on the most effective concentration of sodium hypochlorite. based on a study, the utilization of sodium hypochlorite at concentrations of 0.5% and 3% resulted in a substantial reduction of germs within the root canal [20]. a separate investigation revealed that after employing chemomechanical endodontic preparation in conjunction with 2.5% sodium hypochlorite, there was a significant reduction in the bacterial variety within the root canal [21]. the exceptional organic solvent properties of sodium hypochlorite enhance its effectiveness as an irrigant agent by killing microorganisms. however, sodium hypochlorite can potentially cause irritation to the tissues surrounding the apex of the tooth, especially when used in large amounts [22,23]. the study’s findings revealed no statistically significant disparities in the antimicrobial efficacy between sodium hypochlorite solution and chlorhexidine. this finding was documented in the research [24,25]. prosthodontics is recognized as one of the dental specialties that routinely neglects to employ cross-infection control measures during clinical and laboratory operations. the surveyed dentists expressed skepticism over the probability of cross-contamination between the dental clinic and laboratory. dental laboratory environments might be susceptible to microbial contamination through many routes, such as the utilization of felt discs and pumice during the polishing procedure, as well as direct contact with contaminated hands. additional forms of contamination arise when prostheses are brought to dental clinics for alterations or repairs, as bacteria from the patient’s mouth may contaminate these materials during certain stages of treatment [26]. variances in prosthetic surfaces affect the ability of microbes to adhere. the presence of surface roughness on prosthetic surfaces can lead to small injuries in oral tissues. surface roughness also facilitates the colonization of microbes, which indirectly leads to tissue injury [27]. it is crucial to prioritize the use of disinfectant to maintain cleanliness and decontaminate dental prints or other equipment. the crucial aspects are the procedures employed and the specific chemicals that provide rapid and precise disinfection. conclusion this study determined that the chemical solution may effectively reduce bacterial development in a short period of time, providing the most convenient, rapid, and precise approach for disinfecting dental materials. study the effect of chemical solu3ons on disinfec3on in prosthodon3cs clinic using digital ultrasonic cleaner vol 13 no 1 (2025) doi 10.5195/d3000.2025.818 h#p://den*stry3000.pi#.edu references 1. ettinger r. oral health needs of the elderly--an international review. commission of oral health, research and epidemiology report of a working group. int dent j. 1993; 43(4): 348-54. available from: https:// pubmed.ncbi.nlm.nih.gov/8276519. 2. lim t. w., burrow m. f., mcgrath c. efficacy of ultrasonic home-care denture cleaning versus conventional denture cleaning: a randomized crossover clinical trial. j. of dent. 2024; 148, 105215. https://doi.org/10.1016/j.jdent.2024. 105215 3. saraç d, saraç ys, kurt m, yüzbaşioğlu e. the effectiveness of denture cleansers on soft denture liners colored by food colorant solutions. j prosthodont. 2007; 16(3): 185-91. doi: https://doi.org/10.1111/j.1532849x.2 006.00170.x. 4. ozyilmaz oy, kara o, akin c. evaluation of various denture cleansers on color stability and surface topography of polyetherketoneketone, polyamide, and polymethylmethacrylate. microsc res tech. 2021; 84(1): 3-11. doi: https://doi.org/10.1002/jemt.23558. 5. zidan s, silikas n, haider j, yates j. effect of cleansers on the colour stability of zirconia impregnated pmma bio-nanocomposite. nanomaterials (basel). 2020; 10(9): 1757. doi: https://doi.org/10.3390/nano100917 57. 6. almoula n., al-omari a. effectiveness of sterilization by microwave irradiation on polyvinyl siloxane contaminated with candida albicans: an in vitro study. al–rafidain dent j. 2024; 24(1), 81-86. http://doi:10.33899/rdenj.2024.134 911.1170 7. almoula, n., & al-omari, a. (2024). effectiveness of microwave sterilization on surface roughness of polyvinyl siloxane impression material. alal–rafidain dent j. 2024; 24(1), 7380. http://doi:10.33899/rdenj.2024.134 961.1171 8. ahmed, m., hasan, n., saeed, m., & abdulmajeed, a. (2024). response of exposed pulp to capping with mineral trioxide aggregate mixed with hyaluronic acid as a water subshtute. al–rafidain dent j. 2024; 24(1), 191202. hip://doi: hips://doi.org/10.33899/rdenj.2024. 147417.1250 9. marei hf, alshaia a, alarifi s, almasoud n, abdelhady a. effect of steam heat sterilization on the accuracy of 3d printed surgical guides. implant dent. 2019; 28(4): 372-77. doi: https://doi.org/10.1097/ id.0000000000000908. 10. matsuda jk, grinbaum rs, davidowicz h. the assessment of infection control in dental practices in the municipality of são paulo. braz j infect dis. 2011; 15(1): 45-51. doi: https://doi.org/10.1016/s14138670(11)70139-8. 11. block ss. disinfection, sterilization, and preservation. lippincott williams & wilkins; 2001. 12. macwilliams mp, liao mk. luria broth (lb) and luria agar (la) media and their uses protocol. 2006. available from:https://asm.org/getattachment/ 5d82aa34b514-4d85-8af3aeabe6402874/lb-luria-agarprotocol3031.pdf. 13. araujo mw, andreana s. risk and prevention of transmission of infectious diseases in dentistry. quintessence int. 2002; 33(5): 376-82. available from: https://www.quintessencepublishing.com/deu/en/ article/839204/quintessenceinternational/2002/05/riskandprevention-of-transmission-ofinfectious-diseasesin-dentistry. 14. williams hn, johnson a, kelley ji, baer ml, king ts, mitchell b, hasler jf. bacterial contamination of the water supply in newly installed dental units. quintessence int. 1995; 26(5): 331-7. available from: https://www.quintessencepublishing.com/deu/en/ article/838352/quintessenceinternational/1995/05/ bacterialcontamination-of-the-water-supplyin-newlyinstalled-dental-units. 15. bock jj, fuhrmann ra, setz j. the influence of different disinfectants on primary impression materials. quintessence int. 2008; 39(3): e93-8. available from: https:// www.quintessencepublishing.com/deu/en/article/83997 5. 16. kohn wg, harte ja, malvitz dm, collins as, cleveland jl, eklund kj. guidelines for infection control in dental health care settings--2003. j am dent assoc. 2004; 135(1): 33-47. doi: https://doi.org/10.14219/jada.archi ve.2004.0019. 17. mupparapu m, kothari krm. review of surface disinfection protocols in study the effect of chemical solu3ons on disinfec3on in prosthodon3cs clinic using digital ultrasonic cleaner vol 13 no 1 (2025) doi 10.5195/d3000.2025.818 h#p://den*stry3000.pi#.edu dentistry: a 2019 update. quintessence int. 2019; 50(1): 58-65. doi: https://doi.org/10.3290/j.qi.a41337. 18. subha n, prabhakar v, koshy m, abinaya k, prabu m, thangavelu l. efficacy of peracetic acid in rapid disinfection of resilon and guttapercha cones compared with sodium hypochlorite, chlorhexidine, and povidone-iodine. j endod. 2013; 39(10): 1261-4. doi: https://doi.org/10.1016/j.joen.2013 .06.022. 19. zehnder m. root canal irrigants. j endod. 2006; 32(5): 389-98. doi: https://doi.org/10.1016/j.joen.2005 .09.014. 20. wong dt, cheung gs. extension of bactericidal effect of sodium hypochlorite into dentinal tubules. j endod. 2014; 40(6): 825-9. doi: https://doi.org/10.1016/j.joen.2013 .09.045. 21. rôças in, siqueira jf, jr. identification of bacteria enduring endodontic treatment procedures by a combined reverse transcriptasepolymerase chain reaction and reverse-capture checkerboard approach. j endod. 2010; 36(1): 4552. doi: https://doi.org/10.1016/j. joen.2009.10.022. 22. gernhardt cr, eppendorf k, kozlowski a, brandt m. toxicity of concentrated sodium hypochlorite used as an endodontic irrigant. int endod j. 2004; 37(4): 272-80. doi: https://doi.org/10.1111/j.01432885.2004.00804.x. 23. de sermeño rf, da silva la, herrera h, herrera h, silva ra, leonardo mr. tissue damage after sodium hypochlorite extrusion during root canal treatment. oral surg oral med oral pathol oral radiol endod. 2009; 108(1): e46-9. doi: https://doi.org/10.1016/j.tripleo.200 8.12.024 24. rôças in, siqueira jf, jr. comparison of the in vivo antimicrobial effectiveness of sodium hypochlorite and chlorhexidine used as root canal irrigants: a molecular microbiology study. j endod. 2011; 37(2): 143-50. doi: https://doi.org/10.1016/j.joen.20 10.11.006. 25. jiang yp, liu x, zhu k. letter to the editor: extracorporeal shock wave therapy combined with oral medication and exercise for chronic low back pain. arch phys med rehabil. 2022; 103(7): 1499. doi: https://doi.org/10.1016/j.apmr.2 022.02.022. 26. cotrim lef, santos emd, jorge aoc. procedimentos de biossegurança realizados por cirurgiõesdentistas e laboratórios durante a confecção de próteses dentárias. rev odontol unesp. 2013; 30(2): 233-44. available from: https://revodontolunesp.com. br/journal/rou/article/5880179a7f8c 9d0a098b47f3. 27. williams dw, lewis ma. isolation and identification of candida from the oral cavity. oral dis. 2000; 6(1): 3-11. doi: https://doi.org/10.1111/j.16010825. 2000.tb00314.x. 28. pitt wg, ross sa. ultrasound increases the rate of bacterial cell growth. biotechnol prog. 2003;19:1038-44 29. shay k. denture hygiene: a review and update. j contemp dent pract. 2000; 1:1-8 952 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.952 http://dentistry3000.pitt.edu knowledge, attitudes and practices of dental and medical students about hepatitis b noor na&k raheem, abeer salah salman, athraa ali mahmood, wassan nori, hashim mueen hussein college of den*stry, mustansiriyan university, iraq abstract objec&ve: this study aimed to evaluate and compare hepajjs b's knowledge, aptudes, and pracjces among dental and medical students in iraqi universijes. material and methods: a cross-secjonal observajonal study was conducted from 1 july to 1 september 2024 among second, third, fourth, and fixh-stage medical and dental students. for survey distribujon, the quesjonnaire consisjng of four domains submized to an internet pla[orm (google forms). undergraduate students were invited to parjcipate in the research via a survey link published on specific groups of students in the social networking pla[orms viber and whatsapp. results: 426 students responded to the quesjonnaire. there were stajsjcally significant differences in frequency were seen (p < 0.001) regarding source of your knowledge about hbv, route of infecjon transijon, receiving vaccinajon, doses of vaccine, and periodic checking of jter values of the vaccine. frequency of answers for quesjons regarding immunizajon programs and strategies against hb infecjon were not stajsjcally different. addijonally, dental students had more frequent posijve responses for use of gloves, changing gloves, use of face mask, changing face masks, use of disposable gown, and use of disposable instruments for the pajents posijve or suspected hb virus (95.4%, 94.6%, 93.8%, 73.0%, 74.3%, and 96.3%, respecjvely) whereas medical students less frequently used those (67.6%, 62.7%, 59.5%, 24.9%, 47.6%, and 85.9%, respecjvely). conclusion: the study revealed that although both medical and dental students were knowledgeable about hbv transmission, vaccinajon uptake, parjcularly among medical students, was inadequate. open access cita%on: raheem nn, et al. (2025) knowledge, a<tudes and prac%ces of dental and medical students about hepa%%s b. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.952 received: may 29, 2025 accepted: july 6, 2025 published: august 13, 2025 copyright: ©2025 raheem nn, et al. this is an open access ar%cle licensed under a crea%ve commons auribu%on work 4.0 united states license. email: noorna%kraheem@uomustansiriyah.edu.iq introduc)on hepatitis is a potentially life-threatening liver disease and a global healthcare concern. hepatitis b virus (hbv) is the leading cause of this disease [1]. acute viral hepatitis is a disease that starts with bodily aches, darker urine, nausea/vomiting, loss of appetite, low-grade fever, and general malaise before developing into jaundice [2]. at the same time, chronic infection can lead to liver cirrhosis and hepatocellular carcinoma [3]. an estimated 296 million persons globally were believed to have a chronic hbv infection, and 1.5 million new infections occurred each year [4]. in general, hbv is infectious and can be transferred by transfusion therapy, the improper use of treatment-based injections, tattooing, transmission from maternal-to-infant, and sexual activity [5]. the practice of modern medicine has contributed signioicantly to the rise and spread of bloodborne diseases such as human immunodeoiciency virus (hiv) and hepatitis b (hbv) due to olaws in instrument sterilization techniques and poor handling of hospital waste. ten to 20% of healthcare waste is considered risky [6]. occupational accidents that expose healthcare providers to bloodborne viruses are a signioicant public health risk. dental healthcare professionals are especially vulnerable to hepatitis because of their frequent contact with sharps [7, 8]. because the dental clinic is a high-risk setting for cross-infection, controlling the dissemination of hepatitis b is critical. dental students should know the dangers of conducting dental processes and take adequate precautions when practicing [9]. direct contact with blood, saliva, or other bodily oluids, needle injury, or mucosal contact from splashes, oluid droplets, and aerosols, in addition to indirect contact with infected tools, equipment, or instruments, could all result in infection transmission during dental procedures [7]. hygienic hand care, correct glove use, and safe disposal of sharp tools are all part of the centers for disease control and prevention's (cdc) recommended measures for reducing the spread of bloodborne diseases such as hbv [10]. because some exposures cannot be avoided, the primary sources of protection are immunization and good post-exposure treatment. as a result, understanding hbv infection, transmission, and schemes for disease prevention is critical for future dentists' safety [11]. knowledge, aptudes and pracjces of dental and medical students about hepajjs b vol 13, no 1 (2025) doi 10.5195/d3000.2025.952 http://dentistry3000.pitt.edu 2 inadequate knowledge among healthcare workers in low/middle-income nations leads to inefoicient attachment to safety standards, provoking the hbv burden [12]. improving understanding of hbv is critical for lowering rates of infection among healthcare workers [13]. knowledge assessments are often utilized for evaluating population awareness of illness concepts like causes and symptoms. conversely, attitudes are determined by a convoluted interconnection of emotions, values, and individual beliefs. at the same time, practice is described as the cumulative behaviors taken to prevent the incidence or spread of illness [14]. students in medicine and dentistry, integral to the health care system, are the oirst line of connect between patients and medical care, and their contact with patients and contaminated instruments may increase the risk of hbv infection, particularly if safety cautions are not stalked accurately. at the start of their clinical years, they are required to engage in patient care activities. few studies have been conducted to discover medical and dental students' knowledge, practice, and attitude about hbv in iraq. this study aimed to assess and compare students' awareness of hbv and attitude and practice among different medical and dental colleges in many iraqi universities. material and methods this cross-sectional study included dental and medical undergraduate students in government and private colleges in baghdad city (mustansiriyah, baghdad, al iraqia, alraheed, alhadi, alfarahidi colleges of dentistry) and mustansiriyah, baghdad and ashur medical colleges. this study was conducted from 1 july 2024 to 1 september 2024 among the second, third, fourth, and oifthstage students. the total number of dental students (group 1) was 241 (40 males, 201 females), and the total number of medical students (group 2) was 185 (63 males, 122 females) (figure 1). ethical approval approval was received from the ethical committee, college of medicine, mustansiriyah university with irb number 63 on 27 june 2024. all students were given details about the study and guaranteed conoidentiality. the online questionnaire used to collect data for this study included an opening sentence regarding the study's aim and approval that it did not have any personal information; hence, answering the questionnaire was considered an agreement to participate in the study. it included 4 domains, consisting of 23 items. the domains included sociodemographic information (3 items) including age, gender, and stage; the second domain consisted of 8 items to determine awareness of hbv infection and vaccination [15]; the third one consisted of 6 items about attitudes toward hbv infection and prevention, and the last one (6 items) is clinical practices about hbv infection and prevention [16]. study sample the present study uses the following formula that is used commonly in statistics to calculate the sample size for an inoinite population: n= !".$ ('($) *" here, n denotes the required number size. z: the z-score (1.96) associated with the desired conoidence level (95%). p: estimated prevalence or proportion in the target population (set at 0.5). e: margin of error (0.05). n = ('.+,) !.-.. .('(-..) (-.-.)! n= /.01', .-.". -.--". = 384.16 thus, the required sample size was approximately 385 students. all 426 study participants were encouraged to reply willingly, using digital devices via online survey link published on specioic groups of students on social networking platforms (viber and whatsapp). for survey distribution, the questionnaire was submitted to an internet form google forms. students were asked to answer yes or no to attitude and practice questions. concerning student awareness of hbv transmission and vaccination-related questions, some answers were yes or no, while the others were short choices, and incomplete responses were excluded. a pilot study was carried out involving around 10% of the target population to evaluate the clarity, practicality, and reliability of the survey instrument. participants were selected from similar student demographics and were not included in the main study. the objectives of the pilot study included assessing the comprehensibility of the questions, the time taken to respond, and the internal consistency of the survey, especially in relation to knowledge and attitude areas. feedback from participants regarding any unclear or ambiguous questions was collected, which guided subsequent revisions aimed at enhancing the content validity of the instrument. statistical analysis after examining all the responses sent by the students, the collected data was sent to excel for analysis and then exported to the data editor of spss (statistical package for the social sciences version 28, usa). a descriptive statistical data analysis and a chi-square test were conducted to compare categorical variables. a p-value of ≤ 0.05 was considered signioicant for statistical analysis. results a total of 23 questionnaires were collected from 426 dental and medical students [females (56.6%) and males (43.4%)] among the 2nd, 3rd, 4th, and 5th year students. the age range of the participants was from 18 to 24. the number of dental students (group 1) was 241 [females=201 (83.4%), males = 40 (16.6%)], and the number of medical students (group 2) was 185 [females = 122 (65.9%), males = 63 (34.1%)]. the demographic data are shown in table 1. table 2 shows that the student's awareness of h.b. infection and vaccination was good for most questions. however there are statistically highly signioicant differences in frequency were seen among student groups (p < 0.001) regarding the following: source of your knowledge about hbv, route of infection transition, how it is transmitted, receive vaccination, doses of vaccine, and the periodic checking of the titer values of h.b. vaccine in the blood; while there are statistically nonsignioicant difference in frequency among students groups for the following: immunization programs and strategies against hb infection, necessary to get immunized against hepatitis b even if infection control policies and sterilization process is carried out properly, and essential to get immunization against hepatitis. 93 % of the medical students and 94.2 % of the dental students gave a positive reply when asked, "it is necessary to get immunized against hbv even if proper infection control and sterilization process is carried out." however, only 29.7% of medical students receive vaccinations. on the other hand, approximately 57.3% receive vaccination among dental students. among respondents, 74.1% of the medical students and 91.3% of the dental students think that periodic checking of the titer values of the vaccine is necessary to prevent infection. additionally, 97.8% of the medical students and 96.3% of the dental students think immunization is required. the percentage of responses on attitude questionnaires between groups is shown in table 3. the fear of infection with hbv from practice was the highest (84.9%) among medical students and were (79.3%) among dental students, with nonsignioicant differences among groups (p=0.138). as for treating the patients infected with hbv, the percentages were as follows (59.5% ) among medical students and (43.6%) among dental students, with highly signioicant differences among student groups (p=0.001). knowledge, aptudes and pracjces of dental and medical students about hepajjs b vol 13, no 1 (2025) doi 10.5195/d3000.2025.952 http://dentistry3000.pitt.edu 3 in addition, the result showed that 45.4% of the respondents said medical students treat patients infected with hbv safely, while merely 41.9% of the participants' dental students thought this. 85.9% of medical students and 90% of dentists worry about being infected with hbv by patients. thus, 84.9% of the medical students and 90% of the dental students, with signioicant differences (p < 0.001), consider asking the patient about his infection with hbv a primary priority when taking his medical history in the case sheets before starting treatments. under the domain of questions on clinical practice relating to the protective and preventive measures to avert transmission of h.b. infection, the percentage of responses is shown in table 4. the result showed that for taking protective measures during diagnosis, treatment procedures, and working on patients, dental students scored the highest with positive responses of 95.4%, 94.6%, 93.8%, 73.0%, 74.3%, and 96.3%, and medical students had the minimum percentage with 67.6%, 62.7%, 59.5%, 24.9%, 47.6%, and 85.9% of the respondents (for used gloves, change gloves, used face mask, change face mask, disposable gown, and used disposable instrument for the patients positive or suspected for hb, respectively). discussion this study highlights the knowledge and attitude of both dental and medical students regarding the silent killer or silent epidemic hbv by public health professionals, students’ knowledge about chronic infection, safety measures, and precautions that should be taken in their clinical practice to minimize the increased risk burden of infections. female participation for both dental and medical dental students reported a high percentage (83.4%, 65.9%) respectively, as females are more likely to respond to onlinetype surveys and tend to adopt more healthseeking behavior than males [17,18]; most respondents are dental students, as they seem more interested than medical students. awareness of hbv infection and vaccination status: the dental students have suitable knowledge about hbv from different sources and use safety precautions that raise awareness regarding the route of infection and transmission [19,20]. dentists could be at higher risk of acquiring viral infections, hepatitis b along with other infections as hepatitis c, herpes simplex virus infection, tuberculosis, inoluenza, rubella, and human immunodeoiciency virus [21]. as dentists had more contact with the saliva, blood, and secretions of nasopharyngeal by mouth-nose absorption or accidental needle injury and other cutting instruments used for various dental procedures are the most common exposure routes to hbv; thus, dentist had a 3 to 4-times more likely to be at risk of hbv infection than in the overall population; in spite of that the present study shows a low prevalence of dental students receiving vaccination against hbv, similar to the oindings of [22], but it remains better than the medical students who have a 2–10 fold greater risk to get the hbv infection compared to others. nearly half of the iraqi dental students received vaccination more than in other studies which is higher than previous studies that reported only 42% of dental students were hbv immunized, compared to a lower number of vaccinated medical students.; disagreeing with bhattarai et al. 2014 [23], holenarasipur et al. 2013 [24] stated that the majority of dental students felt it was necessary to get immunization against hbv during practice as dental and oral surgeons had the highest rates of hbv infection among all healthcare workers both groups showed high responses to the necessity of routine check of titers value of post-vaccination blood tests to check for a sufoicient antibody response to hbv [25]. two important methods for preventing hepatitis virus are vaccination and the use of properly sterilized instruments, but most of the study participants did not follow the vaccination schedule properly due to their negligence and other reasons as knowledge and attitude mentioned in our study [26]. a dentist considers every patient as a potential carrier of hepatitis this explains the high percentage of responses of both groups about fear of infection with hbv from their practice. in the current study, the overall oindings demonstrated the gaps in preventive measures between the studied groups in taking personal protective measures during routine diagnosis and treating patients, as the dental students reported the highest responses. this oinding is consistent with [27]; straub et al. [28] stated that the challenges of insufoicient personal protective equipment (ppe) availability and the insufoicient time for supervision make medical students face weaknesses in the proper utilization of ppe. the dentistry clinic must ensure routine and regular disposal of needles, sterilize instruments, and follow all safety protocols (such as wearing ppe and disinfecting all surfaces, tools and equipment after each patient) (29) in teaching dental hospitals the dental students and staff always advised to secure primary protective gears as disposable surgical mask, cap, disposable gloves and scrubs with protective eye/face shield. study limitations and confounders other limitation of this study was that online surveys were only done by students with internet access, but not all of them might have looked intently, realized, and provided honest and accurate responses; specioically, there was no direct supervision of students or face-to-face questionnaires. increasing the number of dental and medical colleges, besides increasing the number of students, increases the necessity to conduct future similar studies for all students. this study offers important insights into the differences in knowledge, practices, and attitudes regarding hbv among medical and dental students in iraqi universities. a notable strength of the research is its identioication of signioicant disparities in infection control practices, with dental students showing greater compliance with protective measures. conclusions the present study concludes a good level of awareness and knowledge of the hbv: route of infection and transmission of both dental and medical students, but unsatisfactory oindings about receiving vaccination and completing the vaccine schedule, especially in medical students. dental and medical schools should reolect more on hbv teaching and advise students to complete the vaccine schedule in preclinical years so that they can protect themselves and their patients and play a critical role in increasing awareness among healthcare staff. conflict of interest none. funds none. acknowledgements the authors thank mustansiriyah university (www.uomustansiriyah.edu.iq), baghdad, iraq, for its support in the present work. author’s contribu)ons a: conception and design: nnr, ass, aam b: analysis and interpretation of the data: nnr, ass, aam c: drafting of the article: nnr, ass d: critical revision of the article for important intellectual content: aam, hmh e: final approval of the article: nnr, ass, aam, wn, hmh f: provision of study materials or samples: nnr, ass, wn g: statistical expertise: nnr, wn h: obtaining of funding: nnr, ass i: administrative, technical, or logistic support: aam, wn, hmh knowledge, aptudes and pracjces of dental and medical students about hepajjs b vol 13, no 1 (2025) doi 10.5195/d3000.2025.952 http://dentistry3000.pitt.edu 4 j: collection and assembly of data: nnr, ass references 1. systematic review and meta-analysis of hiv, hbv and hcv infection prevalence in sudan. badawi mm, atif ms, mustafa yy. virol j. 2018; 15, 148. 2. hepatitis b awareness among the dental professionals, students and dental hygienists in a dental school – an epidemiological study. tripati s, kamala bk, kiran k. int j contemp dent. 2011;2:4550. 3. chronic hepatitis b infection: a review. tang lsy, covert e, wilson e, kottilil s. jama. 2018;319(17):1802-13. 4. world health organization. hepatitis b geneva: who; 2022 [[online] available at: https://www.who.int/news-room/factsheets/detail/hepatitis-b. accessed 23 may 2023. 5. effect of gender and age on the knowledge, attitude and practice regarding hepatitis b and c and vaccination status of hepatitis b among medical students of karachi. pakistan. khan n, ahmed sm, khalid mm, siddiqui sh, merchant aa. j. pak. med. assoc. 2010; 60(6): 450-455. 6. indian perspective journal of hospital infection. taneja n, biswal m. safe disposal of infectious waste. 2009 ;62(4): 525–26. 7. competency assessment for infection control in the undergraduate dental curriculum. milward mr, cooper pr. eur. j. dent. educ.2007; 11(3): 148-154. 8. infection control practices among undergraduate students from a private dental school in india. kumar s, sharma j, duraiswamy p, kulkarni s. rev. odonto ciênc. 2009; 24(2): 124-128. 9. knowledge, attitudes, and practice regarding infection control measures among dental students in central india. singh a, purohit bm, bhamba a, saxena s, singh a, gupta a. j. dent. educ. 2011; 75(3): 421-427. 10. hepatitis b and c infection among dentists– risk, prevention, and management. sheikh an, siddiquee b, chahande j. journal of advances in dental practice and research. 2022;1(1):31-6. 11. hepatitis b: knowledge, preventive attitude and vaccine status of dental students and interns at umm al-qura university, makkah, saudi arabia. aiiii ik, al-zahrani ak, al-sulimani rs, alqahtani m. journal of umm al-qura university for medical sciences.2020; 6. 10.54940/ms45403892. 12. awareness and prevalence of needle stick injury among nursing students at king abdulaziz university. almuslami ok, fadul ss, munshi ss, sangoof fm, mersal na. evidence-based nursing research. 2022;5(1):14-23. 13. sero-prevalence of hepatitis b virus infection and associated factors among health care workers and medical waste handlers in primary hospitals of north-west ethiopia. yizengaw e, getahun t, geta m, mulu w, ashagrie m, hailu d, tedila s. bmc res notes. 2018 jul 3;11(1):437. doi: 10.1186/s13104-018-3538-8. 14. seroprevalence of hepatitis b among healthcare workers in asia and africa and its association with their knowledge and awareness: a systematic review and meta-analysis. maamor nh, muhamad na, mohd dali ns, abdul mutalip mh, leman fn, aris t, lai nm, abu hassan mr. front public health. 2022 apr 28;10:859350. doi: 10.3389/fpubh.2022.859350. 15. knowledge and attitude of dental students toward hepatitis b virus and its vaccination a cross-sectional study. benarji ka, anitha a, suresh b, aparna v, praveena a, penumatsa la. j oral maxillofac pathol. 2021 sep-dec;25(3):553. 16. knowledge, attitudes, and practices toward the prevention of hepatitis b virus infection among medical students in medina city, kingdom of saudi arabia. albadrani ms, abdulaal am, aljabri am, aljohani ss, aljohani sf, sindi ma, jan hk, alsaedi h, alamri wm, alharbi am, alraddadi aa. cureus. 2023 nov 15;15(11):e48845. 17. "does gender iniluence online survey participation? a record-linkage analysis of university faculty online survey response behavior" smith g. eric document reproduction service no. ed 501717 (2008). 18. an event history analysis of the gender effects of survey participation in a probability-based multi-wave panel study with a sequential mixedmode design. becker r. gender and survey participation: methods, data, analyses,2022 16(1), 30. doi:https://doi.org/10.12758/mda.2021.08 19. a. knowledge, awareness and practice towards hepatitis b infection among dental students in the international university for science and technology, syria kadou m, dannan. j comm med and pub health rep.2024; 5(01): https://doi.org/10.38207/jcmphr/2024/jan. 20. knowledge about hepatitis b virus and relevant safety precautions among dental students in kurdistan region, iraq. kareem fa, mohammad rf, zardawi fm, gul ss. j environ public health. 2022 sep 18;2022:8516944. doi: 10.1155/2022/8516944. pmid: 36172400; pmcid: pmc9510674. 21. hepatitis b in health care workers: indian scenario. singhal v, bora d, singh s. j lab physicians 2009; 1:41–8. 22. hepatitis b vaccination and immunization of dental students,lira als, martins krj. rev odontol bras central 2021; 30(89): 180-194. doi 10.36065/robrac.v30i89.1445. 23. hepatitis b vaccination status and needle-stick and sharps-related injuries among medical school students in nepal: a cross-sectional study. bhattarai skcs, pradhan pm, lama s, rijal s. bmc res notes. 2014; 7, 774. https://doi.org/10.1186/1756-0500-7-774 24. awareness regarding hepatitis b immunization among preclinical indian dental students. vasanthakumar ah, d’cruz am. j oral health oral epidemiol 2013; 2(2): 97-101. 25. dentists and hepatitis b vaccination – an in vivo national study. yared g, al khatib a, al manhal w, yared n, younes r. international arab journal of dentistry. 2023; vol. 14: iss. 2, article 17. 26. knowledge, attitude and awareness of hepatitis b vaccine among medical and dental students, karthiknv, rani l, brundha mp. biosc. biotech. res. comm. 2020; special issue vol 13 no (7) pp120-124. 27. dental professionals’ use of personal protective equipment during covid-19: a cross-sectional study in china. wan q, han l, yang x, yu s, zheng x. front. public health. 2023; 11:1183580. doi: 10.3389/fpubh.2023.1183580. 28. personal protective equipment and medical students in times of covid-19: experiences and perspectives from the iinal clerkship year. straub j, franz a, holzhausen y, schumann m, peters h. bmc med educ.2023; 23, 806. doi.org/10.1186/s12909-023-04784-2. 29. transmission of blood-borne pathogens in us dental health care settings: 2016 update. cleveland jl, gray sk, harte ja, robison va, moorman ac, gooch bf. j am dent assoc. 2016;147(9):729738. doi:10.1016/j.adaj.2016.03.020. knowledge, aptudes and pracjces of dental and medical students about hepajjs b vol 13, no 1 (2025) doi 10.5195/d3000.2025.952 http://dentistry3000.pitt.edu 5 figure 1. flowchart of study participants' recruitment. table 1. distribujon of groups in the study and demographic data. student groups no. (%) age (%) gender stage dental students (g1) 241(56.6 %) 18= 11(4.6%), 19= 45(19.0%), 20=74(31.2%), 21=48(20.3%), 22= 33(13.9%), 23= 14(5.9%), 24= 12(5.1%) females = 201 (83.4%) males = 40 (16.6%) 2= 71(29.5%), 3=75(31.1%), 4=65(27.0%), 5=30(12.4%) medical students (g2) 185 (43.4 %) 18= (31.5%), 19= 16(8.0%), 20=49(24.4%), 21=82(40.8%), 22= 33(16.4%), 23= 12(6.0%), 24= 6(3.0%) females = 122 (65.9%) males = 63 (34.1%) 2=21(11.4%), 3=30(16.2%), 4=111(60.0%), 5=23(12.4%) no: number, %: percentage knowledge, aptudes and pracjces of dental and medical students about hepajjs b vol 13, no 1 (2025) doi 10.5195/d3000.2025.952 http://dentistry3000.pitt.edu 6 table 2. comparison of percentages of awareness of hbv infecjon and vaccinajon quesjonnaire between groups. ques&onnaire choices no. (%) of groups x2 p-value g1 g2 1. the source of your knowledge about hbv infecjon and vaccinajon syllabus 86 (35.7%) 113(61.1%) 29.326 <0.001 *** media 103 (42.7%) 40(21.6%) others 52 (21.6%) 32(17.3%) 2. the most common route of infecjon is a transijon from denjst /doctor to pajent 20 (8.3%) 23(12.4%) 16.730 <0.001 *** pajent to pajent 30(12.4%) 48(25.9%) pajent to denjst/doctor 191(79.3%) 114(61.6%) 3. how it (hbv) transmized saliva 55 (22.8%) 16 (8.6%) 30.986 <0.001 *** blood 135 (56.0%) 143(77.3%) sneezing and cough 27 (11.2%) 5 (2.7%) from mother to infant 10 (4.1%) 6 (3.2%) sexual route. 14 (5.8%) 15(8.1%) 4. do you receive vaccinajon yes 138 (57.3%) 55(29.7%) 32.015 <0.001 *** no 103(42.7%) 130(70.3%) 5. no. of doses of vaccine you receive if you have it one 55 (32.0%) 30(16.2%) 30.499 <0.001 *** two 71 (41.3%) 130(70.3%) three 43 (25.0%) 23(12.4%) four 3 (1.7%) 2(1.1%) 6. if you did not menjon the possible reason lack of opportunity 118 (49.2%) 118(63.8%) 16.577 0.002*** lack of awareness 28 (11.7%) 16(8.6%) fear of side effects 37(15.4%) 19(10.3%) the vaccine is non-available. 12(5.0%) 16(8.6%) non-suscepjbility of infecjon 45(18.8%) 16(8.6%) 7. with proper infecjon control and sterilizajon strategies. do you think is it necessary to get immunized against hbv? yes 226(94.2%) 172(93.0%) 0.250 0.617ns no 14(5.8%) 13(7.0%) yes 220 (91.3%) 137(74.1%) 22.896 <0.001 *** knowledge, aptudes and pracjces of dental and medical students about hepajjs b vol 13, no 1 (2025) doi 10.5195/d3000.2025.952 http://dentistry3000.pitt.edu 7 8. do you think it is necessary to check the jter values of the hb vaccine in the blood periodically? no 21 (8.7%) 48(25.9%) ***: p < 0.001 using the chi-squared test, ns: nonsignificant, x2: chi-square table 3. comparison of percentages of aptude quesjonnaire between groups. ques&onnaire choices no. (%) of group x2 p-value g1 g2 9. do you fear about infecjon with hbv from your pracjce yes 191(79.3%) 157(84.9%) 2.204 0.138 ns no 50(20.7%) 28(15.1%) 10. can you treat pajents infected with hbv yes 105(43.6%) 110(59.5%) 10.573 <0.001 *** no 136(56.4%) 75(40.5%) 11. is it safe to treat pajents infected with hbv safely? yes 101(41.9%) 84(45.4%) 0.521 0.470 ns no 140 (58.1%) 101(54.6%) 12. do you have the right to know your pajent's hbv infecjon status? yes 207(85.9%) 165(89.2%) 1.028 0.311 ns no 34(14.1% ) 20(10.8% ) 13. do you worry about being infected with hbv by pajents? yes 217(90.0%) 159(85.9%) 1.695 0.193 ns no 24(10.0% ) 26(14.1% ) 14. do you consider asking the pajent about his infecjon with hbv as a primary priority when taking his medical history in the case sheets before starjng treatments? yes 230(95.4%) 157(84.9%) 14.062 <0.001 *** no 11(4.6%) 28(15.1%) ***: p < 0.001 using the chi-squared test, ns: nonsignificant, x2: chi-square knowledge, aptudes and pracjces of dental and medical students about hepajjs b vol 13, no 1 (2025) doi 10.5195/d3000.2025.952 http://dentistry3000.pitt.edu 8 table 4. comparison of percentages of items on your clinical pracjce quesjonnaire between groups. ques&onnaire choices no. (%) of group x2 p-value g1 g2 15. do you wear latex gloves during diagnosis and treatment?? yes 230(95.4%) 125(67.6%) 58.523 <0.001 *** no 11(4.6%) 60(32.4%) 16. do you change gloves between pajents during diagnosis and treatment? yes 228(94.6%) 116(62.7%) 68.532 <0.001 *** no 13(5.4%) 69(37.3%) 17. do you wear a face mask during diagnosis and treatment?? yes 226(93.8%) 110(59.5%) 73.964 <0.001 *** no 15(6.2%) 75(40.5%) 18. do you change face masks between pajents? yes 176(73.0%) 46(24.9%) 97.289 <0.001 *** no 65(27.0%) 139(75.1%) 19. using disposable gown during diagnosis and treatment?? yes 179(74.3%) 88(47.6%) 31.909 <0.001 *** no 62(25.7%) 97(52.4%) 20. using disposable instruments for the pajents suspected or posijve for hbv?? yes 232(96.3%) 159(85.9%) 14.780 <0.001 *** no 9(3.7%) 26(14.1%) ***: p < 0.001 using the chi-squared test, ns: nonsignificant, x2: chi-square 967 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.967 http://dentistry3000.pitt.edu radiographic study of osteoporosis detection in the jaw hussein haleem jasim college of den*stry, university of wasit, iraq abstract objec0ve: to invesagate the reliability of panoramic radiographs in detecang osteoporosis disease. material and methods: three hundred paaents, regardless of gender, were included, aged between 40 and 50 years. one hundred fiqy paaents had confirmed systemic osteoporosis by dxa scan (experimental group), while the other 150 paaents had no history of osteoporosis (healthy control group). the study used the ramus index to calculate the grey mean values on panoramic images, which correlates with bone mineral density (bmd) compared with the data of dual-energy x-ray absorpaometry (dxa) as the gold standard reference. results: the findings demonstrated that panoramic images and the ramus index were a reliable indicator of osteoporosis (p<0.001) (80% sensiavity, 90% specificity, and 85% accuracy). conclusion: quanataave greylevel evaluaaon of panoramic images demonstrated diagnosac validity. open access cita%on: jasim hh. (2025) radiographic study of osteoporosis detec%on in the jaw. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.967 received: june 21, 2025 accepted: june 26, 2025 published: august 6, 2025 copyright: ©2025 jasim hh. this is an open access ar%cle licensed under a crea%ve commons arribu%on work 4.0 united states license. email: halmhanawi@uowasit.edu.iq introduc)on “osteoporosis is a metabolic-related disease marked by decreased bone mineral density (bmd)”, leading to elevated bone fragility and an elevated risk of fractures [1]. this disorder is caused by disequilibrium between bone formation and resorption, reducing bone mineral composition in the whole body, considerably affecting bone microstructure and increasing vulnerability to fractures in the hip, femur, wrist, spine and other situations [2,3] states that osteoporosis is a condition where the value of bone mineral density is equal to 2.5 standard deviations (or more) below the mean level for young adults. the diagnosis is based on several risk factors, with evaluation of the lumbar spine and femur considered the most signikicant regions [3,4]. over 200 million individuals are influenced by “osteoporosis” worldwide, with one-fifth of males and one-third of females over the age of fifty vulnerable to fractures [5]. with the continued aging of populations worldwide, the rate of osteoporosis will increase, creating a serious economic challenge [5]. osteoporosis reflects a classic multicausal disorder where genetic tendency, exogenous effects, endogenous biological variables, and lifestyle preferences contribute to differentiate individual vulnerability. [6] the normal path of bone mass follows a predictable manner, peaking about age 30, with men typically achieving greater peak bone mass than women, due to this peak, both genders facing an age-related regression in bone density at a rate of 0.5 to 1% per year [7]. on the other hand, the clinical presentation of osteoporosis shows high gender predilection, with epidemiological studies suggesting the prevalence rates of 2 to 8% in men over 50 years compared to rates of 33 to 47% in women for the same age group [8]. the physiopathology of osteoporosis, especially in post-menopausal women, has often been related to endocrine disturbances. the main role of estrogen insufficiency and subsequent secondary hyperparathyroidism has been strongly supported, with these hormonal alterations commonly aggravated by insufficient dietary intake and the frequent prevalence of vitamin d inadequacy in aging people [6]. estrogen exerts its protective effects on bone mainly through estrogen receptor-alpha, which keeps the critical equilibrium between bone formation and resorption. decreased signaling of estrogen receptor-alpha leads to interruption of this balance, causing inordinate bone resorption [9]. the fundamental pathway for this process includes complicated interactions between erα and the primary osteogenesis pathway, including “insulin-like growth factor (igf) and wnt/β radiographic study of osteoporosis detecaon in the jaw vol 13, no 1 (2025) doi 10.5195/d3000/2025.967 http://dentistry3000.pitt.edu 2 catenin signaling systems”. estrogen insufficiency-induced negative regulation of erα diminishes numerous activities of stem cells in bone marrow, decreasing their proliferative efficiency, f-actin stress fiber formation and alkaline phosphatase activity. moreover, erα acts as a crucial controller of primary osteogenic genes such as osteocalcin, osteopontin and osterix [10-12]. experimental evidence from ovariectomized rat samples has supported these findings, showing an apparent association between osteoporosis due to sex hormones insufficiency and decreased mineral density of the jawbone, with major consequences for quality and regenerative capability of alveolar bone [13-17]. in addition to these hormonal mechanisms, several additional risk factors promote the development of osteoporosis, long term glucocorticoid use causes bone loss through osteoblast inhibition [18,19], furthermore, smoking negatively influences bone metabolism through several pathways [20]. insufficient calcium use disrupts bone mineralization [4], and diabetes mellitus changes bone quality due to high blood sugar, creating harmful glycated proteins. [20]. osteoporosis of the jaw has several adverse effects on oral health, delaying healing following tooth extraction, orthodontic treatment, periodontal disease progression and dental implant success [5,21,22]. osteoporosis treatments such as bisphosphonates can enhance jawbone mineral density, which in turn has risks involving osteonecrosis of the jaw related to bisphosphonates [23]. the mandible, as a component of the axial skeleton, revealed exclusive properties that allow the identification of osteoporosis-related alterations through radiographic analysis. studies confirm that osteoporosis causes a decline in bone mineral density, influencing densitometric, morphometric and architectural qualities of the jaw. frequent radiographic signs include extensive radiolucency of the mandible and maxilla, reduced thickness of the mandibular inferior cortex, cortical weakening, and amplified prominence of anatomical landmarks like the nasal cavity and maxillary sinus [24]. radiography is used as a diagnostic procedure for the assessment of dentition and jawbone anomalies in practical dentistry [25,26]. dental radiographs, especially intraoral and panoramic images, have displayed the capability of identifying early signs of bone diseases. panoramic radiography is one of the most prevalent radiographic method due to its ability to get extensive visualization of the maxillofacial structure [27,28]. radiographic bone measurement indices, such as the panoramic mandibular index, mandibular cortical width, and cortical index have been suggested as reliable indices for evaluating low bone mineral density using dental panoramic radiographs [28]. the current study was designed to assess the reliability of panoramic radiographs in detecting “osteoporosis”. material and methods the study was carried out at several specialized dental centers, in baghdad, iraq, between april 2024 and may 2025. the study included 300 paaents of both genders aged between 40 and 50 years who were referred for panoramic images. one hundred fiqy paaents had confirmed systemic osteoporosis by dxa scan (experimental group), while the other 150 paaents had no history of osteoporosis (healthy control group). all panoramic images were analyzed to assess bone density using imagej soqware [version 2.3.0, 2023; nih, usa]. [29] the ramus index was used for bone density analysis of the jaws by measuring the grey values on panoramic images through histogram analysis of pixel intensiaes. this was achieved by calculating the mean grey values (mgv) in the selected region of the mandible on panoramic images. a square (box) of 30 x 30 mm was drawn on the panoramic image in the mandibular region, and then the mean grey values within this box were analyzed. the superior side of this square was drawn to be tangent to the mandibular sigmoid notch, the anterior side extended toward the anterior edge of the ramus, the posterior side extended toward the posterior margin of the ramus, and the inferior side of the square reached close to the mid-ramus region. radiographic data from panoramic images were analyzed to compare localized bone density with systemic dxa results (figure 1). the parameters of the dxa scan used in the study as the gold standard reference are dxa sensiavity: 90%, specificity: 95%, and accuracy: 93%. [30, 31] all parameters of the panoramic x-ray system were adjusted to be similar to all panoramic images (kvp: 80 kv, ma: 10 ma and exposure ame: 15 seconds). bone density evalua0on aparameters of localized bone density on panoramic images: • normal bone density (high density radiopaque): mean grey values range between 160255. • osteopenia (moderate density less radiopaque): mean grey values range between 100159. • osteoporosis: low density (more radiolucent): mean grey values range between 0–99. b“dual-energy x-ray absorpaometry (dxa)” [32]: the diagnosis of osteoporosis via “dxa (dualenergy x-ray absorptiometry)” was based on t-scores, according to criteria of the world health organization (who) as follows: • normal: t-score ≥ -1.0. bone density is within 1 standard deviation (sd) of the young adult mean. • osteopenia or early osteoporosis (low bone mass): t-score between -1.0 and -2.5. bone density is 1 to 2.5 sd below the young adult mean. indicates increased fracture risk. • osteoporosis: t-score ≤ -2.5 bone density is 2.5 sds or more below the young adult mean. high fracture risk. figure 1. ramus index (ri) applied in the study. inclusion criteria o paaents of both genders aged between 40 and 50 years. o paaents had confirmed systemic osteoporosis by dxa test, included in the experimental group. o paaents had no history of osteoporosis in the healthy control group. exclusion criteria o paaents with metabolic bone disorders other than osteoporosis (e.g., paget’s disease, osteomalacia). radiographic study of osteoporosis detecaon in the jaw vol 13, no 1 (2025) doi 10.5195/d3000/2025.967 http://dentistry3000.pitt.edu 3 paaents with systemic diseases that may affect bone metabolism or turnover (e.g., diabetes mellitus and chronic kidney disease). menopausal women. pregnant or lactaang women. paaents on coracosteroids or other medicaaons known to alter bone metabolism within the past 6 months. paaents treated with osteoporosis medicaaons within the past 6 months. paaents with periodonaas, trauma, malignancy or pathology affecang the maxilla or mandible. paaents with dental implants, prostheses, or restoraaons interfering with bone evaluaaon. alcoholic paaents and heavy smokers. paaents on chemotherapy or radiotherapy. sta0s0cal analysis chi-square test: used to assess the reliability of panoramic images in detecang osteoporosis compared to dxa (gold standard). cramér’s v test: to measure the strength of associaaon. roc curve analysis – determine the sensiavity and specificity of using grey values to classify osteoporosis by plorng a receiver operaang characterisac (roc) curve. confidence intervals (95%): to quanafy the precision of diagnosac performance metrics (sensiavity, specificity, and accuracy). results the findings demonstrated a strong correlaaon between the mean grey values (mgv) on panoramic radiographs and the actual bone density status measured by dxa (the gold standard). [chi-square staasac (χ²) =169.28, p-value < 0.001]. the cramér’s v test = 0.75, confirming the high reliability for bone density measurements on panoramic images for the detecaon of osteoporosis. in the osteoporoac group, 80% of paaents had low grey mean values (more radiolucent), consistent with the expected low bone density in osteoporosis. this means that panoramic images correctly idenafied a large number of osteoporoac paaents. in addiaon, 16.7% of osteoporoac paaents had moderate grey mean values (less radiolucent), suggesang osteopenia or expected future osteoporosis, and only 3.3% of osteoporoac paaents had high grey mean values, suggesang a normal density despite having osteoporosis according to dxa. in the control group, 66.7% % of individuals had high grey mean values, confirming their normal bone density. only 23.3% showed moderate grey mean values, and 10% demonstrated low grey mean values, suggesting a small rate of false posiaves where the panoramic images suggested bone loss, even though the dxa did not. the staasacs test revealed that the diagnosac efficiency of the procedure has moderate sensiavity, high specificity, and good accuracy, but it is staasacally less effecave than dxa in all parameters (p < 0.05). sensiavity for panoramic imaging (80%) is significantly lower than dxa (90%) [z = −3.16, p < 0.00], specificity for panoramic imaging (90%) is lower compared to dxa (95%) [z = −2.24, p = 0.025], confirming reduced specificity. in the same way, accuracy for panoramic images (85%) is staasacally lower than dxa’s (93%) [z = −2.89, p = 0.004]. confidence intervals 95% enhanced confirmaaon of these findings. sensiavity for panoramic images (73.6–86.4%) does not reach dxa’s standard sensiavity (~ 90%), proving its inferiority. specificity (85.2–94.8%) minimally overlaps with dxa’s specificity (~ 95%), indicaang possible similarity in an ideal situaaon, although sall mostly lower. accuracy (81.0–89.0%) is sall less than dxa’s accuracy (~ 93%), supporang that panoramic imaging is less accurate. in addiaon, auc = 0.95% indicated excellent diagnosac performance. discussion “osteoporosis” is a systemic disorder idenafied by reduced bone mineral density and disturbance of bone architecture. systemic osteoporosis affects the radial, spinal, femoral, craniofacial bones and oral structures, directly influencing various oral condiaons and dental procedures [33,34]. osteoporosis is primarily associated with increased risk of complicaaons in dental pracace due to advanced alveolar bone resorpaon, causing delayed healing aqer tooth extracaons and a greater risk of pathological fractures during dental procedures, in addiaon to the progression of the periodontal disease much faster. orthodonac treatments encounter difficulaes in tooth movement, and denture wearers oqen exhibit an improper fit resulang from jawbone reducaon. anaresorpave medicaaons such as bisphosphonates also have a marked risk of bisphosphonate-related jaw osteonecrosis aqer aggressive dental surgery. in dental implants, the osteoporoac bones of the jaw mostly lead to impaired osseointegraaon, raising the danger of implant failure too early. osteoporosis also alters the mechanobiology through the bone-implant integraaon, causing more challenges, as the bone may struggle to withstand normal masacaaon forces, possibly leading to peri-implant bone loss and implant overload with ame. therefore, comprehensive pre-surgical evaluaaon is criacal, involving bone density assessments to invesagate the quality of the jawbone, and denasts should consider less invasive techniques. the associaaon between oral health status and osteoporosis is sall controversial. the denast can monitor paaents for osteoporosis, aims to examine people at osteoporosis risk and support their urgent referral [35]. some studies reported the associaaon between the mandibular bone mineral content and many skeletal locaaons frequently ualized for bone densitometry indicators in the idenaficaaon of osteoporosis [36]. the assessment of dental radiographs might contribute to the idenaficaaon of osteoporosis [37]. many studies have reported that panoramic radiography may act as a dependable screening method for osteoporosis detecaon [38,39]. various radio-morphometric indices have been suggested to evaluate the associaaon of bone loss in the mandible, such as panoramic mandibular index (pmi), mandibular coracal index (mci), mental index (mi), mandibular coracal width (mcw) and antegonial index (ai) [35,40-42]. many researchers suggested using the mandibular coracal index (mci), the mental index (mi), and a visual esamaaon index on panoramic images for bone mineral density evaluaaon, and they concluded that these three indices presented as helpful tools for the detecaon of osteoporosis [43]. many studies used panoramic images to detect osteoporosis and have proven their efficiency in that regard. therefore, the current study relied on panoramic imaging to evaluate the reliability of panoramic images using grey values for detecang osteoporosis compared to the measurements of dual-energy x-ray absorpaometry (dxa), which was used as the gold standard reference. the study evolved the ramus index (ri) to calculate the grey mean values on panoramic images, which correlate with bone mineral density (bmd) using imagej soqware. the study showed an evident relaaonship between osteoporosis idenafied through panoramic images (using grey mean values) and the diagnosis, which relied on dxa. the study found that the vast majority of individuals in the control health group (93.3%) showed high grey mean values consistent with normal bone density. a minor proporaon of them (4%) revealed moderate mean grey values; in addiaon, a diminished percentage (2.7%) showed low mean grey values, which could propose radiographic study of osteoporosis detecaon in the jaw vol 13, no 1 (2025) doi 10.5195/d3000/2025.967 http://dentistry3000.pitt.edu 4 signs of early or exisang osteoporosis. in contrast, most paaents (80%) in osteoporoac paaents showed low mean grey values, a suggesave result of decreased bone mineral density or osteoporosis. a small percentage of them (16.7%) revealed moderate mean grey values, proposing osteopenia, while only a few cases (3.3%) had high mean grey values, which could be due to human variaaon. these results reported that panoramic images (based on grey mean value analysis) have a good sensiavity (80%), high specificity (90%), and good accuracy (85%). therefore, on panoramic images, most osteoporoac paaents were correctly idenafied by low mean grey values, and most healthy control individuals were correctly idenafied by high mean grey values. on the opposite side, the current study showed some misclassificaaons (false negaaves and false posiaves), suggesang that although panoramic imaging showed great potenaal as a screening tool, it should not replace dxa for definiave diagnosis. however, the sensiavity of radiographic images in idenafying early bone loss is sall restricted, and bone density reducaon may not be observable unal marked bone loss. in addiaon, the radiographic findings of osteoporosis can interfere with other condiaons, like periodonaas or other metabolic bone disorders, causing it difficult to differenaate from osteoporosis, on the other hand, populaaons at higher risk for osteoporosis (like post-menopausal women), dental radiographs can serve as a helpful adjunct in the early detecaon for paaents. denasts must be trained to idenafy minimal alteraaons in bone density and refer paaents for addiaonal assessment if necessary. so, integraaon of radiographic features with clinical predisposing factors such as a history of fractures, gender, and age can enhance diagnosac accuracy. conclusions panoramic image analysis using grey values can be considered a reliable adjuncave tool for disanguishing osteoporoac paaents from healthy controls, paracularly in situaaons where dxa scans are not readily available. although useful as a screening method, its performance in sensiavity, specificity, and accuracy is staasacally less effecave than dxa. therefore, it should not replace the dxa method for definiave diagnosis, and it should be used as part of a mulamodal approach rather than a standalone diagnosac criterion. in addiaon, denasts must remain careful and analyze panoramic findings within the wider clinical serng. factors such as anatomical variaaons, paaent posiaoning and image quality can affect grey values evaluaaon and may cause misclassificaaon. however, because of its common availability in dental and maxillofacial pracaces, panoramic radiography presents a cost-efficient and accessible iniaal screening tool. this is paracularly valuable in areas with limited services or access to advanced imaging technologies. moreover, integraang ai-based or machine learning models with the method used in this study may further enhance the diagnosac possibility of panoramic images and reduce examiner variability. ethical approval the study was conducted following the ethical standards of the ethics commi}ee for scienafic research at the college of denastry, university of wasit, ref. no. 122024 on 25/4/2024. informed consent was obtained from all individual paracipants included in the study. financial support and sponsorship none. conflicts of interest none to declare. references 1. m.m. pinheiro, r.m. ciconelli, l.a. martini, m.b. ferraz. clinical risk factors for osteoporotic fractures in brazilian women and men: the brazilian osteoporosis study (brazos) osteoporos. int., 20 (3) (2009), pp. 399-408. 2. j. e. compston, m. r. mcclung, and w. d. leslie, “osteoporosis,” lancet, vol. 393, no. 10169, pp. 364-376, 2019, doi: 10.1016/s01406736(18)32112-3. 3. world health organization, “who scientific group on the assessment of osteoporosis at primary health,” world health, vol. may, no. may 2004, pp. 1-13, 2007, doi: 10.1016/s01406736(02)08761-5. 4. e. m. lochmüller, r. müller, v. kuhn, c. a. lill, and f. eckstein, “can novel clinical densitometric techniques replace or improve dxa in predicting bone strength in osteoporosis at the hip and other skeletal sites?” j. bone miner. res. off. j. am. soc. bone miner. res., vol. 18, no. 5, pp. 906-912, may 2003, doi: 10.1359/jbmr.2003.18.5.906. 5. b. yu, c.y. wang. osteoporosis and periodontal diseases an update on their association and mechanistic links. periodontology 2000, 89 (1) (2022), pp. 99-113, 10.1111/prd.12422. 6. clarke bl, khosla s. physiology of bone loss. radiol clin north am. 2010;48:483–495. doi: 10.1016/j.rcl.2010.02.014. 7. f.a. castro da rocha, a.r. ribeiro. low incidence of hip fractures in an equatorial area. osteoporos. int., 14 (6) (2003), pp. 496-499. 8. d.m. black, l. palermo, j. pearson, t. abbott, o. johnell. a simple, useful risk factor system can identify the large majority of women with osteoporosis. bone, 23 (suppl. 5) (1998), p. 605. 9. c.h. cheng, l.r. chen, k.h. chen. osteoporosis due to hormone imbalance: an overview of the effects of estrogen dejiciency and glucocorticoid overuse on bone turnover. int. j. mol. sci., 23 (3) (2022), 10.3390/ijms23031376. 10. l.t. wang, l.r. chen, k.h. chen. hormone-related and drug-induced osteoporosis: a cellular and molecular overview. int. j. mol. sci., 24 (6) (2023), 10.3390/ijms24065814. 11. l. deng, y. guo. estrogen effects on orthodontic tooth movement and orthodontically-induced root resorption. arch. oral. biol., 118 (2020), 10.1016/j.archoralbio.2020.104840. 12. m. zhang, f.m. chen, a.h. wang, y.j. chen, x. lv, s. wu, r.n. zhao. estrogen and its receptor enhance mechanobiological effects in compressed bone mesenchymal stem cells. cells tissues organs, 195 (5) (2012), pp. 400413, 10.1159/000328003. 13. a.c. da cunha, m. marquezan, i. lima, r.t. lopes, l.i. noji ma, e.f. sant'anna. injluence of bone architecture on the primary stability of different mini-implant designs. am. j. orthod. dentofac. orthop.: off. publ. am. assoc. orthod., its const. soc., am. board orthod., 147 (1) (2015), pp. 4551, 10.1016/j.ajodo.2014.09.011. 14. h.s. lee, h.a. heo, s.h. park, w. lee, s.w. pyo. injluence of human parathyroid hormone during orthodontic tooth movement and relapse in the osteoporotic rat model: a preliminary study. orthod craniofacial res. (2018), 10.1111/ocr.12226. 15. m. salazar, l. hernandes, a.l. ramos, o. salazar.bde, k.r. micheletti, l.r. paranhos, m.r. de mendonça, o.a. cuoghi. effect of alendronate sodium on tooth movement in ovariectomized rats. arch. oral. biol., 60 (5) (2015), pp. 776781, 10.1016/j.archoralbio.2015.02.003. 16. m. seiji, b. ezzati, s. saedi, m. hedayati. the effect of ovariectomy and orchiectomy on orthodontic tooth movement and root resorption in wistar rats. j. dent, 16 (4) (2015), pp. 302-309. 17. y. xu, t. zhao, w. xu, y. ding. periodontal microstructure change and tooth movement pattern under different force magnitudes in ovariectomized rats: an in-vivo microcomputed tomography study. am. j. orthod. dentofac. orthop.: off. publ. am. assoc. orthod., const. soc., am. board orthod., 143 (6) (2013), pp. 828836, 10.1016/j.ajodo.2013.01.020. 18. g.d. mishra, m.c. davies, s. hillman, h.f. chung, s. roy, k. maclaran, m. hickey. optimising health after early menopause. lancet, 403 (10430) (2024), pp. 958-968, 10.1016/s0140-6736(23)02800-3. 19. m. martiniakova, r. biro, n. penzes, a. sarocka, v. kovacova, v. mondockova, r. omelka. links among obesity, type 2 diabetes mellitus, and osteoporosis: bone as a target int. j. mol. sci., 25 (9) (2024), 10.3390/ijms25094827. 20. i. foessl, h.p. dimai, b. obermayer-pietsch. long-term and sequential treatment for osteoporosis.nat. rev. endocrinol., 19 (9) (2023), pp. 520533, 10.1038/s41574-023-00866-9. 21. t. toriya, t. kitahara, h. hyakutake, m. todo, i. takahashi. analysis for predictors of failure of orthodontic mini-implant using patient-specijic jinite element models. ann. biomed. eng., 51 (3) (2023), pp. 594-603, 10.1007/s10439-02203067-z. 22. b.b. só, f.m. silveira, g.s. llantada, l.c. jardim, t. calcagnotto, m.a.t. martins, m.d. martins. effects of osteoporosis on alveolar bone repair after tooth extraction: a systematic review of preclinical radiographic study of osteoporosis detecaon in the jaw vol 13, no 1 (2025) doi 10.5195/d3000/2025.967 http://dentistry3000.pitt.edu 5 studies. arch. oral. biol., 125 (2021), article 105054, 10.1016/j.archoralbio.2021.105054. 23. k. balachandran, r. ramli, s.a. karsani, m. abdul rahman. identijication of potential biomarkers and small molecule drugs for bisphosphonate-related osteonecrosis of the jaw (bronj): an integrated bioinformatics study using big data. int. j. mol. sci., 24 (10) (2023), 10.3390/ijms24108635. 24. b. cakur, s. dagistan, a. sahin, a. harorli, and a. yilmaz. “reliability of mandibular cortical index and mandibular bone mineral density in the detection of osteoporotic women.,” dentomaxillofac. radiol., vol. 38, no. 5, pp. 255-261, jul. 2009, doi: 10.1259/dmfr/22559806. 25. taguchi a, suei y, sanada m, ohtsuka m, nakamoto t,sumida h, et al. validation of dental panoramic radiographymeasures for identifying postmenopausal women with spinalosteoporosis. ajr am j roentgenol 2004; 183: 1755-60. 26. güngör k, akarslan z, akdevelioglu m, erten h, semiz m. the precision of the panoramic mandibular index. dentomax-illofac radiol 2006; 35: 442-6. 27. schulze r, krummenauer f, schalldach f, d’hoedt b. precision and accuracy of measurements in digital panoramic radio-graphy. dentomaxillofac radiol 2000; 29: 52-6. 28. whaites e. essentials of dental radiography and radiology. 3rded. edinburgh: churchill livingstone; 2002. p. 161-76. 29. schneider, c. a., rasband, w. s., & eliceiri, k. w. (2012). nih image to imagej: 25 years of image analysis. nature methods, *9*(7), 671–675. https://doi.org/10.1038/nmeth.2089. 30. blake, g. m., & fogelman, i. (2007). the role of dxa bone density scans in the diagnosis and treatment of osteoporosis. postgraduate medical journal, 83(982), 509517. https://doi.org/10.1136/pgmj.2007.057505 . 31. kanis, j. a., mccloskey, e. v., johansson, h., et al. (2013). frax® with and without bone mineral density. osteoporosis international, 24(1), 23-57. https://doi.org/10.1007/s00198-012-2074-y. 32. world health organization. assessment of fracture risk and its application to screening for postmenopausal osteoporosis: technical report series 843. geneva: who, 1994. 33. dervis e. oral implications of osteoporosis. oral surg oral med oral pathol oral radiol endod 2005;100:349-356. 34. aguirre ji, akhter mp, kimmel db, pingel j, xia x, williams a, jorgensen m, edmonds k, et al. enhanced alveolar bone loss in a model of non-invasive periodontitis in rice rats. oral diseases 2012;18:459-468. 35. d. ledgerton, k. horner, h. devlin, h. worthington. radiomorphometric indices of the mandible in a british female population. dentomaxillofacial radiol, 28 (1999), pp. 173-181. 34. siminoski k, o'keeffe m, brown jp, burrell s, coupland d, dumont m, ganguli sn, hanley da, et al. canadian association of radiologists technical standards for bone mineral densitometry reporting. can assoc radiol j 2013; 64:281-294. 37. mohajery m, brooks sl. oral radiographs in the detection of early signs of osteoporosis. oral surg oral med oral pathol 1992;73:112-117. 38. a. taguchi. triage screening for osteoporosis in dental clinics using panoramic radiographs. oral dis, 16 (2010), pp. 316-327. 39. k.a. balto, m.m. gomaa, r.m. feteih, n.m. alamou di, a.z. elsamanoudy, m.a. hassanien. dental panoramic radiographic indices as a predictor of osteoporosis in postmenopausal saudi women. j bone metab, 25 (3) (2018), pp. 165-173. 40. klemetti e, kolmakov s, kröger h. pantomography in assessment of the osteoporosis risk group. scand j dent res. 1994;102(1):68-72. doi:10.1111/j.1600-0722.1994.tb01156.x. 41. benson bw, prihoda tj, glass bj. variations in adult cortical bone mass as measured by a panoramic mandibular index. oral surg oral med oral pathol. 1991;71(3):349-356. doi:10.1016/00304220(91)90314-6. 42. horner k, devlin h. the relationship between mandibular bone mineral density and panoramic radiographic measurements. j dent. 1998;26(4):337-343. doi:10.1016/s03005712(97)00019-0. 43. o. s. kim et al., “digital panoramic radiographs are useful for diagnosis of osteoporosis in korean postmenopausal women,” gerodontology, vol. 33, no. 2, pp. 185-192, 2016, doi: 10.1111/ger.12134. 1071 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1071 http://dentistry3000.pitt.edu connection of some growth parameters and teething in infants sulafa khair al-deen banoosh college of den*stry, university of tikrit, iraq abstract objec7ve: eruption of teeth can be de1ined as a normal physiological process of movement of the teeth through the alveolar bone and it takes its place in the oral cavity. the aim of this study was to 1ind if there was any relation between growth parameters and eruption of teeth in infants. materials and methods: 77 infants and their families accepted to be in this study. 11 questions were asked to their parents related to the normal growth of the children. oral examination was done by using dental probe and mirror. results: in this study, there was a signi1icant delay of teething in children that had a cesarean delivery in comparison to normal delivery (p< 0.001), and a signi1icant delay in males compared to females (p< 0.001). conclusions: the type of delivery is associated with differences in the eruption of teeth. open access cita%on: banoosh ska. (2025) connec%on of some growth parametrs and teething in infants. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1071 received: october 11, 2025 accepted: october 15, 2025 published: november 10, 2025 copyright: ©2025 banoosh ska. this is an open access ar%cle licensed under a crea%ve commons asribu%on work 4.0 united states license. email: sulafa.khairy@yahoo.com introduc)on most parents view an infant's first tooth as one of several major developmental milestones, and an "old wives' tale" claims that its early eruppon indicates high intelligence [1]. during the development of deciduous teething, a variety of genepc and environmental factors, including race, gender, systemic condipons, environmental condipons, socioeconomic level, maternal breasueeding, infant nutriponal status, and physical development, interact to affect the pming of dental eruppon. none of these factors act independently [2,3]. the pme of teeth eruppon in a healthy infant may depend on growth factors and feeding habits [4]. according to certain research, the development of the primary teeth is predominantly influenced by the gestaponal age, the severity of the neonatal sickness, postnatal nutripon, and the degree of prematurity. according to other studies, low and normal birth weight are associated to the primary denppon's eruppon pa\ern [5]. nutriponal aspects have an impact on tooth emergence and odontogenesis [6]. lack of nutrients, such as a calcium deficit, can impact the cellular architecture of the organic matrix, including the maturapon process of amelogenesis and calcificapon, as well as the morphology and pa\ern of teeth eruppon [7], during the development of teeth. the eruppon of deciduous teeth can also be delayed by other disorders like intrauterine growth restricpon, inadequate size for gestaponal age, and long-term parenteral feeding [9]. when preterm and low birth weight (lbw) combine, the delay makes the condipon worse, especially in those who are in a key developmental stage when they are diagnosed with a systemic disease at birth [10,11]. the aim of this study was to assess the relaponship between gestaponal age, birth weight, neonatal sickness, and the first deciduous tooth's eruppon pming. materials and methods this was a cross-secponal study that focused on children aged between one and two years who were carefully selected by methodical arbitrary specimen from the children a\ending ibn rushd, ibn sina, and al-razy primary health care centers in tikrit city. any child with a serious illness that could affect the scoring presentapon of children, or the eruppon of the teeth was excluded from the study. the period of employment included march 2017 unpl the end of january 2018. the study's inclusion criteria were met by 77 youngsters. as a screening tool for developmental delays, the third edipon of the age and stages quesponnaire was employed [12]. these invespgapons focused on 10 main issues. to ascertain the tooth eruppon, an oral examinapon was conducted in a well-lit area connecpon of some growth parameters and teething in infants vol 13, no 1 (2025) doi 10.5195/d3000.2025.1071 http://dentistry3000.pitt.edu 2 using a dental mirror and probe. the stapspcal analysis was done using microsoi excel 2013, with a p-value of 0.05 as showing significance. results table 1 shows summary stapspcs of teething and some factors related to infant life. delayed teething was seen more among those born by cesarean secpon than normal delivery, and among males more than females. discussion tooth eruppon is a series of metabolic events in alveolar bone defined by bone resorppon and creapon on opposite sides of the dental follicle. when it comes to the producpon of teeth, some vitamin and hormone deficits can have a negapve impact on the formapve cells and the matrix they create. producpon of hypoplaspc pssue could occur if the organic matrix content is reduced. the end consequence would be a hypoplaspc matrix that is also hypomineralized [13]. in this study, we discovered that newborns whose mothers deliver them via cesarean secpon had significantly higher stapspcal tooth eruppon delays than kids whose mothers’ birth normally. finding the cause of these results is challenging because any changes during a cesarean secpon are only temporary (like general anesthesia and medicapons), and teeth usually don't erupt unpl at least six months aier the baby is born. however, the authors believe that calcium or vitamin d deficiency may be one of the causes of cesarean delivery or lower their blood levels. because vitamin d is essenpal for maintaining calcium homeostasis, the greater serum calcium level contributed to the mechanism of labor's onset [14]. the infant's development and the emergence of the baby's deciduous teeth may be impacted by this condipon's potenpal to induce the same shortage in the body aier delivery [7,8]. this fact needs to be demonstrated by analyzing the serum calcium levels in both the mother and the newborn following the delivery by marine infecpon. another conclusion in this study is that boys experience a stapspcally significant teething delay compared to girls. this finding is consistent with research done in 2001 on korean newborns by choi and yang, who found that males experience tooth emergence earlier than females [15]. this result can be explained by the fact that males have thicker mandibular alveolar bones than females do [16], previous data report on the subject were not clear [16-20]. no one pa\ern can adequately define sex variapons in the pa\ern and pme of emerging globally, according to tanguay et al.'s hypothesis that ethnic characterispcs may underlie sex differences in tooth development [21]. in addipon, tanguay et al. noted that, except for the first deciduous molar, males' teeth typically appeared one month earlier than girls' teeth. according to holman and jones, there is less support for the idea that sex differences in the eruppon of deciduous teeth are mediated by ethnicity. however, they agreed with demirijian and levesque's theory of a developmental crossover in which females lead males for the posterior teeth and vice versa [22]. this trend starts in utero, when males acquire their teeth more quickly than females [23]. according to holman et al., the pa\ern seems to correspond to a spapal gradient in tooth posipon that emerges early in dental development and corresponds to sex differences [24]. even in several trials where sex differences were not determined to be stapspcally significant, this overall developmental crossover trend persisted [25]. acknowledgments all praise and appreciapon to the community medicine department of tikrit university college of medicine. references 1. g. t. mcintyre1 and g. m. mcintyre, teething troubles? bri<sh dental journal volume 192 no. 5 march 9 2002. 2. duarte meq, andrade ma, faria pc, marques ls, jorge mlr. fatores associados à cronologia de erupção de dentes decíduos revisão de literatura: erupção de dentes decíduos e fatores associados. rev uni vale do rio verde. 2011; 9 (1): 139-51. 3. lynch rj. the primary and mixed den<<on, pos erup<ve enamel matura<on and dental caries: a review. int dent j. 2013; 63 (suppl. 2): 3-13. 4. haddad ae, correa ms. the rela<onship between the number of erupted primary teeth and the child’s height and weight: a crosssec<onal study. j pediatr dent 2005;29:357–62. 5. aktoren o, tuna eb, guven y, gokcay g. birth weight and teeth erup<on a retrospec<ve study. community dent health 2010;27:52–6. 6. thomaz eb, alves cm, ribeiro cc, ba<sta rf, simões vm, cavalli r, rasaiva mdac, cardoso vc, bejol h, barbieri ma, da silva aa. perinatal outcomes and changes in the oral cavity: brazilian cohorts of ribeirão pret 7. fernandes neto pg, falcão mc. cronologia de erupção dos primeiros dentes decíduos em crianças nascidas prematuras com peso inferior a 1500g. rev paul pediatr. 2014; 32 (1): 17-23. 8. peedikayil fc. delayed tooth erup<on. e-journal of den<stry. 2011; 1 (4): 81-6. 9. fernandes neto pg, falcão mc. cronologia de erupção dos primeiros dentes decíduos em crianças nascidas prematuras com peso inferior a 1500g. rev paul pediatr. 2014; 32 (1): 17-23. 10. al-sayagh gd, qasim aa, al-rawi ba. the effect of premature birth on the primary den<<on. al–rafidain dent j. 2008; 8 (1): 18-22. 11. zaidi i, tahyath mn, singh s, sinha a. preterm birth: a primary e<ological factor for delayed oral growth and development. int j clin pediatric dent. 2015; 8 (3): 215-9. 12. ages and stages ques<onnaires 3rd ed. brookes publishing co. 2009. available at: hop://www.brookespublishing.com/resource-center/screening-and assessment/ asq/asq-3. 13. wise g. cellular, molecular, and gene<c determinants of tooth erup<on. crit rev oral biol med 2002;12(4):323–35. 14. anne merewood, supriya d. mehta, tai c. chen, howard bauchner, and michael f. holick, associa<on between vitamin d deficiency and primary cesarean sec<on, j clin endocrinol metab. 2009 mar; 94(3): 940–945. 15. n.k. choi, k.h. yang. a study on the erup<on <ming of primary teeth in korean children asdc j dent child, 68 (2001), pp. 244-249. 16. v. sairam, gareema raju poouri, b. praveen, and g vikas, assessment of effect of age, gender, and dentoalveolar changes on mandibular morphology: a digital panoramic study, contemp clin dent. 2018 jan-mar; 9(1): 49–54. 17. afrin mohamed khalifa reyad atef el gendy mohamed mahmoud abd el-mohsen ahmad alsayed hammour rasha sabry abd el lateef aly, rela<onship between gesta<onal age, birth weight and deciduous tooth erup<on, egyp<an pediatric associa<on gazeoe, volume 62, issue 2, june 2014, pages 41-45. 18. r.m. viscardi, e.d. romberg, r.g. abrams. delayed primary tooth erup<on in premature infants: rela<onship to neonatal factors pediatr dent, 16 (1) (1994), pp. 23-28. 19. n. sajjadian, h. shajari, jahadi ramin, g. michaeldental erup<on and birth weight iran pediatr neonatol, 51 (4) (2010), pp. 235237. 20. el-mougi mt, abu shebika r, el ahmady a, hammour a, mohammed sm. teething, age of erup<on, factors affec<ng and knowledge of mothers about it [m.sc. thesis]. al azhar faculty of medicine, 2011. 21. tanguay r, buschang ph, demirjian a. sexual dimorphism in the emergence of deciduous teeth: its rela<onship with growth components in height. american journal of physical anthropology, 1986, 69(4):511–515. connecpon of some growth parameters and teething in infants vol 13, no 1 (2025) doi 10.5195/d3000.2025.1071 http://dentistry3000.pitt.edu 3 22. demirjian a, levesque gy. sexual differences in dental development and predic<on of emergence. journal of dental research, 1980, 59:1110–1122. 23. burdi ar, garn sm, miller rl. development advancement of the male in the first trimester. journal of dental research, 1970, 49:889. 24. holman dj, jones re. longitudinal analysis of deciduous tooth emergence: iv. covariate effects in japanese children . american journal of physical anthropology, 2005, 126(3):352–358. 25. saleemi m et al. early child health in lahore, pakistan: xiii. primary teeth emergence. acta paediatrica supplement, 1994, 82(suppl. 390):159–167. table 1. effect of growth affecpng factors on teething. domain teething chi-square p-value delayed normal gestaponal age normal 22 53 0.397 0.52 low 1 1 birth weight normal 16 43 0.912 0.33 low 7 11 maturity preterm 1 1 0.397 0.52 full term 22 53 mode of delivery normal delivery 11 44 8.952 0.002 cesarean secpon 12 10 sex male 18 26 5.972 0.01 female 5 28 feeding bo\le 10 8 2.382 0.12 breast 44 15 ofc normal 45 16 1.857 0.17 low 9 7 1039 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1039 http://dentistry3000.pitt.edu zno nanoparticles and pepper fruit extract effect on streptococcus mutans and staphylococcus aureus sura a. jaber college of den*stry, university of almaarif, al anbar, iraq abstract objec.ves: to test the effect on streptococcus mutans and staphylococcus aureus isolates from dental cavihes of different amounts of zinc oxide (zno) nanoparhcles and capsicum annuum l. extract. methods: zinc oxide nanoparhcles (zno, purity: 99.8%, d50: 10–30 nm) were synthesised from skyspring nanomaterials. pepper fruits were procured from local markets in babylon. the fruits were cleansed using dishlled water to eliminate soil, air-dried, and subsequently pulverised using an electric grinder. the extract was produced with soxhlet equipment. results: the inhibihon zones for s. mutans at doses of 0.2, 0.4, 0.6, and 0.8 mg/ml were 2.12, 2.27, 2.58, and 2.98 mm, respechvely. the inhibitory zones for s. aureus measured 2.02, 2.2, 2.45, and 2.68 mm, respechvely. significant anhbacterial achvihes of the pepper fruit extract were demonstrated against both s. mutans and s. aureus. the inhibihon zones for s. mutans at 100, 200, 3000, and 350 mg/ml doses were 1.24, 1.46, 1.54, and 1.7 mm, respechvely. the inhibitory zones for s. aureus measured 1.35, 1.56, 1.78, and 1.89 mm, respechvely. conclusion: both pepper fruit extract and zno nanoparhcles possess anhbacterial properhes. zno nanoparhcles at a dose of 1.0 mg/ml exhibited the most substanhal inhibihon zone against s. mutans. in contrast, pepper fruit extract at 300 mg/ml demonstrated the most significant inhibihon zone against s. aureus. open access cita%on: jaber sa. (2025) zno nanopar%cles and pepper fruit extract effect on streptococcus mutans and staphylococcus aureus. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1039 received: september 6, 2025 accepted: september 24, 2025 published: october 8, 2025 copyright: ©2025 jaber sa. this is an open access ar%cle licensed under a crea%ve commons avribu%on work 4.0 united states license. email: dr.sura.ameer@gmail.com introduc)on for millennia, nature has served as a source of medicinal cures since the dawn of humanity. despite this, synthetic drugs often have adverse effects, and it has become more and more apparent that microbes are resistant to these substances. consequently, numerous developing countries have begun to emphasise herbal treatments and nanotechnology [1]. zinc oxide nanoparticles (zno nps) are important microelements that play important roles in many bodily functions. consumption of water and food leads to the acquisition of zinc, which the small intestine primarily absorbs and transports to the blood plasma. many businesses and drug companies use zinc oxide nanoparticles because they might be able to kill fungi, bacteria, diabetes, and inflammation, speed up wound healing, and protect cells from damage. researchers have generated these nanoparticles using green synthesis techniques that use plants, fungi, bacteria, and algae [2,3]. the fruits of the pepper plant (capsicum annuum l.) contain the alkaloid capsaicin, which imparts its spicy flavour. capsaicin is present in elevated proportions in the fruits of spicy peppers. alkaloids are essential molecules in the process of making medicines and pharmaceuticals. they also have big effects on the structure and function of cell membranes [4,5]. paprika powder often comprises less than 0.5% essential oil. pepper fruits possess a volatile oil level that is comparatively modest, varying from 0.1% to 2.6%, contingent upon the cultivar and the maturity stage at harvest. the primary chemical classes of compounds present in capsicum oil encompass terpenes and their derivatives, alcohols, aldehydes, ketones, carboxylic acids, esters of carboxylic acids, benzene derivatives, naphthalene derivatives, hydrocarbons, sulphur and nitrogencontaining compounds, phenolic compounds, and carotenoid derivatives [6]. dental caries (cavitation) is notably severe and persistent among numerous dental issues. it is an irreversible microbial illness that impacts the calcified tissues of the teeth, marked by the demineralisation of the inorganic component and the degradation of the organic matrix. dental caries recognises streptococcus mutans and other zno nanoparhcles and pepper fruit extract effect on streptococcus mutans and staphylococcus aureus vol 13, no 1 (2025) doi 10.5195/d3000.2025.1039 http://dentistry3000.pitt.edu 2 streptococci as the principal causative agents [7]. dental plaque, which develops on the tooth surface, particularly in the cervical region, comprises bacteria encased within an organic matrix. this plaque is associated with gingivitis, periodontal disease, and tooth caries [8]. previous studies have demonstrated the management of dental plaque through physical removal and antimicrobial toothpaste and mouthwashes [9,10]. new types of bacteria, like staphylococcus mutans and staphylococcus aureus, are often linked to cavities and other periodontal diseases in the mouth [11,12]. these bacteria are becoming resistant to antibiotics, which is a major global health problem. this is especially applicable in the context of oral infections. these bacteria cause tooth decay and are also linked to the development of systemic illnesses. this shows the importance of effective antimicrobial treatments in dental care. even though conventional antibiotics have been utilised in the fight against these diseases, the growing resistance to these medications has made it necessary to investigate alternative treatments [13]. nanomaterials, including zinc oxide (zno) nanoparticles, are among the most promising options for antibacterial treatments. in oral health, zinc oxide (zno) has attracted significant attention due to its broad-spectrum antibacterial properties. these include the ability to combat both gram-positive and gram-negative bacteria, making it a potential therapeutic agent [14]. recent studies have demonstrated that zno nanoparticles can effectively inhibit the growth of s. mutans and s. aureus. this could offer a new approach to treating oral microbial infections. however, the optimal concentration of zno nanoparticles needed for effective antibacterial activity while minimizing harm to human cells remains under investigation [15]. researchers have long recognised plant-derived chemicals for their antibacterial characteristics and nanomaterials. numerous researchers are investigating the possibility of natural extracts serving as adjuncts or replacements to traditional antibiotics [16,17]. pepper fruit (capsicum annuum), which is full of bioactive substances like capsaicin, flavonoids, and alkaloids, might be able to change the activity of microbes. this includes antibacterial actions against a wide range of pathogens. using zno nanoparticles in conjunction with plant extracts, such as pepper fruit extract, could increase antibacterial action. this would reduce the requirement for higher doses of either agent on its own, potentially lowering the likelihood of resistance development. this study aims to _ind out what happens to strains of staphylococcus aureus and staphylococcus mutans that have been taken from dental cavities when different amounts of zno nanoparticles and pepper fruit extract are mixed together. the study aims to help the ongoing search for long-lasting alternatives to traditional antibiotics in dental care by looking at how well these agents kill bacteria on their own and when mixed together. this research aims to provide insights into the potential of these agents as effective treatments for oral infections. materials and methods we synthesised zinc oxide nanoparticles/nanopowder (speci_ications: zno, purity: 99.8%, d50: 10–30 nm, colour: white to light yellow) from skyspring nanomaterials (fig. 1). the common contaminants in the nanoparticles consist of cu (25 ppm), cd (25 ppm), mn (25 ppm), pb (20 ppm), and as (20 ppm). figure 1. zinc oxide nanoparticle. we obtained the pepper fruits from local markets in babylon. we rinsed the samples with distilled water to remove dirt and then dried them in the shade. after drying, we pulverised the samples using an electric mill [18]. the extraction was conducted via soxhlet equipment. one hundred grams of the ground sample were placed in the thimble, and methanol at a 70% concentration was added to the round-bottom flask. the system was thereafter maintained at 60°c for 24 hours. following the extraction process, the methanol was evaporated with a rotary evaporator to isolate the alcohol from the crude extract. the resultant extract was refrigerated until utilised [19]. analysis of the impact of varied doses on the proliferation of specific pathogenic microorganisms we synthesised pepper fruit extract and zno nanoparticles using an msa medium and incubated them anaerobically for 48 hours at 37°c before evaluating their antagonistic activity. ten bacterial colonies were transferred to create the bacterial inoculum. to get a 0.5 mcfarland standard turbidity, which is equal to 1.5 x 10⁸ cells/ml, the bacterial suspension was put into a test tube with 5 ml of brain heart infusion (bhi) broth in a clean environment. the infected plates with msa media were prepared by dispersing 0.1 ml of the suspension onto the plates. the plates were maintained at room temperature for 15 minutes to facilitate inoculum absorption. a sterile cork borer with a diameter of 7 mm was employed to form wells in the inoculated agar plates. a micropipette was used to dispense 50 microliters of different concentrations of the test chemicals into the wells (0.2, 0.6, 0.8, and 1.0 mg/ml for zno nanoparticles; 100, 200, 300, and 350 mg/ml for the crude extract). control plates were made by substituting the crude extract with 50 µl of sterilised distilled water in the wells. the plates were kept at 37°c for 48 hours, and the results were judged by measuring the size of the inhibition zone, which is the area around the well where bacteria can't grow [20]. statistical analysis the tests were executed as factorial experiments with three replications, employing a completely randomised design (crd). the data were analysed with the least signi_icant difference (l.s.d.) at a 1% probability threshold (p ≤ 0.01) results table 1 demonstrates the strong impact of zno nanoparticles on staphylococcus mutans and staphylococcus aureus. at 0.3, 0.5, 0.7, and 0.9 mg/ml, the inhibition zones for s. mutans are 2.12, 2.27, 2.58, and 2.98 mm, and for s. aureus they are 2.02, 2.2, 2.45, and 2.68 mm. table 2 indicates a substantial impact of pepper fruit extract on s. mutans and s. aureus. at 100, 200, 300, and 350 mg/ml, the zones that stop s. mutans from growing are 1.24, 1.46, 1.54, and 1.7 mm, and the zones that stop s. aureus from growing are 1.35, 1.56, 1.78, and 1.89 mm. discussion the study shows that zno nanoparticles can kill staphylococcus mutans and staphylococcus aureus bacteria, especially when 0.8 mg/ml of them are present. nanoparticles may be responsible for this action. these nanoparticles may target subcellular compartments of the cell membrane, resulting in cellular damage and ultimately leading to cell death. zinc oxide nanoparticles, also known as zno nps, have demonstrated the ability to alter the construction of bacterial cell walls. zno nps achieve this by influencing the linkage between n-acetylglucosamine and nacetylmuramic acid in glycans. this, in turn, leads to the formation of pits and a weakening of the cell wall. along with this, zno zno nanoparhcles and pepper fruit extract effect on streptococcus mutans and staphylococcus aureus vol 13, no 1 (2025) doi 10.5195/d3000.2025.1039 http://dentistry3000.pitt.edu 3 nanoparticles can stick to the surface of peptides, which breaks down the glycan structure of the bacterial cell wall [21,22]. the results of this observation are similar to those of earlier research [23], which showed how important the concentration of zno nanoparticles and the size of their particles are to antibacterial activity. numerous studies show that the antibacterial activity of zno nanoparticles is concentration-dependent. these nanoparticles are better at killing microbes because they have a larger surface area and are more reactive. the increased surface area results in an increase in the number of reactive sites that are available for interactions with bacterial cells. some people also think that zno nanoparticles interact with the plasma membrane of bacteria, changing the surface chemistry and function of the membrane, lowering the amount of adenosine triphosphate (atp), and messing up the cell's energy metabolism. in the end, these alterations have an effect on the stability of the bacterial cell membrane, which finally results in the membrane's disruption [24]. some types of bacteria, like staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa, can be killed by zinc oxide nanoparticles (zno nps). researchers have found that 0.8%, 1%, and 1.5% of s. aureus bacteria survived at different concentrations of zno nps; 1%, 1.5%, and 5% of p. aeruginosa bacteria survived; and 2%, 3.7%, and 6% of e. coli bacteria survived [25]. researchers found that the presence of zno nps was associated with these percentages. compared to gram-positive bacteria, which have thicker and more resistant cell walls, gram-negative bacteria, such as e. coli, have thinner cell walls, which makes them more sensitive to metal nanoparticles. this is because gram-negative bacteria are more susceptible to metal nanoparticles. because of this structural difference, nanoparticles can more easily penetrate gram-negative bacteria cells [26]. nanoparticles can target various bacterial structures in addition to their action on the cell membrane [27]. these structures include respiratory chain dehydrogenases and bacterial chromosomes. it has also been shown that metallic nanoparticles like zno can make reactive oxygen species (ros) that kill bacteria. these ros are produced by creating oxidative stress and inhibiting the oxidation of metal ions that have been freed [28]. this research also indicates that pepper fruit extract possesses powerful antibacterial characteristics. since phenolic chemicals allow the extract to penetrate bacteria cell walls, these properties may be linked to them. the findings of this study are consistent with those of other studies [29], which suggested that polyphenols are important bioactive chemicals that are responsible for antibacterial activity. there are hydroxyl groups or phenolic rings in these compounds that might interact with the proteins and membranes of bacteria. this could lead to the formation of complexes that stop the cell from working properly [30,31]. researchers think that bioactive phytochemicals like _lavonoids, alkaloids, and tannins, known to kill bacteria, make pepper extract bacteria-killing. these components are believed to be responsible for what makes pepper extract so effective. it has been observed in previous research [32, 33] that plant extracts abundant in phenolic compounds are particularly effective against bacteria because they disrupt their growth and metabolism. the active ingredients in pepper fruit extract, especially _lavonoids and alkaloids, help stop the growth of bacteria. this is more proof that pepper fruits can be used as a natural antibacterial. conclusion the conclusion is that zno nanoparticles and pepper fruit extract exhibit strong antibacterial effects. microparticles of zno specifically target the cell walls and membranes of bacteria. on the other hand, bioactive chemicals like phenols, flavonoids, and alkaloids in pepper fruit extract make it work. these results show that zno nanoparticles and plantbased extracts may be useful in the fight against bacterial infections. they also offer a viable route for developing antimicrobial medicines based on natural materials and nanomaterials. references 1. sakoulas, g., & moellering, r. c. (2004). increasing antibiotic resistance among methicillinresistant staphylococcus aureus strains. clinical infectious diseases, 46(suppl), 360-367. 2. skalniy, a. v., & rudakov, i. a. (2004). bioelements in medicine. onix vol. 21:272 (in russian). 3. ramesh, p., rajendran, a., & meenashisundaram, m. (2014). green synthesis of zinc oxide nanoparticles using ulower extract of cassia auriculata. journal of nanoscience and nanotechnology, 41, 41-45. 4. serrano-martıńez, a., fortea, f. m., del amor, f. m., & núñez-delicado, e. (2008). kinetic characterisation and thermal inactivation study of partially puriuied red pepper (capsicum annuum) peroxidase. food chemistry, 107, 193-199. 5. al-ghazali, l. h., al-masoody, i. h., ismael, m. h., & al-ibrahemi, n. (2023). effect of alcohol extract, volatile oil, and alkaloid isolated from capsicum frutescens l. fruits on candida albicans. iop conference series: earth and environmental science, 1225(1), 012075. 6. kocsis, n., markus, f., mednyanszky, z. s., amtmann, m., & korany, k. (2003). recognition experiments of the vintage year 1997 hot and red paprika (capsicum annuum) varieties grown in kalocsa. acta alimentaria, 32(1), 63-75. 7. winston, a. e., & bhaskar, s. n. (1998). low sucrose levels promote extensive s. mutans-induced dental caries. journal of the american dental association, 129(11), 1463-1468. 8. tanzer, j. m. (2002). bacterial genomics— streptococcus mutans. proceedings of the national academy of sciences of the united states of america, 99(22), 14434-14439. 9. jensen, j. l., & barkvoll, p. (1998). clinical implications of dry mouth: oral mucosal diseases. annals of the new york academy of sciences, 842(1), 156-162. 10. caglar, e., kavaloglu, s. c., kuscu, o. o., sandalli, n., holgerson, p. l., & twetman, s. (2007). effect of chewing gums containing xylitol or probiotic bacteria on salivary mutans streptococci and lactobacilli. clinical oral investigations, 11, 425429. 11. koo, h., et al. (2020). pathogenesis of dental caries and periodontal disease. journal of clinical periodontology, 47(2), 129-140. 12. lima, r. g., et al. (2021). staphylococcus aureus and its role in oral infections: implications for treatment. journal of microbiology and biochemistry, 16(4), 245-259. 13. lópez, m. e., et al. (2022). oral health and systemic disease: the role of staphylococcus in chronic infections. microbial pathogenesis, 156, 104905. 14. bączek, k., et al. (2022). zinc oxide nanoparticles in antibacterial applications. materials science and engineering c, 131, 112486. 15. zhang, z., et al. (2023). antibacterial activity of zinc oxide nanoparticles against staphylococcus spp. and their mechanism of action. applied microbiology and biotechnology, 107(12), 4357-4367. 16. barlow, j., et al. (2023). antibacterial properties of capsicum annuum fruit extract: a comprehensive review. phytotherapy research, 37(4), 1109-1120. 17. silva, a. b., et al. (2024). capsicum-derived natural compounds: antimicrobial properties and therapeutic potential. food and chemical toxicology, 169, 113218. 18. salman, t. a.-s., ahmed, a. t., oleiwi, g., & al-ibrahemi, n. (2023). study of the effect of oil extract of dill (anethum graveolens l.) plant on oxidative stress parameters of liver enzymes in male rats. iop conference series: earth and environmental science, 1215(1), 012059. 19. al-ibrahemi, n., al-yassiry, a., al-laith, z. n., & al-musawi, b. h. (2023). phytochemical study of volatile oils for ocimum basilicum l. and mentha spicata by gas chromatography technique. iop conference series: earth and environmental science, 1158(5), 1-8. 20. tepsorn, r. (2009). antimicrobial activity of thai traditional medicinal plant extracts incorporated into alginate-tapioca starch-based edible uilms against food-related bacteria, including foodborne pathogens. (phd thesis). university of hohenheim, thailand. 21. li, x., hu, z., ma, j., wang, x., zhang, y., wang, w., & yuan, z. (2018). the systematic evaluation of size-dependent toxicity and multi-time biodistribution of gold nanoparticles. colloids and surfaces b: biointerfaces, 167, 260-266. 22. alnuaimi, m. t. a., al-hayanni, h. s. a., & aljanab, z. z. (2023). green synthesis of gold nanoparticles from sophora ulavescens extract and their antibacterial effect against some pathogenic zno nanoparhcles and pepper fruit extract effect on streptococcus mutans and staphylococcus aureus vol 13, no 1 (2025) doi 10.5195/d3000.2025.1039 http://dentistry3000.pitt.edu 4 bacteria. malaysian journal of microbiology, 19(1), 74-82. 23. peng, x., palma, s., fisher, n. s., & wong, s. s. (2011). effect of morphology of zno nanostructures on their toxicity to marine algae. aquatic toxicology, 102(3), 186-196. 24. ashe, b. (2011). a detailed investigation to observe the effect of zinc oxide and silver nanoparticles in biological systems. (m.sc. thesis, national institute of technology). 25. khitam, s. s., alhtheal, e. d., & azhar, j. b. (2018). effect of zinc oxide nanoparticles prepared from zinc sulfate (znso₄) against gram-negative and gram-positive microorganisms in vitro. the iraqi journal of veterinary medicine, 42(1), 18-22. 26. alfahad, m. a., al-hayanni, h. s. a., & alnuaimi, m. t. (2022). antibacterial activity of biologically synthesised silver nanoparticles using seeds extract of nigella sativa coated on antibiotics against antibiotic-resistant salmonella typhi. indian journal of ecology, 49(special issue 19), 396-402. 27. yougbare, s., chang, t. k., tan, s. h., kuo, j. c., hsu, p. h., su, c. y., & kuo, t. r. (2019). antimicrobial gold nanoclusters: recent developments and future perspectives. international journal of molecular sciences, 20(12), 2924. 28. iswarya, v., manivannan, j., de, a., paul, s., roy, r., johnson, j. b., kundu, r., chandrasekaran, n., mukherjee, a., & mukherjee, a. (2016). surface capping and size-dependent toxicity of gold nanoparticles on different trophic levels. environmental science and pollution research, 23, 4844-4858. 29. jafeer, k. h. a., & kheirallah, e. a. (2016). effect of piper nigrum fruit and syzygium aromaticum rose extract on bacterial and fungal isolate growth. journal of medicinal plants research, 2(21), 123-128. 30. ruiz, c., falcocchio, s., xoxi, e., villo, l., nicolosi, g., pastor, f. j., dıáz, p., & saso, l. (2006). inhibition of candida rugosa lipase by saponins, ulavonoids, and alkaloids. journal of molecular catalysis b: enzymatic, 40, 138-143. 31. gumgumjee, n. m., & hajar, a. s. (2012). antimicrobial efuicacy of casuarina equisetifolia extracts against some pathogenic microorganisms. journal of medicinal plants research, 6(47), 58195825. 32. zongo, c., savadogo, a., somda, k. m., koudou, j., & traore, a. s. (2011). in vitro evaluation of the antimicrobial and antioxidant properties of extracts from the whole plant of alternanthera pungens. international journal of phytomedicine, 3, 182-191. 33. brantner, a., males, z., papeljnjak, s., & antolic, a. (1996). antimicrobial activity of paliurus spina-christi mill. journal of ethnopharmacology, 52, 119-122. table 1. effect of different concentrations of zno nanoparticles on s. mutans and s. aureus. control s. aureus s. mutans concentrations mg/ml 0 2.02 2.12 0.2 0 2.2 2.27 0.4 0 2.45 2.58 0.6 0 2.68 2.98 0.8 1.25 1.50 lsd = table 2. effect of different concentrations of pepper fruit extract on s. mutans and s. aureus. control s. auruas s. mutans concentrations mg/ml 0 1.35 1.24 100 0 1.56 1.46 200 0 1.78 1.54 300 0 1.89 1.7 350 1.76 1.37 lsd = 938 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.938 http://dentistry3000.pitt.edu emerging role of viral infection in periodontal disease alaa w. izzat1, batool hassan al-ghurabi1, lina sami adham1, zahraa f. shaker2 1college of den*stry, university of baghdad, baghdad, iraq 2college of den*stry, university of al-mustansiriyah, baghdad, iraq abstract periodontal disease is primarily linked to a bacterial infection, such as dental plaque, which harbors a broad microbial community and can induce numerous in:lammatory responses in periodontal tissue. in numerous instances, the local bacterial incursion and host-mediated immune reactions result in severe alveolar bone resorption. while researchers mainly concentrate on identifying periodontopathogenic bacteria, recent investigations have suggested that several viruses may also play a role in the onset and advancement of periodontitis. human viruses can induce cytokine release from both in:lammatory and non-in:lammatory cells, hence compromising periodontal immune protection. this review investigates the potential role of viral infection in the pathogenesis of periodontal disease. open access cita%on: izzat aw, et al. (2025) emerging role of viral infec%on in periodontal disease. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.938 received: may 7, 2025 accepted: may 31, 2025 published: june 27, 2025 copyright: ©2025 izzat aw, et al. this is an open access ar%cle licensed under a crea%ve commons asribu%on work 4.0 united states license. email: batoolamms@codental.uobaghdad.edu. introduc)on periodontal disease, or gum disease, comprises a series of in5lammatory disorders that impact the tissues encircling the teeth [1,2]. in its initial phase, termed gingivitis, the gums exhibit swelling, redness, and potential bleeding. it is regarded as the primary cause of tooth loss among adults globally. in its advanced stage, known as periodontitis, the gums may retract from the tooth, resulting in bone loss, and potentially causing teeth to become loose or dislodge [3]. halitosis (bad breath) may also occur [4]. periodontal disease results from microbial infection, which causes in5lammation and loss of connective tissue attachment as well as alveolar bone around the teeth [3]. bacterial plaque is a primary etiologic factor of periodontitis. in most cases, the bacteria are proteolytic, gram-negative species that produce pathogenic factors that trigger host defense responses, causing in5lammation and tissue damage. microbiological culture and cultureindependent genetic investigations have recognized around 1200 bacterial species and 18,000 phylotypes in the oral cavity and a minimum of 400 bacterial species in subgingival locations. nevertheless, despite the extensive enumeration of many bacteria associated with periodontitis, fewer than 20 species are classi5ied as principal periodontal pathogens. plaque contains non-bacterial microorganisms, including viruses, mycoplasma, yeasts, and protozoa [5]. multiple studies indicate that human viruses may contribute to the etiology of periodontitis by modifying immune defenses, triggering harmful host responses, or exerting direct lytic effects on periodontal tissues [6]. several immune-compromising factors, including smoking, in5lammation, stress, trauma, and immunosuppressive disorders, may reactivate latent viruses residing in the body [7]. in numerous clinical studies, human viruses such as human cytomegalovirus (hcmv), epstein-barr virus (ebv), herpes simplex virus (hsv), and human immunode5iciency virus (hiv) have been implicated in the pathogenesis of periodontal disease [8]. in this research, we chose to shed light on the involvement of viruses in periodontal disease due to the scarcity of previous studies on them and their importance in understanding the basic mechanisms of immune responses to viral infection in the etiopathogenesis of the disease. material and methods for this review, the authors executed an electronic search across many pertinent scienti5ic databases, including scopus, google scholar, pubmed, and web of science. the preliminary evaluation comprised 46 publications published between 2015 and 2025 that had the terms “periodontal disease,” “herpes viruses,” “periodontopathogenic emerging role of viral infeceon in periodontal disease vol 13, no 1 (2025) doi 10.5195/d3000/2025.938 http://dentistry3000.pitt.edu 2 bacteria,” and “in5lammatory response.” eligibility criteria: this study included recent papers, reviews, and case-control studies; all articles included in this review were published in english. results/discussion figure 1 shows the work5low of this work. 46 articles were analyzed. periodontal diseases periodontal infections represent one of the world's most prevalent chronic infectious conditions. periodontitis is still one of the most common causes of tooth loss in adults. the primary etiological component of periodontitis is subgingival bacteria and their toxins; however, the destruction of tissue that occurs is a result of host response activities that are induced by microbial factors [9]. periodontitis is characterized by bone resorption, loss of attachment, and tooth mobility [10]. the periodontal diseases affect 20%-50% of the population in the world. they are associated with a wide spectrum of chronic systemic disorders, including diabetes, cardiovascular, renal, neurodegenerative diseases, in addition to immune-mediated rheumatic diseases, obesity, neoplastic disorders, hypertension, and adverse pregnancy, chronic obstructive pulmonary disease [11]. according to several researchers, untreated periodontal diseases has been linked to higher medical care costs for nonoral diseases. a periodontal disease is a risk factor for a variety of systemic diseases, including cardiovascular diseases and diabetes. the mechanisms underlying this observation are unknown, but it is apparent that in5lammatory cytokines represent a significant link between periodontal and systemic diseases [12]. many variables, including genetic, metabolic, immunological, and in5lammatory elements, are linked to the progression of periodontitis. chronic periodontitis is an in5lammatory disease induced by bacteria, wherein the immune response signi5icantly contributes to the degradation of alveolar bone. appropriate cytokine production is thought to result in protective immunity, whereas inappropriate cytokine production results in tissue death and disease development [13]. moreover, it is widely acknowledged that the regulation of the t helper 1 (th1)/t helper 2 (th2) cell equilibrium is critical to periodontal disease immunoregulation [11]. most cases of periodontitis are historically classi5ied as either aggressive periodontitis or chronic periodontitis. recently, it has become clear that insuf5icient evidence supports this classi5ication based on factors such as varying clinical presentation and scienti5ic investigations. a de5iciency of distinct pathobiology-based differentiations among the speci5ied categories, diagnostic ambiguity, and obstacles in therapy execution. in periodontology, a new classi5ication was introduced despite the 1999 classi5ication have been used for the previous 17 years [14]. biological and clinical discoveries have revised the periodontal, peri-implant illness, and status classi5ication system, resulting in the 2017 world workshop classi5ication [15]. pathophysiology of periodontal diseases dental plaque is a microbial bio5ilm that accumulates on the teeth and gingiva, which is the 5irst stage of gingivitis. in the absence of treatment, gingivitis can advance to periodontitis, a condition characterized by deep periodontal pockets that re5lect the illness and may result in the loss of teeth [16]. interestingly, studies estimate that a human host may have up to 150 different types of microbes in their bio5ilm. in addition, human dental plaque contains over a thousand species [17]. research on the healthy residential microbiome using current dna methods reveals an overabundance of gram-positive microbes, despite the existence of periodontal pathogens [18]. this makes them symbionts, or obligate inhabitants of the subgingival and submarginal regions of the dental bio5ilm formation. further spirochetes, viruses, and gram-negative anaerobic bacteria are all possible causative pathogens [19]. particularly, treponema denticola, porphyromonas gingivalis (p. gingivalis), and tannerella forsythia are members of the red complicated collection of anaerobic microorganisms thought to be the effective bacterial species affecting the onset and progression of periodontal disease within the many different and extensive range of bacteria residing in subgingival bio5ilm formation. it is possible that microbial bio5ilm presence alone is insuf5icient for pd development. the disease develops when a microbial bio5ilm and its host have lost their equilibrium due to dysbiosis brought by the presence of "keystone" bacteria or an excessive response of the immune system in the host to the existence of microbes [20]. moreover, in5lammation is triggered when the host reacts to the typical residing plaque as it builds, which alters the microbial ecology and dysbiosis. an "ecological catastrophe" can be brought by a pathological malfunctioning of the immune response. an upward cycle of escalating dysbiosis feeds on itself. in particular, the immune cell remnants (after apoptosis), the complete spectrum of host immune elements encompasses immunoglobulins, complement, serum proteins, and cytokines. chemokines and collagen breakdown products are elevated during in5lammation. furthermore, serum exudates are present in in5lamed pockets due to enhanced capillary permeability. there is an increase in anaerobic bacteria because an anoxic environment occurs in in5lammatory situations. therefore, given the new environmental circumstances, certain symbionts become pathobionts, i.e., commensals become potentially pathogenic due to an aberration in homeostasis. pathobionts' proliferation (such as p. gingivalis) can further provoke and exacerbate host in5lammatory responses. a transcriptome investigation of subgingival microbiomes in periodontitis demonstrated elevated expression of genes encoding proteolytic enzymes, iron acquisition, and lipopolysaccharide synthesis; this strongly suggests that numerous saccharolytic, anaerobic, and gram-negative bacteria in periodontal in5lammation take advantage of ecological alterations for their nourishing and enormous requirements [21]. as periodontal in5lammation worsens, the bacterial biomass of bio5ilms is associated with human periodontitis development. thus, the dysbiosis and in5lammation encourage one another, creating a vicious cycle that could continue periodontal in5lammation and the disease's development [14]. the altered ecology then promotes the outgrowth of pathobionts, which might exacerbate in5lammatory reactions and prolong the disease's course. in addition to the subgingival bacterial bio5ilm on the tooth root surface and epithelial lining (a), genetic predispositions and epigenetic modi5ications (b), lifestyle-related risk factors (c), systemic diseases (d), and other factors (e) have been recognized as causal risk factors for periodontitis [22]. regarding the degradation of bone and connective tissue in periodontitis, the host response is thought to play a crucial role. antigens of microbes and virulent elements trigger in5lammation and immunological responses. these reactions include both innate and adaptive immune responses. furthermore, there is individual diversity in response due to differences in cytokine and other antimicrobial responses and the individual's environmental circumstances and genetic predispositions. the host's reaction to bacterial invasion entails activating and stimulating various in5lammatory cell types and resident tissue cells [23]. role of viruses in periodontal disease herpesviruses the herpesviridae family, including ebv-1, hcmv, and herpes simplex virus (hsv), has been associated with several types of periodontal disease [24,25]. research indicates that these viruses can infect and modify the functioning of polymorphonuclear leukocytes, lymphocytes, and macrophages, resulting in a reduced capacity to combat emerging role of viral infeceon in periodontal disease vol 13, no 1 (2025) doi 10.5195/d3000/2025.938 http://dentistry3000.pitt.edu 3 bacterial challenges [26]. this may augment the virulence of periodontopathogenic microbiota. in contrast, the dysfunction of polymorphonuclear leukocytes at periodontal sites can promote the proliferation of periodontopathic bacteria and the following progression of destructive periodontal disease. periodontal sites associated with the herpes virus typically exhibit increased concentrations of periodontopathic bacteria, such as p. gingivalis, tannerella forsythia, dialister pneumosintes, dialister invisus, prevotella intermedia, prevotella nigrescens, treponema denticola, campylobacter rectus, and aggregatibacter actinomycetemcomitans [27]. the presence of periodontal hcmv, ebv, and maybe other viruses, local host immune responses, and periodontopathic bacteria constitutes a fragile equilibrium that may result in periodontal damage [28]. the pathogenicity of the herpes virus is intricate, either through direct viral infection and reproduction or by a virally mediated modi5ication of the host's immunological system. the initial stages of periodontitis in immunologically naive hosts may primarily entail cytopathogenic occurrences, while most clinical manifestations in immunocompetent individuals are attributable to cellular or humoral immune responses [29]. herpes viruses may exhibit periodontopathic potential through the following mechanisms: herpes viruses can directly elicit cytopathic effects on keratinocytes, endothelial cells, 5ibroblasts, and in5lammatory cells, including polymorphonuclear leukocytes, lymphocytes, macrophages, and possibly osteocytes. ebv and cmv can infect and alter the functions of monocytes, macrophages, and lymphocytes in periodontal lesions. aggressive periodontitis lesions, potentially due to a herpes virus periodontal infection, exhibit a reduced number of viable cells, an increased presence of t-suppressor lymphocytes, and a higher concentration of b lymphocytes (ebv impact) compared to chronic periodontitis lesions or healthy periodontal sites. cytopathic effects generated by the herpes virus may impede tissue turnover and repair [30]. 2. an infection with the periodontal herpes virus may augment the virulence of the periodontal microbiota. proteins of the herpes virus synthesized on eukaryotic cell membranes may function as innovative bacterial binding sites. cytomegalovirus can enhance the adherence of a. actinomycetemcomitans to primary epithelial cells from periodontal pockets and hela cells [31,32]. herpes viruses may cause dysfunctions in the phagocytosis, adherence, chemotaxis, and bactericidal functions of polymorphonuclear leukocytes, which are crucial for managing periodontopathic bacteria. active epstein-barr virus infection can produce anti-neutrophilic antibodies, resulting in neutropenia, and can polyclonally increase the proliferation and differentiation of b cells. the pathogenic processes of herpesviruses synergistically worsen illness; hence, a dual periodontal infection involving cytomegalovirus and ebv, or cmv and hsv, is likely to manifest in severe forms of periodontitis [33]. epstein-barr virus (ebv) the epstein-barr virus, a common γ-hhv, infects around 95% of individuals asymptomatically and is generally transmitted by blood or oral secretions. the virus proliferates in oropharyngeal epithelial cells or b lymphocytes. nearly all seropositive individuals actively excrete viruses in their saliva [34]. while ebv infection in children is typically subclinical, ebv infection in adults causes infectious mononucleosis. fever, lymphadenopathy, and pharyngitis are the predominant symptoms of infectious mononucleosis. oral ulcers, palatal petechiae, and, less frequently, gingival ulcerations may arise [33]. the most common ebv-related lesion is oral hairy leukoplakia. its presence usually indicates a relatively advanced stage of immunosuppression, such as in hiv infection. hiv-infected patients’ t cells suppress ebv-infected b cells less effectively than immunocompetent people’s t cells. thus, hiv patients have 10–20 times the number of circulating ebv-infected b cells as healthy people [26]. according to the ebv status, there may be two dissimilar mechanisms involved in the pathogenesis of bl: virus-driven and mutational coding [35]. it induces latent infection in b-cells, with occasional lytic reactivation linked to the terminal differentiation of b-cells into plasma cells. chronic latent ebv infection is associated with lymphomas and carcinomas [36]. ebv is associated with several autoimmune disorders [37], serving as a direct cause of multiple sclerosis [38]. recent studies revealed substantial insights into ebv's involvement in periodontitis [39]. they found that ebv commonly infects periodontal junctional epithelial cells [40]. furthermore, it was determined that periodontitis lesions harbor signi5icant plasma cells, predominantly exhibiting ebv positivity [41]. these 5indings provided the initial evidence of active ebv presence in periodontal lesions, indicating that it exacerbates local in5lammation and contributes to the etiology of periodontitis [39]. cytomegalovirus (cmv) cytomegalovirus (cmv) infects t lymphocytes, periodontal monocytes, and macrophages [42]. the global seroprevalence of cmv infection varies from 0% to 95%, contingent upon the geographic region (developed versus developing countries) [43]. the infection typically initiates during childhood or even before birth, as the placenta plays a crucial role in the transmission of cmv to the fetus. evidence indicates that cmv has developed several techniques to circumvent the host immune response and has utilized the host immunological response to facilitate reactivation from dormancy and propagate infection [25,44]. acute cmv infection is characterized by increased viral replication and dissemination to many organs. the pathogenesis of acute infection illustrates a correlation among viral replication levels, organ dysfunction, and disease in patients [32]. the pathogenesis of chronic infection involves a bi-directional relationship between viral gene expression and the host's in5lammatory response: the host's in5lammatory response facilitates viral reactivation, while the virus provokes the host's in5lammatory response. in this case, the disease could be attributed to viral and host functions [45]. consequently, cmv is frequently reactivated, leading to temporary immunosuppression and the proliferation of periodontal pathogenic bacteria. while it has been presumed that the pathogenesis of periodontitis is primarily due to bacterial infection on the tooth surface and within the gingival sulcus, numerous studies have demonstrated that host response factors, including in5lammatory responses and activation of the innate immune system, are essential to the development of periodontal disease [26]. human immunode:iciency virus (hiv) periodontal pathology in hiv-infected patients falls into three categories: (i) linear gingival erythema, (ii) necrotizing ulcerative periodontal diseases, and (iii) enhanced progression of chronic adult periodontitis [26]. in the mid-1980s, several reports described necrotic periodontitis and intense gingival erythema in hiv-infected patients. hiv-associated periodontitis (hiv-p) was named after these acute necrotic lesions, characterized by the loss of the underlying bone, and hiv-associated gingivitis (hiv-g) was termed for a speci5ic kind of gingival erythema that proved resistant to conventional plaque treatment. the contemporary terminology utilized by the american academy of periodontology classi5ies hiv-g lesions as linear gingival erythema and hiv-p lesions as necrotizing ulcerative periodontitis. in addition to linear gingival erythema and necrotizing ulcerative periodontitis, these conditions were more common in patients with hiv. necrotizing ulcerative gingivitis and necrotizing ulcerative periodontitis are two periodontal conditions that have been identi5ied in both hiv-positive and hiv-negative emerging role of viral infeceon in periodontal disease vol 13, no 1 (2025) doi 10.5195/d3000/2025.938 http://dentistry3000.pitt.edu 4 patients. necrotizing ulcerative gingivitis is characterized by interdental papilla ulceration, gingival bleeding, and severe pain. the interproximal papilla is usually described as punched out and a 5ibrinous pseudo membrane typically covers the affected area [46]. the only difference between necrotizing ulcerative periodontitis and necrotizing ulcerative gingivitis is that lesions of necrotizing ulcerative periodontitis in5iltrate the alveolar bone. necrotizing ulcerative periodontitis is distinguished by exposed bone, gingival recession, and tooth movement. additional manifestations of necrotizing ulcerative gingivitis or necrotizing periodontitis are halitosis, lymphadenopathy, pyrexia, and general malaise [26]. conclusions herpes viruses may have a role in the etiopathogenesis of destructive periodontal disease. based on research completed over the last 15 years. active epstein-barr virus or cytomegalovirus infections are statistically correlated with aggressive periodontitis, while latent herpes virus infections are predominantly located in chronic periodontitis and gingivitis sites. herpes viruses are probably not independent periodontopathic agents but instead cooperate with bacteria in the degradation of periodontal tissue. papillomaviruses and other mammalian viruses frequently reside in periodontitis lesions. however, their involvement in the pathophysiology of the disease remains unclear. the recurrent and prolonged activation of periodontal herpes viruses in immunologically naive and immunocompromised individuals may elevate the incidence and severity of clinical exacerbations of periodontal disease. references 1. goker, f., larsson, l., del fabbro, m. & asa’ad, f. j. gene delivery therapeutics in the treatment of periodontitis and peri-implantitis: a state of the art review. international journal of molecular science. 2019;20: 3551. 2. ali, c. j., imran, n. k., abdul, m., & ahmed, a. evaluation of leukocytes cells types counts in blood from patients with different severities of periodontal diseases. 2023; 3(2), 45– 53. 3. bhuyan, r. et al. periodontitis and its inylammatory changes linked to various systemic diseases. a review of its underlying mechanisms. 2022:1–18. 4. gupta, c. and dhruvakumar, d. role of periodontopathogenic virus in periodontal disease: a review. tanta dental journal (january 2017). 2018; 14:51–55. 5. martıńez-garcıá, m. and hernándezlemus, periodontal inylammation and systemic diseases: an overview. frontiers in physiology journal. 2021; 12(october): 1–26. 6. picolo, m. et al. association of herpesvirus and periodontitis: a clinical and laboratorial case – control study. european journal of dentistry 2023;17(4): 1300–1308. 7. chapple, i.l.c. et al. periodontal health and gingival diseases and conditions on an intact and a reduced periodontium: consensus report of workgroup 1 of the 2017 world workshop on the classiyication of periodontal and peri-implant diseases and conditions. journal of periodontology. 2018; 89(march):74–84. 8. azodo, c. c., & erhabor, p. (2015). the roles of viruses in periodontal diseases. journal of dental research and review. 2015; 2(1): 37–41. 9. kwon, t. h. et al. current concepts in the management of periodontitis’, journal of the international clinical dental research organization. 2021;21(2): 53. 10. palwankar, p. et al. diabetes and periodontitis – a socioeconomic disease? journal of evolution of medical and dental sciences. 2021; 10(30):2320–2324. 11. hirtz, c. et al. the potential impact of salivary peptides in periodontitis. critical reviews in clinical laboratory sciences. 2021; 58(7):479– 492. 12. daily za, al-ghurabi bh, al-qarakhli am, moseley r. microrna-155 (mir-155) as an accurate biomarker of periodontal status and coronary heart disease severity: a case–control study. bmc oral health. 2023 nov 16;23(1):868. 13. karam, t. a. an evaluation of serum and salivary adipokines (leptin and resistin) levels in periodontal health and disease. 2015;27(december): 119–124. 14. loos, b. g. & van dyke, t. e. the role of inylammation and genetics in periodontal disease. periodontology journal. 2020;83(1): 26-39. 15. dietrich, t., ower, p., tank, m., west, n., walter, c., needleman, i., hughes, f., wadia, r., milward, m. & hodge, p. j. periodontal diagnosis in the context of the 2017 classiyication system of periodontal diseases and conditions–implementation in clinical practice. british dental journal. 2019;226: 16-22. 16. al-ghurabi bh: the role of soluble tlr-2 in the immunopathogenesis of gingivitis. intern. med. 2021; 28(1): 37–39. 17. gomes, t., lourenço, b., heller, d., martins, r., silva-senem, m. x., varela, v. m., cynesia, m., torres, b., feres-filho, e. j., paula, a., & colombo, v. long-term evaluation of the antimicrobial susceptibility and microbial proyile of subgingival bioyilms in individuals with aggressive periodontitis. brazilian journal of microbiology. 2015;46(2): 493–500. 18. cui z, wang p and gao w (2025) microbial dysbiosis in periodontitis and peri-implantitis: pathogenesis, immune responses, and therapeutic. front. cell. infect. microbiol. 2025; 15:1-19 19. gonçalves, c. et al. association of three putative periodontal pathogens with chronic periodontitis in brazilian subjects’, journal of applied oral science. 2016;24(2):181-185. 20. xu j, yu l, ye s, ye z, yang l, xu x. oral microbiota-host interaction: the chief culprit of alveolar bone resorption. front immunol. 2024 feb 22;15:1-16. 21. hajishengallis, g. porphyromonas gingivalis virulence factors involved in subversion of leukocytes and microbial dysbiosis. virulence journal. 2015; 6(3): 236-243 22. murakami, s. et al. dental plaque – induced gingival conditions. journal of clinical periodontology. 2018;45(february 2017):17–27. 23. moutsopoulos, n.m. and konkel, j.e. tissue-speciyic immunity at the oral mucosal barrier. trends in immunology. 2018; 39(4): 276–287. 24. kustner, e. c., & medeiros, r. are herpes virus associated to aggressive periodontitis? a review of literature. journal of oral and maxillofacial pathology. 2015 ;19(3): 348–355. 25. mahmood hk, al-ghurabi bh. association between anti-cmv igg and salivary levels of il6 and tnf-α in chronic periodontitis. j bagh coll dent. 2020;32(2):5–11. 26. gupta, m. et al. inyluence of viruses in periodontal disease: a review, (april). journal of research and advancement in dentistry. 2022;13(3):1-5 27. golda, a. et al. organotypic model of the gingiva for studying bacterial and viral pathogens implicated in periodontitis. journal of oral microbiology. 2024; 16(1). 28. chandran, w. and dharmadhikari, s. viruses in periodontal disease: a literature review’, international journal of applied dental sciences. 2022; 8(2): 242-245. 29. zhu, s. and viejo-borbolla, a. pathogenesis and virulence of herpes simplex virus.virulence. 2021; 12(1): 2670–2702. 30. gupta, c. and dhruvakumar, d. role of periodontopathogenic virus in periodontal disease: a review, tanta dental journal (january 2017).2018; 14:51–55. 31. zhu, c., li, f., chun, m., wong, m., feng, x., lu, h., & xu, w. association between herpesviruses and chronic periodontitis: a meta-analysis based on case-control studies. 2015.10(2):1-13. 32. nakamura, m. oral human cytomegalovirus prevalence and its relationships with periodontitis and porphyromonas gingivalis in japanese adults: a crosssectional study. international journal of applied dental sciences. 2020: 1–10 33. mahmood, m.k. et al. role of viruses in periodontitis: an extensive review of herpesviruses, human immunodeyiciency virus, coronavirus-19, papillomavirus and hepatitis viruses. world journal of virology. 2024; 13(4): 1–18. 34. kuri, a. et al. epidemiology of epsteinbarr virus infection and infectious mononucleosis in the united kingdom. bmc public health. 2020;20(912): 1–9. 35. izzat, a. w., al-drobie, b., & ismael, f. j. (2024). jaw vs abdominal burkitt’s lymphoma: an analysis of 25 iraqi cases. 107(4), 1–12. 36. ayee, r., ekua, m., ofori, o., wright, e., & quaye, o. epstein barr virus associated lymphomas and epithelia cancers in humans. journal of cancer. 2020; 11(7): 1737-1750. 37. houen, g., trier, n. h., & rockwell, c. epstein-barr virus and systemic autoimmune diseases. frontiers in immunology. 2021; 11(january): 1–13. 38. bjornevik, k., cortese, m., healy, b. c., kuhle, j., mina, m. j., leng, y., elledge, s. j., niebuhr, d. w., & scher, a. i. longitudinal analysis reveals high prevalence of epstein-barr virus associated with multiple sclerosis. 2022;301(january): 296– 301. 39. tonoyan, l. et al. unveiling the etiopathogenic role of epstein-barr virus in periodontitis’. journal of dental research. 2025;104(4) :449 –458 emerging role of viral infeceon in periodontal disease vol 13, no 1 (2025) doi 10.5195/d3000/2025.938 http://dentistry3000.pitt.edu 5 40. tonoyan, l., olivieri, c. v, chevalier, m., marsault, r., olivieri, c. v, chevalier, m., marsault, r., & doglio, a. detection of epstein-barr virus infection in primary junctional epithelial cell cultures. journal of oral microbiology. 2024; 16(1). 41. olivieri, c. v, tonoyan, l., abid, s., doglio, a., hospitalier, c., nice, u. de, hospitalier, c., & nice, u. de. (n.d.). epstein-barr virus-infected plasma cells in periodontitis lesions. microbial pathogenesis. 2020.143:123. 42. sánchez, a.h. and darbinyan, e.h. prevalence of epstein barr virus, cytomegalovirus, and periodontopathic bacteria in patients with periodontitis: a case – control study, clinical and experimental dental research. 2025;11(1): 1–8. 43. fowler, k. et al. a systematic literature review of the global seroprevalence of cytomegalovirus: possible implications for treatment, screening, and vaccine development, bmc public health. 2022: 1–15. 44. chiopris, g. et al. (2020). congenital cytomegalovirus infection: update on diagnosis and treatment. microorganisms journal (cmv). 2020: 1–17. 45. silva, n. et al. host response mechanisms in periodontal diseases, journal of applied oral science. 2015;23(3):329–355. 46. cirib, m. et al. the management of necrotizing gingivitis in paediatric patients: a scoping review and two case reports. children journal. 2024;11(8):1019 figure 1. progression of the search and selection procedure. identi5ication screening illegibility included exclusion based on the year, title and abstract 14 excluded after full text reading 12 distinct titles, abstracts, and recent articles after 2015 72 full text reading 58 final article selected 46 1050 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1050 http://dentistry3000.pitt.edu analysis of different symptoms associated with temporomandibular joint complaints natheer ayed jassem college of den*stry, al-bayan university, baghdad, iraq abstract objec2ve: to determine the frequency of symptoms of pakents who have tmj discomfort. materials and methods: an electronic queskonnaire in the google plasorm and distributed to people with temporomandibular joint disease. results: 420 responses were obtained. 312 people indicated they were tense or nervous and 219 people answered they had headache, and 207 indicated having a problem in the joints. 183 people answered they heard noises in their tmj while chewing or opening their mouth. 78 pakents followed up their symptoms for further inveskgakon and treatment. conclusion: a high frequency of individuals experiences tmd symptoms. open access cita%on: jassem na. (2025) analysis of different symptoms associated with temporomandibular joint complaints. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1050 received: september 18, 2025 accepted: september 24, 2025 published: october 15, 2025 copyright: ©2025 jassem na. this is an open access ar%cle licensed under a crea%ve commons asribu%on work 4.0 united states license. email: natheer.ayed@albayan.edu.iq introduc)on the temporomandibular joint (tmj) is amongst most complex, fragile and frequently used joints in the body, connecting the jaw to the skull via muscles, ligaments and tendons. the temporomandibular joint is very susceptible to abnormalities or disorders due to its unique anatomical and functional relationship with the masticatory muscles and ligaments. the term “temporomandibular disorders” (tmd) refers to a variety of medical conditions that affect the oral muscles and/or joints and tissues. tmd is more frequently occur in female than male and occurs most often in adults between the ages of 20 and 40. tmd shares symptoms with many other chronic conditions. problems with the functioning of the activity of this system explain complexity of the system itself. although many treatments are recommended, a multidisciplinary team is still necessary because tmd is a complex disease. temporomandibular disorders (tmd) typically manifest through a wide range of symptoms, such as pain, limited jaw movement, abnormal joint sounds, and functional difliculties including problems with chewing and speaking. these are frequently associated with psychological disturbances. the most prevalent types of temporomandibular disorders have complicated and mainly unresolved etiologies. although psychogenic factors have been mentioned, they are frequently viewed as aggravating rather than causative factors for temporomandibular disorders, like trauma and malocclusion. as a result, it has been hypothesized that only a subset of patients who are predisposed to temporomandibular disorders will experience pain and dysfunction after an aggravating event like a recent physical or psychological trauma. this raises the possibility of an underlying genetic predisposition to tmd, though this remains a mystery [1-3]. patients with temporomandibular disorders frequently report a variety of nonspecilic symptoms, including headaches, earaches, tinnitus, neck, and shoulder pains; however, since these symptoms are not thought to be specilically diagnostic for temporomandibular disorders, other causes should be looked for and ruled out [4,5]. tmd treatment depends on a proper diagnosis, which in turn needs the patient's medical history, a clinical examination, and the right investigations to add to the information needed to conlirm the diagnosis [1]. orofacial pain, joint noises, limited mouth opening, or a mix of these, along with other less specilic issues like headache and tinnitus, may be the main complaint. it is important to consider how the pain interacts with other characteristics like joint noise and limited mandibular movements. pain should be carefully assessed in terms of its onset, nature, intensity, site, duration, aggravating and relieving factors, and more. additionally, analysis of different symptoms associated with temporomandibular joint complaints vol 13, no 1 (2025) doi 10.5195/d3000.2025.1050 http://dentistry3000.pitt.edu 2 during the consultation, it is necessary to conlirm any underlying factors like stress, anxiety, depression, or a major life event to develop a better picture of the psychogenic basis of the temporomandibular disorder. in general, the likelihood that a patient will react favorably to additional treatment decreases with the length of the symptoms and the number of treatments, particularly "failed treatments," unless a psychogenic cause can be identilied [5,6]. tmd classilication can be generally divided into two categories: extra-articular (affecting the surrounding tissues) and intra-articular (occurring within the joint) [7]. the patients with tmds often have onset of their symptoms during periods of psychological stress (i.e., anxiety) and exacerbation of symptoms during periods of stressful situations. myofacial pain is a symptom usually precipitated by stress and is usually noticed aggravated during the exams period among the university students [8]. temporomandibular joint (tmj) clicking was the most common indication and symptom of tmj dysfunction 73.6%, followed by tmj pain 71.8%, headache 59.4%, bruxism 51.3%, deviation 29.1%, trismus 22.6% and the least one is dislocation 12.3% however, no signilicant difference between females and males, while there was signilicant difference with deviation and highly signilicant difference with headache between females and males [9]. materials and methods an electronic questionnaire was conducted on the google platform. the questionnaire was circulated on 21/2/2024. all people spoke arabic. 420 responses were obtained and participants ranged from 20 to 60 years of age. results table 1 summarizes the frequency of answers. 312 people answered they were tense or nervous. 219 people answered yes for headache and 207 had problem in the joints. 183 people said yes for noise in their tmj while chewing or opening their mouth. 78 patients chose to follow up for further investigation and treatment. discussion we found the biggest number of patients considered themselves nervous and this agrees with previous work [10]. when tmd and headache occur together [11], the treatment of one may have a benelicial therapeutic effect on the other as well. from the 78 patients who came to teaching clinic for follow up, 42 patients had headaches, and 3 patients had been diagnosed previously with migraines and were under medication. malocclusion is also associated with tmd [12]. 60% of the respondents visited the clinic for orthodontic treatment. we found that 39.3 of responders had pain and previous work showed that 59.09% of patients have pain [13]. neck pain was found in 38.3% of the responders in this study and previous work [14] showed a higher frequency (70%). ethical approval scientific research ethics committee of albayan dentistry college 20230s003/21/9/2023. conflict of interest none. data availability the raw data collected by the researcher from which the statistical analysis obtained was saved completely and can be forward at any time under your request. funding none. references 1. ahmad m, schiffman el. temporomandibular joint disorders and orofacial pain. dental clinics. 2016 jan 1;60(1):105-24. https://doi.org/10.1016/j.cden.2015.08.004 2. glaros ag, marszalek jm, williams kb. longitudinal multilevel modeling of facial pain, muscle tension, and stress. journal of dental research. 2016 apr;95(4):416-22. https://doi.org/10.1177/0022034515625216 3. ghurye s, mcmillan r. pain-related temporomandibular disorder–current perspectives and evidence-based management. dental update. 2015 jul 2;42(6):533-46. https://doi.org/10.12968/denu.2015.42.6.533 4. forssell h, kotiranta u, kauko t, suvinen t. explanatory models of illness and treatment goals in temporomandibular disorder pain patients reporting different levels of pain-related disability. j oral facial pain headache. 2016 jan 1;30(1):14-20. doi: 10.11607/ofph.1482 5. graff-radford sb, abbott jj. temporomandibular disorders and headache. oral and maxillofacial surgery clinics. 2016 aug1;28(3):335-49. https://doi.org/10.1016/j.coms.2016.03.004 6. ismail f, eisenburger m, lange k, schneller t, schwabe l, strempel j, stiesch m. identibication of psychological comorbidity in tmd-patients. cranio®. 2016 may 3;34(3):1827. https://doi.org/10.1179/2151090315y.0000000 008 7. gopi i, muthukrishnan a, maragathavalli g. clinical practice guidelines for the management of temporomandibular joint disorders--a review. journal of evolution of medical and dental sciences. 2021 aug 16;10(33):280916. 8. hasan rm, ahmed jn. determination of the effect of stress on the salivary cortisol level among sample university students having myofacial pain. journal of baghdad college of dentistry. 2013;25(3):87-90. 9. hasan rm, diajil ar, ali nm, aswad f, tariq lz. clinical characteristics of temporomandibular disorders in a sample of iraqi patients. wiadomości lekarskie has been published since 1928. 2025;78(1):143-7. doi: 10.36740/wlek/197186 10. santos ea, peinado br, frazao dr, né yg, fagundes nc, magno mb, maia lc, lima rr, souza-rodrigues rd. association between temporomandibular disorders and anxiety: a systematic review. frontiers in psychiatry. 2022 oct 13;13:990430. doi: 10.3389/fpsyt.2022.990430 11. yakkaphan p, smith jg, chana p, renton t, lambru g. temporomandibular disorder and headache prevalence: a systematic review and meta-analysis. cephalalgia reports. 2022 may 16;5:25158163221097352. https://doi.org/10.1177/25158163221097352 12. rustam g. correlation between temporomandibular disorders and malocclusions: a retrospective observational study can malocclusions or previous orthodontic treatments affect temporomandibular disorders? hi ohi-s, 36 min de lectura24 julio 2023. 13. sanchla ad, shrivastav s, bharti l, kamble r, shrivastav sr s. comparative evaluation and correlation of pain pattern in neck musculature observed in mild, moderate, and severe temporomandibular joint disorder cases as compared to non-temporomandibular joint disorder cases. cureus. 2022 oct 9;14(10). doi: 10.7759/cureus.30099 14. alana b. neck pain and tmj disorders: are they related? 2023. ttps://www.medicalnewstoday.com/articles/can-tmj-cause-neck-pain. analysis of different symptoms associated with temporomandibular joint complaints vol 13, no 1 (2025) doi 10.5195/d3000.2025.1050 http://dentistry3000.pitt.edu 3 table 1. questionnaire used. questionnaire questions no (%) yes (%) do you have difliculty in opening your mouth wide? 78.6 21.4 do you have difliculty moving your jaws to the sides? 85.6 14.4 do you feel tired/ have muscle pain when you chew? 60.7 39.3 do you have frequent headaches? 47.8 52.1 do you have neck pain / stiff neck? 61.2 38.3 do you have earaches or pain in that area (tmj)? 68.8 31.2 have you ever noticed any noise in your tmj while chewing or opening your mouth? 56.4 43.6 do you have any habits such as clenching/grinding your teeth? 66.4 33.6 do you feel that your teeth do not articulate well? 50.7 49.28 do you consider yourself a tense (nervous person)? 25.7 74.2 975 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.975 http://dentistry3000.pitt.edu impact of fixed orthodontic appliances on staphylococcus aureus and candida albicans maryam aljbouri, nebras al-dabbagh, thaer jaber al-khafaji college of den*stry, university of babylon, babylon, iraq abstract objec<ve: this study invesegated the prevalence of salivary s. aureus and c. albicans in paeents with fixed orthodonec appliances and assessed their resistance profiles to selected anebacterial and anefungal agents over three-eme intervals. material and methods: a prospeceve cohort of 40 paeents (20 males, 20 females) undergoing orthodonec treatment was followed across three eme points: baseline (t0), two weeks post-applicaeon (t1), and four weeks post-applicaeon (t2). unsemulated saliva samples were collected and cultured for microbial ideneficaeon. organisms were confirmed via vitek biochemical teseng. anemicrobial suscepebility was determined using the disc diffusion method against six anebacterial and six anefungal agents. data were analyzed using two way-anova and chi-square tests. results: among the 80 total samples analyzed, 73.75% were posieve for s. aureus and/or c. albicans. s. aureus was most prevalent, with clindamycin and vancomycin showing the strongest anebacterial acevity. resistance to amoxicillin, ampicillin, and erythromycin was notably high. for c. albicans, fluconazole and amphotericin b demonstrated the highest efficacy, while clotrimazole and nystaen showed poor inhibieon. resistance pa^erns might suggest a biofilm-associated microbial adaptaeon and reduced suscepebility over eme. conclusion: fixed orthodonec appliances significantly influence oral microbial ecology by facilitaeng colonizaeon of resistant strains of s. aureus and c. albicans. the findings highlight the importance of rouene microbial surveillance and personalized anemicrobial strategies in orthodonec care to miegate infeceon risks and manage emerging resistance. open access cita%on: aljbouri m, et al. (2025) impact of fixed ortohon%c appliances on staphylococcus aureus and candida albicans. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.975 received: june 30, 2025 accepted: july 7, 2025 published: august 21, 2025 copyright: ©2025 aljbouri m, et al. this is an open access ar%cle licensed under a crea%ve commons atribu%on work 4.0 united states license. email: mariamaljbori1999@gmail.com introduc)on orthodontic appliances significantly modify the physiological environment of the oral cavity by hindering natural cleaning mechanisms, leading to increased plaque accumulation and impaired oral hygiene. these devices create retention sites that facilitate biofilm formation composed of diverse bacteria and fungi, which colonize various oral surfaces. consequently, orthodontic biofilms contribute to microbial overgrowth and increase the risk of infection if hygiene is not properly maintained [1]. fixed orthodontic components such as bands, wires, and brackets complicate oral hygiene practices. their complex structure and the extended surface area they cover hinder effective cleaning, encouraging plaque accumulation. inadequate oral hygiene in orthodontic patients can lead to gingival inflammation and progressive periodontal disease. therefore, maintaining high standards of cleanliness during orthodontic treatment is crucial to preventing long-term oral health complications [2]. the oral cavity hosts the second most diverse microbial community in the human body, following the gut. it comprises numerous bacteria, fungi, and viruses that coexist in a balanced microbiome under normal conditions. this ecosystem plays a vital role in oral health but is vulnerable to disruption through external influences such as orthodontic treatment [3]. orthodontic therapy has been shown to alter the oral microbiota composition, increasing the prevalence of streptococci, staphylococci, lactobacilli, and yeasts. the physical presence of orthodontic appliances fosters bacterial plaque accumulation, exacerbates gingival inflammation, and deepens probing depth. these conditions create an ideal niche for pathogenic bacteria, potentially escalating to periodontitis if left unchecked [4,5]. staphylococcus aureus is commonly found on the skin and mucosal surfaces, including the impact of fixed orthodonec appliances on staphylococcus aureus and candida albicans vol 13, no 1 (2025) doi 10.5195/d3000.2025.975 http://dentistry3000.pitt.edu 2 oropharynx. though typically harmless in carriers, it can become pathogenic and cause a wide range of infections, from minor skin conditions to severe systemic diseases like osteomyelitis and endocarditis. around 30% of individuals are asymptomatic carriers, but the spread of antibiotic-resistant strains such as methicillin-resistant s. aureus (mrsa) poses a significant clinical challenge [6-8]. antibiotic resistance in s. aureus, particularly mrsa, has made treatment increasingly difficult. these strains have developed resistance to several frontline antibiotics, including penicillin, methicillin, and in some cases, daptomycin and vancomycin. this resistance complicates therapeutic options and demands the development of novel approaches to bacterial infection control [9,10]. as bacterial resistance accelerates faster than antibiotic development, the potential for a post-antibiotic era looms. understanding bacterial resistance mechanisms such as changes in cell wall composition and virulence is vital for designing new treatments. the peptidoglycan-rich cell wall of s. aureus is a critical target due to its role in maintaining bacterial integrity and pathogenicity [11]. candida species, particularly c. albicans, naturally colonize the skin, mucosa, and gastrointestinal tract without causing harm. however, under certain conditions like immunosuppression or poor oral hygiene, they can overgrow and lead to candidiasis. symptoms vary depending on the infection site [12-14]. candida albicans predominates in the oral cavity, with colonization reported in up to 60% of young adults. however, nonalbicans species such as c. glabrata and c. krusei are becoming more common. these species often display resistance to commonly used antifungals like azoles and polyenes, emphasizing the importance of targeted antifungal therapy [15-17]. this study aimed to evaluate the impact of xixed orthodontic appliances on the salivary prevalence of staphylococcus aureus and candida albicans, as well as their resistance to various antimicrobial agents. the objectives included isolating and identifying both microorganisms using culture media, quantifying their presence at three different treatment intervals, and assessing their resistance patterns to antibacterial and antifungal drugs over time. subjects and methods a prospective cohort study was followed in which 40 individuals (20: females, 20: males) have undergone orthodontic treatment in orthodontic department, nasser almousawi specialized center, najaf, iraq. eligible study participants: included individuals aged 18 to 35 years with class i malocclusion and mild to moderate dental crowding, characterized by an anb angle between 0º and 4º. subjects also exhibited a normal vertical relationship (sn-mp angle of 32º ± 2), a straight facial proxile, and a full set of teeth, with or without third molars. excluding criteria: participants were excluded if they had a history of orthodontic treatment, orthognathic surgery, systemic diseases, craniofacial anomalies, or traumatic injuries. prior to treatment, all necessary patient information was recorded. saliva samples were collected at three time points: before the application of fixed orthodontic appliances (t0), two weeks after (t1), and four weeks after treatment began (t2). each participant underwent comprehensive intraoral and extraoral examinations, including panoramic and lateral cephalometric radiographs, to establish a diagnosis and determine the appropriate treatment plan. sample collection: unstimulated saliva samples were collected from participants between 9 and 11 a.m. using the passive drooling method.[18] subjects abstained from food, drink, and oral hygiene for at least one hour prior. after rinsing with 150 ml distilled water and resting, they allowed approximately 3 ml of saliva to collect in labeled containers. samples were immediately stored on ice and transported to the microbiology laboratory in najaf governorate [19]. ethical approval: this study was conducted in accordance with the ethical principles of the declaration of helsinki and the scientixic committee in college of dentistry/ university of babylon was approved under protocol number 123, dated 03/10/2024. isolation and identi<ication of s. aureus: the specimens were transferred directly to the laboratory. at 37°c for 24 hours, blood agar media was used then mannitol salt agar (msa) for the cultivation of s. aureus. the grown bacterial colonies were subjected to various biochemical tests by api system (api 20 -vitek apparatus) to ensure their identity [20]. antibiotic sensitivity test: the antibiotic sensitivity test was done following the disk diffusion method.[21] using a sterile loop, 45 colonies were suspended in 2 ml of sterile normal saline. the bacterial suspension's turbidity was adjusted to a mcfarland standard of 0.5. the bacteria were then inoculated into the mueller-hinton agar plate by using a sterile swab. the plates were left at room temperature for about 5 minutes. the following antibiotics were used: clindamycin (2μg), erythromycin (15µg), azithromycin (15µg), ampicillin (10µg), amoxicillin (10µg) and vancomycin (30μg). using sterile forceps, the antibiotic discs were placed and xixed on the surface of the cultivated agar. finally, the plates were inverted and incubated at 37°c for 18 h. preparation of specialized culture media for candida albicans sabouraud's dextrose agar medium (sda) this medium (65 g) was dissolved in (1000 ml) distilled water and heated to dissolve in a glass beaker, after which the medium was sterilized in an autoclave, distributed to petri dishes, and (0.05 g) anti-chloramphenicol was added after cooling the medium to (45°c) to prevent bacterial growth [22]. chromo agar candida medium the medium was prepared according to the manufacturer's instructions by suspending 42.72 g of powder in 1000 ml of distilled water, and the solution was heated to boiling point to completely dissolve the medium. isolation and identi<ication of candida diagnosis using biochemical test (vitek apparatus) it is a selective and differential chromogenic medium intended for the identixication of candida species and to determine color and colony morphology well when chromium was used for candida isolate directly and xilamentous fungi and of clinical specimens. c. albicans grow as green colonies after incubation for 48 hours at 37°c. this medium does not require sterilization in an autoclave.[23] diagnosed by genus and species using biochemical tests, the vitek system consists of cassette and reagent cards that contained 64 pits (every pit represents the substrate to conduct the test), and plastic pipes, as well as the densi chek device and the unity of the input and output of information.[24] diagnosis of candida species isolates using biochemical tests diagnosis using the vitek® apparatus (vitek® 2 densichek; biomérieux, france). yeast isolates were diagnosed as genus and species using biochemical tests. study the resistance of oral c. albicans to the antifungal drugs: a clinical isolates of candida species were tested for antifungal susceptibility against six antifungal drugs [clotrimazole (10µg), fluconazole (10µg), nystatin (50µg), 5-flucytosine (1µg), caspofungin (5µg), and amphotericin b (100µg)]; the antifungal susceptibility of oral c. albicans was tested by disc diffusion. statistical analysis: data were analyzed using spss (version 26, spss inc. chicago, illinois, usa). descriptive statistics (mean, standard error), as the results was expressed as %. statistical analysis was carried out using chi-square. differences w ere compared by two-way anova at p≤0.05 using duncan’s multiple range test [25]. impact of fixed orthodonec appliances on staphylococcus aureus and candida albicans vol 13, no 1 (2025) doi 10.5195/d3000.2025.975 http://dentistry3000.pitt.edu 3 results out of the total 40 participants enrolled in this study, the culture results showed a signixicant presence of staphylococcus aureus (80%) and candida albicans (68%) among individuals undergoing xixed orthodontic treatment. among male participants (n = 20) in three period times (t0, t1 and t2), staphylococcus aureus was detected in 16 cases in each period time (40% vs total no.) while candida albicans was found in 13 cases in each period time (32.5% vs total no.). in the female group (n = 20) in three period times (t0, t1 and t2), 16 participants (40% vs total no.) tested positive for staphylococcus aureus in each period time, while only 14 cases (35% vs total no.) were positive for candida albicans in each period time. overall, 59 out of 80 samples (73.75%) were culture positive for either s. aureus or c. albicans, whereas 21 samples (26.25%) were negative for both. the prevalence appeared consistent across genders (table 1). antibiotics susceptibility test for staphylococcus aureus isolates vancomycin and clindamycin showed the largest inhibition zones (14–15 mm), suggesting strong antibacterial activity against s. aureus. the difference is statistically signixicant (*p < 0.001) compared to all other antibiotics while azithromycin showed intermediate inhibition (10–11 mm), which is statistically less than clindamycin but still more effective than ampicillin and erythromycin. the weakest activity were amoxicillin, ampicillin, and erythromycin that demonstrated smaller inhibition zones (5–8 mm), indicating poor antimicrobial effectiveness. these results may rexlect the emergence of resistance to β-lactam and macrolide antibiotics among oral s. aureus isolates (figure 1). antifungals susceptibility test for candida albicans isolates fluconazole and amphotericin b produced the largest inhibition zones (13–14 mm), indicating the strongest antifungal activity against c. albicans. their effect is statistically signixicant (p < 0.001) compared to all other agents while 5-flucytosine showed moderate inhibition (11 mm) and was signixicantly better than nystatin and clotrimazole but slightly less effective than fluconazole and amphotericin. clotrimazole and nystatin had the smallest inhibition zones (5–6 mm), suggesting limited antifungal effectiveness against oral c. albicans isolates. the statistical differences between clotrimazole, nystatin, and caspofungin were not signixicant (figure 2). discussion this study highlighted a significant increase in the prevalence of staphylococcus aureus and candida albicans in the saliva of patients undergoing treatment with fixed orthodontic appliances. the components of these appliances, such as brackets, wires, bands, and ligatures, create multiple niches that favour the accumulation of dental plaque and biofilm. these structures hinder natural self-cleansing mechanisms and complicate routine oral hygiene, fostering a micro-environment that promotes microbial colonization and growth. consequently, the oral microbial load and diversity are notably altered, creating conditions conducive to the proliferation of pathogenic organisms, especially during the early phases of orthodontic treatment [1,2,4,5]. this observation is in line with existing research indicating that fixed appliances function as reservoirs for microbial biofilms. these devices hinder effective plaque removal and increase the oral burden of opportunistic pathogens. both s. aureus and c. albicans are part of the normal oral flora but can become pathogenic when the microbial balance is disrupted such as by reduced immunity, increased substrate availability, or structural changes in the oral environment. this microbial shift can lead to localized infections, including oral candidiasis and gingival inflammation, as the pathogens exploit the altered oral ecology [3,6,26-28]. antibiotic and antifungal susceptibility tests revealed patterns of resistance that pose a clinical concern. methicillin-resistant s. aureus (mrsa) and antifungal-resistant c. albicans are increasingly identified in orthodontic patients, raising treatment challenges. resistance mechanisms such as gene mutations, efflux pumps, and biofilmrelated protection contribute to diminished therapeutic outcomes. this highlights the need for ongoing microbiological surveillance and tailored antimicrobial strategies in orthodontic practice to prevent complications arising from resistant infections [8,9,29-32]. the antibiotic sensitivity analysis in this study showed limited efficacy of commonly used agents such as amoxicillin, ampicillin, and erythromycin, likely due to β-lactamase production and other resistance mechanisms. in contrast, clindamycin and vancomycin demonstrated strong inhibitory effects, reflecting their distinct modes of action. clindamycin by targeting the 50s ribosomal subunit and vancomycin by inhibiting cell wall synthesis with binding to the terminal d-alanyl-d-alanine residues of peptidogly can precursors, specifically lipid ii [9,29,33]. likewise, antifungal testing demonstrated varied efficacy. clotrimazole and nystatin showed minimal inhibition zones, suggesting possible resistance due to frequent topical use. conversely, fluconazole and amphotericin b were highly effective against c. albicans, supporting their continued use in oral candidiasis. amphotericin b’s mechanism of binding to ergosterol and disrupting fungal membranes underpins its potency, especially in drugresistant strains [16,28,3034,35]. 5flucytosine, though moderately effective, remains valuable in combination therapies due to its rapid resistance development when used alone [36,37]. overall, these findings emphasize the importance of incorporating routine susceptibility testing and individualized treatment approaches into orthodontic care. as fixed appliances significantly alter the oral microenvironment, they increase the risk of harboring resistant microbial species. regular microbial assessments and targeted antimicrobial interventions are essential for preventing and managing persistent or recurrent oral infections in orthodontic patients [1]. acknowledgements we would like to extend our heartfelt thanks to the orthodontic department, nasser almousawi specialized center, najaf, iraq, and special thanks to orthodontist dr. almustafa alhumadi for their support and the assistance provided. conflict of interest statement none to declare. references 1. lucchese a, bondemark l. the influence of orthodontic treatment on oral microbiology. biological mechanisms of tooth movement, 2021: 139–158. 2. mazin h, salman sa, salah r. the effect of fixed orthodontic appliances on gingival health diode laser versus scalpel gingivectomy view project periodontology view project. iosr journal of dental and medical sciences (iosr-jdms), 15(11), 2016: 82–88. available at: https://doi.org/10.9790/0853-1517078288. 3. deo pn, deshmukh r. oral microbiome: unveiling the fundamentals. journal of oral and maxillofacial pathology. jomfp. 2019; 23(1), p. 122. 4. kado i, hisatsune j, tsuruda k, tanimoto k, sugai m. the impact of fixed orthodontic appliances on oral microbiome dynamics in japanese patients. sci rep. 2020;10(1):21989. doi: 10.1038/s41598-020-78971-2. 5. kim sh, choi ds, jang i, cha bk, jostbrinkmann pg, song js. microbiologic changes in subgingival plaque before and during the early period of orthodontic treatment. angle orthod. 2012 ;82(2):254-60. doi: 10.2319/030311-156.1. 6. rojo p et al. (2010). communityassociated s. aureus infections in children. expert rev anti infect ther, 8, 541–554. 7. ryu,s.; song, p.i.; seo, c.h.; cheong, h.; park, y. colonization and infection of the skin by s. impact of fixed orthodonec appliances on staphylococcus aureus and candida albicans vol 13, no 1 (2025) doi 10.5195/d3000.2025.975 http://dentistry3000.pitt.edu 4 aureus: immune system evasion and the response to cationic antimicrobial peptides. int. j. mol. sci. 2014, 15, 8753–8772. 8. kale, p., dhawan, b. (2016). the changing face of community-acquired methicillin-resistant staphylococcus aureus. indian j. med. microbiol., 34, 275–285. 9. casanova, g.n.; ruiz, s.m.; bellido, m.j.l. mechanisms of resistance to daptomycin in staphylococcus aureus. rev. española quimioter. (2017), 30, 391–396. 10. sirichoat, a.; lulitanond, a.; kanlaya, r.; tavichakorntrakool, r.; chanawong, a.; wongthong, s.; thongboonkerd, v. phenotypic characteristics and comparative proteomics of staphylococcus aureus strains with different vancomycin-resistance levels. diagn. microbiol. infect. dis. 2016, 86, 340–344. 11. romaniuk, j.a.; cegelski, l. bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell nmr. philos. trans. r. soc. b biol. sci. 2015, 370. 12. al-tekreeti ar, al-halbosiy mmf, dheeb bi, hashim aj, al-zuhairi afh. molecular identification of clinical candida isolates by simple and randomly amplified polymorphic dna-pcr. arab j. sci. eng. (2017); doi 10.1007/s13369-0172762-1. 13. hussain af, sulaiman gm, dheeb bi, hashim aj, seddiq sh. improving conditions for gliotoxin production by local isolates of aspergillus fumigatus. journal of biotechnology research center. (2017); 11(2):14-24. 14. ayesha a. prevalence, antifungal susceptibility and biofilm characterization of candida species isolated from tertiary care hospitals, 2016; quaid-i-azam university, islamabad, pakistan (2018); 1-22. 15. khanpayeh e, jafari aa, tabatabaei z. comparison of salivary candida profile in patients with fixed and removable orthodontic appliances therapy. iran j microbiol. 2014 aug;6(4):263-8. 16. al-harba, huda reyad; nasir-alla, nebrass; jabir, azher abdulhafidh. the correlation between oral candida albicans and interlukine-23 in diabetic patients in hilla city, iraq. medical journal of babylon 21(4):p 915-920, october-december 2024. | doi: 10.4103/mjbl.mjbl_517_23. 17. alhamadi w, al-saigh rj, al-dabagh nn, al-humadi hw. oral candida in patients with fixed orthodontic appliance: in vitro combination therapy. biomed res int. (2017); 2017:1802875. doi: 10.1155/2017/1802875. 18. navazesh, m., kumar, s.k. 2008. measuring salivary flow: challenges and opportunities. the journal of the american dental association, 139, 35s-40s. 19. kaushik, r., yeltiwar, r. k., pushpanshu, k. 2011. salivary interleukin-1β levels in patients with chronic periodontitis before and after periodontal phase i therapy and healthy controls: a case-control study. journal of periodontology, 82, 1353-1359 20. james c, natalie s. microbiology. a laboratory manual. pearson education, 2014. available from: https://lib.hpu.edu.vn/handle/123456789/289 98 21. hudzicki j. kirby-bauer disk diffusion susceptibility test protocol. american society for microbiology. 2009;15:55-63. 22. scognamiglio t, zinchuk r, gumpeni p, larone dh. comparison of inhibitory mold agar to sabouraud dextrose agar as a primary medium for isolation of fungi. j clin microbiol. 2010 may;48(5):1924-5. doi: 10.1128/jcm.01814-09. 23. nadeem, s.g., hakim, s.t., kazmi, s.u. (2010). use of chromagar candida for the presumptive identification of candida species directly from clinical specimens in resourcelimited settings, libyan journal of medicine, 5(1). 24. abdulbaqi nj, dheeb bi, irshad r. expression of biotransformation and antioxidant genes in the liver of albino mice after exposure to aflatoxin b1 and an antioxidant sourced from turmeric (curcuma longa). jordan journal of biological sciences. (2018); 11(2) 89 – 93. 25. cohen, j. (2013). statistical power analysis for the behavioral sciences. routledge. 26. wade, w.g. the oral microbiome in health and disease, pharmacological research, (2013); 69(1), pp. 137–143. 27. salman, s.a., salah, r. (2016). the effect of fixed orthodontic appliances on gingival health diode laser versus scalpel gingivectomy view project periodontology view project, iosr journal of dental and medical sciences (iosr-jdms), 15(11), pp. 82–88. available at: https://doi.org/10.9790/08531517078288. 28. moyes dl, wilson d, richardson jp, mogavero s, tang sx, wernecke j, höfs s, gratacap rl, robbins j, runglall m, murciano c, blagojevic m, thavaraj s, förster tm, hebecker b, kasper l, vizcay g, iancu si, kichik n, häder a, kurzai o, luo t, krüger t, kniemeyer o, cota e, bader o, wheeler rt, gutsmann t, hube b, naglik jr. candidalys in is a fungal peptide toxin critical for mucosal infection. nature. 2016 apr 7;532(7597):64-8. doi: 10.1038/nature17625. 29. ali t, basit a, karim am, lee j-h, jeon j-h, lee sh. mutation-based antibiotic resistance mechanism in methicillin-resistant staphylococcus aureus clinical isolates. pharmaceuticals. https://doi.org/10.3390/ph14050420 18. (2021);14(5):420. 30. nobile cj, jjhnson ad. (2015). candida albicans biofilms and human disease. annu rev microbiol, 69:71–92. 31. parastan r, kargar m, solhjoo k, kafilzadeh f. a synergistic association between adhesion-related genes and multidrug resistance patterns of staphylococcus aureus isolates from different patients and healthy individuals. journal of global antimicrobial resistance. 2020;22:37985. https://doi.org/10.1016/j.jgar.2020.02.025 32. abbasi montazeri, e., khosravi, a.d., khazaei, s. et al. prevalence of methicillin resistance and superantigenic toxins in staphylococcus aureus strains isolated from patients with cancer. bmc microbiol 21, 262 (2021). https://doi.org/10.1186/s12866-02102319-7 33. gardete s, tomasz a. mechanisms of vancomycin resistance in staphylococcus aureus. j clin invest. 2014 jul;124(7):2836-40. doi: 10.1172/jci68834. 34. achkar jm, fries bc. candida infections of the genitourinary tract. clinical microbiology reviews. (2010); 23(2), 253–273. 35. abdalwahd n., al-saigh r.j., al-humadi h.w. assessment of antifungal drugs’ activity against some candida albicans isolates in the presence or absence of human albumin: a study employing an in vitro pharmacokinetics / pharmacodynamics model. rev. clin. pharmacol. pharmacokinet. int. ed. 38(sup2): 39-42 (2024). https://doi.org/10.61873/sexh5182 36. bhavan, p., rajkumar, r, subramanian, radhakrishnan, seenivasan, dr. chandrasekar & kannan, soundarapandian. culture and identification of candida albicans from vaginal ulcer and separation of enolase on sds-page. international journal of biology. (2010); vol 2. 10.5539/ijb.v2n1p84. 37. abbas, nada fadhil. comparison between conventional methods and molecular diagnosis for candida albicans and candida dubliniensis isolated from cancer patients infected with oral candidiasis. medical journal of babylon 21(suppl 2):p s276-s281, november 2024. | doi: 10.4103/mjbl.mjbl_1079_23 impact of fixed orthodonec appliances on staphylococcus aureus and candida albicans vol 13, no 1 (2025) doi 10.5195/d3000.2025.975 http://dentistry3000.pitt.edu 5 table 1. culture positive and culture negative for staphylococcus aureus and candida albicans. type of microorganisms gender culture positive culture negative total staphylococcus aureus male 16 32 4 8 40 female 16 4 candida albicans male 13 27 7 13 40 female 14 6 total 59 21 80 impact of fixed orthodonec appliances on staphylococcus aureus and candida albicans vol 13, no 1 (2025) doi 10.5195/d3000.2025.975 http://dentistry3000.pitt.edu 6 figure 1. the bar graph illustrates the inhibition zones (mm) for staphylococcus aureus isolates at three-time intervals (t0 (a), t1 (b), t2 (c)), when exposed to six different antibiotics: amoxicillin, vancomycin, ampicillin, erythromycin, azithromycin, and clindamycin. statistical comparisons between groups are indicated with asterisks (* for p < 0.05, ** for p < 0.01, *** for p < 0.001) and "ns" for non-signixicant differences. impact of fixed orthodonec appliances on staphylococcus aureus and candida albicans vol 13, no 1 (2025) doi 10.5195/d3000.2025.975 http://dentistry3000.pitt.edu 7 figure 2. the bar graph illustrates the inhibition zones (mm) for candida albicans isolates at three-time intervals (t0 (a), t1 (b), t2 (c)), when exposed to six antifungal agents: clotrimazole, fluconazole, nystatin, 5-flucytosine, caspofungin, and amphotericin b. statistical comparisons between groups are annotated with *** for p < 0.001 and “ns” for non-signixicant differences. 846 final studying water absorp3on, solubility, and uv absorp3on of room temperature vulcanized maxillofacial silicone a@er the addi3on of stron3um titanate nano-powder vol 13 no 1 (2025) doi 10.5195/d3000.2025.846 h#p://den*stry3000.pi#.edu studying water absorption, solubility, and uv absorption of room temperature vulcanized maxillofacial silicone after the addition of strontium titanate nano-powder safwan abid-alhameed suliman, nusaiba mustafa muhammad, rusal saad ahmed college of dentistry, tikrit university, tikrit, iraq abstract objec&ve: the study's primary goal is to assess how adding stron6um 6tanate (srtio3) nanopar6cles to vst-50 room temperature-vulcanized silicone elastomer affects water absorp6on, solubility and uv light absorp6on. materials and method: two weight percentages (1% and 1.5% srtio3) were chosen and incorporated into the vst-50 silicone. sixty specimens were collected and split in half. there were 30 samples tested for water and solvent solubility and 30 samples tested for uv light penetra6on. applying a one-way anova and post hoc p < 0.05. scanning electron microscope and fourier transform infra-red were also used in the study. results: there was an increase in the solubility and amount of water absorbed by the experimental groups (1% and 1.5%). also, the uv light absorp6on was significantly increased for the experimental groups. conclusion: srtio3 nanopar6cle's addi6on into the vst-50 silicone increased water absorp6on, solubility, and uv light absorp6on. keywords: stron6um 6tanate, maxillofacial silicone (vst-50), water absorp6on, solubility, uv absorp6on. cita;on: suliman sa, et al. (2025) studying water absorp;on, solubility, and uv absorp;on of room temperature vulcanized maxillofacial silicone aser the addi;on of stron;um titanate nano-powder. den;stry 3000. 1:a001 doi:10.5195/d3000.2025.846 received: february 8, 2025 accepted: april 1, 2025 published: april 3, 2025 copyright: ©2025 suliman sa, et al. this is an open access ar;cle licensed under a crea;ve commons a[ribu;on work 4.0 united states license. email: safwanalsabaawi@tu.edu.iq introduction patients might be affected by facial defects and deformities because of traumatic accidents, congenital diseases, or tumor surgeries. a maxillofacial prosthesis is used to treat such patients with craniofacial and combined intra/extra oral abnormalities [1]. the number of patients needing facial prostheses has increased in recent years, owing to an aging population and rising survival rates among patients with cancers in the craniofacial regions [2]. different materials can be used in the field of maxillofacial restorations such as chlorinated polyethylene, polymethylmethacrylate, acrylic, polyurethanes, polyvinyl chlorides and silicone elastomers. in recent years, silicone has emerged as the material of choice for maxillofacial prosthetics. the primary reasons for this are its low maintenance requirements, high strength, and extended lifespan., chemical inertness and comfortable to the patient [3]. in clinical use, silicone's physical and mechanical characteristics deteriorate, resulting in textural changes, poorly fitting edges, and color changes [4]. these changes are related to the patient's care and maintenance of the prosthesis' cleanliness and exposure to uv radiation, air pollution, and temperature changes [5,6]. since the silicone elastomer comes into touch with the wound surface and mucosa, germs from a patient's saliva and blood tend to infect elastomers. so that the washing and cleaning of the silicone prosthetic device is important [7]. in terms of how long a maxillofacial prosthesis will last and how well it studying water absorp3on, solubility, and uv absorp3on of room temperature vulcanized maxillofacial silicone a@er the addi3on of stron3um titanate nano-powder vol 13 no 1 (2025) doi 10.5195/d3000.2025.846 h#p://den*stry3000.pi#.edu will function, there are several aspects to consider. many researchers have proposed what they believe are the most important aspects over the yearschemical stability, dimensional stability, and little to almost no absorption of water, to name just a few [8]. elasticity and elongation have also been brought up as important factors in discussions about dimensional stability. on the other hand, the prosthesis may need to be replaced if the material absorbs so much water that it distorts or dissolves. be rendered useless [8]. water absorption has been demonstrated to influence the look of various silicone compounds in previous research [9]. water absorption tests would be very helpful in determining the source of the change in appearance. they may be watersoluble if they can take it on. since nanotechnology is becoming a key component of dentistry nanomaterials are more efficient than conventional materials. the development of modified nanoparticles would aid in the elimination of many of the flaws in dental materials [10] over the last several years, research has concentrated on developing a new and better material by fusing nanofillers with silicone material that uses both the rigidity of the polymer matrix and the pliability of the nano-oxides (12). this study used the chemical formula srtio3 for strontium titanate oxide. hulterström et al. investigated water sorption and solubility of seven silicones and concluded that condensation-type polymers can have significant changes in volume as they exposed to fluids [11]. salih et al. used a blend consisting of silicone rubber and pmma as the matrix to which they added several natural and synthetic fibers and nanoparticles (tio2, sewak fibers and uhmwpe fibers) [12]. the results showed an increase in water absorption. water absorption and solubility of mdx10 silicone were also investigated after the addition of pigments and opacifiers. the results showed a non-significant increase in both properties after artificial aging [13]. material and methods materials vst-50 is a combination of strontium titanate nano-powder with an average particle size of 80100 nm and rtv silicone (factor ii inc., usa) is a vulcanized silicone that may be used at room temperature (nanoshel, usa). these two materials were utilized in this work. two weight percentages of strontium titanate were utilized in the study (1% and 1.5%). specimens fabrication sixty samples were produced and randomly assigned to one of three groups (group a: control; group b: 1%; and group c: 1.5%). twenty samples were used for each group, with each sample being tested for solubility and water absorption in water. we tested ten samples for uv absorbance. mold fabrication acrylic sheets 3 ±0.2 mm (for water absorption and solubility) and 2mm (for uv absorption) in thickness were cut by a laser engraving machine (jl-1612, jinan link manufacture and trading co., ltd., china). the cutting was done by the predetermined standards of the autocad (computer aided design) program. bolts and nuts hold the mold's three components—the base, the matrix, and the cover—together. in addition, g-clamps utilized around the edges to tighten things up even further. mixing according to vst-50 silicone is blended per vst-50 silicone manufacturer's recommendations. a 10:1 ratio (ten parts base to one part catalyst). to avoid air entrapment, a vacuum mixer was utilized to mix the silicone. the vst-50 silicone base and catalyst were weighed to an accuracy of 0.0000 on an electronic scale studying water absorp3on, solubility, and uv absorp3on of room temperature vulcanized maxillofacial silicone a@er the addi3on of stron3um titanate nano-powder vol 13 no 1 (2025) doi 10.5195/d3000.2025.846 h#p://den*stry3000.pi#.edu before being combined in a vacuum mixer at a speed of 360 rpm and a pressure of 10 bar for 5 minutes. for 1% and 1.5% specimens, the appropriate srtio3 concentration is measured on a digital balance and then the base part is added. in the vacuum mixer, the srtio3 and the base were mixed for three minutes, without air evacuation to prevent the suction of nanoparticles. after that, while the room is being sucked dry of air, the nano-powder and the base are combined for 7 minutes. the catalyst was introduced after the liquid had cooled for 5 minutes. was subjected to a 5-minute vacuum mixing [14]. preparation and storage of specimens the silicone mixture was applied gently and slowly metal spatula until all matrix specimen regions were filled; specimens were somewhat overloaded to prevent deficiency. after placing the matrix section on top of the cover, we applied moderate, continuous hand pressure to the cover's center until the bolts and nuts were tightened at the corners and the cover was secured in place. secured around the mold boundaries with the g-clamps. the pressure force of bolts and g clamps should eliminate the air bubbles and excess silicone material after mold is closed. specimens with included bubbles were excluded. after 24 hours at room temperature (23°c � 2°) the molds were removed gently, and the specimens were placed in a storage box (the same one used for the vaccination). the specimens were stored for 16 hours at 20-25°c, 50 10 humidity, and in the storage box [15]. testing procedures water absorption specimens for water absorption test were made according to astm 570: 2018 [16]. the specimen dimensions are 50.8mm in diameter and 3.2mm in thickness as shown in figure 1 and image 1. the specimen is first conditioned in a drying oven for 24 hours at 50 ±3 °c, cooled inside a desiccator and then weighed immediately on 0.0001 g balance (image 2a, b, c). figure 1. specimen dimensions for water absorption and solubility test. image 1. specimens for water absorption and solubility tests. image 2. a, drying inside oven. b, specimens placed inside distilled water. c, weighing on a four-digit electronic balance. a long-term immersion procedure was used to measure the water absorption of the specimens. the conditioned specimen is placed in distilled water maintained at 23 ±1 °c and should be immersed entirely for 24 hours (image 2b). at the end of 24 hrs. the specimen is taken from the distilled water and wiped from surface water with clean and dry cloth, weighed on 0.0001g balance and replaced immediately inside distilled water. these weightings are repeated at the end of 7 days and every 2 weeks until the specimen is considered saturated when no more change in weight is achieved. this is to measure the increase in weight. the increase in weight is calculated using the following formula: increased in weight, %=(wet weight-conditioned weight)/(conditioned weight)*100 for the solubility test the specimens, after immersion in studying water absorp3on, solubility, and uv absorp3on of room temperature vulcanized maxillofacial silicone a@er the addi3on of stron3um titanate nano-powder vol 13 no 1 (2025) doi 10.5195/d3000.2025.846 h#p://den*stry3000.pi#.edu distilled water, were weighed again, and then reconditioned for another drying session at the same temperature and duration as the first. the samples were weighed again after being chilled in a desiccator. water solubility was determined by doing an immersion test and subtracting the reconditioned weight from the conditioned weight. solubility is calculated using the following formula: solubility = (conditioned weightreconditioned weight)/(conditioned weight)*100 according to astm 570: 2018, the water-absorption value for such materials shall be taken as the sum of the increase in weight on immersion and the weight of the water-soluble matter. uv absorption the specimen's absorption of uv light was measured using a uv vis spectrophotometer. a disk-shaped specimen with 2mm in thickness and 20mm in diameter was fabricated [17] as shown in image 3. the device measured the amount of uv light absorption as a function of wavelength. the disk-shaped specimens were located over the light source and exposed to light, then the readings of the absorbed light were captured from the computer’s screen attached to the spectrophotometer, as shown in images 4a, b, c, d. image 3. specimens to be tested inside spectrophotometer. image 4. (a) uv spectrophotometer setup; (b) specimen placement inside the device; (c) closer look at the specimen inside the device; d, uv probe software. sem the dispersion of srtio3 nanoparticles inside the vst-50 silicone matrix was analyzed using a scanning electron microscope (tescan mira3, france). ftir ftir device (shimadzu, japan) was used to distinguish any chemical reaction among the silicone material and nanosized srtio3 nanoparticles. the statistical analysis: done using tukey's hsd for post hoc testing after a one-way anova. results sem the results obtained for the control group revealed the lack of nanoparticles, while the 1% srtio3 group exhibited a random distribution with little aggregation of srtio3 nanoparticles. however, nanoparticles cause the 1.5% srtio3 cluster within the silicone matrix to grow into bigger aggregates as shown in images 5a, b, and c. 1. 2. 3. image 5. sem picture of (a) vst-50 silicone, (b) control specimen at 10 m magnification. b, a sample of 1% srtio3 and, (c) srtio3 sample with 1.5% purity. ftir b a studying water absorp3on, solubility, and uv absorp3on of room temperature vulcanized maxillofacial silicone a@er the addi3on of stron3um titanate nano-powder vol 13 no 1 (2025) doi 10.5195/d3000.2025.846 h#p://den*stry3000.pi#.edu the absence of new peaks in the silicone elastomers microstructure, suggesting that nanoparticle addition did not cause chemical changes as showed in figure 2a, b. figure 2. a, ftir analysis of the standard sample. b, ftir analysis of the sample after 1% srtio3 nanoparticles were added. water absorption the tested group (c) showed the highest mean value (0.2656) followed by (b) group with a (0.2470) mean value while the lowest mean value was for the group (a) with (0.1229) as shown in table 1. table 1. descriptive statistics, oneway anova and tukey’s hsd of water absorption test. water absorption anova tukey hsd group min max mean ±sd f pvalue groups pvalue control(a) 0.0917 0.1772 0.1229 0.0248 120.676 0.000 a b 0.000 1 % (b) 0.2027 0.2831 0.2470 0.0247 a c 0.000 1.5 % (c) 0.2432 0.2912 0.2656 0.0162 b c 0.171 levene’s statistics=0.751, p-value=0.482 to compare the mean values among all study groups, post-hoc tukey’s test was conducted. a highly significant difference was found between group (a) and groups (b and c) while a nonsignificant mean difference was found between group (b) and group (c). solubility group c had the highest average (0.0303), group b had the second highest average (0.0288), and group a had the lowest average (0.0217) as shown in table 2. studying water absorp3on, solubility, and uv absorp3on of room temperature vulcanized maxillofacial silicone a@er the addi3on of stron3um titanate nano-powder vol 13 no 1 (2025) doi 10.5195/d3000.2025.846 h#p://den*stry3000.pi#.edu table 2. descriptive statistics, one way anova and tukey’s hsd of solubility test. solubility avova tukey hsd group min max mean ±sd f p-value groups p-value control(a) 0.0129 0.0354 0.0217 0.00675 6.829 0.004 a b 0.000 1 % (b) 0.0209 0.0386 0.0288 0.00600 a c 0.000 1.5 % (c) 0.0255 0.0356 0.0303 0.00338 b c 0.014 levene’s statistics=1.590, p-value=0.222 group (a) was significantly different from group (b), while group (a) was significantly different from group (c). the difference in mean scores between groups b and c was not statistically significant. between group (b) and group (c). was not statistically significant. uv absorption the experimental group (1.5%) showed the highest uv absorption (3.1796) followed by group (1%) with (3.14913), followed by the control group with (1.06509) as shown in table 3. there was a significant difference between the control group and groups (1% and 1.5% srtio3). also, a significant difference between group (1%) and group (1.5%) was shown by tukey’s hsd post-hoc test. table 3. descriptive statistics, one-way anova and tukey’s hsd of uv absorption. uv absorption anova tuckey hsd group min max mean ±sd f p-value groups p-value control(a) 16.98 19.21 1.06509 0.01865 91174.274 0.000 a b 0.000 1 % (b) 23.56 26.64 3.14913 0.00622 a c 0.000 1.5 % (c) 21.23 24.27 3.17960 0.00983 b c 0.000 levene’s statistics =1.6440, p-value=0.212 discussion none of the commercially available silicones may provide the desirable properties (19). thus, efforts are always being made to better silicone materials by refinement of their formulation or the addition of fillers. [18]. since maxillofacial prosthetic material is cleaned by the patient with saliva, water, or other liquids, water absorption is an essential physical feature. the look, mechanical strength, and dimensions of a material may all be studying water absorp3on, solubility, and uv absorp3on of room temperature vulcanized maxillofacial silicone a@er the addi3on of stron3um titanate nano-powder vol 13 no 1 (2025) doi 10.5195/d3000.2025.846 h#p://den*stry3000.pi#.edu affected by the amount of liquid it absorbs [16]. the literature [21] suggests that prolonged storage of silicone materials that allow water absorption due to both the load component in their composition and a low degree of adhesion among the silicone polymers may account for the higher water absorption rates observed for (1% and 1.5%) srtio3 specimens compared to control specimens. furthermore, this might owing to the fact that nano particles are dry, causing the silicone to absorb more water [19]. this is likely related to the type of filler and the degree to which the filler is attached to the silicone rubber. this agreed with braden and wright who stated that the nature of filler may influence the rubber’s water absorption [20]. dos santos et al. in 2012 [13] discovered that mdx4-4210 face silicone absorbed and dissolved more water after 502 hours of storage than it did after 252 hours of storage. it was also mentioned that the little improvements in water solubility and water absorption readings seen after 502 hours could not indicate a clinical issue. since silicone rubbers are closed-cell materials and naturally hydrophobic, this low water absorption rate makes sense. the fact that vst-50 silicone is just another kind of silicone, and not a condensation silicone. the addition silicone curing occurs without byproduct formation. condensation silicone cures with byproducts that later exit from the structure of the polymer leading to more porous structure than the additional silicone [11]. this agreed with canay et. al. and parker et. al. who found that the silicone–type materials absorbed less water than plasticized acrylics because of their highly hydrophobic nature [21,22]. in addition, the high number of cross-linking agents in the silicone can minimize absorption owing to a strong association between the load and the elastomeric material molecules decreasing the space among the polymer chains where water could penetrate [23,24]. for solubility, the results showed significant increase in the amount of soluble matter lost. this increase in solubility could be related to the release of formaldehyde over time, a peculiar characteristic of elastomeric materials polymerized at room temperature. the loss of alcohol generated as a reaction byproduct and extraction of the metallic salt catalyst, which is left unchanged in the reaction, are thought to be responsible for the water solubility of silicones crosslinked at room temperatures [20]. it should be emphasized that silicone-based materials generally exhibit much lower sorption and solubility than acrylic-based lining materials because they do not contain components such as plasticizers that are rinsed out by water and consequently allow absorption and solubility [25]. the uv absorption test results revealed a highly significant increase in the amount of uv light absorbed by the reinforced specimens (1 % and 1.5% srtio3) when compared to the control groups. currently, titanium oxide and zinc oxide nanoparticles are used widely in sunscreen applications because they are more effective than microparticles [26]. the uv light absorbing ability of srtio3 can be related to the wide band gap of the material which is 3.25 ev (in the typical range of semiconductors) [27]. wide band gap materials are known for their ability to absorb light because their electrons need to absorb more photon energy to transfer from the valance band (h) to the conduction band (e). it has shown that srtio3 had a peak at 354 nm wavelength with the absorbance of 0.86, which means that it exhibits good absorbance at the uv region [28]. conclusion the addition of 1% and 1.5% of srtio3 nanoparticles into vst-50 studying water absorp3on, solubility, and uv absorp3on of room temperature vulcanized maxillofacial silicone a@er the addi3on of stron3um titanate nano-powder vol 13 no 1 (2025) doi 10.5195/d3000.2025.846 h#p://den*stry3000.pi#.edu increases water absorption, solubility, and uv absorption. conflicts of interest the authors declare no competing interest. source of funding completely self-funded. ethical clearance none. references 1. maxillofacial prostheqcs, k. phasuk and s. p. haug, oral and maxillofacial surgery clinics, 2018, oct., vol. 30, no. 4, pp. 487-497. pmid: 30266191. 2. maxillofacial prostheses challenges in resource constrained regions, s. teseh, r. j. bibb, and s. j. marqn, disability and rehabilitaqon, 2019, jan., vol. 41, no. 3, pp. 348-356. pmid: 29050577. 3. maxillofacial prostheqc materials-an inclinaqon towards silicones, a. mitra, s. choudhary, h. garg, and h. jagadeesh, journal of clinical and diagnosqc research: jcdr, 2014, dec., vol. 8, no. 12, p. ze08. pmid: 25654035. 4. maxillofacial prostheqc rehabilitaqon in the uk: a survey of maxillofacial prostheqsts’ and technologists’ attudes and opinions, m. hatamleh, c. haylock, j. watson, and d. wass, internaqonal journal of oral and maxillofacial surgery, 2010, dec., vol. 39, no. 12, pp. 1186-1192. pmid: 20869164. 5. avaliação da dureza e rugosidade de superwcie de dois silicones maxilofacial seguido de desinfecção, m. goiato, a. pesqueira, d. santos, and s. dekon, brazilian oral research, 2009, janmar., vol. 23, no. 1, pp. 49-53. pmid: 19488474. 6. color stability of polymers for facial prosthesis, d. m. dos santos, m. c. goiato, m. a. c. sinhoreq, a. ú. r. fernandes, p. do prado ribeiro, and s. f. de carvalho dekon, journal of craniofacial surgery, 2010, jan., vol. 21, no. 1, pp. 54-58. pmid: 20072025. 7. disinfecqon of maxillofacial silicone elastomer using a novel anqmicrobial agent: recombinant human beta-defensin-3, y. shi et al., european journal of clinical microbiology & infecqous diseases, 2009, apr., vol. 28, no. 4, pp. 415-420. pmid: 19005689. 8. evaluaqon of polymeric materials for maxillofacial prostheqcs, d. j. moore, z. r. glaser, m. j. tabacco, and m. g. linebaugh, the journal of prostheqc denqstry, 1977, sep., vol. 38, no. 3, pp. 319-326. pmid: 332618. 9. changes in appearance of silicone elastomers for maxillofacial prostheses as a result of aging, a. k. hulterström and i. eystein ruyter, internaqonal journal of prosthodonqcs, 1999, nov-dec., vol. 12, no. 6, pp. 498504. pmid: 10895668. 10. nanoparqcles used in denqstry: a review, s. priyadarsini, s. mukherjee, and m. mishra, journal of oral biology and craniofacial research, 2018, jan-apr., vol. 8, no. 1, pp. 58-67. pmid: 29628756. 11. wesability, water sorpqon and water solubility of seven silicone elastomers used for maxillofacial prostheses, a. k. hulterström, a. berglund, and i. ruyter, journal of materials science: materials in medicine, 2008, jan., vol. 19, no. 1, pp. 225-231. pmid: 17520360. 12. the anqfungal potenqal of cinnamon oil incorporated into a heat-polymerized so{ liner, a. s. ahmed, r. s. ahmed, and l. n. arab, journal of dental materials & techniques, 2024, vol. 13, no. 3. 13. effect of addiqon of pigments and opacifier on the hardness, absorpqon, solubility and surface degradaqon of facial silicone a{er arqficial ageing, d. m. dos santos, m. c. goiato, a. moreno, a. a. pesqueira, s. f. de carvalho dekon, and a. m. guiot, polymer degradaqon and stability, 2012, aug., vol. 97, no. 8, pp. 1249-1253. pmid: 28832312. 14. effect of nano silicon dioxide addiqon on some properqes of heat vulcanized maxillofacial silicone elastomer, m. tukmachi and m. moudhaffer, iosr journal of pharmacy and biological sciences, 2017, may-jun., vol. 12, no. 3, pp. 37-43. studying water absorp3on, solubility, and uv absorp3on of room temperature vulcanized maxillofacial silicone a@er the addi3on of stron3um titanate nano-powder vol 13 no 1 (2025) doi 10.5195/d3000.2025.846 h#p://den*stry3000.pi#.edu 15. effect of stronqum titanate nano powder addiqon on some mechanical properqes of room temperature vulcanized maxillofacial silicone, a. s. ahmed and m. m. m. ali, journal of research in medical and dental science, 2021, dec., vol. 9, no. 12, pp. 59-65. 16. standard test method for water absorpqon of plasqcs, astm d57098, annual book of astm standards, 2018. 17. effect of nano-oxide concentraqon on the mechanical properqes of a maxillofacial silicone elastomer, y. han, s. kiatamnuay, j. m. powers, and y. zhao, the journal of prostheqc denqstry, 2008, dec., vol. 100, no. 6, pp. 465473. pmid: 19041086. 18. handbook of plasqcs, elastomers, and composites, c. a. harper, mcgraw-hill educaqon, 2002. 19. influence of some plant extracts on anqfungal properqes, hardness, and peel bond strength of heat-cured denture so{ liner, a. s. ahmed et al., diyala journal of medicine, 2024, vol. 26, no. 2, pp. 14–28. 20. water absorpqon and water solubility of so{ lining materials for acrylic dentures, m. braden and p. wright, journal of dental research, 1983, jun., vol. 62, no. 6, pp. 764768. pmid: 6574160 21. ahmed, ali saad; kamil, saif saad; arab, luma nasrat. assessment of peel bond strength between two facial prostheqc materials a{er surface treatment methods: in vitro study. journal of internaqonal oral health, 2024, 16.1: 63-68. 22. so{ acrylic resin materials containing a polymerisable plasqciser ii: water absorpqon characterisqcs, s. parker, d. marqn, and m. braden, biomaterials, 1999, jan., vol. 20, no. 1, pp. 55-60. pmid: 9916767 23. alwan, sohaib qais, et al. evaluaqon of microhardness of self-adhesive pediatric filling. denqstry 3000, 2025, 13.1. 24. evaluaqon of varying amounts of thermal cycling on bond strength and permanent deformaqon of two resilient denture liners, j. r. r. pinto, m. f. mesquita, m. a. de arruda nóbilo, and g. e. p. henriques, the journal of prostheqc denqstry, 2004, sep., vol. 92, no. 3, pp. 288-293. pmid: 15343244 25. bond strength of so{ liners to fiber-reinforced denture-base resin, l. v. lassila, m. m. mutluay, a. tezvergil-mutluay, and p. k. vallisu, journal of prosthodonqcs: implant, estheqc and reconstrucqve denqstry, 2010, aug., vol. 19, no. 8, pp. 620-624. pmid: 20456030 26. nano-sized cosmeqc formulaqons or solid nanoparqcles in sunscreens: a risk to human health?, g. j. nohynek and e. k. dufour, archives of toxicology, 2012, jul., vol. 86, no. 7, pp. 10631075. pmid: 22526641 27. bulk electronic structure of srtio3: experiment and theory, k. van benthem, c. elsässer, and r. french, journal of applied physics, 2001, dec., vol. 90, no. 12, pp. 6156-6164. pmid: 11770399 28. synthesis and characterizaqon of nano-tio2 via different methods, r. vijayalakshmi and v. rajendran, archives of applied science research, 2012, mar., vol. 4, no. 2, pp. 1183-1190. pmid: not available. 847 localiza(on of e-cadherin and vimen(n in mucosal ulcer healing treated by vi(s vinifera seeds oil in rat model vol 13 no 1 (2025) doi 10.5195/d3000.2025.847 h#p://den*stry3000.pi#.edu localization of e-cadherin and vimentin in mucosal ulcer healing treated by vitis vinifera seeds oil in rat model russol faik, nada m.h. al-ghaban college of dentistry, university of baghdad, baghdad, iraq abstract objec&ve: the an(-inflammatory components generated by vi(s vinifera (grape) seeds oil (gso) help reduce inflamma(on and accelerate the healing process when applied topically to an ulcer. the current study aimed to assess the expression of e-cadherin and vimen(n during oral ulcer healing treated by gso. materials and methods: twenty-four albino male rats (350–450 g) were used for this study. the rats were slaughtered on the 5th and 10th days aker an ulcer induc(on on either their lek or right cheek mucosa. the rats in the control group received sterile dis(lled water, whereas those in the experimental group received gso for ulcers treatment. the (ssue samples were subsequently u(lized for histological and immunohistochemical inves(ga(ons of e-cadherin and vimen(n. results: compared with the control group, the gso-treated ulcer group presented significantly higher expression of e-cadherin and vimen(n at days 5 and 10 than the control group, with some evidence of epithelial-mesenchymal transi(on (emt) at 5 days in both groups. conclusion: with the limita(on of this study, gs may accelerate the healing of mucosal ulcers, as demonstrated by increased expression of e-cadherin and vimen(n. clinical relevance: gso can be used as a topical herbal modality for mucosal ulcer healing. keywords: vi(s vinifera seed oil (gso), mucosal ulcer, vimen(n, e-cadherin, rats, healing, emt. cita;on: faik r, et al. (2025) localiza;on of e-cadherin and vimen;n in mucosal ulcer healing treated by vi;s vinifera seeds oil in rat model. den;stry 3000. 1:a001 doi:10.5195/d3000.2025.847. received: february 12, 2025 accepted: march 31, 2025 published: april 18, 2025 copyright: ©2025 falk r, et al. this is an open access ar;cle licensed under a crea;ve commons a[ribu;on work 4.0 united states license. email: rusul.abd2206@codental.uobaghdad.edu.iq introduction the development of the extracellular matrix (ecm), migration, adhesion, proliferation, and differentiation are all steps in the multicellular process that constitutes wound healing [1]. the complex connections among many cell types, cytokines, and the circulatory system are involved in the wound-healing process. the initial cascade of vasoconstriction and platelet aggregation stops bleeding, and an influx of inflammatory cells, led by neutrophils in the initial stages, occurs. to encourage angiogenesis and re-epithelialization, fibroblasts then construct a framework by laying down extracellular components [2]. phenolic compounds are plant secondary metabolites found in fruits, vegetables and beverages, including grapes, the beneficial health effects of grape and grape products and the antiinflammatory and protective effects of traditional grape preparations on the cardiovascular system are related to their bioactive phenolic compounds. the biological activities of grape phenolic compounds include among others anti-inflammatory, antioxidant and cardioprotective effects [3]. proanthocyanidin in grape seeds can target human keratinocytes and cause them to produce the vascular endothelial growth factor vefg [4]. owing to the high concentrations of vitamins, fatty acids, and phenolic compounds, grape seed oil (gso) is beneficial to the food, pharmaceutical, and esthetic sectors. grape seed oil may interact with cellular and molecular pathways; it has anti inflammatory, antibacterial, and anticancer properties [5]. it is localiza(on of e-cadherin and vimen(n in mucosal ulcer healing treated by vi(s vinifera seeds oil in rat model vol 13 no 1 (2025) doi 10.5195/d3000.2025.847 h#p://den*stry3000.pi#.edu composed of polyunsaturated fatty acids (pufas), with linoleic acid (c18:2) accounting for 58– 78% of the total [6]. this may inhibit oxidative stress by preventing the release of proinflammatory mediators by oxidized low-density lipoproteinstimulated macrophages [5]. the adhesion of cells in human epithelial tissues relies on adherent junctions, which are enhanced by the cell adhesion protein e-cadherin (e-cad) [7]. ecadherin is crucial in the wound healing process, including mitosis, maturation, and migration. in vitro research revealed that the upregulation of keratinocyte migration leads to the downregulation of e-cadherin expression [8]. vimentin is an intermediate filament protein that contributes to cellular processes such as migration, morphology, plasticity, and organelle anchoring. in addition to its structural and cytoskeletal functions. lymphocytes, fibroblasts, endothelial cells, macrophages, and melanocytes are mesenchymal cells that express vimentin [9]. vimentin located on the cell surface has several functions in cell‒cell interactions, wound healing, migration, and immune system activation [10]. the epithelial‒mesenchymal transition (emt) is caused by metabolic alterations that enable polarized epithelial cells to adopt a phenotype like that of mesenchymal cells. during this process, the cell's basal surface interacts with the basement membrane [11]. typically, studies related to emt have investigated the expression of epithelial markers (e.g., e-cadherin) and mesenchymal markers (e.g., ncadherin or vimentin) to characterize the process; however, conclusive experimental evidence for a real mesenchymal state as the endpoint is absent in several cases where the term emt is employed [12]. the benefits of grape seed oil on the healing of oral ulcers have not been widely researched. therefore, the purpose of this study is to investigate the curative process of an experimentally induced oral ulcer treated with grape seed oil in a rat by assessing the expression of e-cadherin and vimentin monoclonal antibodies. material and methods the current study was approved by the institutional ethical committee of animal experimentation of the (blinded for review). the animals were kept under nursing and supervision from the staff of the animal house of the medical research institute, city, for approximately 3 months, starting in november 2023 and ending in february 2024. in this study, the authors followed the animal research: reporting in vivo experiments (arrive) guidelines [13]. grape seed extract has been approved as generally recognized as safe (gras) by the food and drug administration (fda) and is sold commercially as a dietary supplement listed in the everything added to food in the united states (eafus) database. bentivegna and whitney reported that the no-observedadverseeffect level (noael) of grape seed extracts determined in rats was 1.78 g/kg body weight/day, and the normal amount of grape seed extract used in food applications was 0.01–1% [14]. the rats were housed in polypropylene enclosures within the experimental animal facility, maintaining consistent environmental conditions such as 12-hour light‒dark cycles and a temperature of 24 ± 2 °c. the animals were given a rat chow pellet meal and were allowed free access to tap water. before ulcer induction, the rats were allowed a one-week acclimation period to adjust to the laboratory environment. localiza(on of e-cadherin and vimen(n in mucosal ulcer healing treated by vi(s vinifera seeds oil in rat model vol 13 no 1 (2025) doi 10.5195/d3000.2025.847 h#p://den*stry3000.pi#.edu a total of twenty-four male albino rats, weighing between 250 and 300 grams and aged two to three months, were used as experimental models. twelve rats were allocated for each study duration (five and ten days). the rats were then randomly divided into two groups, a control group and a group treated with gso. female rats were specifically excluded because of cyclic hormonal variations and the potential for pregnancy throughout the experimental study. to determine the precise dosage of general anesthesia, the rats were weighed. then, general anesthesia was induced via intraperitoneal injection of xylazine (5 mg/kg) and ketamine (50 mg/kg) [15]. after disinfection with 70% alcohol, ulcers were induced on both the right and left buccal mucosa via sterile round diamond burs, creating lesions 5 mm in diameter and 1 mm deep (figure 1), which were measured via a digital vernier caliper [16]. figure 1. a photograph showing traumatic ulcer induction in the buccal mucosa. the treatment involved daily application of 30 µl of gso (us organic, amazon)" usda certified organic & 100% pure: cold-pressed for maximum purity and nutrient retention, sourced from premium quality grapes, antioxidant-rich: rich in essential fatty acids like oleic and linoleic" (figure 2) on the right side via a micropipette, whereas the left side received distilled water as a control. figure 2. vitis vinifera seed oil (gso). the animals were euthanized on days 5 and 10 through the administration of an excessive amount of anesthetic solution [17]. the ulcerated regions were excised and extended 5 mm beyond the ulcer edge. after fixation in 10% neutral buffered formalin for 24 hours, the samples were washed [18]. the samples were dehydrated by placing them in increasing alcohol concentrations, then clarified in xylene, and finally embedded in paraffin wax. sections (4 µm) were then subjected to hematoxylin and eosin (h&e) staining (figure 3) and immunohistochemical analysis, which was performed under a light microscope, to evaluate mucosal ulcer healing. figure 3. histological progression of oral ulcers at two time points (day 5 and day 10) under 10x magnification. a) control group on day 5, b) gso-treated group on day 5, c) control group on day 10, d) gso-treated group on day 10. immunohistochemical analysis of e-cadherin and vimentin was carried out via monoclonal mouse anti-human e-cadherin, clone nch-38 (dako company), england, and monoclonal mouse anti vimentin, clone vim 3b4 (daco company), england. localiza(on of e-cadherin and vimen(n in mucosal ulcer healing treated by vi(s vinifera seeds oil in rat model vol 13 no 1 (2025) doi 10.5195/d3000.2025.847 h#p://den*stry3000.pi#.edu under a light microscope at x40, five fields were chosen from the epithelium area, and another five fields from the lamina propria from each tissue section were captured by a digital camera (5 pixels), and the images evaluated were imported to a computer. the evaluation of the staining results was achieved by applying the aperio positive pixel count algorithms program (from aperio image scope software v11.1.2.760 developed by aperio technologies inc., usa), and we neglected the yellow color, which represents a weak positive reading [1,19,20]. the average mean positive percentages for each of the five areas from the epithelium and lamina propria were obtained and considered the value of the expression of both e-cadherin and vimentin. the data obtained from the immunohistochemical examination were assessed for normality via the shapiro‒wilk test. numeric values are presented as a range encompassing the minimum and maximum values, along with the mean, standard deviation, and median. when the variables exhibited a normal distribution, student’s t test was used to compare two distinct groups. the predetermined level of significance for the findings was established at p=0.05. the data were analyzed via the ibm spss software package version 20.0. results the control group on days 5 and 10 presented mild to moderate membranous morphology with positive expression of e-cadherin in the newly formed basal, spinosum, and granulosum cells of the epithelium and negative expression in the keratinized layer. on the other hand, the endothelial cells and fibroblasts weakly positively expressed e-cadherin (figure 4, a and b). the study group at both time points presented moderate to strong positive expression in all layers of the newly regenerated epithelium around the ulcer margin except the keratinized layer (figure 3, c and d). in lamina propria, endothelial and fibroblasts presented moderate positive expression of e-cadherin, especially on day 5, in the control group (figure 5). the expression levels of e-cadherin in the examine tissue samples are summarized in (table 1). the control group at two time points (day 5 and day 10) after ulcer induction presented moderate cytoplasmic expression of vimentin in fibroblasts, endothelial cells, and inflammatory cells (figure 6, a and b). however, the study group exhibited strong cytoplasmic expression of vimentin in fibroblasts, endothelial cells, and inflammatory cells at both time points (figure 6, c and d). on the other hand, moderate positive expression was detected in the cytoplasm of the granulosum epithelial cell layer, especially in both groups, on day 5 (figure 7). the distribution and intensity of vimentin expression in the studied samples are presented in (table 2). a comparative analysis of ecadherin and vimentin expression patterns is provided in (table 3) demonstrate an inverse relationship between these markers. the correlation analysis between e-cadherin and vimentin expression levels is summarized in (table 4). localiza(on of e-cadherin and vimen(n in mucosal ulcer healing treated by vi(s vinifera seeds oil in rat model vol 13 no 1 (2025) doi 10.5195/d3000.2025.847 h#p://den*stry3000.pi#.edu figure 4. a: photomicrograph showing membranous positive expression of e-cadherin in the epithelial layer, dab stain (40x), a) control day 5 b) control day 10, c) study day 5, d) study day 10. figure 5. photomicrograph of the lamina propria of the rat oral mucosa showing moderate positive expression of e-cadherin in a and weak positive expression in b, c, and d. dab staining at 40x. a) control day 5. b) control day 10. c) study day 5. d) study day 10. a b c d d c b a localiza(on of e-cadherin and vimen(n in mucosal ulcer healing treated by vi(s vinifera seeds oil in rat model vol 13 no 1 (2025) doi 10.5195/d3000.2025.847 h#p://den*stry3000.pi#.edu figure 6. immunohistochemical image showing strong positive cytoplasmic expression of vimentin in inflammatory cells, fibroblasts, and blood vessels in the lamina propria; vimentin dab stain (40x). a) control day 5, b) control day 10, c) study day 5, d) study day 10. figure 7. photomicrograph showing weakly positive expression of vimentin in the granulosa layer of the epithelium in a, b, and c and strongly positive expression in d. dab staining (40x): a) control day 5 b) control day 10 c) study day 5 d) study day 10. d c a b d c b a localiza(on of e-cadherin and vimen(n in mucosal ulcer healing treated by vi(s vinifera seeds oil in rat model vol 13 no 1 (2025) doi 10.5195/d3000.2025.847 h#p://den*stry3000.pi#.edu table 1. comparison of e-cadherin expression in the epithelium and lamina propria between the studied groups. table 2. comparison of the expression of vimentin in the epithelium and lamina properties between the studied groups. day site control study t test p-value mean sd mean sd 5th epithelium 27.29 3.5 49.05 4.32 -13.56 0.000*1 lamina properia 41.09 2.93 71.09 3.17 -24.059 0.000*1 10th epithelium 36.70 1.83 56.51 3.55 -4.158 0.000*2 lamina properia 54.93 3.27 82.77 1.67 -26.233 0.000*2 day site control study t test p value mean sd mean sd 5th epithelium 56.95 3.26 71.31 3.57 -10.291 0.000*2 lamina properia 26.71 4.73 19.10 4.53 3.292 0.000*1 10th epithelium 60.74 2.28 80.37 1.79 -23.434 0.000*2 lamina properia 12.36 1.66 13.63 2.22 -1.585 0.127*2 localiza(on of e-cadherin and vimen(n in mucosal ulcer healing treated by vi(s vinifera seeds oil in rat model vol 13 no 1 (2025) doi 10.5195/d3000.2025.847 h#p://den*stry3000.pi#.edu table 3. comparison of e-cadherin and vimentin markers. day group layer mean differnce±se t test p value day 5 control epithelium 29.66±1.38 21.495 0.000*1 lamina properia -14.38±1.61 -3.927 0.000*2 study epithelium 22.27±1.62 13.768 0.000*1 lamina properia -51.99±1.60 -32.569 0.000*1 day 10 control epithelium 24.04±0.85 28.446 0.000*1 lamina properia -42.57±1.06 -40.187 0.000*1 study epithelium 23.86±1.15 4.158 0.000*2 lamina properia -69.15±0.8 -86.141 0.000*1 table 4. correlations between e-cadherin and vimentin marker. day group layer r p value day 5 control epithelium 0.035 0.9151 lamina properia -0.165 0.6092 study epithelium 0.148 0.6461 lamina properia 0.031 0.9251 day 10 control epithelium 0.028 0.9321 lamina properia 0.241 0.4511 study epithelium 0.329 0.2962 lamina properia 0.417 0.1771 localiza(on of e-cadherin and vimen(n in mucosal ulcer healing treated by vi(s vinifera seeds oil in rat model vol 13 no 1 (2025) doi 10.5195/d3000.2025.847 h#p://den*stry3000.pi#.edu discussion the current study chose laboratory albino rats because of their physiological similarity to humans, making them a practical model for evaluating the dynamics of the mucosal healing process [21]. traumatic ulcers were induced with a diamond bur 5 mm in diameter, which reflects the common clinical similarity with mucosal injury [22]. the development of mucosal inflammation has frequently been suggested to be influenced by a loss of epithelial barrier function [23]. currently, herbal medicine represents a significant aspect of complementary and alternative medical practices. the therapeutic use of herbs in wound and injury management has been wellestablished since the dawn of ancient civilizations [24]. previous studies have shown that the therapeutic efficacy of the gso accelerates mucosal healing by enhancing wound closure, reducing inflammation, and accelerating the restoration of epithelial integrity [25]. this finding is consistent with our finding that ulcers treated topically with gso accelerated wound healing better than that in the control one. moreover, gso significantly increased the expression of ecadherin and vimentin compared with the control group. this may be because of the phenolic compounds, fatty acids, and vitamins present in the oil, which act as anti-inflammatory and antioxidant agents [26]. the present study revealed strong membranous expression of ecadherin in almost all the epithelial layers except the keratinized layer. this finding coincided with previously observed moderate to strong (2+/3+) membranous ecadherin staining observed in the epithelial cells of the oral cavity [27]. in a previous study [28], ecadherin was highly expressed in normal mucosal tissue. intense staining of ecadherin was observed in the spinous and basal cell layers, this finding correlates with our results. vimentin is a type iii intermediate filament protein exclusively expressed in mesenchymal cells. because vimentin is naturally observed in the lamina propria, vimentin facilitates the integration, response, and perception of microenvironmental stimuli by cells [29]. greater expression was observed in the gso group than in the control group, which was confirmed previously [30]. the immortalized keratinocyte cell line exhibited significantly elevated levels of vimentin when cocultured with both normal and keloid-derived fibroblasts in the extracellular matrix (ecm). however, several investigations have recorded the temporary stimulation of vimentin expression in epithelial cells in response to certain pathological or physiological situations that require epithelial cell motility. consequently, vimentin has been used as an indicator of the migratory state of epithelial cells [31]. our results demonstrate that vimentin is expressed in the granulosum layer of the epithelium, which matches previous findings [30], with epidermal cells not differentiating into fibroblasts but having a fibroblast-like phenotype. additionally, abnormal vimentin expression in the oral epithelium of premalignant lesions and weak vimentin immunoreactivity in the cytoplasm of suprabasal epithelial cells was previously reported [32]. the production of vimentin in epithelial cells may induce early molecular alterations in both the epithelial and mesenchymal compartments. the expression of vimentin in the epithelial layer may be linked to localiza(on of e-cadherin and vimen(n in mucosal ulcer healing treated by vi(s vinifera seeds oil in rat model vol 13 no 1 (2025) doi 10.5195/d3000.2025.847 h#p://den*stry3000.pi#.edu the appearance of cells exhibiting a mesenchymal phenotype inside an epithelial sheet, which is generally referred to as epithelialto-mesenchymal transition (emt) which results from the transformation of endogenous epithelial cells [33,34]. the epithelial-mesenchymal transition (emt) is a crucial process requiring two key proteins, ecadherin and vimentin, known for their varied expression and unique roles in this transition. the cytoplasm was positively stained with vimentin, whereas the cell membrane was positively stained with e-cadherin. during emt, cellular phenotypes change quickly and may be reversed. in contrast, epithelial cells change their polarity and cytoskeleton by losing adhesion junctions. e-cadherin is recognized for maintaining the integrity of epithelial cells and plays a crucial role in cell differentiation. vimentin is a cytoskeletal protein present in endothelial cells, fibroblasts, and leukocytes, among other cell types. it is absent in normal epithelial cells and contributes to emt [35]. in the present study, after reepithelization, complete ulcer healing, and strong expression of vimentin in the epithelial layers on day 10 in the gso group may be due to mesenchymal-toepithelial transition (met) may occur to help in wound healing and restores epithelial layers. as an organism's body plan develops, the met plays a role in several stages of morphogenesis and organogenesis. it occurs in intermittent alternation with the emt. met controls epithelial creation throughout embryonic development, whereas mesenchymal cells gradually achieve apicobasal polarity [36]. conclusion the topical application of gso effectively treated experimentally induced buccal mucosal ulcers in rats by promoting their proliferation, as shown by alterations in the expression of ecadherin and vimentin. continuous treatment for 10 days increased the expression of ecadherin and vimentin, with significant differences observed between the control and study groups. in the current study, the expression of the epithelial marker e-cadherin was detected strongly in epithelial cells with weak intensity in the lamina properia. in contrast, the mesenchymal marker vimentin was identified in lamina properia with weak intensity in epithelial cells. emt and met may explain this; however, this finding requires validation via several future investigations. conflicts of interest the authors declare no competing interest. references 1. abd ss, al-ghaban nmh. the epidermal growth factor receptor expression in leptin-treated traumatic oral ulcer in rats. jgpt. 2020. doi:10.13140/rg.2.2.33703.7824. 2. ozgok kangal mk, regan jp. wound healing. treasure island (fl): statpearls publishing; 2023 may 1. 3. ardid-ruiz a, harazin a, barna l, walter fr, bladé c, suárez m, et al. the effects of vitis vinifera l. phenolic compounds on a bloodbrain barrier culture model: expression of leptin receptors and protection against cytokineinduced damage. j ethnopharmacol. 2020;247:112253. doi:10.1016/j.jep.2019.112253. 4. abd alrazaq mh, al-ghaban nmh. immunohistochemical localization of bone morphogenic protein-2 in extracted tooth socket treated by local application of grape seeds oil in rabbits. biochem cell arch. 2020;20:581. doi:10.35124/bca.2020.20.1.581. 5. garavaglia j, markoski mm, oliveira a, marcadenti a. grape seed oil compounds: biological and chemical actions for health. localiza(on of e-cadherin and vimen(n in mucosal ulcer healing treated by vi(s vinifera seeds oil in rat model vol 13 no 1 (2025) doi 10.5195/d3000.2025.847 h#p://den*stry3000.pi#.edu nutr metab insights. 2016;9:59– 64. doi:10.4137/nmi.s32910. 6. bellili s, jazi s, nasr s, et al. grape seed oil: chemical composition, biological properties and health benefits. 2018. isbn: 978-153613561-9. 7. abe-yutori m, chikazawa t, shibasaki k, murakami s. decreased expression of ecadherin by porphyromonas gingivalis-lipopolysaccharide attenuates epithelial barrier function. j periodontal res. 2016;52(1):42–50. doi:10.1111/jre.12367. 8. grabenstetter a, mohanty as, rana s, zehir a, brannon ar, d'alfonso tm, et al. e-cadherin immunohistochemical expression in invasive lobular carcinoma of the breast: correlation with morphology and cdh1 somatic alterations. hum pathol. 2020;102:44–53. doi:10.1016/j.humpath.2020.06.0 02. 9. arrindell j, desnues b. vimentin: from a cytoskeletal protein to a critical modulator of immune response and a target for infection. front immunol. 2023;14:1224352. doi:10.3389/fimmu.2023.122435. 10. ramos i, stamatakis k, oeste cl, pérez-sala d. vimentin as a multifaceted player and potential therapeutic target in viral infections. int j mol sci. 2020;21(13):4675. doi:10.3390/ijms21134675. 11. kalluri r, weinberg ra. the basics of epithelial–mesenchymal transition. j clin invest. 2009;119(6):1420–8. doi:10.1172/jci39104. 12. haensel d, dai x. epithelial-tomesenchymal transition in cutaneous wound healing: where we are and where we are heading. dev dyn. 2018;247(3):473–80. doi:10.1002/dvdy.24561. 13. kilkenny c, browne w, cuthill ic, emerson m, altman dg. animal research: reporting in vivo experiments: the arrive guidelines. br j pharmacol. 2010;160(7):1577–9. 14. ma zf, zhang h. phytochemical constituents, health benefits, and industrial applications of grape seeds: a mini-review. antioxidants. 2017;6(3):71. doi:10.3390/antiox6030071. 15. giroux mc, hélie p, burns p, vachon p. anesthetic and pathological changes following high doses of ketamine and xylazine in sprague dawley rats. exp anim. 2015;64(3):253–60. doi:10.1538/expanim.14-0088. 16. wang x, yan l, tang y, he x, zhao x, liu w, et al. antiinflammatory effect of hgf responses to oral traumatic ulcers using an hgf-tg mouse model. exp anim. 2022;71(2):204–13. doi:10.1538/expanim.21-0141. 17. mohamed if, ghani ba, fatalla aa. histological evaluation of the effect of local application of punica granatum seed oil on bone healing. int j biomater. 2022;2022:426658. 18. dantas ts, de barros silva pg, verde meql, júnior alr, cunha ms, mota mrl, et al. role of inflammatory markers in the prognosis of oral squamous cell carcinoma. asian pac j cancer prev. 2019;20(12):3635. 19. takahiro t, sushil k, rohan n, vahid a, guillaume t, hwan c, et al. quantitative multiplex immunohistochemistry reveals myeloid-inflamed tumor immune complexity associated with poor prognosis. cell rep. 2017;19:203– 17. 20. rizzardi a, johnson a, vogel r, pambuccian s, henriksen j, skubitz a, et al. quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring. diagn pathol. 2012;7:42. 21. al-ghaban nm, jasem gh. histomorphometric evaluation of the effects of local application of red clover oil (trifolium pratense) on bone healing in rats. j bagh coll dent. 2020;32(2):26–31. 22. abed a, al-ghaban nmh. acceleration of ulcer healing by local application of myrrh oil: an experimental study. al anbar med j. 2023. doi:10.33091/amj.2023.140046.11 31. 23. elsherif na, abdelhamid mm, taha a. histopathological evaluation of cancer tendency in localiza(on of e-cadherin and vimen(n in mucosal ulcer healing treated by vi(s vinifera seeds oil in rat model vol 13 no 1 (2025) doi 10.5195/d3000.2025.847 h#p://den*stry3000.pi#.edu the buccal mucosa of aged albino rats (histological and immunohistochemical study). ain shams dental journal. 2023 dec 1;32(4):37-48. 24. kamil n, al-ghaban n. evaluation of effect of local exogenous application of myrrh oil on healing of wound incisions of facial skin. j bagh coll dent. 2019;31. doi:10.26477/jbcd.v31i4.2724. 25. cavalcante gm, sousa de paula rj, souza lp, et al. experimental model of traumatic ulcer in the cheek mucosa of rats. acta cir bras. 2011;26(3):227–34. doi:10.1590/s010286502011000300012. 26. youssry g, sabet ne, abbas it, negm im, al boghdady dm. evaluation of nasolabial angle changes after dermal fillers injection of skeletal class iii orthodontically treated females. ain shams dental journal. 2023 dec 1;32(4):1-5.. 27. burandt e, lübbersmeyer f, gorbokon n, et al. e-cadherin expression in human tumors: a tissue microarray study on 10,851 tumors. biomark res. 2021;9(1):44. doi:10.1186/s40364021-00299-4. 28. sridevi u, jain a, nagalami v, kumar uv, goyal s. expression of e-cadherin in normal oral mucosa, in oral precancerous lesions and in oral carcinomas. eur j dent. 2015;9:364–72. 29. ostrowska-podhorodecka z, ding i, norouzi m, mcculloch ca. impact of vimentin on regulation of cell signaling and matrix remodeling. front cell dev biol. 2022;10:869069. doi:10.3389/fcell.2022.869069. 30. kuwahara h, tosa m, egawa s, murakami m, mohammad g, ogawa r. examination of epithelial mesenchymal transition in keloid tissues and possibility of keloid therapy target. plast reconstr surg glob open. 2016;4(11):e1138. doi:10.1097/gox.0000000000001 138. 31. chai j, norng m, modak c, et al. ccn1 induces a reversible epithelial–mesenchymal transition in gastric epithelial cells. lab invest. 2010;90:1140–51. doi:10.1038/labinvest.2010.101. 32. sawant s, vaidya m, chaukar d, alam h, dmello c, gangadaran p, et al. clinical significance of aberrant vimentin expression in oral premalignant lesions and carcinomas. oral dis. 2013;20(5):453–65. doi:10.1111/odi.1215. 33. baum b, settleman j, quinlan mp. transitions between epithelial and mesenchymal states in development and disease. semin cell dev biol. 2008;19:294–308. 34. lee jm, dedhar s, kalluri r, thompson ew. the epithelial– mesenchymal transition: new insights in signaling, development, and disease. j cell biol. 2006;172:973–81. 35. tian my, li y. the expression of e-cadherin and vimentin in nonsmall cell lung cancer and their correlation with epithelial– mesenchymal transition. j biosci med. 2018;6:1–8. 36. yuksel h, ocalan m, yilmaz o. e-cadherin: an important functional molecule at respiratory barrier between defense and dysfunction. front physiol. 2021;12:720227. doi:10.3389/fphys.2021.720227. 1074 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1074 http://dentistry3000.pitt.edu variation in maxillary sinus height and length among skeletal types i-iii maha esam college of den*stry, tikrit university, iraq abstract objec-ve: the purpose of this study was to evaluate the effect of malocclusion in skeletal class i, ii, iii on maxillary sinus dimensions. materials and methods: 30 cone-beam computed tomography (cbct) images of 30 patients were analyzed. msh (maxillary sinus height) and msl (maxillary sinus length) were measured using the mimics 19.0 software (materialise hq technologielaan, leuven, belgium). skeletal patterns (angle class i-ii-iii) were defined. results: the mean value of the msh in class i was 23.27±4.01 mm for the right side and 23.03± 4.07 mm for the left side. for msl, the values for class i were 29.20±3.26 for the right and 30.04± 3.13mm for the left. the mean value of the msh in class ii was 21.73±3.44 mm for the right and 19.61± 2.22 mm for the left. msl in class ii was 26.57±2.31 for the right and 26.96±3.41mm for the left. the average right msh for class iii was 22.90±0.62 mm, whereas the left side had a mean of 19.61±2.22 mm. conclusion: class i individuals exhibited the greatest msh and msl values bilaterally compared to class ii and iii. overall, maxillary sinus dimensions showed varia[on across skeletal classes, with class ii presen[ng the lowest averages. open access cita%on: esam m. (2025) varia%on in maxillary sinus height and length among skeletal types i-iii. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1074 received: october 12, 2025 accepted: october 15, 2025 published: november 10, 2025 copyright: ©2025 esam m. this is an open access ar%cle licensed under a crea%ve commons awribu%on work 4.0 united states license. email: maha1974@tu.edu.iq introduc)on maxillary sinus is an air-filled, pyramidalshaped structure present in the body of the maxilla [1]. it is the largest of the four pranasal sinuses [2] it is a cavity or space filled with air in the body of maxilla [3]. consisting of the maxillary, frontal, sphenoid, and ethmoidal sinuses, which perform critical functions such as air filtration and providing immune barrier. for the sinuses, the inner surfaces are covered with a thin mucus layer, which ensures maintenance of moisture and health [4-5]. the maxillary sinus is a multifunctional bony cavity that begins at the ethmoidal infundibulum in the third month of fetal development [6,7]. after birth maxillary sinus continues to extend both laterally and inferiorly during the rapid growth period from birth to 3 years of age [8]. it measures approximately 144 mm3 at birth and grows pyramidally into adulthood. anatomically, the maxillary sinus described as a quadrangular pyramid form with an internal base that formed by the lateral wall of the nasal cavity [9]. the size of the maxillary sinus is important in the nield of dentistry during placement of implants, mini-screws, augmentation procedures, mesialisation of second molars in place of nirst molars, and intrusion of maxillary molars [10]. craniofacial growth and development have a complex, multifactorial structure. according to the functional matrix theory, soft tissues affect the development of hard tissues [11]. the maxillary sinus can be affected by factors such as head posture, functional anterior displacement, the vertical–sagittal skeletal relationship, and surgical mandibular advancement [12]. orthodontic treatment plan is affected by the size and position of the maxillary sinus. likewise, the maxillary sinus may be affected by different malocclusions either dental or skeletal, anteroposterior or vertical in terms of size and position [13]. with the emergence of cone beam computed tomography (cbct) and its advantages in the world of radiography appreciated, orthodontists are using it increasingly frequently. studying the maxillary sinus becomes more accurate and with a 3d approach and the volume of the sinus could be evaluated [14]. materials and methods the sample of the study was collected from the patients treated at the orthodontic clinic of the department of orthodontic in college of dentistry, tikrit university, from december 2023 to february 2024. out of 50 varia[on in maxillary sinus height and length among skeletal types i-iii vol 13, no 1 (2025) doi 10.5195/d3000.2025.1074 http://dentistry3000.pitt.edu 2 subjects, 30 patients were selected that fulnilled the following selection criteria: 1. the subjects were 18 to 25 years. 2. no history of abnormal habits (mouth breathing) and no apparent facial disharmony or cleft lip and palate. 3. no history of orthodontic, orthopedic or facial surgical treatment. 4. fully erupted permanent dentition (including upper third molar). 5. symmetrical faces. 6. no maxillary sinus pathology. 7.no pathologies like cysts or tumors. 8. no nasal or sinus polyposis. ten patients for each angle’s class were included [15]. in the class ii malocclusion group, individuals were characterized by class ii molar-canine relationship and convex pronile, in the creation of the class iii malocclusion group, attention was paid to the fact that the individuals were characterized by class iii molar-canine relationship, concave or nlat pronile [16]. our study included cbct images in which the boundaries of the maxillary sinus region were clearly observed in 3d within the nield of view (fov) area [17]. cbct images were obtained in the standard sitting position using the device (3d accuitomo cbct instrument (j morita manufacturing corp, kyoto, japan 17.5-second exposure, 90 kv voltage, 5 ma current, and 140 x 100 mm fov). patients were asked to bite with maximum intercuspation and not move their heads or tongues during scanning. all images had a full nield of view so that the maxillary sinus was fully observed. to compute msv, images were changed into dicom format obtained from 0.25 mm thick axial slices [18]. variables were denined as follows (figure 1): maxillary sinus length (msl): this line extends from an to po. the maximum distance between the most lateral and medial points of each sinus was identinied as the width [19]. maxillary sinus height (msh): this line extends from su to in. the height was denined as the maximum distance between the bottom and the highest points of the sinus on each side [20]. figure 1. cephalometric landmarks, planes and variables used in the study. statistics consisted of descriptive statistics including mean values for merged sample were calculated. all the groups were subjected to one way anova test to assess the signinicance of association between maxillary size measurements and three malocclusion groups in male sample, female sample and merged sample (alpha of 0.05). results/discussion the mean value of right msl for class i was 29.20±3.26 mm and was 30.04± 3.13 mm for the left side (table 1). on the other hand, the mean value of right msh for class i was 23.27±4.01 mm and for the left side the mean value was 23.03± 4.07 mm. also, according to the present study the mean value of right msl for class ii was 26.57±2.31 mm and was 26.96±3.41mm for the left side. on the other hand, the mean value of right msh for class i was 21.73±3.44 mm and for the left side the mean value was 19.61± 2.22 mm. on the other hand, the mean value of right msl for class iii was 23.96± 2.38mm and was 29.26±2.57 mm for the left side. while the mean value of right msh for class iii was 22.90±0.62 mm and for the left side the mean value was 19.61± 2.22 mm. maxillary sinus dimensions vary signinicantly across skeletal classes i to iii, with class i showing the highest values. references 1. mariya qadir, mohammad mushtaq. maxillary sinus size and malocclusion: is there any relation? international journal of applied dental sciences 2017; 3(4): 333-337. 2. blanton pl, biggs nl. eighteen hundred years of controversy: the paranasal sinuses. am j anat. 1969; 124(2):135-47. 3. stammberger h. history of rhinology: anatomy of the paranasal sinuses. rhinology. 1989; 27(3):197-210. 4. chunduru r, rachel p, kailasam v, padmanabhan s. the evaluation of maxillary sinus dimensions in different craniofacial patterns: a systematic review and meta-analysis. turk j orthod. 2023; 36(3): 208-215. 5. emirzeoglu m, sahin b, bilgic s, celebi m, uzun a. volumetric evaluation of the paranasal sinuses in normal subjects using computer tomography images: a stereological study. auris nasus larynx. 2007;34:191–5. 6. killey hc, kay lw. the maxillary sinus and its dental implications. bristol: j. wright; 1975. 7. porter, g., quinn, f.b., francis, b., quinn, j. r., matthew, w. ryan. paranasal sins anatomy and function, 2002. 8. ahn nl, park hs. differences in distances between maxillary posterior root apices and the sinus gloor according to skeletal pattern. am j orthod dentofacial orthop. 2017;152(6):811-819. 9. preuschoft h, witte h, witzel u: pneumatized spaces, sinuses and spongy bones in the skulls of primates. anthropol anz. 2002, 60:67-79. 10. said ot, rossouw pe, fishman ls, feng c: relationship between anterior occlusion and frontal sinus size. angle orthod. 2017, 87:752-8. 10.2319/010617-18.1 11. tanaka e, yamada h, higashino m, et al. (february 01, 2024) ingluence of orthodontic treatment on changes in the maxillary sinus dimensions. cureus 16(2): e53363. doi 10.7759/cureus.53363 12. uchida y, goto m, katsuki d, akioshi t. a cadaveric study of maxillary sinus size as an aid in bone grafting of maxillary sinus gloor. j oral maxillofac surg 1998;56:1158-63. 13. munhoz l, abdala júnior r, abdala r, arita es: diffusion-weighted magnetic resonance imaging of the paranasal sinuses: a systematic review. oral surg oral med oral pathol oral radiol. 2018, 126:521-36. 10.1016/j.oooo.2018.07.004 14. shah rk, dhingra jk, carter bl, rebeiz ee: paranasal sinus development: a radiographic study. laryngoscope. 2003, 113:205-9. 10.1097/00005537-200302000-00002 15. da silva y, munhoz l, filho jr, et al.: maxillary sinus volumetric changes in jet aircraft pilots: a multislice computed tomography pilot study. imaging sci dent. 2023, 53:53-60. 10.5624/isd.20220173 16. natheer h, uthman bd, canturk n. evaluation of maxillary sinus dimensions in gender determination using helical ct scanning. forensic sciences j 2011;56(2):403-8. 17. sharan a, madjar d. maxillary sinus pneumatization following extractions: a radiographic study. int j oral maxillofac implants 2008;23(1):48-56. 18. uchida y, goto m, katsuki d, akioshi t. a cadaveric study of maxillary sinus size as an aid in bone grafting of maxillary sinus gloor. j oral maxillofac surg 1998;56:1158-63. 19. weir n. otolaryngology an illustrated history. boston: butterworth-heinemann; 2010. 20. prabhat m, rai s, kaur m, prabhat k, bhatnagar p, panjwani s. computed tomography based forensic gender determination by measuring the size and volume of the maxillary sinuses. j forensic dent sci 2016; 8:40-46. varia[on in maxillary sinus height and length among skeletal types i-iii vol 13, no 1 (2025) doi 10.5195/d3000.2025.1074 http://dentistry3000.pitt.edu 3 table 1. descriptive statistics and anova for overall sample. groups r h r l l h l l class i 23.27±4.01 ab 29.20±3.26 a 23.03± 4.07 a 30.04± 3.13 a class ii 21.73±3.44 b 26.57±2.31 b 19.61± 2.22 b 26.96±3.41 b class iii 24.64±3.50 a 23.96± 2.38 22.90±0.62 a 29.26±2.57 a p-value 0.05 0.01 0.01 0.05 1038 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1038 http://dentistry3000.pitt.edu correlation between a vegf marker and patients with mucoepidermoid carcinoma asmaa h. ali college of medicine, university of fallujah, iraq abstract objec/ve: the aim of this work was to examine the expression of vegf and its correlanon with the grade of mucoepidermoid carcinoma. subjects and methods: a cohort of males with mean age 47.1±13.15 and 13 females with mean age 38.65±22.15 was studied. eight cases had high, 11 moderate, and 11 had low grade lesions based on histopathology. fiuy formalinfixed, paraffin-embedded nssue blocks of these panents with mucoepidermoid disease were used to confirm the diagnosis in this study using secnons stained with hematoxylin and eosin. results: vegf was expressed in all cases. cytoplasmic expression varied from +1 in two cases to +4 in 28 cases. vegf was expressed at high intensity in 27 cases. a significant difference (p < 0.05) was found between the intensity and expression of vegf, according to the chi-square test. there was a significant correlanon between mucoepidermoid carcinoma and grade of cancer. conclusion: vegf was expressed in malignant tumors and may serve as a marker for the disease. open access cita%on: ali ah. (2025) correla%on between a vegf marker and pa%ents with mucoepidermoid carcinoma. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1038 received: september 6, 2025 accepted: september 24, 2025 published: october 8, 2025 copyright: ©2025 ali ah. this is an open access ar%cle licensed under a crea%ve commons avribu%on work 4.0 united states license. email: mmss2006@uofallujah.edu.iq introduc)on the lymphatic and blood vascular systems are formed and maintained in large part by the vascular endothelial growth factor (vegf) [1]. vegf affects the vascularization of tumors, the vascularization that occurs naturally during the healing of wounds, and the pathologic neovascularization that results from local tissue ischemia [2]. mucoepidermoid carcinoma, or mec, is a common salivary gland cancer that mostly affects the parotid gland. in a clinical setting, mec may manifest as a damaging mass that is rapidly growing or as a mass that grows slowly. histologic grade and clinical stage are usually associated with the prognosis of mucoepidermoid carcinoma [3]. immunohistochemistry studies have been produced aiming to aid in both the prognosis of tumors in salivary gland malignant and the differential diagnosis of cancers in salivary gland but paid little attention to the expression of vegf marker and its signimicance [4,5]. evaluations of the expression in healthy salivary glands next to cancers were conducted [6]. expression levels in mucoepidermoid carcinoma and small salivary gland papillary cystadenoma were also assessed [6]. identifying an immunohistochemistry marker is essential for determining the development of malignancies in mucoepidermoid patients and its correlation marker [7]. the objective of this work was to examine immunohistochemically the expression of vegf and the correlation of that expression with the grade of cancer. subjects and methods fifty formalin-mixed, parafmin-embedded tissue blocks of 30 patients with mucoepidermoid disease were used to conmirm diagnosis in this study using sections stained with hematoxylin and eosin. the madenat altib hospital's pathology department archives were used to choose the patients. after being taken from the patient miles, the case slides were examined to verify the clinicopathologic data, which included age, sex, and histologic grade. partitions measuring 4 μm were cut out of each parafmin-covered sample block and the blocks were deparafminized using 100% xylene. subsequently, blocks were rehydrated using increasing concentrations of ethanol and submerged in tbs with a ph of around 6.0 tris buffer saline and for antigen retrieval the samples heated at 750 watts in a microwave oven. samples were let cool down at room temperature and incubated in primary antibodies. after being diluted to a 1:12,000 ratio with envision, vegf (anti-human clone: kdi5, leicca mexi, in usa polyclonal rabbit) was left to sit for one hour [8]. after a wash in saline tris solution, a secondary antibody treatment was administered. samples correlanon between a vegf marker and panents with mucoepidermoid carcinoma vol 13, no 1 (2025) doi 10.5195/d3000.2025.1038 http://dentistry3000.pitt.edu 2 were stained after chromogenic treatment [9]. we counted the positive cytoplasmic staining cells to quantify the studied marker in each slide of mucoepidermoid carcinoma. furthermore, a score corresponding to the staining intensity was assigned, as indicated in table 1. at the same time, two pathologists assessed each slide [10-14]. table 1. scoring system of vascular endothelial growth factor (vegf). number (score) 0 (negative) 1%– 25% (1) 26%– 50% (2) 51%– 75% (3) 76%– 100% (4) intensity 0 = no positive cells + = mild ++ = moderate + 3 = strong + 3 = strong results the main characteristics of the case study show that included 17 males with the mean age 47.1±13.15 and 13 females with the mean age 38.65±22.15. eight cases had a high grade, 11 moderate, and 11 a low grade. vegf was expressed in all cases as displayed in tables 2 and 3. cytoplasmic expression +1 was found in 2 cases and +4 in 28 cases. expression and intensity of vegf between these two types of cases were different (figure 2). table 2. vascular endothelial growth factor expression in mucoepidermopid carcinoma via immunohistochemistry. score 0 +1 +2 +3 +4 malignant mucoepidermoid 0 2 0 0 28 table 3. intensity of vegf expression in mucoepidermoid carcinoma via immunohistochemistry. mild moderate strong total 0 3 27 30 seventeen mucoepidermoid carcinomas were classimied as having high grade, 11 moderate, and only 4 low grade. figure 1. mucoepidermoid carcinoma secnons showing strong vegf expression [peroxidade (brown) immunostaining, counterstained with hematoxylin (blue). discussion/conclusion a role for vegf in the transformation of malignant cells is suggested by the elevated expression of the marker in malignant tumors. reducing vegf synthesis may contribute to the understanding of the signimicant correlation observed between vegf expression and tumor angiogenesis. references 1. neville bw, damm dd, allen cm, bouguot je. oral & maxillofacial pathology. (4th ed) 2016:444–8. 454–7, 462–4. 2. shamloo n, taghavi n, yazdani f, shalpoush s, ahmadi s. cd44 expression in pleomorphic adenoma, carcinoma ex pleomorphic adenoma and their adjacent normal salivary glands. dent res j (isfahan) 2018;15:361–6. 3. fonseca fp, de andrade ba, lopes ma, pontes ha, vargas pa, de almeida op, et al. p63 expression in papillary cystadenoma and mucoepidermoid carcinoma of minor salivary glands. oral surg oral med oral pathol oral radiol. 2021;115:79–86. 4. akbarzadeh baghban a, taghavi n, shahla m. combined analysis of vascular endothelial growth factor expression with cyclooxygenase-2 and mast cell density in oral squamous cell carcinoma. pathobiology. 2017;84:80–6. 5. sakurai k, urade m, noguchi k, kishimoto h, ishibashi m, yasoshima h, et al. increased expression of cyclooxygenase-2 in human salivary gland tumors. pathol int. 2001;51:762–9. 6. gallo o, franchi a, magnelli l, sardi i, vannacci a, boddi v, et al. cyclooxygenase-2 pathway correlates with vegf expression in head and neck cancer. implications for tumor angiogenesis and metastasis. neoplasia. 2001;3:53–61. 7. lequerica-fernández p, astudillo a, de vicente jc. expression of vascular endothelial growth factor in salivary gland carcinomas correlates with lymph node metastasis. anticancer res. 2007;27:3661–6. 8. fonseca fp, basso mp, mariano fv, kowalski lp, lopes ma, martins md, et al. vascular endothelial growth factor immunoexpression is increased in malignant salivary gland tumors. ann diagn pathol. 2024;19:169–74. 9. lim jj, kang s, lee mr, pai hk, yoon hj, lee ji, et al. expression of vascular endothelial growth factor in salivary gland carcinomas and its relation to p53, ki-67 and prognosis. j oral pathol med. 2003;32:552–61. 10. gnepp dr. diagnostic surgical pathology of the head and neck. philadelphia: saunders; 2019. 11. auclair pl, goode rk, ellis gl. mucoepidermoid carcinoma of intraoral salivary glands, cancer 69:2021–2030. 12. aoki t, tsukinoki k, kurabayashi h, sasaki m, yasuda m, ota y, et al. hepatocyte growth factor expression correlates with cyclooxygenase-2 pathway in human salivary gland tumors. oral oncol. 2022;42:51–6. 13. cho np, han hs, soh y, son hj. overexpression of cyclooxygenase-2 correlates with cytoplasmic hur expression in salivary mucoepidermoid carcinoma but not in pleomorphic adenoma. j oral pathol med. 2007;36:297–303. 14. merza ms. comparative imuunohistochemical analysis of k-ras oncogene and cyclooxygenase 2 enzyme expression in pleomorphic adenoma and adenoid cystic carcinoma of salivary glands. eur sci j. 2023;9:1857–7881. 957 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.957 http://dentistry3000.pitt.edu effectiveness of calcium sulfate bone graft with platelet rich fibrin in oroantral fistula closure mahmoud altabba, issa wehbeh, ahmad alnada faculty of den.stry, damascus university, syria abstract objec6ve: oroantral communicadon is a common complicadon observed following the extracdon of posterior maxillary teeth, ooen causing concern for both padents and general pracddoners. various methods are employed to close these communicadons. to idendfy a closure technique with minimal complicadons that is rapid and easily applicable by general pracddoners, we evaluated the effecdveness of using calcium sulfate grao material mixed with platelet-rich fibrin, followed by clinical and radiographic assessment of closure success. material and methods: a moderate-sized oroantral communicadons resuldng from recent tooth extracdons was radiographed to confirm the size and dimensions of the communicadon. subsequently, a 20 ml blood sample was collected from the padent and centrifuged to obtain two tubes containing platelet-rich fibrin. using the first tube, a platelet-rich fibrin plug was formed, applied, and sutured at the site of the communicadon. calcium sulfate grao material was then applied aoer being mixed with the platelet-rich fibrin clot from the second tube. the padent was followed up at intervals of: day one, day three, one week, two weeks, and six months. results: we showed successful closure of oroantral communicadons in this case, with clinical and radiographic follow-up for 6 months post-procedure. conclusion: within the limitadons of this study, it can be concluded that the technique of using calcium sulfate grao material with platelet-rich fibrin is a successful, cost-effecdve, and easily applicable method for managing moderate-sized oroantral communicadons. open access cita%on: altabba m, et al. (2025) effec%veness of calcium sulfate bone grac with platelet rich fibrin in oroantral fistula closure. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.957 received: june 6, 2025 accepted: june 26, 2025 published: august 13, 2025 copyright: ©2025 altabba m, et al. this is an open access ar%cle licensed under a crea%ve commons axribu%on work 4.0 united states license. email: mahmoud.tabba@damascusuniversity.edu.sy introduc)on oroantral communication (oac) is an abnormal communication that results from the loss of both soft and hard tissues separating the oral cavity from the maxillary sinus [1]. the birst signs that may indicate the occurrence of an oroantral communication include the presence of signibicant bleeding or an air bubble in the extraction socket of the removed tooth. the valsalva test is a conbirmatory diagnostic tool for this communication, as it causes air to escape through the alveolar socket [2]. there are numerous techniques described in the medical literature for the closure of this communication, each with its own indications, advantages, and disadvantages. these include vestibular blap displacement, rotational palatal blap, and cheek fat pad with a pedicle blap [3]. the sizes of oroantral openings have been classibied by james hupp into small (diameter < 3 mm), medium (3–6 mm), and large (> 6 mm) openings [4]. small openings can heal spontaneously if their diameter is less than 2 mm [5], and the use of a gelfoam, secured with sutures in the socket containing the communication in an "8" shape, may also help ensure complete closure [6]. on the other hand, openings larger than 2 mm but smaller than 5 mm have not been shown to close spontaneously without any intervention, according to a systematic review conducted by visscher [7]. if the oroantral communication remains open for more than 48 hours, it may lead to the formation of a oroantral bistula, as the tract becomes lined with epithelial tissue, which increases the risk of maxillary sinus infections [1]. several techniques have been proposed for closing oroantral communications, all of which primarily involve the closure of soft tissues [8]. most of the methods for closing maxillary sinus openings have focused on achieving closure and preventing the formation of an oroantral bistula. however, with the development of biologically acceptable materials and the improvement of their physical and biological properties, it has become possible to focus on controlling both the short and long-term outcomes of such closures, while minimizing the number of surgical procedures required and reducing their complications and negative effects. effecdveness of calcium sulfate bone grao with platelet rich fibrin in oroantral fistula closure vol 13, no 1 (2025) doi 10.5195/d3000.2025.957 http://dentistry3000.pitt.edu 2 material and methods intervention a non-smoker 30-year-old-female patient was referred to the department of oral and maxillofacial surgery at the faculty of dentistry, damascus university in 8/2024. the main complaint was pain related to the tooth 26. intraoral examination revealed badly decayed tooth indicated for extraction. radiographic examination (figure 1) revealed the presence of a long palatal root that might have been involved within the maxillary sinus. after taking the patient's medical history, it was determined that there was no contraindication to extracting the affected tooth. the roots were separated individually, and then the roots were extracted sequentially. to ensure the integrity of the maxillary sinus, we performed the valsalva maneuver, which showed the release of air bubbles from the extraction site. when the area was rinsed with saline, bluid leakage from the oral cavity into the maxillary sinus and then into the nose was observed (figure 2). a 3d image was taken, which revealed an opening in the maxillary sinus from the palatal root measuring an average of 4 mm. the case was reviewed and approved by the local research ethics committee at damascus university (no: 032024866). a written informed consent was obtained for publication of this report and accompanying images. pre-surgical phase patient interview a comprehensive clinical history was obtained from patients visiting the oral and maxillofacial surgery department at the faculty of dentistry, damascus university, who had undergone extraction of a maxillary posterior tooth and developed an oroantral communication. this included the patient's personal information, general medical history, and dental history. clinical and radiographic examination before the procedure a thorough examination of the extraction site was performed, and oroantral communication was clinically conbirmed by conducting the valsalva maneuver, along with a test for bluid passage from the oral cavity to the nasal cavity using a syringe and saline solution. the size of the communication was then assessed, and its compliance with inclusion criteria was veribied radiographically through three-dimensional cone beam computed tomography (cbct) imaging (figure 3). surgical phase patient preparation and site preparation (figure 4) initially, 20 ml of venous blood was drawn from the patient and divided into two glass tubes, each with a capacity of 10 ml. these tubes are specibically designed for the preparation of platelet-rich bibrin (prf). the tubes were placed in a centrifuge and spun at 2700 rpm for 12 minutes [9]. after centrifugation, each tube separates into three distinct layers: • red blood cell layer (rcl): the layer of red blood cells appears at the bottom of the tube. • platelet-rich fibrin (prf) layer: the middle layer, which contains the bibrin matrix enriched with platelets. • platelet-poor plasma (ppp): the top layer, which is plasma with a low platelet count. the prf gel is then extracted from the tube and separated from the red blood cells. to prepare the prf plug, the birst tube is used, and the prf gel is placed in a cylinder within the prf box. it is gradually compressed using a piston to obtain the binal prf plug. after anesthetizing the patient, the area of oroantral communication is examined. granulation tissue is removed if more than 24 hours have passed since the tooth extraction. the edges of the extraction socket are carefully inspected, and the socket is irrigated using a saline solution. preparation of calcium sulfate graft and mixing with platelet-rich fibrin (prf) (figure 5) the calcium sulfate graft is prepared by mixing the fast-set liquid with the powder to achieve the desired paste-like consistency, according to the manufacturer's instructions. the mixture is then placed into the designated grafting tray. subsequently, the graft is combined with pieces of platelet-rich bibrin (prf) obtained from the second tube after compressing the prf gel using the prf box. application of platelet-rich fibrin plug and mixed graft the prf plug is prepared birst. using 3/0 polyglactin (vicryl) suture, a needle is passed through the vestibular gingiva, then through one side of the plug, and binally through the palatal gingiva without tying any knots, ensuring no blaps are raised. the prf plug is then gently inserted into the area of oroantral communication. the ends of the suture are held to prevent the plug from displacing into the maxillary sinus. (figure 6). after sealing the communication with the prf plug, the mixed graft is applied gently over it until the entire socket is billed. the suture is then tied to secure the graft in place. then, we wait for 2 to 4 minutes while applying proper isolation with sterile gauze until the graft solidibies. afterward, the graft is left exposed to the oral cavity without being covered by any membrane. the patient is instructed to bite down on a sterile gauze pad for one hour. post-surgical phase in addition to the conventional post-extraction instructions, the following strict guidelines are provided: 1. avoid eating or drinking during the jirst two hours post-surgery. after that, only liquids should be consumed for the birst 48 hours (liquid diet), ensuring that they are not hot. 2. avoid rinsing the mouth for the jirst 48 hours and stay away from all irritant odors. 3. avoid using plastic straws for drinking during the week following surgery to prevent negative pressure, which can negatively affect the surgical site. 4. avoid violent sneezing and forceful expulsion of nasal secretions. if this is necessary, the patient should keep the mouth open during sneezing to prevent sudden pressure increases within the maxillary sinus. 5. do not use wind instruments or whistle. 6. regular follow-up appointments are necessary for monitoring. the patient is given a prescription for a set of medications, provided there are no contraindications. the prescription includes: rx i. augmentin (amoxicillin + clavulanic acid) 1000mg tab, twice daily. take one tablet every 12 hours for seven days. ii. ibuprofen 400mg tab, three times daily. take one tablet every 8 hours after meals for two days. iii. oxymetazoline hcl 0.05%. spray in the nostril corresponding to the side of the oroantral communication, three times a day for bive days. effecdveness of calcium sulfate bone grao with platelet rich fibrin in oroantral fistula closure vol 13, no 1 (2025) doi 10.5195/d3000.2025.957 http://dentistry3000.pitt.edu 3 clinical and radiological follow-up the patient is clinically evaluated at intervals (second day, third day, one week, and two weeks) post-surgery(figure 7). healing is monitored and graded on a scale from 1 to 5 according to the landry criteria. radiological evaluation is performed using cone-beam computed tomography (cbct) imaging 6 months after the procedure. results the application of this method showed successful closure of oroantral communication clinically and radiographically after 6 months of follow-up. discussion maxillary sinus perforation is considered one of the potential complications following the extraction of maxillary posterior teeth. the incidence of oroantral communication (oac) following extractions in the posterior maxilla ranges from 0.31% to 4.79% [5]. platelet-rich bibrin (prf) is a bibrin-based biomaterial that promotes angiogenesis and directs epithelial cell migration across its surface. it accelerates wound healing and provides protection for the surgical site [10]. the objective of this case report was to evaluate the efbicacy of platelet-rich bibrin combined with calcium sulfate graft material in the closure of oroantral communication post-extraction of maxillary posterior teeth, as well as in the preservation of the alveolar ridge. prf was chosen due to its richness in growth factors, cytokines, and platelets that enhance both soft and hard tissue healing [11]. calcium sulfate was selected as a biocompatible material with a well-documented history of clinical use. it has been employed in periodontal regeneration, endodontic repair, alveolar bone resorption cases, and sinus augmentation procedures. however, to date, it has not been reported in the closure of oroantral communication [12]. moreover, calcium sulfate forms a barrier against bibroblast migration, eliminating the need for membrane application or blap elevation following graft placement [12,13]. the success of oac closure was conbirmed using the following criteria: • valsalva maneuver test with observation of air bubble leakage. • presence of bluid leakage through the nose. • necessity for a second surgical intervention. • development of an oroantral bistula. cone-beam computed tomography (cbct) imaging was performed at two time points: pre-operatively and six months post-procedure. changes in alveolar ridge height and width were monitored, in addition to assessing radiographic bone density at the oac site. after six months, radiographic density ranged between 570 hounsbield units (hu) and 919 hu, with a mean value of 785.91 hu, consistent with d3 bone quality. this indicates a substantial improvement in bone radiodensity attributed to the combined use of prf and calcium sulfate. this radiodensity enhancement may be due to prf’s contribution to increasing bone density through the presence of growth factors, such as transforming growth factor (tgf), which stimulates osteoblast proliferation during normal socket healing. furthermore, the bibrin scaffold promotes neovascularization, supports immune cell activity, and reduces inblammation. calcium sulfate also plays a critical role, as its dissolution increases local calcium ion concentrations, stimulating osteoblastic activity [14]. additionally, it lowers the ph upon resorption, facilitating the release of bone morphogenetic proteins (bmps) from adjacent bone. this is followed by the formation of hydroxyapatite carbonate layers like natural bone mineral, which later remodels during bone maturation [12]. historadiographic studies have shown that calcium sulfate grafts are fully resorbed within nine months and replaced by mature lamellar bone [15], indicating its signibicant role in bone regeneration [13]. minor reductions in both vertical and horizontal dimensions of the alveolar ridge were observed six months post-closure compared to pre-operative values. this suggests that the combination of calcium sulfate and prf effectively minimized, but did not entirely prevent, post-extraction bone resorption. finally, a significant reduction in the radiographic diameter of the oroantral communication was observed six months post-grafting, decreasing from 4 mm to 0 mm. statistical analysis revealed a significant difference between preand post-grafting oac diameters, confirming the clinical effectiveness of the applied treatment. conclusions the application of calcium sulfate and prf clot led to a complete closure when treating oroantral communication. references 1.krishanappa, s. k. k., prashanti, e., sumanth, k. n., naresh, s., moe, s., aggarwal, h., & mathew, r. j. (2016). interventions for treating oro-antral communications and nistulae due to dental procedures. cochrane database of systematic reviews (5). ,116. 2.fragiskos, f. (2007). perioperative and postoperative complications oral surgery (pp. 181-203): springer. 3.fatani, b., fatani, a., & alomar, a. (2020). oroantral communication and fistula: a review of the literature, saudi journal of oral and dental research, e3-e9. 4.hupp, j. r., tucker, m. r., & ellis, e. (2013). contemporary oral and maxillofacial surgery-e-book: elsevier health sciences.,123-131. 5. mourão, c. f. d. a. b. (2019). which treatments are best for oro-antral nistulae? evidence-based dentistry, 20(2), 44-45. 6. le, b. t., & woo, i. (2007). management of complications of dental extractions. a peer reviewed publication.,4-16. 7. visscher, s. h., van minnen, b., & bos, r. r. (2010). closure of oroantral communications: a review of the literature. journal of oral and maxillofacial surgery, 68(6), 1384-1391. 8. sandhya, g., reddy, p. b., kumar, k. j., reddy, b. s., prasad, n., & kiran, g. (2013). surgical management of oro-antral communications using resorbable gtr membrane and fdmb sandwich technique: a clinical study. journal of maxillofacial and oral surgery, 12(3), 254-259. 9.ghanaati, s., booms, p., orlowska, a., kubesch, a., lorenz, j., rutkowski, j., landes, c., sader, r., kirkpatrick, c., choukroun, j. 2014. advanced plateletrich nibrin: a new concept for cell-based tissue engineering by means of innlammatory cells. j oral implantol, 40, 679-89. 10.shah, r., thomas, r., & mehta, d. (2017). an update on the protocols and biologic actions of platelet rich fibrin in dentistry. the european journal of prosthodontics and restorative dentistry, 25(2):64-72. 11.caruana, a., savina, d., macedo, j. p., & soares, s. c. (2019). from platelet-rich plasma to advanced platelet-rich fibrin: biological achievements and clinical advances in modern surgery. eur j dent, 13(2):280-286. 12.gupta h, pandey a, agarwal r, mehra h, gupta s, gupta n, et al. (2024). application of calcium sulfate as graft material in implantology and maxillofacial procedures: a review of literature. natl j maxillofac surg; 15:183-7. 13.yahav, a., kurtzman, g. m., katzap, m., dudek, d., & baranes, d. (2020). bone regeneration: properties and clinical applications of biphasic calcium sulfate. dental clinics; 64(2), 453-472. 14.boyle kk, sosa b, osagie l, turajane k, bostrom mp, yang x. (2019). vancomycin-laden calcium phosphate-calcium sulfate composite alloes bone formation in a rat infection model. plos one;14:e0222034. 15.laino, l., troiano, g., giannatempo, g., graziani, u., ciavarella, d., dioguardi, m., lo muzio, l., lauritano, f., cicciu, m. (2015a). sinus lift augmentation by using calcium sulphate. a retrospective effecdveness of calcium sulfate bone grao with platelet rich fibrin in oroantral fistula closure vol 13, no 1 (2025) doi 10.5195/d3000.2025.957 http://dentistry3000.pitt.edu 4 12 months radiographic evaluation over 25 treated italian patients. open dent j, 9: 414-9. figure 1. a panoramic image before the extraction of tooth 26. figure 2. an image after the extraction and the occurrence of sinus perforation. effecdveness of calcium sulfate bone grao with platelet rich fibrin in oroantral fistula closure vol 13, no 1 (2025) doi 10.5195/d3000.2025.957 http://dentistry3000.pitt.edu 5 figure 3. a cbct image in the coronal section before the procedure, showing the sinus perforation at the palatal root. figure 4. patient preparation and site preparation. a. platelet-rich fibrin (prf) gel after separation, b. platelet-rich fibrin (prf) plug, c. the perforation site after curettage. effecdveness of calcium sulfate bone grao with platelet rich fibrin in oroantral fistula closure vol 13, no 1 (2025) doi 10.5195/d3000.2025.957 http://dentistry3000.pitt.edu 6 figure 5. preparation of calcium sulfate graft and mixing with platelet-rich fibrin (prf); a. graft mixing according to the manufacturer's instructions, b. graft mixed with pieces of platelet-rich bibrin (prf). figure 6. application of platelet-rich fibrin plug and mixed graft: a. passing the suture initially through the buccal gingiva, b. passing the suture through the platelet-rich bibrin (prf) plug, c. application of the calcium sulfate graft mixed with platelet-rich bibrin (prf), d. graft hardening and performing the x-shaped suture. effecdveness of calcium sulfate bone grao with platelet rich fibrin in oroantral fistula closure vol 13, no 1 (2025) doi 10.5195/d3000.2025.957 http://dentistry3000.pitt.edu 7 figure 7. post-surgical phase. a: follow-up on the next day, b: follow-up after 3 days, c: follow-up after one week, d: follow-up after two weeks. 978 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.978 http://dentistry3000.pitt.edu stability assessmernt with immediate loading using blx slactive dental implant ahmed abdulkareem mahmood college of den*stry, tikrit university, iraq abstract objec2ve: to assess the primary and secondary stability of dental implants with immediate loading protocol. material and methods: thirty palents between the ages of 18 and 45 years who received forty dental implants were study. these cases involved bone level x modified sand blast large grit acid etch blx sla aclve implant. ater surgery, the primary stability was examined while secondary stability was evaluated 3 months later. the spss program was used to evaluate and analyze the results employing the paired t test and independent t test at p <0.05. results: thirty pa'ents, 20 females and 10 males, par'cipated in this study and had a mean age of 32 years. in comparison to the primary stability baseline value, the mean implant stability quolent values for secondary stability ater three months showed a stalslcally significant improvement. sex and jaws did not differ significantly in terms of stability. conclusion: when compared to the inilal primary stability baseline values, the study found that secondary implant stability measured values for the type of dental implant studied increased significantly. open access cita%on: mahmood aa. (2025) stability assessment with immediate loading using blx slac%ve dental implant. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.978 received: july 3, 2025 accepted: july 4, 2025 published: august 21, 2025 copyright: ©2025 mahmood aa. this is an open access ar%cle licensed under a crea%ve commons auribu%on work 4.0 united states license. email: ahmedabdulkareem@tu.edu.iq introduc)on restoring missing teeth with the placement of endosseous dental implants is a popular therapeutic option for achieving good cosmetic and functional outcomes [1]. brånemark >irst suggested that the implants be submerged and unloaded for three to six months [2]. due to this lengthy duration, a different loading protocol had to be implemented to reduce time. immediate loading is when the prosthesis is placed within three days of the implant being placed. a satisfactory level of osseointegration is anticipated following the surgical implantation of dental implants. the initial protocol called for a two-step surgical process for branemark implants to become osseo integrated [3]. lack of mobility is regarded as a clinical condition of implant stability [4]. it is typically separated into two categories: primary stability, or mechanical engagement, and secondary stability, or biological osseo integration. the >irmness that arises from the mechanical interaction between the implant and the bone is known as primary stability. secondary stability is the outcome of new bone cells growing around the biocompatible implant leading to osseo integration [5]. more stability has been the aim of many implant designs [6]. the straumann blx is one such implant design. this kind of dental implant is superior because it is entirely tapered and has strong stability, especially in low quality of bone. it is constructed using roxolid® and the surface treatment of dental implants made with a large grit acidetched surface with blx modi>ied sandblast (blx slactive) [7]. the titanium surface is given a macro-roughness by employing a big grit sandblasting process with corundum particles to create the sla surface of the implant. after that, there is a vigorous acidetching bath at a higher temperature for a few minutes. the topography that results provides the perfect framework for cell attachment [8]. similar to sla, the chemically modi>ied sandblasted, large grit, and acid etched surface (slactive) was applied, but with the addition of washing under protective n2 conditions and packing in isotonic solution (nacl). this produced almost 60% more bone formation than sla implants by improving surface chemistry and signi>icantly enhancing hydrophilic qualities [9]. by preventing infection and encouraging a quicker recovery, antibiotics administered after surgery lessen discomfort and accelerate healing. the most notable effects on pain relief and healing were shown by augmentin and azithromycin, which also successfully controlled infection and improved recovery [10]. stability assessmernt with immediate loading using blx slaclve dental implant vol 13, no 1 (2025) doi 10.5195/d3000.2025.978 http://dentistry3000.pitt.edu 2 materials and methods 30 iraqi patients between the ages of 18 and 45 years —ten men and twenty women— who met the study's eligibility requirements were enrolled. to evaluate the primary and secondary stability, these patients were contained inside a group of 40 dental implants (blx slactive implant). clinical and radiographic examinations of the hard and soft tissues were conducted at the dental implant surgery site using orthopantomography (opg). eligibility criteria good overall health free of local or systemic disorders such >ibrous dysplasia, hyperparathyroidism, heavy smoking, etc. that could impair bone healing ability. patients gender of both male and female, aged > 18 years. according to the sac classi>ication, straight forward cases involving partially edentulous maxilla or mandible (short span – one or two teeth loss) were handled as delayed implant implantation protocols (at least 6 months after tooth extraction). exclusion criteria patients were excluded if any of the following conditions were present: psychological disease, impractical expectations, or current pregnancy, uncontrolled systemic disorders such as uncontrolled diabetes, head and neck radiation or chemotherapy within the last >ive years, or patients receiving bisphosphate treatment can all impair natural healing or make it more dif>icult for a patient to recover from surgery. local problems in the implant zone include acute or chronic infections, poor oral hygiene, and local pathological diseases. cases that were advanced and complex based on the sac classi>ication, as well as any clinical or historical evidence of parafunctional behaviors (such as clenching or bruxism), were excluded. surgical procedure using the in>iltration technique, local anesthetic of the intended surgical >ield was achieved using lidocaine 2%, starting with one tooth prior to and after the implantation site. depending on the relevant criteria and the surgeon's evaluation, the implantation procedure was performed using either the >lapped or >lapless technique. the drilling procedure was carried out in compliance with the manufacturer's recommendations. as seen in figure 1, the implant bed was prepared using spiral drills, serial drilling, and copious amounts of normal saline irrigation in accordance with the blx implant system's recommendations until the desired diameter was reached. a surgical micro-motor hand piece with a torque of 35 ncm and a speed of 15 rpm was used to introduce the implants, as shown in figure 2. figure 1. the blx drill during osteotomy. figure 2. installation of the blx slactive implant in the prepared site. a torque ratchet up to 50 ncm was used to manually seat dental implants into their ultimate position, and the penguin rfa tool with smart peg type 38 was used to measure the isq immediately as a primary stability and secondary stability were also evaluated and recorded. as shown in figure 3, the average of the measurements taken in the buccopalatal and mesiodistal directions was recorded. figure 3. measurement of the isq using penguin rfa and smart peg type 38. for the >lapping technique, the incision was closed using interrupted 3/0 braided black silk sutures. co-amoxiclav tab 625 mg were to be taken three times daily for >ive days, along with a 250 mg tablet of metronidazole three times daily. when necessary, 50 mg tabs of diclofenac potassium are given as an effective pain reliever. within 3 days after implant insertion, the patients underwent the immediate loading protocol with screwretained restorations in non-functional occlusion shown in figure 4. figure 4. screw retained restoration. follow up and measurement of secondary stability three months after loading, the screw-retained restoration was disconnected to assess secondary stability in a manner identical to that of the primary stability, and a screwdriver and a ratchet with a torque of 35 ncm tightened the screw-retained restoration and re inserted into its functional position. a te>lon piece was inserted into the screw hole, and the composite >illing material (light cured) with functional occlusion was packed into it. statistical analysis ibm spss (statistical package for social sciences) version 26 was used to evaluate data. these data were tested using the paired t test and independent t test. p<0.05 indicates signi>icance. results 24 dental implants were placed in the mandible and 16 implants in the maxillary bone. the average isq values for the implants' secondary stability three months after loading were noticeably higher than those for the primary stability (72.63 versus 81.75). after three months, there is a statistically signi>icant difference between primary and secondary stability (<0.05). on the other hand, the study showed no signi>icant differences between primary stability and secondary stability among male and female patients as noticed in table 1. there were no signi>icant differences in primary and secondary stability in relation to implant site in maxillary and mandibular arches (table 2). stability assessmernt with immediate loading using blx slaclve dental implant vol 13, no 1 (2025) doi 10.5195/d3000.2025.978 http://dentistry3000.pitt.edu 3 discussion in this study, gender and implant had no effect on stabilit y. but, considering additional elements includi ng surgical technique, implant design, and b one quality, individual evaluations are still e ssential. the means of secondary stability isq values in the current research were substantially h igher than the baseline values for primary st ability. additionally, the means of primary and seco ndary stability for the maxillary and mandib ular arches, as well as between male and fe male patients, did not differ signi>icantly [11 ]. the study's >inding that primary and second ary stability differ signi>icantly is consistent with previous studies showing that these tw o types of stability are separate. while secondary stability is necessary for lo ngterm success, primary stability is critical for the >irst post-implantation phase [12]. gender had no effect on implant stability, which is in line with work by others [13]. however, work indicated that the isq value for female patients was higher than that of male patients [14], and this disagreed with men that had a greater implant stability value than women [15]. contrary to some existing literature, the study's findings showed that implant stability is unaffected by the implant site in both arches. in general, greater primary stability is linked to the mandible's denser bone than the maxilla. the results of this study, however, could be explained by elements like implant design, surgical method, or bone quality in a particular patient. a study by lang et al. highlighted the impact of bone density and quality by reporting variations in implant stability across the maxilla and mandible [16]. however zhang et al. did a metaanalysis which revealed that the implant sit e had no significant impact on implant stabil ity. this suggests that other parameters, incl uding implant length and implantation time, are more important in determining stability than location [17]. similarly, a study conducted by abd el-hady et al. assessed how implant materials affected the stability according to the study's findings, the material composition of the implant had a greater impact on its stability than the precise location within the maxilla or mandible [18]. conclusions after three months, there was a statistically signi>icant increase in implant stability in comparison to primary stability. references 1. manzano-moreno, f., herrera-briones, f., bassam, t., et al. factors affecting dental implant stability measured using the ostell mentor device. implant dent 2015; 24:565-577. 2. sommer, m., zimmermann, j., grize, l. et al. marginal bone loss one year after implantation: a systematic review of different loading protocols. int j oral maxillofac surg 2020; 49:121-134. 3. misch ce, wang hl, misch cm, sharawy m, lemons j, judy kw. rationale for the application of immediate load in implant dentistry: part i. implant dent. 2004; 13: 207-17. 4. andreotti, a., goiato, m., nobrega, a., et al. relationship between implant stability measurements obtained by two different devices: a systematic review. j periodontol 2020; 88:281-288. 5. kittur, n., oak, r., dekate, d.,et al. dental implant stability and its measurements to improve osseointegration at the bone-implant interface: a review. materials today: proceedings. 2021; 43:1064-1070. 6. cooper, l., de kok, i., reside, g., et al. immediate fixed restoration of the edentulous maxilla after implant placement. j oral maxillofac surg 2005; 63:97-110. 7. kordusky, benjamin andrew. accuracy of blx and blt guided implants in the edentulous maxilla: an in vivo study. graduate theses, dissertations, and problem reports. 2020; 7544. 8. preshaw, p. summary of: implant surface characteristics and their effect on osseointegration. br dent j 2015; 218:292-293. 9. nicolau p, guerra f, reis r, et al. 10-year outcomes with immediate and early loaded implants with a chemically modibied sla surface. quintessence int j 2019; 50: 114-124. 10. ahmed, a., saber, m., ahmed, a., sohaib, q., saif, s., ali, s. the inbluence of antibiotics administration on infection subsequent to dental extraction. (2025). international journal of medical science and dental health, 11(02), 72-85. https://doi.org/10.55640/ijmsdh-11-02-05. 11. ahmed, ali saad, et al. evaluation of peel bond strength between heat cured acrylic based denture soft lining material and heat polymerized acrylic after different acrylic surface treatment methods. 2024. 12. miri r, shirzadeh a, kermani h, khajavi a. relationship and changes of primary and secondary stability in dental implants: a review. int j contemp dent med rev. 2017;2017:03011. 13. degidi m, daprile g, piattelli a. primary and secondary stability of implants in different bone types. j prosthet dent. 2019;121(5):611-617. 14. amer a, et al. (2025) evaluation of soft tissue in rabbit utilizing suture and tissue glue. dentistry 3000. 1:a001 doi:10.5195/d3000.2025.882. 15. zix, j., kessler-liechti, g., & mericske-stern, r. stability measurements of 1-stage implants in the maxilla by means of resonance frequency analysis: a pilot study. int j oral maxillofac implants 2005; 20. 16.ahmed, ali saad; ahmed, rusal saad; arab, luma nasrat. the antifungal potential of cinnamon oil incorporated into a heat-polymerized soft liner. journal of dental materials & techniques, 2024, 13.3. 17. zhang y, tang x, zhang y, cao c. a network meta-analysis comparing treatment modalities of short and long implants in the posterior maxilla with insufbicient bone height. bmc oral health. 2024 dec 31;24(1):1574. doi: 10.1186/s12903024-05377-1. pmid: 39741292; pmcid: pmc11686903. 18. el-hady aia, eid hi, mohamed sl, fadl sm. inbluence of titanium and titanium-zirconium alloy as implant materials on implant stability of maxillary implant retained overdenture: a randomized clinical trial. bmc oral health. 2024 aug 6;24(1):902. doi: 10.1186/s12903-024-04692-x. pmid: 39107737; pmcid: pmc11305035. stability assessmernt with immediate loading using blx slaclve dental implant vol 13, no 1 (2025) doi 10.5195/d3000.2025.978 http://dentistry3000.pitt.edu 4 table 1. males vs. females using independent t-test. test comparison mean ± sd t-statistic p-value signi>icance independent t-test primary stability (male) 72.00 ± 3.30 -0.69 0.50 not signi>icant independent t-test primary stability (female) 72.83 ± 3.40 independent t-test secondary stability (male) 81.70 ± 2.83 -0.07 0.95 not signi>icant independent t-test secondary stability (female) 81.77 ± 2.37 table 2. maxilla vs. mandible using independent ttest. test comparison mean ± sd t-statistic p-value signi>icance independent t-test primary stability (maxilla) 71.93 ± 3.63 -0.81 0.42 not signi>icant independent t-test primary stability (mandible) 72.88 ± 3.15 independent t-test secondary stability (maxilla) 82.07 ± 2.64 0.19 0.85 not signi>icant independent t-test secondary stability (mandible) 81.92 ± 2.10 stability assessmernt with immediate loading using blx slaclve dental implant vol 13, no 1 (2025) doi 10.5195/d3000.2025.978 http://dentistry3000.pitt.edu 5 figure 1. the bar graph illustrates the inhibition zones (mm) for staphylococcus aureus isolates at three-time intervals (t0 (a), t1 (b), t2 (c)), when exposed to six different antibiotics: amoxicillin, vancomycin, ampicillin, erythromycin, azithromycin, and clindamycin. statistical comparisons between groups are indicated with asterisks (* for p < 0.05, ** for p < 0.01, *** for p < 0.001) and "ns" for non-signi>icant differences. stability assessmernt with immediate loading using blx slaclve dental implant vol 13, no 1 (2025) doi 10.5195/d3000.2025.978 http://dentistry3000.pitt.edu 6 figure 2. the bar graph illustrates the inhibition zones (mm) for candida albicans isolates at three-time intervals (t0 (a), t1 (b), t2 (c)), when exposed to six antifungal agents: clotrimazole, fluconazole, nystatin, 5-flucytosine, caspofungin, and amphotericin b. statistical comparisons between groups are annotated with *** for p < 0.001 and “ns” for non-signi>icant differences. 960 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.960 http://dentistry3000.pitt.edu assessment of color change after adding nano aluminum oxide to heat-cure acrylic wafaa jasem, nisreen mahdi saleh, hussein falah hasan, ahmed jamal ibrahim college of den*stry, university of al maarif, al anbar, iraq abstract objective: this study investigates the effect of introducing nano-sized aluminum oxide (al2o3) particles into heat-cure acrylic resin on color stability. color changes are an important cosmetic element in dental materials. the purpose of this work was to examine the color change of polymethyl methacrylate reinforced by varied weight percentages wt.% of nano al2o3 dillersmaterials on degree of conversion and polymerization of dual cure resin cement after 24 hours. material and methods: this study was 40 disc-shaped samples (no. 10 per group) were prepared. group a control, group b pmma with al2o3 0.5%, group c pmma with al2o3 1%, group d pmma with al2o3 2%. results: the results showed that group d with al2o3 2% had a greater mean value of color change than the control group. while group b 0.5% had the lowest mean value of color change. conclusion: nano-al₂o₃ can be incorporated into heat-cure acrylic to improve material properties, but concentration is critical for maintaining color stability. open access cita%on: jasem w, et al. (2025) assessment of color change a<er adding nano aluminum oxide to heatcure acrylic. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.960 received: june 12, 2025 accepted: june 26, 2025 published: august 13, 2025 copyright: ©2025 jasem w, et al. this is an open access ar%cle licensed under a crea%ve commons asribu%on work 4.0 united states license. email: wafaa.atallah@uoa.edu.iq introduc)on heat-cure acrylic resins are widely used in dental prosthetics due to their esthetic properties, cost-effectiveness, and ease of handling. however, one major drawback is their susceptibility to color change over time due to environmental exposure, aging, and chemical degradation. nanotechnology has emerged as a solution to improve the mechanical and physical properties of dental polymers. nano-al2o3 is commonly used for reinforcement due to its hardness, thermal stability, and biocompatibility. however, its effect on optical properties, particularly color stability, needs thorough investigation. polymethyl methacrylate (pmma) is commonly regularly fabricated denture ingredients. despite fact that pmma is the most often used denture material, it falls short of highest materials criteria [1]. the pmma prostheses have poor surface quality and mechanical properties, such as hardness, dlexural strength as well as impact strength. consequently, the pmma materials must be strengthened. nanomaterials has been used in dental applications, and numerous studies have been carried out to determine its anticipated benedits and applications. nanoscale dillers encapsulated in a polymer matrix make up polymer nanocomposites [2]. according to recent research, nanoscale toughening agents can lead to new physical, mechanical, and biological functions, resulting in nanocomposites. pmma is also strengthened by the grafting of multiple nano dillers [2]. nanoparticles of aluminum oxide (al2o3) (99.9%) [2]. al2o3 is utilized in the formulation of high-impact acrylic resin's physical characteristics. combination of al2o3 into different biomaterials has been investigated besides enhancing mechanical properties [3]. furthermore, al2o3 is a lowcost multifunctional metal oxide nanoparticle with excellent chemical, optical, electrical, and biocompatibility properties. the dielectric properties of alumina nano diller and pmma blend nanocomposite, for example, have been observed to change. the type, form, and size of the nanoparticles dedine the characteristics of the new composite. nanomaterials, despite having higher physical features, for example density, fall short in relationships of mechanical features. because there is a growing need to improve materials' physical besides mechanical structures [4]. material and methods materials -heat-cure acrylic resin (pmma) -nano-aluminum oxide (al₂o₃) with particle size ~20–50 nm -spectrophotometer (to measure color using the cie lab* system) -artidicial aging chamber (uv light, heat, humidity) sample preparation 40 disc-shaped samples (10 per group) were prepared. table 1. percentage of al2o3 assessment of color change ager adding nano aluminum oxide to heat-cure acrylic vol 13, no 1 (2025) doi 10.5195/d3000.2025.960 http://dentistry3000.pitt.edu 2 group nano-al2o3 concentration group a 0% (control) group b 0.5% by weight group c 1.0% by weight group d 2.0% by weight fill the bottom half of the cuvette with stones (type 4) (100 g/25 ml) (p/w) according to the manufacturer's instructions, then covered with plastic patterns [5]. using a large brush, apply the releasing agent. once the release medium has dried, stack the top and bottom halves of the dlask and add a second layer of stamps on top of the dirst layer, with the clamps open after setting. warped a towel around the dlask. stamp his second layer of plungers after removing the plastic design [6]. following that, the mold is dinished, and is ready for packing in two groups, nano-diller powder was added by weight, with 1% and 1.5% to powder being added [9]. high precision sensitive electronic balance (0.0000g, mettle type ae260-s snr h50193) was utilized [7]. to achieve homogenous mixing, the diller was well distributed in the powder using amalgamator device dirm (sinalident) produced in china utilizing (300vibration/minute) for two minutes [8]. color measurement color values (l*, a*, b*) were measured: before aging and after artificial aging (300 hours exposure to uv and moisture).the total color difference (δe) was calculated using: δe=(l2−l1)2+(a2−a1)2+(b2−b1)2\delta e = \sqrt{(l_2 l_1)^2 + (a_2 a_1)^2 + (b_2 b_1)^2}δe=(l2−l1)2+(a2−a1)2+(b2−b1)2 a δe ≥ 3.3 is considered visually perceptible by most observers [9]. results as shown in table 2, anova analysis revealed a highly signidicant differences (pvalue p<0.01) of color stably values between the 4 (a, b, c, d groups, respectively). discussion the dindings indicate that: -low concentrations (≤1%) of nano-al₂o₃ have a tolerable effect on color change. higher concentrations (2%) result in significant visual color differences, potentially due to nanoparticle agglomeration, which affects light reflection and scattering. -the increase in δe values could be due to surface irregularities or heterogeneous distribution of nanoparticles. this underscores the need to optimize nanoparticle dispersion and concentration to preserve esthetic quality while enhancing physical properties. conclusions -nano-al₂o₃ can be incorporated into heatcure acrylic to improve material properties, but concentration is critical for maintaining color stability. -0.5% to 1.0% nano-al₂o₃ is the recommended range for minimizing color change. references 1. satyanarayan, a., singh, r., sharma, r., lunkad, h., mishra, r., & sandhu, h. s. (2022). assessing the outcomes of aluminium oxide on surface roughness and flexural strength of heat cure denture base resin. journal of pharmaceutical negative results, 13, 1116-1118. 2. ali sabri, b., satgunam, m., abreeza, n. m., & n. abed, a. (2021). a review on enhancements of pmma denture base material with different nanowillers. cogent engineering, 8(1), 1875968. 3. gad, m. m., fouda, s. m., al-harbi, f. a., näpänkangas, r., & raustia, a. (2017). pmma denture base material enhancement: a review of wiber, willer, and nanowiller addition. international journal of nanomedicine, 3801-3812. 4. al-harbi, f. a., abdel-halim, m. s., gad, m. m., fouda, s. m., baba, n. z., alrumaih, h. s., & akhtar, s. (2019). effect of nanodiamond addition on wlexural strength, impact strength, and surface roughness of pmma denture base. journal of prosthodontics, 28(1), e417-e425. 5. motawea, i., gamal el deen, h., & eltayeb, h. (2017). effect of denture cleansers on wlexural strength, color stability and surface roughness of wlexible and microwave cured acrylic resin. alazhar dental journal for girls, 4(3), 311-323. 6. yeh, h. c. (2014). effect of silica filler on the mechanical properties of silicone maxillofacial prothesis (doctoral dissertation). 7. gad, m. m., abualsaud, r., rahoma, a., althobity, a. m., akhtar, s., & fouda, s. m. (2022). double-layered acrylic resin denture base with nanoparticle additions: an in vitro study. the journal of prosthetic dentistry, 127(1), 174-183. 8. ghosh, p. k., kumar, k., & kumar, a. (2019). udm processed superior acrylic resin based zro2 nanoparticle reinforced composite for dental application. materials research express, 6(10), 105404. 9. deste, g., durkan, r., & oyar, p. (2020). evaluation of the transverse strength and elastic modulus of high impact denture base material. journal of dental materials & techniques, 9(3). 10. rizzatti-barbosa, c. m., gabriotti, m. n., joia, f. a., machado, c., & ribeiro, m. c. (2006). surface roughness of acrylic resins processed by microwave energy and polished by mechanical and chemical process. 11. asar, n. v., albayrak, h., korkmaz, t., & turkyilmaz, i. (2013). inwluence of various metal oxides on mechanical and physical properties of heatcured polymethyl methacrylate denture base resins. the journal of advanced prosthodontics, 5(3), 241-247. 12. sasaki, h., hamanaka, i., takahashi, y., & kawaguchi, t. (2016). effect of long-term water immersion or thermal shock on mechanical properties of high-impact acrylic denture base resins. dental materials journal, 35(2), 204-209. 13. hamed, q. a. (2017). investigation some mechanical properties of self-cured pmma resin reinforced by different types of nanoparticles. iraqi journal of mechanical and material engineering, 13(3). 14. jehan, a., ahila, s., & muthukumar, b. (2022). evaluation of wlexural strength and surface hardness of heat activated provisional pmma resin reinforced with nanoparticles–an in vitro study. j med pharm allied sci, 11, 4340-8. 15. quezada, m. m., salgado, h., correia, a., fernandes, c., & fonseca, p. (2022). investigation of the effect of the same polishing protocol on the surface roughness of denture base acrylic resins. biomedicines, 10(8), 1971. 16. kitao, n., kakimoto, k., masuda, t., kusunoki, t., utoh, e., matuo, k., ... & komasa, y. (2015). mechanical properties of artiwicial porcelain denture teeth. journal of osaka dental university, 49(2), 245-251. 17. chowdhury, a. r., kaurani, p., padiyar, n., meena, s., sharma, h., & gupta, a. (2021). effect of addition of titanium oxide and zirconium oxide nanoparticles on the surface roughness of heat cured denture base resins: an in-vitro study. svoa mater sci technol, 3(3), 36-44. 18. basima, m. a., & aljafery, a. m. (2015). effect of addition zro2-al2o3 nanoparticles mixture on some properties and denture base adaptation of heat cured acrylic resin denture base material. journal of baghdad college of dentistry, 27(3), 1521. 19. bhat, a., budholiya, s., aravind raj, s., sultan, m. t. h., hui, d., md shah, a. u., & safri, s. n. a. (2021). review on nanocomposites based on aerospace applications. nanotechnology reviews, 10(1), 237-253. 20. abdel-karim, u. m., el-safty, s. m., & kenawy, e. r. (2018). surface roughness, hardness, color stability, water sorption and water solubility of pmma denture base material reinforced with synthesized inorganic, organic, and hybrid nanowibers. egyptian dental journal, 64[4-october (fixed prosthodontics, dental materials, conservative dentistry & endodontics)], 3593-3608. 21. atallah, w., alfuraiji, n., & hussein, o. h. (2023). nanocomposites for prosthetic dental technology: a systemic review. journal of techniques, 5(1), 129-136. assessment of color change ager adding nano aluminum oxide to heat-cure acrylic vol 13, no 1 (2025) doi 10.5195/d3000.2025.960 http://dentistry3000.pitt.edu 3 table 2. color stability of all groups with means in micrometer (µm). group n δe (mean) sd visual perception anova test a (0%) 10 1.25 0.28 not perceptible p = 0.00 highly sign. (p<0.01) b (0.5%) 10 1.60 0.35 slight c (1%) 10 2.35 0.40 noticeable d (2%) 10 3.85 0.50 clearly perceptible *color stability decreased with increasing nano-al₂o₃ content, especially above 1%. the 0.5% group showed minimal change. 866 final the impact of thermocycling on the physiomechanical proper6es of sla and dlp 3d-printed dental ceramics: a compara6ve study vol 13 no 1 (2025) doi 10.5195/d3000.2025.866 h#p://den*stry3000.pi#.edu the impact of thermocycling on the physio mechanical properties of sla and dlp 3d-printed dental ceramics: a comparative study nadia h hasan college of dentistry, university of mosul, mosul, iraq abstract objec&ve: three-dimensional (3d) prin4ng technology is highly promising for producing nanoceramic resin dental restora4ons. however, the effects of environmental stressors on the structural integrity and clinical performance of these restora4ons require further elucida4on. to inves4gate the effects of stereolithography (sla) and digital light processing (dlp) 3d prin4ng technologies on the physicalmechanical proper4es of a 3d-printed resin material used in dental applica4ons. methods: a total of 120 resin specimens (senertek p-crown v2) were fabricated using sla and dlp technologies. the microhardness, flexural strength, and surface roughness of the specimens were evaluated under control and thermocycling condi4ons to evaluate their long-term performance. to assess sta4s4cal significance a two independent sample t-tests (p < 0.05) were used to analysis the data. results: sla samples exhibited significantly higher microhardness (p = 0.001) and flexural strength than dlp samples, both in the control state and aqer thermocycling. aqer thermocycling, the microhardness of sla samples increased, whereas that of dlp samples decreased. surface roughness values increased significantly in both sla and dlp samples aqer thermocycling, with sla samples exhibi4ng higher roughness values. conclusion: sla-printed resin demonstrated superior microhardness and flexural strength compared to dlp-printed resin. however, its long-term durability is affected by immersion and thermocycling. this study highlights the impact of water sorp4on, polymeriza4on mechanisms, and surface morphology on material performance. keywords: 3d prin4ng, dental materials, digital light processing, flexural strength, microhardness, stereolithography, surface roughness, thermocycling. cita:on: hasan nh. (2025) the impact of thermocycling on the physiomechanical proper:es of sla and dlp 3dprinted dental ceramics: a compara:ve study. den:stry 3000. 1:a001 doi:10.5195/d3000.2025.866 received: february 27, 2025 accepted: march 31, 2025 published: april 21, 2025 copyright: ©2025 hasan nh. this is an open access ar:cle licensed under a crea:ve commons awribu:on work 4.0 united states license. email: nadiahameed@uomosul.edu.iq. introduction stereolithography (sla) and digital light processing (dlp) are 3d printing dental techniques requiring high resolution and accuracy. despite their similarities in using volume-accelerated technology (vat) to create accurate dental restorations, but differ in speed, resolution, and cost-effectiveness [1]. ultraviolet light uses to solidify a resin in both sla and dlp but different method [2,3]. a resistance to daily exposure to an intense condition of the oral environment, is a significant factor for the successful clinical use of a nano ceramic materials that impact it’s the morphological, physical, mechanical properties cause surface integrity degradation [4,5]. a higher microhardness enhances wear resistance, while excessive surface roughness can weaken flexural strength by creating stress concentration points [6,7], and an optimal balance through material selection and techniqueprocessing is essential to improving ceramic durability, structural reliability [8]. the study aimed to compare the sla and dlp methods with regards to their physicalmechanical properties and the influence of artificial aging. the the impact of thermocycling on the physiomechanical proper6es of sla and dlp 3d-printed dental ceramics: a compara6ve study vol 13 no 1 (2025) doi 10.5195/d3000.2025.866 h#p://den*stry3000.pi#.edu null hypothesis stated that both 3d printing techniques showed no difference in such properties. material and methods the samples used in this study were divided as depicted in figure 1. ethical considerations all study participants provided informed consent, and the study design was performed solely on laboratory models; therefore, informed consent was not required confidentiality, and procedures for withdrawal from the research. measures a total of 120 rectangular bars (25 × 5 × 1 mm) were prepared using the senertek p-crown v2 resin material (libral traders, india). to design the bar geometry, autocad software was used and saved as a standard tessellation language (stl) file. the stl file was utilized in the anycubic photon mono 2 and anycubic photon d2 3d printers (lux creo inc., chicago, il, usa), which use sla and dlp technologies, respectively, to produce 60 bars from each technique (figure 1). the stl file was processed using a slicer program that converted it into g-code for machine readability [9]. the samples were constructed with resolutions as fine as 50 microns, ensuring precise detailing. 120 bars preperd by 3d-additive manufacturing technologies stereolith ography (sal) n=60 control group n=30 microhardness test n=10 fluxral strength test n=10 surface ruoghness test n=10 thermocycling group n=30 digital light processing (dlp) n=60 figure 1. schematic diagram of the study design. the impact of thermocycling on the physiomechanical proper6es of sla and dlp 3d-printed dental ceramics: a compara6ve study vol 13 no 1 (2025) doi 10.5195/d3000.2025.866 h#p://den*stry3000.pi#.edu after printing, the samples were cured in an ultraviolet oven for 8 minutes in a clear jar filled with glycerin, to improve the mechanical properties and ensure complete solidification. postprocessing steps included sanding, painting, polishing, tumbling, highpressure air cleaning, and coloring to make the 3d model ready for application [10]. in sla, a perforable platform was positioned adjacent to the vat containing liquid polymer resin. a laser beam was directed onto the platform, solidifying the first layer of resin. the platform was then incrementally lowered to form subsequent layers, repeating until the 3d object was fully constructed, taking approximately 38 minutes. sla requires support structures to stabilize the model during printing. dlp is like sla, with the primary distinction being the light source. dlp employs a conventional light source and digital micromirrors to print all the layers simultaneously. this approach allows faster printing compared to sla, where the laser traces the cross-sectional area point by point. [11,12]. sixty rectangular bars (30 from each technique) underwent thermocycling in a thermocycling unit (dorsa apparatus, tehran, iran) from 5 °c to 55 °c for 1,000 cycles with a 30 second dwell time, to simulate approximately 1 year of intraoral use [13]. the microhardness, flexural strength, and surface roughness of the nanoceramic resin material were evaluated in 40 rectangular bars per test (20 bars from each 3d printing technique: 10 as control group and 10 after thermocycling). the microhardness of the nanoceramic resin bars was evaluated by subjecting the centre of each bar to three indentations using a diamond pyramid indenter. a load of 300 g was applied for 10 s to determine the vickers hardness number in kg/mm² [13]. the mean values of these measurements were calculated using a dm 8/dm 2 microhardness tester (yang yi technology co., ltd, tainan city 70960, taiwan) (figure 2). using a universal testing machine, flexural strength of the nanoceramic resin bars was assessed. on two supporting bearers each bar was placed with its intaglio surface facing downward, following iso standards [14]. a 1.6 mm spherical-tipped indenter applied force centrally on a 5-mm wide face, perpendicular to the bar’s longitudinal axis. the load required to fracture each bar was recorded to the nearest 0.1 n, with a force application rate not exceeding 0.5 n/s (figure 3). a b figure 2: (a) vickers micro-indentation testing machine, (b) image of indentation. a b b figure 3: (a) schematic diagram of the three-point flexural strength test apparatus, (b) image of universal testing machine and testing bar. the impact of thermocycling on the physiomechanical proper6es of sla and dlp 3d-printed dental ceramics: a compara6ve study vol 13 no 1 (2025) doi 10.5195/d3000.2025.866 h#p://den*stry3000.pi#.edu tests were conducted in a dry environment, and flexural strength was calculated using the standard equation [14,15]. σ = 3 fl/2 wd2 n/mm2 or mpa where σ (sigma) represents stress in mpa; f, the applied force (n); l, length or distance over which the force is applied (mm); w, width of the element affected by the stress (mm); and d, depth or thickness of the element (mm). using the touch approach of an atomic force microscope (flexafm, nanosurf ag, liestal, switzerland ) , an intaglio surfaces of the nanoceramic resin bars were analysed for surface topography. the three central readings per bar were recorded, and surface roughness value (sa) was calculated. a 3d image covering an area of 20,000 × 20,000 nm was captured (figure 4). statistical analysis for statistical analysis of data distribution, the microhardness, flexural strength, and surface roughness values were statistically analyzed using spss statistics 27.0 (ibm corp., armonk, ny, usa). parametric independent sample t-tests were used to compare the effect of each variable and determine statistical differences between groups, with 95% confidence level and p ≤ 0.05 considered statistically significant. results participant characteristics to determine the sample size a software (g*power) used, at α = 0.05 and 1-β = 0.80, with an expected effect size of 0.3. descriptive statistics the impact of thermocycling on the physical-mechanical figure 4. roughness parameters. the impact of thermocycling on the physiomechanical proper6es of sla and dlp 3d-printed dental ceramics: a compara6ve study vol 13 no 1 (2025) doi 10.5195/d3000.2025.866 h#p://den*stry3000.pi#.edu properties of 3d-printed senertek p-crown v2 resin material was determined. tables 1 and 2 present the descriptive statistics, including mean, standard deviation, and campers’ mean, of the microhardness, flexural strength, and surface roughness of 3d-printed resin samples to assess the impact of control and thermocycling states on sla and dlp technologies and vice versa, respectively. two independents sample t-tests (p < 0.05) were conducted to assess the significance of the results. correlation among variables as shown in table 1, after thermocycling, the microhardness values were higher in sla samples (0.71153 kg/mm²) compared to dlp samples, whereas dlp samples had significantly higher values in the control state (0.32602 kg/mm²). higher mean flexural strength values (24.5080 mpa, 5.3940 mpa) were obtained for sla samples compared to dlp samples, respectively, in both the control state and after thermocycling. finally, the highest mean value for surface roughness (sa value) was obtained for sla samples that underwent thermocycling. dlp, digital light processing; sla, stereolithography test n mean std. deviation std. error mean t-test p value flexural strength value dlp control 10 19.7040 0.95789 0.30291 0.298 0.772 thermocycling 10 20.1170 4.27474 1.35179 sla control 10 24.5080 1.04583 0.33072 1.258 0.225 thermocycling 10 25.3940 1.96681 0.62196 microhardness value dlp control 10 18.6250 1.03098 0.32602 3.463 0.004** thermocycling 10 16.3200 1.83473 0.58019 sla control 10 16.9720 0.73976 0.23393 2.351 0.039* thermocycling 10 18.7330 2.25007 0.71153 surface roughness value dlp control 10 4.8087 0.66203 0.20935 3.974 0.002** thermocycling 10 7.1051 1.71642 0.54278 sla control 10 5.3585 0.70850 0.22405 9.148 0.000** thermocycling 10 22.5296 5.89325 1.86361 table 1. descriptive statistics and camper mean of flexural strength, microhardness, and surface roughness values for both stereolithography and digital light processing 3d printing technologies. the impact of thermocycling on the physiomechanical proper6es of sla and dlp 3d-printed dental ceramics: a compara6ve study vol 13 no 1 (2025) doi 10.5195/d3000.2025.866 h#p://den*stry3000.pi#.edu additionally, in shown in table 2, among dlp samples, higher microhardness values were observed in the control state (18.6250 kg/mm²), whereas the sla group showed higher values after thermocycling (18.7330 kg/mm²). no significant differences in flexural strength were observed between the control and thermocycling groups for both sla and dlp technologies. finally, the increase in sa values was more pronounced after thermocycling than in the control state for both dlp and sla technologies (5.3585 µm and 22.5296 µm, respectively). dlp, digital light processing; sla, stereolithography discussion in this in vitro comparative study, we investigated the effects of sla and dlp 3d printing on the physicalmechanical properties of a 3dprinted resin material used in dental applications. in clinical dentistry, sla and dlp have become pivotal for fabricating indirect restorations, offering efficiency and material conservation. both processes enable the production of intricate designs with minimal material waste compared to traditional subtractive manufacturing techniques, yet each exhibits distinct advantages and test n mean std. deviation std. error mean t-test p value flexural strength value control dlp 10 19.7040 0.95789 0.30291 10.712 0.000** sla 10 24.5080 1.04583 0.33072 thermocycling dlp 10 20.1170 4.27474 1.35179 3.546 0.002** sla 10 25.3940 1.96681 0.62196 microhardness value control dlp 10 18.6250 1.03098 0.32602 4.119 0.001* sla 10 16.9720 0.73976 0.23393 thermocycling dlp 10 16.3200 1.83473 0.58019 2.628 0.017* sla 10 18.7330 2.25007 0.71153 surface roughness value control dlp 10 4.8089 0.66178 0.20927 1.793 0.09 sla 10 5.3585 0.70850 0.22405 thermocycling dlp 10 7.1051 1.71642 0.54278 7.947 0.000** sla 10 22.5296 5.89325 1.86361 table 2. descriptive statistics and camper mean of flexural strength, microhardness, and sa surface roughness values for each 3d printing technologies in control and thermocycling states. the impact of thermocycling on the physiomechanical proper6es of sla and dlp 3d-printed dental ceramics: a compara6ve study vol 13 no 1 (2025) doi 10.5195/d3000.2025.866 h#p://den*stry3000.pi#.edu limitations depending on the application [16,17]. for instance, challenges such as weak interlayer adhesion and anisotropic material behavior underscore the importance of understanding the nuances of each technology. choosing between sla and dlp requires not only technical expertise but also the guidance of knowledgeable partners to optimize production outcomes and achieve high-quality results [18]. during daily use within oral environment, restorations are subjected to thermal stresses and evaluating the effects of 3d-printed resins in simulated oral conditions are important studies to assess the clinical performance of these newly introduced technologies. bardia et al. [12], reported that when a resin restoration subjecting a to 1,000 cycles of a thermal variation to simulates 1 year of clinical dental use. a plasticizer effect of water sorption of resin affects its mechanical properties. moreover, increased temperature accelerates water sorption, thereby interfering with its mechanical properties [19]. salivary ph usually ranges from 6.8 to 7.2; however, dietary acids can cause a drop in salivary ph . a dietary acids hydroxyl ion may diffuse through saliva water molecules, react with resin ceramic oxygen and caused ceramic degradation [5]. surface hardness is an indicator of a material’s abrasion resistance and surface strength. restorations with low surface hardness are more prone to scratches, damage to the resin surface, and dimensional changes during mechanical dental brushing or chewing hard foods [20]. in our study, dlp samples exhibited low hardness values after thermocycling, probably due to water sorption and alterations in the composition of the material and printing layers under thermal stress. in contrast, the sla group exhibited a significant increase in hardness with aging compared to the bulkier dlp group, which may be attributed to its layering technique and enhanced polymerization rate, depth of polymerization, and degree of conversion. the higher flexural strength of the sla group compared to that of the dlp group indicates its greater resistance to surface deformation and higher flexibility, which may be explained by the differences in the curing mechanisms of dlp and sla, coupled with differences in surface morphologies [21]. additionally, a non-significant positive effect in flexural strength after artificial aging was observed for both the dlp and sla groups, possibly owing to reduced water sorption, which alters a material’s characteristics by creating internal tensions that are damaging to a resin’s long-term usefulness [22]. reducing water sorption can prevent breaches and cracks and enhance the longevity of dental restorations. moreover, remnant monomers in the resin have been linked to dimensional fragility of restorations and may adversely affect oral tissues [23]. surface roughness is one of the most important properties affecting the longevity of dental restorations. rougher surfaces increase microbial adhesion, leading to dental stomatitis, and promote surface staining and discoloration, which can compromise dental aesthetics [18]. the surface topography of additively manufacturing materials requires further investigation, as the layering technology impacts the the impact of thermocycling on the physiomechanical proper6es of sla and dlp 3d-printed dental ceramics: a compara6ve study vol 13 no 1 (2025) doi 10.5195/d3000.2025.866 h#p://den*stry3000.pi#.edu porosity and morphological features of printed objects [24]. significant difference in surface roughness existed between the control and thermocycling groups for both the dlp and sla technologies. according to al-dulaijan et al., this difference may be attributable to the deterioration that occurs in the organic matrix or at the matrix-filler interface of dental resins during thermocycling and to the layering technology, which involves multiple steps between adjacent layers[25]. therefore, the null hypothesis, which posited no differences in microhardness, flexural strength, and surface roughness between slaand dlp-printed nanoceramic resin bars even after artificial aging, was rejected. conclusion to conclude, sla printed resins are preferable for strength, but their roughness value increases with time and their long-term performance needs further study focusing on reducing water sorption and improving resin stability. conflicts of interest the author declares no competing interest. references 1. mechanical and biocompapbility properpes of 3d-printed dental resin reinforced with glass silica and zirconia nanoparpcles: in vitro study. alshamrani a, alhotan a, kelly e, ellakwa a polymers 2023; 15:2523. 2. an overview of modern metal additive manufacturing technology. armstrong m, mehrabi h, naveed n. j manuf process 2022; 84:10011029. 3. additive manufacturing by digital light processing: a review. chaudhary r, fabbri p, leoni e, mazzanti f, akbari r, antonini c. prog addit manuf 2023; 8:331-351. 4. lithium disilicate and zirconia reinforced lithium silicate glassceramics for cad/cam dental restorations: biocompatibility, mechanical and microstructural properties after crystallization. mavriqi l, valente f, murmura g, sinjari b, macrì m, trubiani o, et al. j dent 2022; 119:104054. 5. investigation of the influence of gastric acid on the surface roughness of ceramic materials of metal-ceramic restorations. an in vitro study. matsou e, vouroutzis n, kontonasaki e, paraskevopoulos km, koidis p. int j prosthodont 2011; 24:269. 6. gastric acid challenge of lithium disilicate–reinforced glass– ceramics and zirconia-reinforced lithium silicate glass–ceramic after polishing and glazing— impact on surface properties. hjerppe j, shahramian k, rosqvist e, lassila lvj, peltonen j, närhi to. clin oral investig 2023; 27:6865–6877. 7. effect of simulated gastric acid exposure on the hardness, topographic, and colorimetric properties of machinable and pressable zirconia-reinforced lithium silicate glass-ceramics. alhotan a, alaqeely r, al-johani h, alrobaish s, albaiz s. j prosthet dent 2024; 132:625. e1-625.e7. 8. flexural strength, fatigue behavior, and microhardness of three-dimensional (3d)printed resin material for indirect restorations: a systematic review. abadcoronel c, durán urdiales d, benalcázar arias mv, córdova ak, medina ms, et al. materials 2025; 18:556. 9. additive manufacturing: a comprehensive review. zhou l, miller j, vezza j, mayster m, raffay m, justice q, et al. sensors 2024; 24: 2668. the impact of thermocycling on the physiomechanical proper6es of sla and dlp 3d-printed dental ceramics: a compara6ve study vol 13 no 1 (2025) doi 10.5195/d3000.2025.866 h#p://den*stry3000.pi#.edu 10. evaluating oxygen shielding effect using glycerin or vacuum with varying temperature on 3d printed photopolymer in post-polymerization. lim jh, lee sy, gu h, jin g, kim je. j mech behave biomed mater 2022; 130:105170. 11. 3d printing in dentistry. jambhule s, palandurkar m, shewale a. int j adv res 2022; 10:742-750. 12. the effect of build angle and artificial aging on the accuracy of slaand dlp-printed occlusal devices. saadat sb, schmidt f, beuer f, metin ds, simeon p, nicic r, et al. polymers 2024; 16:1714. 13. effect of 3d printing system and post-curing atmosphere on microand nano-wear of additive-manufactured occlusal splint materials. wada j, wada k, garoushi s, shinya a, wakabayashi n, iwamoto t, et al. j mech behav biomed mater 2023; 142:105799. 14. iso/astm 52900: additive manufacturing — general principles — fundamentals and vocabulary. 2nd ed. international organization for standardization, 2021. 15. hibbeler rc. mechanics of materials. 8th ed. pearson prentice hall, boston, usa; 2010. 16. additive manufacturing technologies used for processing polymers: current status and potential application in prosthetic dentistry. revillaleón m, özcan m. j. prosthodont 2019; 28:146-158. 17. material anisotropy in additively manufactured polymers and polymer composites: a review. zohdi n, yang r. polymers 2021; 13:3368. 18. fracture load of 3d-printed fixed dental prostheses compared with milled and conventionally fabricated ones: the impact of resin material, build direction, post-curing, and artificial aging: an in vitro study. reymus m, fabritius r, keßler a, hickel r, edelhoff d, stawarczyk b. clin oral investig 2020; 24:701-710. 19. strength and surface properties of a 3d-printed denture base polymer. gad mm, fouda sm, abualsaud r, alshahrani fa, althobity am, khan sq, et al. j prosthodont 2022; 31:412-418. 20. effect of post-curing light exposure time on the physico– mechanical properties and cytotoxicity of 3d-printed denture base material. aati s, akram z, shrestha b, patel j, shih b, shearston k, et al. dent mater 2021; 38:57-67. 21. evaluating and comparing flexure strength of dental models printed using fused deposition modelling, digital light processing, and stereolithography apparatus printers. atwal n, bhatnagar d. cureus 2024; 16:54312. 22. factors affecting flexural strength of 3d-printed resins: a systematic review. gad mm, fouda sm. j. prosthodont 2023; 32 supp 1:96-110. 23. porosity, water sorption and solubility of denture base acrylic resins polymerized conventionally or in microwave. figuerôa rms, conterno b, arrais cag, sugio cyc, urban vm, neppelenbroek kh. j appl oral sci 2018; 26: e20170383. 24. the impact of fabrication techniques and materials used on the surface integrity of ceramic dental crowns. salman za, hasan nh, abdulmajeed aa. nat j clin orthop 2025; 9:10-14. 25. comparative evaluation of surface roughness and hardness of 3d printed resins. al-dulaijan ya, alsulaimi l, alotaibi r, alboainain a, alalawi h, alshehri s, et al. materials (basel) 2022; 15:6822. 1052 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1052 http://dentistry3000.pitt.edu tea tree oil addition on surface roughness and strength of high-impact acrylic resin material mohammed hameed hassan, aseel mohammed al-khafaji college of den*stry, university of baghdad, baghdad, iraq abstract objec7ve: polymethylmethacrylate (pmma) is porous and rough in nature; this allows food to sock to it and microbes to proliferate, maybe resulong in oral disease. selecong the right denture cleanser is not enough to prevent all fungi from adhering to the denture or decrease roughness. the essenoal tea tree oil (tto) possesses anofungal, anooxidant, and anobacterial characterisocs. tto will be used to study its effect on the surface roughness and impact strength of high-impact acrylic denture material aver incorporaoon with tto at concentraoons of 0%, 6%, and 9% by volume. materials and methods: 60 specimens were made and categorized into two groups. each group consisted of 30 samples and was divided into three different concentraoons of tto (0%, 6%, and 9% by volume). aver 48 h incubaoon in disolled water, all samples were assessed by two tests, the surface roughness test and the impact strength test. atomic force microscopy (afm) was used to prove the effect of tto on the surface morphology at the nanoscale of high-impact acrylic. results: tea tree oil had a staosocally significant effect on surface roughness, which led to a decrease in surface roughness of high-impact acrylic. also, tto has a significant effect on the impact strength of high-impact acrylic at a 9% concentraoon, which causes a decrease in impact strength (p>0.05), but 6% tto has a non-significant effect on high-impact acrylic (p<0.05). that means 6% tto is the be_er concentraoon to use to improve the property of acrylic. conclusion: the high-impact acrylic resin incorporated with the tea tree oil is effecove. it appeared that 6% vol tto will decrease the surface roughness and not significantly affect the impact strength of high-impact acrylic. open access cita%on: hassan mh, et al. (2025) tea tree oil addi%on on surface roughness and strength of high-impact acrylic resin material. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1052 received: september 19, 2025 accepted: september 24, 2025 published: october 17, 2025 copyright: ©2025 hassan mh, et al. this is an open access ar%cle licensed under a crea%ve commons arribu%on work 4.0 united states license. email: mohammed.hasan2401m@codental.uobaghdad.edu.iq introduc)on polymethyl methacrylate (pmma) was the most widely utilized material for denture resin due to its aesthetically pleasing appearance, minimal water absorption, low toxicity, and excellent color stability [1]. pmma is porous and rough in nature; this allows food accumulation, discoloration of acrylic, compromised oral hygiene, reduced long-term success of prosthetic appliances, and the growth of microorganisms, which increases the risk of developing denture stomatitis, which is mostly caused by candida albicans and can result in candidiasis. therefore, it is essential to clean dentures after meals [2,3]. also, the polymethyl methacrylate remained weak and unable of withstand the forces applied during chewing, and broken dentures are the most frequent complaint from denture users that dentists hear. these can be brought on by inadvertent prosthesis prognosis-induced overload oral stresses or due to the denture base prosthesis becoming excessively rigid with time because of chronic failure induced by recurrent bite forces [4]. research indicates that 67% of dentures suffer from degeneration after a few years of makeup [5]. to prevent fractures, high-impact pmma was developed [6]. particles of butadiene-styrene rubber are mixed with the powdered denture foundation components. the rubber is grafted with a methacrylate group to provide a covalent connection between the particles and the polymer network. pmma with a high impact was developed to resist fractures. the addition of a rubber phase as a butadiene-styrene copolymer has led to elevated costs and diminished transverse strength [7,8]. some oils that are extracted from medicinal plants are utilized in biomaterials as a natural substitute that improves the properties of acrylic and has strong antifungal and antibacterial qualities. according to recent studies, plant oils with strong antifungal properties hold promise as tea tree oil addioon on surface roughness and strength of high-impact acrylic resin material vol 13, no 1 (2025) doi 10.5195/d3000.2025.1052 http://dentistry3000.pitt.edu 2 a treatment for stomatitis brought on by dentures [9,10]. in addition to their economic worth, the primary beneuits of utilizing natural plant extracts include safety, biocompatibility, and the absence of unwanted effects [2]. research is being done on the therapeutic use of essential oils. tea tree oil (tto) is utilized in pmma because it has antibacterial, antifungal, and antioxidant properties [11,12]. the leaves of melaleuca alternifolia, a plant from the area in australia that produced tea tree oil (tto), may be steam-distilled to obtain the oil. tto was composed of several compounds, primarily hydrocarbons of monoterpene and sesquiterpene and their alcohols. tto possessed antiseptic and antibacterial properties, according to multiple studies [13]. the current study aimed to investigate the surface roughness and impact strength of high-impact acrylic's characteristics following its incorporation with tto. materials and methods specimen grouping in this study, 60 specimens of the high-impact pmma (veracril® / opti-cryl high impact, newstetic, colombia) were made; the concentration of tto that is incorporated with high-impact heat-cure acrylic resin is 6 wt.% and 9 wt.% by volume, and the specimens will be created. a total of specimens of high-impact acrylic were carried out and categorized into two sets. each set includes 30 specimens spread among three different groups. (group one: control; group two: 6% tto; and group three: 9% tto), including 10 specimens per group. two tests are used in this study, including the impact strength test and the surface roughness test. before the testing procedures, every specimen was incubated at 37°c for 48 hours after being stored in distilled water [14]. the uirst control group was preserved in distilled water with 0% tto. the second group was the incorporation of high-impact acrylic with a 6% tto. the third group was the incorporation of high-impact acrylic with a 9% tto. a total of 60 specimens were used in the study. the measurement and mixing of high-impact pmma components the preparation was performed for each percentage (0%, 6%, and 9%) of tto by volume (ml) to be able to withdraw with a micropipette, while the powder of high-impact acrylic was calculated by using a digital balance of 0.000 grams. tto was added to the monomer of high-impact acrylic. the proportion of polymer to monomer (powder-toliquid ratio of (2:1) by weight) p|l [15]. general test specimen preparation for surface roughness a carving machine was used to create a round stainless-steel mold and a cover for it. according to iso 20795-1, 2013, the specimen's thickness was 0.5 mm ± 0.1 mm, and its diameter was 50 mm ± 1 mm [16]. in both halves of the ulask, the steel mold and lid were immersed in stone, as indicated in figure 1. following the investment of the stainless-steel mold and cover, the two components that made up the ulask were then separated. the testing was conducted using a prouilometer instrument, and the specimen was positioned on a level surface as shown in figure 2. three measurements were obtained for each specimen: one at the center and two equally from the center towards the boundaries on both sides. the average ra values were computed in micrometers (μm). figure 1. the steel mold and lid were immersed in stone inside ulask. figure 2. a profilometer instrument to measure the surface the roughness of specimen. for the test of impact strength the designs were made using dimensions of 80 mm in length, 10 mm in width, and 4 mm in thickness in accordance with iso 179 [17]. according to the manufacturer's recommendations (water/powder ratio of 25 ml/100 g), the type 4 dental stone (zhermack®, italy) was prepared and put into the ulask's lower section after it had been painted with separating media (izo-sol, zhermack, italy). the mold and plastic design were then inserted into the stone and left to harden. following the stone setting, the whole surface was painted with a separating substance, including the stone and plastic pattern. following that, the ulask's top half was placed over its bottom half, uilled with the new stone mixture, covered, and left to solidify. therefore, the two halves of the ulask were taken apart, and the designs were taken out, as shown in figure 3. the specimens were then constructed, uinished, and polished as shown in figure 4. figure 3. flask and plastic mold for impact strength test. figure 4. finishing and polishing of impact strength specimen. the charpy impact testing apparatus was utilized to perform the impact strength assessment, as shown in figure 5. the charpy impact strength of unnotched specimens was obtained using the following formula: impact strength = e/b.d x 103 [18]. e is the energy absorbed for fracture measured in joules. b represents the samples' width in millimeters. d represents the thickness of the samples in millimeters. tea tree oil addioon on surface roughness and strength of high-impact acrylic resin material vol 13, no 1 (2025) doi 10.5195/d3000.2025.1052 http://dentistry3000.pitt.edu 3 the specimen was positioned vertically and impacted by a freely swinging pendulum of 5 joules, with the scale reading indicating the impact energy in joules. figure 5. specimen of impact test and charpy impact testing machine. atomic force microscope (afm) atomic force microscopy (afm) is a nanoscale imaging technology that offers three-dimensional surface characterization with sub-nanometer resolution. it is extensively utilized in polymer research to assess morphological alterations, surface roughness, and nanomechanical characteristics following the modiuication or incorporation of plasticizers, uillers, or essential oils [19]. afm employs a sharp probe afuixed to a ulexible cantilever to examine a surface. intermolecular interactions between the tip and the surface induce deulections in the cantilever, which are documented to generate highresolution maps (figure 6). figure 6. atomic force microscope device. statistical analysis individual surface roughness and impact strength were calculated and compiled to determine the average and standard deviation for every category; ibm spss version 20 was employed. tukey hsd and one-way anova were used to compare the outcomes between the groups. p < 0.05 was set as the signiuicant level. results the group control demonstrated the greatest mean value (0.1431) upon examination of the pigmented specimens from each speciuic group. the experimental groups came after that, with group 9% showing the minimal average value (0.0851). all the specimens utilized in the experiment showed a statistically signiuicant decrease in surface roughness, according to the uindings shown in table 1. tables 2 to 6 summarize results for all comparisons. atomic force microscope (afm) the afm results show the mean surface roughness (sa) of the control group is 0.03308 µm (figure 7), while the surface roughness after the addition of tto at 6% is 0.024834 µm (figure 8) and at 9% is 0.022768 µm (figure 9). the incorporation of tto into the high-impact acrylic results in a reduction in the surface roughness, as conuirmed by the afm images. a roughness and irregular topography were observed in the control sample; however, the addition of 6% v/v and 9% v/v tto resulted in a reduction in the surface irregularities. revealed that there was a change in the surface morphology. figure 7. the results of the afm surface topography analysis for the high-impact acrylic resin. figure 8. afm surface topography results for high-impact acrylic resin with 6% v/v tto. figure 9. afm surface topography results for high-impact acrylic resin with 9% v/v tto. discussion the oral cavity experiences many alterations when utilizing an artiuicial prosthesis, potentially resulting in microbial colonization. individuals who wear dentures are at an elevated risk of getting denture stomatitis, largely characterized by the presence of candida albicans, which may result in the onset of candidiasis. this results from the roughness of the denture due to the nature of acrylic, leading to the adherence of microbes [2,20]. natural tea tree oil signiuicantly decreased surface roughness when administered to acrylic resin, so the incorporation of tto (6%, 9%) by volume into the high-impact acrylic in the experiment groups in comparison to the control groups. to has strong antioxidant and antimicrobial properties, in which the terpinen-4-ol has signiuicant effects on the growth and function of candida cells (noumi emira), which may change the permeability and membrane characteristics of fungal cells [21]. the impact strength values for the incorporation of 6% tto have no signiuicant difference from the control (p > 0.05), whereas the incorporation of 9% tto has a signiuicant effect (p < 0.05) and decreases impact strength; therefore, 6% tto is the better concentration to have been used because it has no effect on impact strength. these results may be attributed to the concentration of supplementary oil, which functions as an elastomer for high-impact pmma acrylic resin [22,23]. the incorporation of oil elastomer enhanced the material's energy absorption and reduced the likelihood of resin cracking, hence increasing the prosthetic device's resistance to mechanical failure [24]. conclusion this study was performed to examine the surface roughness and impact strength of high-impact acrylic after incorporation with 6% and 9% tto. the 6% and 9% of the oil group that incorporated with high-impact acrylic lead to a decrease in surface roughness of high-impact acrylic and a non-significant effect on impact strength with 6% tto, but with 9% tto it causes a decrease in the impact strength; therefore, the recommended concentration of tea tree oil is 6% tto. conflict of interest none. references tea tree oil addioon on surface roughness and strength of high-impact acrylic resin material vol 13, no 1 (2025) doi 10.5195/d3000.2025.1052 http://dentistry3000.pitt.edu 4 1. noori zs, al-khafaji am, dabaghi f. effect of tea tree oil on candida adherence and surface roughness of heat-cure acrylic resin. journal of baghdad college of denestry. 2023 dec 15;35(4):46-54. 2. hammed ss, al-khafaji am. tea tree oil effect on dimensional change and detail reproduceon of addieon silicon impression material. journal of denestry 3000.2024. 3. pudhupalayam muthukuy g, singh mk, palaniappan sk, vijayananth k, boonyasopon p, mavinkere rangappa s, siengchin s. sustainable polymer composites from agro and municipal green wastes: a comprehensive review of materials, properees, and applicaeons. journal of material cycles and waste management. 2025 jul 15:1-22. 4. al-hiloh sa, ismail ij. a study of the effect of the addieon of silanized zirconium oxide nanoparecles on some properees of high-impact heat-cured acrylic resin. journal of baghdad college of denestry. 2016;28(2):19-25. 5. solhi l, atai m, nodehi a, imani m, ghaemi a, khosravi k. poly(acrylic acid)graded montmorillonite as novel fillers for dental adhesives: synthesis, characterizaeon, and properees of the adhesive. dental materials. 2012 apr 1;28(4):369-77. 6. qanber lm, hamad ti. effect of plasma treatment on the bond of sod denture liner to conveneonal and highimpact acrylic denture materials. journal of baghdad college of denestry. 2021 sep 15;33(3):9-17. 7. meng tr, laha ma. physical properees of four acrylic denture base resins. the journal of contemporary dental pracece. 2007 mar 1;6(4):93-100. 8. craig rg, o'brien wj, powers jm. dental materials: properees and manipulaeon. (no title). 2004. 9. yahya yk, al-khafaji am. the impact of tea tree oil on bacillus subelis and the surface roughness of type iii dental stones. journal of internaeonal dental and medical research. 2024 may 1;17(2):498506. 10. al-mashhadane fa. tea tree oil: a new anefungal against candida albicans cells on heat-cured acrylic resin denture base material. an in vitro study. al-rafidain dental journal. 2007 dec 1;7(3):54-7. 11. noori zs, al-khafaji am. evaluaeon of the effect of tea tree oil denture cleanser on the properees of dental polymers. journal of engineering. 2024 nov 1;30(11):39-49. 12. emira noumi, ms. in vitro effect of melaleuca alternifolia and eucalyptus globulus esseneal oils on mycelia formaeon by oral candida albicans strains. 13. mondello f, de bernardis f, girolamo a, salvatore g, cassone a. in vitro and in vivo acevity of tea tree oil against azole-suscepeble and -resistant human pathogenic yeasts. journal of anemicrobial chemotherapy. 2003 may 1;51(5):1223-9. 14. salman ta, khalaf ha. the influence of adding modified zro2-tio2 nanoparecles on certain physical and mechanical properees of heat-polymerized acrylic resin. journal of baghdad college of denestry. 2015;27(3):33-9. 15. hussain wa, hashim fs. effect of addieves on impact strength of denture base resin. iraqi journal of science. 2017:860-7. 16. al-khafagi ks, mahmood w. evaluaeon of the transverse strength and surface roughness properees of high-impact polymethylmethacrylate ader long-term submergence in clove oil solueon. advanced journal of chemistry" (2025): 278-291. 17. mawlood zs, naji ga. influence of addieon of bergamot esseneal oil on physico-mechanical behavior of heat-cured acrylic denture base. int med j. 2021 jun 2;28(1):21-5. 18. nimer am, jassim rk. studying the effect of ascorbic acid on some properees of autoclaved and heat-cured denture base material. 19. liang x. visualizaeon of nanomechanical properees of polymer composites using atomic force microscopy. polymer journal. 2023 sep;55(9):913-20 20. dhir g. physical properees of denture base resins resistant to candidiasis. 21. emira n, mejdi s, aouni m. in vitro acevity of melaleuca alternifolia (tea tree) and eucalyptus globulus esseneal oils on oral candida biofilm formaeon on polymethylmethacrylate. j. med. plants res. 2013 jun 12; 7 (20): 1461-6. 22. faot f, costa ma, cury aa, garcia rc. impact strength and fracture morphology of denture acrylic resins. the journal of prostheec denestry. 2006 nov 1;96(5):36773. 23. alarifi, im. a comprehensive review on advancements of elastomers for engineering applicaeons. advanced industrial and engineering polymer research. 2023 oct 1;6(4):451-64. 24. al-badr rj, al-huwaizi hf. effect of tea tree, thymus vulgaris, and nigella saeva oils on the eliminaeon of enterococcus faecalis (in vitro study). journal of baghdad college of denestry. 2017 mar 13;29(1):5562. tea tree oil addioon on surface roughness and strength of high-impact acrylic resin material vol 13, no 1 (2025) doi 10.5195/d3000.2025.1052 http://dentistry3000.pitt.edu 5 table 1. descriptive statistics for test of surface roughness. sample n mean standard deviation standard error minimum maximum control 10 0.1431 0.004841 0.001531 0.136 0.149 6% tea tree oil 10 0.1033 0.003713 0.001174 0.097 0.108 9% tea tree oil 10 0.0851 0.002807 0.000888 0.081 0.089 total 30 0.1105 0.024916 0.004549 0.081 0.149 table 2. anova analysis of surface roughness test. sample sum of squares degrees of freedom mean square f p-value between groups 0.018 2 0.009 585.286 0.000 within groups 0.0 27 0.0 total 0.018 29 table 3. comparative analysis of surface roughness among groups utilizing tukey hsd. (i) (j) (i-j) mean difference standard error p-value control 6% tto 0.398 0.001734 0.000 9% tto 0.058 0.001734 0.000 6% tto 9% tto -0.0182 0.001734 0.000 tea tree oil addioon on surface roughness and strength of high-impact acrylic resin material vol 13, no 1 (2025) doi 10.5195/d3000.2025.1052 http://dentistry3000.pitt.edu 6 table 4. descriptive statistics for the impact strength. sample n mean standard deviation standard error minimum maximum control 10 13.925 0.23717 0.075 13.5 14.25 6% tea tree oil 10 13.8 0.2582 0.08165 13.5 14.25 9% tea tree oil 10 13.375 0.3385 0.10704 13 13.75 table 5. anova test for impact strength test. sample sum of squares degrees of freedom mean square f p-value between groups 1.663 2 0.831 10.5 0.000 within groups 2.138 27 0.079 total 3.8 29 table 6. multiple comparisons of impact strength between groups using tukey hsd. a multiple comparison test in table 6 showed a statistically signiuicant difference between control and 9% tto, and 6% and 9% tto (p < 0.05), while between control and 6% tto there was a non-signiuicant difference (p > 0.05), which explains why the 6% had no effect on the impact strength of high-impact acrylic. (i)group (j)group mean difference(i-j) standard error p-value control 6% tto 0.125 0.12583 0.587 9% tto 0.55 0.12583 0.000 6% tto 9% tto -0.425 0.12583 0.006 1063 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1063 http://dentistry3000.pitt.edu survival of immediate-loaded pterygoid implants with multi-unit abutments in the atrophic posterior maxilla mohamad droubi, mazen zena2 faculty of den.stry, damascus university, damascus, syria abstract objec2ve: rehabilitacon of the atrophic posterior maxilla is challenging due to insufficient bone quancty and quality. convenconal approaches, such as sinus floor elevacon or onlay bone grasing, are associated with higher morbidity, longer treatment cmes, and increased complicacons. pterygoid implants offer a less invasive alternacve, uclizing dense corccal bone in the pterygoid apophysis and posterior maxillary tuberosity to provide distal anchorage and full-arch prosthecc support, while avoiding the need for sinus augmentacon. clinical evidence indicates high survival rates, making them a reliable opcon for posterior maxillary rehabilitacon. materials and methods: this prospeccve clinical study included 15 pacents (aged 49–78 years) with unilateral posterior maxillary tooth loss that was unsuitable for convenconal implants. each pacent received one pterygoid implant and two compressive implants. preoperacve planning included cbct scans. surgical procedures involved flap elevacon, precise drilling, and manual insercon with high insercon torque. immediate prosthecc loading was performed within seven days. postoperacve care included ancbioccs, analgesics, chlorhexidine mouthwash, and oral hygiene instruccons. implant failure was defined as detectable mobility or condicons requiring removal. results: all 45 implants (15 pterygoid and 30 compressive) survived over the 12-month follow-up, with no failures observed at 3, 6, or 12 months. survival rates were 100% for both implant types, and no differences were noted across follow-up periods. conclusions: pterygoid implants demonstrate high survival rates and represent a predictable, minimally invasive opcon for posterior maxillary rehabilitacon. proper preoperacve planning and surgical expercse are essencal, and further longterm studies are recommended to confirm standardized protocols and durability. open access cita%on: droubi m, et al. (2025) survival of immediateloaded pterygoid implants with mul%-unit abutments in the atrophic posterior maxilla. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1063 received: october 3, 2025 accepted: october 15, 2025 published: november 4, 2025 copyright: ©2025 droubi m, et al. this is an open access ar%cle licensed under a crea%ve commons avribu%on work 4.0 united states license. email: mohamad.salah.droubi@gmail.com introduc)on rehabilitation of the atrophic posterior maxilla remains one of the most challenging cases of implant dentistry due to the amount and quality of bone that is missing in this location [1,2]. conventional treatment strategies, such as onlay bone grafting or elevation of the sinus aloor, have been widely utilized to circumvent these anatomical deaicits. though these methods have been able to provide adequate bone volume to accommodate implant placement, they are typically associated with increased surgical morbidity, higher treatment cost, longer treatment time, and a high incidence of intraand postoperative complications, including sinus membrane perforation and graft resorption. such drawbacks have prompted the development of alternative treatments that can bypass the utilization of complex grafting protocols [3]. among such alternatives, the use of extramaxillary anchorage, particularly zygomatic implants, has been the subject of signiaicant interest. zygomatic implants have the advantage of involving the engagement of the zygomatic bone, thereby offering stability in the presence of severe maxillary resorption [4,5]. however, the procedure is technically demanding, involves speciaic training, specialized equipment, and has increased rates of complications such as sinusitis, paresthesia, or oroantral communication. due to these limitations, pterygoid implants are a valuable and less invasive choice for posterior maxillary rehabilitation [6,7]. pterygoid implants, airst introduced some decades back, take advantage of the dense cortical bone of the pterygoid apophysis and posterior maxillary tuberosity. their anatomical location provides space to place long implants that can provide excellent posterior survival of immediate-loaded pterygoid implants with mulc-unit abutments in the atrophic posterior maxilla vol 13, no 1 (2025) doi 10.5195/d3000.2025.1063 http://dentistry3000.pitt.edu 2 support, skipping sinus augmentation procedures altogether [8,9]. moreover, they provide for distal anchorage that allows for fullarch prosthetic rehabilitation even when severely atrophic maxillae are present. this singular biomechanical feature shortens cantilever lengths in prostheses and improves load distribution, possibly leading to better long-term results [10]. clinical outcomes and survival rates of pterygoid implants have been examined by an increasing volume of literature over the last decade. araujo et al. (2019), in arguably the most comprehensive systematic review to date, had a survival rate of 94.9% from pooled estimates of different clinical studies and demonstrated the stability of this treatment modality. similarly, bidra (2023) was at 95.5%, highlighting that pterygoid implants are capable of providing outcomes comparable with conventional implants placed in more favorable anatomical locations. these results further support their role as a trustworthy treatment option [3,6]. later studies have conairmed these. d'amario et al. (2024) and raouf and chrcanovic (2024) provided meta-analytic evidence of 97% to 99% survival, further supporting the role of pterygoid implants as a high-survival treatment. their evaluations also revealed that survival outcomes are similar across different follow-up periods, though with slight discrepancies due to differences in study design, experience of operators, and patient selection criteria [10,11]. on the other hand, future clinical evidence remains somewhat restricted. the early results, as noted by mirdah et al. (2025), were good in terms of early survival rates but also highlighted that anatomical challenge, bone quality, and factors related to the patient— oral hygiene compliance and health in general—still play a major role in long-term predictability. this means that while pterygoid implants are predictable, they must be carefully planned preoperatively, and technically skilled surgical capability must be employed in order to attain the best results [12]. collectively, the literature has shown pterygoid implants to be a reliable, minimally invasive option for sinus augmentation procedures during the rehabilitation of the atrophic posterior maxilla. their survival rate is consistently reported to be greater than 94%, with many of these studies attaining or surpassing 97–99% [10,11]. these aindings compare well with conventional implant therapy and highlight the potential of pterygoid implants to reduce treatment complexity, morbidity, and overall rehabilitation time intervals. nonetheless, disparity in success reported highlights the need for discerning patient selection, strict attention to particular surgical protocols, and followup over the long term. subsequent investigations with a focus on standardized surgical techniques, multicenter randomized controlled trials, and extended follow-up periods will be signiaicant to establish conclusive results regarding their predictability and late outcome. this study aimed to evaluate the survival rates of pterygoid implants after immediate loading of single-piece pterygoid implants and multi-unit abutments in the posterior region of the atrophic maxilla. materials and methods the study was designed as a prospective, single-group, interventional clinical study. the research sample consisted of 15 patients, representing 15 cases, each with three implants: one pterygoid implant and two compressive implants. the approval of the ethics committee at damascus university was obtained under the number (dn-15042025-h25), and the study was registered in the (isrctn-biomedcentral) database with the identiaier (isrctn77752182) and the link (https://www.isrctn.com/isrctn7775218 2). the research sample was selected from patients attending the oral and maxillofacial surgery clinic at damascus university. these patients, aged between 49 and 78 years, had unilateral maxillary posterior dentition loss and were found to be unsuitable for conventional implant placement. the study was conducted in the dental implant clinic within the department of oral and maxillofacial surgery at the faculty of dentistry at damascus university between 2022 and 2025. after informing the patients of the purpose and nature of the study in written and verbal form, ensuring their understanding and answering their questions, written informed consent was obtained from the patients participating in the study. for the inclusion criteria, the patient had to have good oral health and a history of tooth loss in the premolar and molar regions of the maxilla. the height of the alveolar ridge between the crest of the alveolar bone and the aloor of the maxillary sinus should be less than 4 mm, with a vestibular bone width in the premolar region greater than 4 mm. the inferior angle of the anterior wall of the maxillary sinus must be anterior to the second premolar. additionally, the last extraction in the included area must have occurred more than two months ago, and there should be sufaicient occlusal space for prostheses. the patient's consent was obtained after a full explanation of the research procedures. for the exclusion criteria, patients with any metabolic diseases affecting normal bone metabolism, such as hyperparathyroidism or osteoporosis were not included. patients with bruxism should be excluded. also, no patients on medications that caused bone metabolism disorders, such as corticosteroids, hormonal treatments, or chemotherapy, and patients receiving radiation therapy to the face or neck were not included. finally, patients that had any contraindications to implants due to systemic diseases such as leukemia or coagulation disorders were not included. a cbct scan was performed before beginning the surgery. this was to verify measurements and dimensions, ensuring accurate planning of the surgical procedure. the mouth was disinfected using a 0.12% chlorhexidine rinse, and the perioral skin was cleaned with a polyvidone iodine solution. local inailtration anesthesia was administered using lidocaine 2% with adrenaline (1:80,000) in the premolar and maxillary canine regions, where the compressive implant would be placed. nerve block anesthesia was then performed using lidocaine 2% with adrenaline (1:80,000) in the tuberosity region, where the pterygoid implant would be located. a full-thickness vestibular mucoperiosteal envelope alap was created to minimize healing time. the alap was carefully elevated while preserving the periosteum, ensuring an optimal healing process. after exposing the alveolar bone, the implants’ sites were prepared according to the protocol followed in this study: two compressive implants. roott m implants ranged in length from 8-12 mm and diameters from 3.5-4 mm, and roott p implants were placed in the posterior maxillary sinus area with the implant tilted at an angle ranging from 1545°. implant diameters range from 3.5 to 4.5 mm and lengths from 18-20 mm. for compressive implants, the initial drill was used to drill the cortical bone. then, we moved on to a pilot drill with a diameter of 2 mm. this completed the preparation for implants with a diameter less than 4 mm. for implants with a diameter of 4 mm, we prepared a second drill with a diameter of 2.5 mm. the implant was then inserted manually with the implant insertion tool, ensuring that the insertion torque is 35 n cm. for pterygoid implants, we began with a hand drill to determine the desired angle of drilling. we then moved on to a 2 mm pilot drill, completing the preparation for 3.5 mm implants. for 4.5 mm implants, we prepared a second 2.5 mm drill, the implant was then inserted using an implant insertion tool, ensuring that the insertion torque is greater than 35 n cm. after the three implants were inserted with an insertion torque greater than 35 n cm, the gingival alap was then repositioned and survival of immediate-loaded pterygoid implants with mulc-unit abutments in the atrophic posterior maxilla vol 13, no 1 (2025) doi 10.5195/d3000.2025.1063 http://dentistry3000.pitt.edu 3 sutured around the abutments. the implant transfer was then placed, and impressions were taken using rubber. the impressions were sent to the lab for prosthetics (ceramic on metal). the healing abutments were then placed on the multi-unit abutments. the patient was advised to avoid rinsing the mouth on the airst day following surgery. cold compresses could be applied to the cheek on the surgical site immediately after the procedure, alternating every 4 hours for the airst few hours. an analgesic might be taken if necessary to manage pain. oral hygiene procedures were suggested to begin the day after surgery. the patient was prescribed the following medications: amoxicillin 875 mg + clavulanic acid 125 mg every 12 hours, and diclofenac potassium 50 mg as needed for pain, not to exceed 150 mg per day. additionally, a 0.12% chlorhexidine mouthwash should be used twice daily (for 60 seconds) for 7 days, starting the day after surgery. after completing suturing, the implant abutments were placed, and an impression was immediately taken using a condensation silicone material in a single stage. the following day, a veriaication jig was prepared by attaching the implant abutments to the stone model using pattern resin, and the jig was tested in the patient's mouth to verify the accuracy of the impression. next, the metal framework for the restoration was designed and 3d-printed using laser technology. the metal framework was then tried in to ensure it aits accurately onto the abutments and the gingiva. the bridge was then veneered without glazing and tried again to adjust the occlusion, ensuring that each tooth has 3 to 4 contact points without any premature contacts, and that the restoration was in proper contact with the gingiva. finally, the restoration was glazed and aixed with screws tightened to 15 n cm, according to the manufacturer's instructions. the screw holes were sealed with composite material, and a tealon barrier was placed. all prosthetic steps were completed within seven days of surgery. failure was deained as the presence of any detectable mobility of the implant and/or any condition requiring implant removal. the implants were followed up for one year after immediate loading. results the study sample included 15 patients, with 15 wing implants and 30 compressive implants. the mean age was 60.07 years, with 8 males and 7 females. the frequencies of implant survival and failure were calculated for the studied implants across the three follow-up periods. the results are summarized in table 1. as shown in table 1, no implant failures were observed among the studied implants at any of the follow-up periods. all applied and studied implants remained fully retained throughout the observation period. consequently, there were no differences in implant survival/failure rates across followup periods, and therefore, the application of the wilcoxon test to compare survival/failure between follow-up stages was not warranted. discussion the present study demonstrated a 100% survival rate for pterygoid implants, which is remarkably high compared to most outcomes reported in the literature. over the past decade, numerous clinical studies and systematic reviews have evaluated the longterm performance of pterygoid implants, generally showing favorable but slightly lower success rates. for example, bidra (2023) [6] conducted a systematic review and reported a cumulative survival of 95.5% over six years, emphasizing that pterygoid implants provide a reliable alternative to zygomatic implants in atrophic maxillae. similarly, araujo et al. (2019) [3], in one of the largest systematic reviews including nearly 1,900 implants, found a survival rate of 94.9%. these aindings reinforce the concept that pterygoid implants are a predictable treatment modality, though outcomes vary slightly among different cohorts and followup durations. meta-analytical data also corroborate these results. d’amario et al. (2024) [11] analyzed 1,882 implants across 1,024 patients and reported a survival rate of 97.4%, further supporting the long-term reliability of pterygoid implants. raouf and chrcanovic (2024) [10] conducted a systematic review comparing pterygoid and tuberosity implants, reporting similar survival outcomes between the two techniques, thereby highlighting the clinical versatility of posterior maxillary anchorage solutions. retrospective series, such as the one by curi et al. (2015) [13], found 99% survival over three years, suggesting that these implants consistently yield excellent results across different populations and clinical settings. on the other hand, prospective studies sometimes demonstrate slightly lower survival rates. for instance, mirdah et al. (2025) [12] reported 88.6% survival at one year, noting that early failures were often related to anatomical limitations and bone quality rather than prosthetic complications. these aindings emphasize the importance of case selection and meticulous surgical planning, as the anatomical complexity of the pterygoid region can inaluence outcomes. similarly, signorini et al. (2021) [14] reported favorable survival rates in a short-term follow-up, but highlighted the need for longer longitudinal studies to fully assess long-term predictability. when comparing these aindings to the present study, the observed 100% survival rate indicates that under controlled clinical conditions, with proper patient selection, careful preoperative planning, and precise surgical execution, pterygoid implants can achieve survival rates equal to or even higher than those previously reported. differences in outcomes may be attributed to multiple factors, including study design (retrospective versus prospective), sample size, operator experience, and follow-up duration. additionally, the prosthetic protocols adopted— such as immediate loading versus delayed loading—may also play a role in inaluencing success rates. the superior outcomes in the present study may also realect advancements in imaging modalities, such as cone-beam computed tomography (cbct), which enhance preoperative assessment and reduce the risk of surgical complications. furthermore, the use of new implant designs may have contributed to the high survival rate observed. these aindings align with the broader body of literature afairming the predictability of pterygoid implants but suggest that under optimized conditions, survival can approach 100% [7,15]. taken together, these results underscore the clinical reliability of pterygoid implants as a viable option for the rehabilitation of the atrophic posterior maxilla. they not only provide a predictable survival rate but also reduce the need for more invasive procedures such as sinus grafting or zygomatic implant placement. future studies, particularly long-term multicenter prospective trials, are necessary to validate these aindings across diverse patient populations and clinical contexts. conclusion within the limitations of this study, pterygoid implants demonstrated high survival rates and represented a predictable, minimally invasive alternative for posterior maxillary rehabilitation. current evidence supports their reliability, yet further long-term prospective studies are warranted to establish standardized protocols and confirm their long-term clinical performance. funding none. data availability the authors can provide data on demand. compe)ng interests none. survival of immediate-loaded pterygoid implants with mulc-unit abutments in the atrophic posterior maxilla vol 13, no 1 (2025) doi 10.5195/d3000.2025.1063 http://dentistry3000.pitt.edu 4 references 1. candel-martı ́ e, carrillo-garcıá c, peñarrocha-oltra d, peñarrocha-diago m. rehabilitation of atrophic posterior maxilla with zygomatic implants. journal of oral implantology. 2012;38(5):653-7. 2. jimoh o, jan m, luc v, henri d. rehabilitation of atrophic maxilla using pterygoid implants: case reports. mod app dent oral health 1 (1)-2018. madohc ms id. 2015;103. 3. araujo rz, júnior jfs, cardoso cl, condezo afb, júnior rm, curi mm. clinical outcomes of pterygoid implants: systematic review and meta-analysis. journal of craniomaxillofacial surgery. 2019;47(4):651-60. 4. chrcanovic br, abreu mhng. survival and complications of zygomatic implants: a systematic review. oral and maxillofacial surgery. 2013;17(2):81-93. 5. vrielinck l, blok j, politis c. survival of conventional dental implants in the edentulous atrophic maxilla in combination with zygomatic implants: a 20-year retrospective study. international journal of implant dentistry. 2022;8(1):27. 6. bidra as, peña-cardelles jf, iverson m. implants in the pterygoid region: an updated systematic review of modern roughened surface implants. journal of prosthodontics. 2023;32(4):285-91. 7. maryam s, oral p, ayub ms. evaluating the success rate of pterygoid implants in the rehabilitation of the posterior maxilla: a comprehensive review and meta-analysis. health affairs. 2024;12(4). 8. szabó ad l, nagy ad l, lászlófy c, gajdács m, bencsik p, kárpáti k, et al. distally tilted implants according to the all-on-four® treatment concept for the rehabilitation of complete edentulism: a 3.5-year retrospective radiographic study of clinical outcomes and marginal bone level changes. dentistry journal. 2022;10(5):82. 9. toti p, marchionni s, menchini-fabris gb, marconcini s, covani u, barone a. surgical techniques used in the rehabilitation of partially edentulous patients with atrophic posterior mandibles: a systematic review and meta-analysis of randomized controlled clinical trials. journal of cranio-maxillofacial surgery. 2017;45(8):123645. 10. raouf k, chrcanovic br. clinical outcomes of pterygoid and maxillary tuberosity implants: a systematic review. journal of clinical medicine. 2024;13(15):4544. 11. d'amario m, orsijena a, franco r, chiacchia m, jahjah a, capogreco m. clinical achievements of implantology in the pterygoid region: a systematic review and meta-analysis of the literature. journal of stomatology, oral and maxillofacial surgery. 2024;125(5):101951. 12. mirdah wf, goyal r, singh a, singh n, laxmi sk, tarpara kj, et al. clinical outcomes and success factors of pterygoid implants in the posterior atrophic maxilla: a prospective study. cureus. 2025;17(4):e82820. 13. curi mm, cardoso cl, braga ribeiro kdc. retrospective study of pterygoid implants in the atrophic posterior maxilla: implant and prosthesis survival rates up to 3 years. international journal of oral & maxillofacial implants. 2015;30(2). 14. signorini l, faustini f, samarani r, grandi t. immediate lixed rehabilitation supported by pterygoid implants for participants with severe maxillary atrophy: 1-year postloading results from a prospective cohort study. the journal of prosthetic dentistry. 2021;126(1):6775. 15. rastelli s, capogreco m, d'amario m, falisi g, severino m, iacomino e. pterigoid implants: a viable alternative for the rehabilitation of the posterior sectors of the atrophic maxilla. oral & implantology. 2024;16(1). table 1. percentage of implant survival/failure across three follow-up periods. implant outcome implant type 3 months 6 months 12 months 3 months (%) 6 months (%) 12 months (%) survival pterygoid 15 15 15 100% 100% 100% survival conventional 1 15 15 15 100% 100% 100% survival conventional 2 15 15 15 100% 100% 100% failure pterygoid 0 0 0 0% 0% 0% failure conventional 1 0 0 0 0% 0% 0% failure conventional 2 0 0 0 0% 0% 0% total – 45 45 45 100% 100% 100% 749 2024 assessment of oral histology knowledge and its applica8on in clinical orthodon8cs, opera8ve den8stry, oral medicine, and oral surgery vol 12 no 2 (2024) doi 10.5195/d3000.2024.749 1 assessment of oral histology knowledge and its application in clinical orthodontics, operative dentistry, oral medicine, and oral surgery selma merza hasan1, zainab a. almashhadi 2, amjed f. hussein3, heider hemeed abbas 4 1college of dentistry, university of kufa, college of dentistry, ahl al bayt university, iraq. email: drsmerza@gmail.com, orcid: https://orcid.org/0009-0002-6442-5254 2college of dentistry, ahl al bayt university, iraq. email:almashhadizainabaj@gmail.com, orcid: https://orcid.org/0009-0004-5819-6170 3college of dentistry, ahl al bayt university, iraq. email:dr.amjedfouad88@gmail.com, orcid: https://orcid.org/0009-0008-5846-9975 4college of dentistry, babylon university, iraq. email: dent.heider.hemeed@uobabylon.edu.iq, orcid: hqps://orcid.org/0000-0003-1691-2728 abstract objec:ve: this study aims to assess the knowledge of oral histology among fi6h-year dental students in iraq and evaluate its relevance to various clinical dental disciplines, including orthodon>cs, opera>ve den>stry, oral medicine, and oral surgery. methods: a cross-sec>onal survey was conducted from february to may 2024 among dental students from both public and private ins>tu>ons in iraq. par>cipants were recruited through targeted online adver>sements on plahorms such as telegram, whatsapp, and facebook. results: the results compare clinical and basic knowledge between younger and older dental students. younger students scored higher in understanding basic concepts like structural changes in the periodontal ligament, bone remodeling, tooth erup>on enamel structure, cavity design and surgical interven>ons. however, older students excelled in clinical areas such as maxillofacial development, orthodon>c appliances, and developmental anomalies. conclusion: this study provides insights into the perceived value of oral histology educa>on among dental students and its applica>on in clinical prac>ce. the findings may be encouraging early clinical involvement while reinforcing basic science concepts can lead to a more well-rounded and confident dental workforce. keywords: oral histology, orthodon>cs, opera>ve den>stry, oral medicine, maxillofacial surgery, student ques>onnaire. cita%on: hasan sm, et al. (2024) assessment of oral histology knowledge and its applica%on in clinical orthodon%cs, opera%ve den%stry, oral medicine, and oral surgery. den%stry 3000. 1:a001 doi:10.5195/d3000.2024.749 received: october 5, 2024 accepted: october 21, 2024 published: november 27, 2024 copyright: ©2024 hasan sm, et al. this is an open access ar%cle licensed under a crea%ve commons asribu%on work 4.0 united states license. email:drsmerza@gmail.com introduction oral histology is a fundamental discipline in dentistry, providing a microscopic understanding of oral tissues. it serves as a cornerstone for various specialties, including orthodontics, operative dentistry, oral medicine, and oral surgery. a comprehensive grasp of oral histology is essential for diagnosing, treating, and preventing oral diseases [1,2]. understanding oral histology is crucial for dental students to comprehend the pathophysiology of oral diseases [3]. instead of rote memorization, theoretical lectures and clinical application help students to understand histologic specimens. then, knowledge of oral histology can help dental practitioners to manage patients with oral diseases in the clinic [4,5]. dental education program has provided valuable suggestions and strategies for the integration of histology in the dental curriculum and for teaching the subject in a way that better integrates preclinical education in the clinical years [6]. some available studies have reported strong correlations between histology students' performance in clinical discipline learning [1]. the competence of dental students in their professional tasks after graduation depends on the acquisition of assessment of oral histology knowledge and its applica8on in clinical orthodon8cs, opera8ve den8stry, oral medicine, and oral surgery vol 12 no 2 (2024) doi 10.5195/d3000.2024.749 2 knowledge and clinical skills needed to deliver evidence-based dental treatment [7]. preclinical basic medical information is important for dental students to become qualified dentists [8]. oral histology is integral to various dental specialties, providing essential insights into tissue structure and function that directly impact clinical practice [9]. in orthodontics, tooth movement relies on the remodeling of alveolar bone and the periodontal ligament, with histological changes in these tissues playing a key role in understanding the effects of orthodontic forces, managing root resorption, and predicting soft tissue responses [10,11]. in operative dentistry, understanding the histology of enamel, dentin, pulp, and cementum is vital for designing effective cavity preparations, selecting appropriate restorative materials, and predicting tissue response and healing [12]. histological analysis of cariesaffected tissues informs the extent of decay and guides intervention strategies [13], while the compatibility of restorative materials with dental tissues ensures long-term success [14]. oral surgery heavily depends on histological knowledge for understanding wound healing processes, the interaction between bone and soft tissues during procedures like extractions and implants [15], and diagnosing pathological conditions requiring surgical intervention, oral histology is crucial for diagnosing oral lesions, differentiating between various types of pathological conditions [16], understanding oral manifestation of systemic disease manifestations in the oral cavity, and assessing the impact of medications on oral tissues, all of which contribute to informed clinical decision-making and patient care [17]. this study will help in recognizing dental education deficiencies and providing insight that may benefit and improve dental students' performance and education. it appears that no previous study has been conducted in iraq examining the proficiency of dental students in oral histology, which is a considerable current deficiency in our academic dental counseling. therefore, our research study will target the fifth year of dental students from specific age groups. the importance of the study arose for measuring dentists' orofacial histological expertise, which has not previously been published, and for assessing the impact of the teaching process in academic dental institutions students for basic science and undergraduate clinical education. material and methods study method a cross-sectional survey was conducted from february to may 2024 among dental students in iraq. participants were recruited through targeted advertisements online by different social media especially telegram, whatsapp and facebook groups, focusing on individuals within the specified age range, different genders and geographic locations. survey design at the beginning of the survey, as part of the demographic data, respondents were asked about their gender in addition to their age, which was divided into two groups. the first group ranged from 22 to 24 years old, considered the fresh group, representing the typical age for graduation. the second group ranged from 25 to 28 years old, considered the old group. this questionnaire was designed for fifth-year dental students in iraq, including both assessment of oral histology knowledge and its applica8on in clinical orthodon8cs, opera8ve den8stry, oral medicine, and oral surgery vol 12 no 2 (2024) doi 10.5195/d3000.2024.749 3 governmental and private educational institutions. the survey contains 40 items addressing key questions that connect oral histology to various clinical dental branches, aiming to evaluate how much students benefit from studying oral histology as a basic science and its application in clinical dentistry. consultation was made with the heads of relevant departments and teaching staff involved in the research to identify the questions they deemed important for the fifth-year stage, which should be addressed before starting the practical component. improving reliability of online surveys is crucial for precise data gathering. the validity of the content was evaluated by professionals in orthodontics, operative dentistry, oral surgery, oral medicine, and oral histology. data collection the validity of the content was evaluated, obscurity was ensured throughout the data collection process. the fifth-year representatives in both public and private universities have been contacted to ensure that the questionnaire reaches the largest number of students. the responses designated as: extensive knowledge (score 4), limited knowledge (score 3), moderate knowledge (score 2) and knowledge unimportant (score 1). statistical analysis the data analysis was conducted using jeffreys’s amazing statistics program (jasp), version 0.19.0. the methods applied included descriptive statistics, such as the mean and standard deviation, along with the independent samples t-test to compare the two categories under study results the sample consisted of approximately 1,265 students from iraqi public and private universities. the number of students aged 22-24 was around 731, representing about 57.7% of the sample, while students aged 25-28 numbered around 534, making up about 53.3%. the female students in the overall responses were approximately 735, representing about 58%, while the male students were around 530, accounting for 41.7%. students’ knowledge in orthodontics the older students consistently demonstrated a higher level of knowledge in clinical areas (table 1), while fresh students, however, displayed a stronger understanding of basic knowledge areas like tooth eruption. the independent samples t-test results show significant differences between fresh and older students across various orthodontic knowledge areas. as shown in table 2. table 1. scores of responses regarding the students’ knowledge in orthodontics. question group valid mean standard deviation to what extent do you know about the structural changes in the periodontal ligament during orthodontic movement? fresh students 731 2.412 0.856 older students 534 2.011 0.893 to what extent do you know about the cells involved in bone remodeling during orthodontic treatment? fresh students 731 2.31 0.848 older students 534 2.11 0.979 to what extent do you know about maxillofacial development? fresh students 731 2.238 0.818 assessment of oral histology knowledge and its applica8on in clinical orthodon8cs, opera8ve den8stry, oral medicine, and oral surgery vol 12 no 2 (2024) doi 10.5195/d3000.2024.749 4 older students 534 2.521 0.976 to what extent do you know about orthodontic appliances? fresh students 731 2.022 0.747 older students 534 2.264 0.943 to what extent do you know about tooth eruption and shedding sequences? fresh students 731 2.23 0.810 older students 534 1.83 0.996 to what extent do you know about developmental anomalies that need orthodontic intervention? fresh students 731 2.093 0.891 older students 534 2.300 1.014 to what extent do you know about the importance of mixed dentition in orthodontic treatment? fresh students 731 2.083 0.769 older students 534 2.272 0.883 to what extent do you know about tooth development and teeth anomalies? fresh students 731 1.973 0.769 older students 534 2.124 0.837 to what extent do you know about procedures to accelerate tooth movement? fresh students 731 2.226 0.826 older students 534 2.331 0.864 to what extent do you know about the effect of age on orthodontic treatment? fresh students 731 1.921 0.832 older students 534 1.963 0.883 table 2. comparison of orthodontic responses between two age groups. questions t degrees of freedom p to what extent do you know about the structural changes in the periodontal ligament during orthodontic movement? -4.786 1263 < .001 to what extent do you know about maxillofacial development? -5.590 1263 < .001 to what extent do you know about the cells involved in bone remodeling during orthodontic treatment? -4.126 1263 < .001 to what extent do you know about orthodontic appliances? -5.092 1263 < .001 to what extent do you know about the effect of age on orthodontic treatment? -0.862 1263 0.389 to what extent do you know about developmental anomalies that need orthodontic intervention? -3.841 1263 < .001 to what extent do you know about procedures to accelerate tooth movement? -2.205 1263 0.028 to what extent do you know about tooth development and teeth anomalies? -3.321 1263 < .001 to what extent do you know about tooth eruption and shedding sequences? -4.305 1263 < .001 to what extent do you know about the importance of mixed dentition in orthodontic treatment? -4.033 1263 < .001 assessment of oral histology knowledge and its applica8on in clinical orthodon8cs, opera8ve den8stry, oral medicine, and oral surgery vol 12 no 2 (2024) doi 10.5195/d3000.2024.749 5 students’ knowledge in operative as shown in table 3, fresh students generally demonstrate higher knowledge in basic concepts such as enamel structure, cavity design while older students tend to have better understanding in more advanced clinical. the independent samples t-test reveals that fresh students have significantly higher knowledge in basic areas such as enamel structure and cavity design compared to older students. in contrast, older students show significantly better understanding of the pulpal response to caries progression and root canal as shown in table 4. table 3. scores of responses regarding the operative knowledge. question group valid mean standard deviation to which extent do you know about the structure of enamel and its significance in cavity preparation? fresh students 731 2.323 0.990 older students 534 1.981 0.777 to what extent do you know about cavity design? fresh students 731 2.361 1.046 older students 534 2.021 0.847 to what extent do you know about the structure of carious dentin? fresh students 731 2.264 0.749 older students 534 2.154 0.786 to what extent do you know about the pulpal response to caries progression? fresh students 731 2.114 0.888 older students 534 2.416 0.808 to what extent do you know about root canal configuration? fresh students 731 2.397 0.867 older students 534 2.599 0.943 to what extent do you know how to read radiographic films in operative procedures? fresh students 731 2.273 0.773 older students 534 2.289 0.777 to what extent do you know about advancements in adhesives? fresh students 731 2.405 0.764 older students 534 2.483 0.774 to what extent do you know about the materials used in crown and bridge? fresh students 731 2.441 0.889 older students 534 2.549 0.849 to what extent do you know about the treatment of discolored teeth? fresh students 731 2.040 0.782 older students 534 2.139 0.868 to what extent do you know about the interaction of restorative materials with the pulp-dentin complex? fresh students 731 2.218 0.780 older students 534 2.290 0.849 table 4. comparison the scores of responses of operative knowledge between two age groups. questions t degrees of freedom p to which extent do you know about the structure of enamel and its significance in cavity preparation? 6.620 1263 < .001 to what extent do you know about cavity design? 6.185 1263 < .001 to what extent do you know about the structure of carious dentin? 2.536 1263 0.011 assessment of oral histology knowledge and its applica8on in clinical orthodon8cs, opera8ve den8stry, oral medicine, and oral surgery vol 12 no 2 (2024) doi 10.5195/d3000.2024.749 6 to what extent do you know about the interaction of restorative materials with the pulpdentin complex? 1.578 1263 0.115 to what extent do you know about advancements in adhesives? 1.789 1263 0.074 to what extent do you know about the treatment of discolored teeth? 2.121 1263 0.034 to what extent do you know about the materials used in crown and bridge? 0.178 1263 0.859 to what extent do you know about the pulpal response to caries progression? 6.209 1263 < .001 to what extent do you know how to read radiographic films in operative procedures? 0.221 1263 0.825 to what extent do you know about root canal configuration? 3.953 1263 < .001 students’ knowledge in oral medicine the responses indicate that fresh students seem to perform better in foundational areas, such as recognizing challenges in reading pathological slides and distinguishing between cysts and tumors. in contrast, older students exhibit a stronger understanding of clinical aspects, particularly in areas like tmj disorder knowledge, as shown in table 5. the comparison of scores through independent samples t-test (table 6), fresh students demonstrated a significantly higher difficulty in reading pathological slides, while older students outperformed in understanding tmj disorders. table 5. scores of responses regarding the students’ knowledge in oral medicine. questions group valid mean standard deviation to what extent do you know about the anatomy and histology of the temporomandibular joint (tmj)? older students 729 2.044 0.841 fresh students 534 2.086 0.911 to what extent do you know about differentiating between cysts and tumors in the oral cavity? fresh students 729 2.303 0.734 older students 534 2.243 0.761 to what extent do you know about the anatomy, histology, and abnormalities of the salivary glands? fresh students 731 2.212 0.697 older students 534 2.155 0.671 to what extent do you know about oral mucosal abnormalities (growths, ulcers, infections, allergies, immune-mediated and autoimmune disorders)? fresh students 731 2.238 0.735 older students 534 2.193 0.716 to what extent do you know about apththous ulceration? older students 731 2.274 0.850 fresh students 534 2.193 0.823 to what extent do you know about tmj disorder? older students 731 2.460 0.737 fresh students 534 2.296 0.724 to what extent do you know about differences between benign and malignant lesions? older students 731 2.408 0.766 assessment of oral histology knowledge and its applica8on in clinical orthodon8cs, opera8ve den8stry, oral medicine, and oral surgery vol 12 no 2 (2024) doi 10.5195/d3000.2024.749 7 fresh students 534 2.446 0.748 to what extent do you know about oral manifestation of systemic diseases? fresh students 731 2.230 0.763 older students 534 2.238 0.759 to what extent do you observe difficulties in reading pathological slides? older students 714 2.444 0.791 fresh students 502 2.654 0.855 based on your previous academic year, to what extent do you have knowledge about common premalignant lesions? fresh students 727 2.329 0.789 older students 532 2.333 0.866 to what extent do you know about gingival tissues in healthy individuals versus those with gingivitis or periodontitis? fresh students 731 2.285 0.786 older students 534 2.365 0.777 table 6. comparison the scores of responses of oral medicine knowledge between two age groups. questions t degrees of freedom p to what extent do you know about the anatomy and histology of the temporomandibular joint (tmj)? 0.248 1261 0.805 to what extent do you know about differentiating between cysts and tumors in the oral cavity? 1.407 1261 0.160 to what extent do you know about differences benign and malignant lesion? 1.811 1263 0.03 to what extent do you know about the anatomy, histology, and abnormalities of the salivary glands? 1.450 1263 0.147 to what extent do you know about apththous ulcers? 1.091 1263 0.276 to which extent you observed difficulties in reading pathological slides? 2.305 1214 0.021 based on your previous academic year, to what extent do you have knowledge about common premalignant lesions? 0.084 1257 0.933 to what extent do you know about tmj disorder? 1.691 1263 0.044 to what extent do you know about oral manifestation of systemic diseases? 0.185 1263 0.853 to what extent do you know about oral mucosal abnormalities? 1.535 1263 0.125 students’ knowledge in oral and maxillofacial surgery the result reveals that fresh students performed better in foundational areas such as knowledge of the histological concept of dental implants, the mechanism of bone remodeling after extraction as shown in table 7. in contrast, older students demonstrated stronger knowledge in areas, including suturing techniques as demonstrated in table 8. assessment of oral histology knowledge and its applica8on in clinical orthodon8cs, opera8ve den8stry, oral medicine, and oral surgery vol 12 no 2 (2024) doi 10.5195/d3000.2024.749 8 table 7. scores of responses regarding the students’ knowledge in oral and maxillofacial surgery. questions group valid mean standard deviation to what extent do you know the regional anatomy of the oral cavity? fresh students 731 1.914 0.804 older students 533 1.961 0.908 to what extent do you know about developmental anomalies that need surgical intervention? fresh students 731 1.885 0.945 older students 534 2.028 1.091 to what extent do you know about the mechanism of bone remodeling after extraction? fresh students 729 2.23 0.792 older students 534 2.092 0.875 to what extent do you know about grafting materials? fresh students 731 1.828 0.844 older students 533 1.816 0.813 to what extent do you know about histopathological correlation with clinical presentation in oral cysts? fresh students 731 2.34 0.681 older students 534 2.12 0.747 to what extent do you know about the number and distribution of roots before extraction? fresh students 730 2.136 0.767 older students 534 2.12 0.829 to what extent do you know about complications and delayed healing after extraction? fresh students 731 1.949 0.820 older students 534 2.131 0.852 to what extent do you know about suturing techniques and types of sutures? fresh students 730 2.036 0.773 older students 534 2.369 1.011 to what extent do you know about the histological concept of dental implants? fresh students 731 2.35 0.795 older students 534 2.067 0.810 to what extent do you know about the stages of healing following a surgical incision in the oral cavity? fresh students 731 2.314 0.756 older students 533 1.681 0.978 table 8. comparison the scores of responses of oral and maxillofacial surgery. questions t degrees of freedom p to what extent do you know the regional anatomy of the oral cavity? -0.967 1262 0.334 to what extent do you know about developmental anomaly needed surgical intervention? -2.489 1263 0.013 to which extent do you know about grafting materials? 0.243 1262 0.808 to what extent do you know about the mechanism of bone remodeling after extraction? -0.288 1261 0.774 assessment of oral histology knowledge and its applica8on in clinical orthodon8cs, opera8ve den8stry, oral medicine, and oral surgery vol 12 no 2 (2024) doi 10.5195/d3000.2024.749 9 to which extent do you know about the histological concept of dental implant? -10.023 1262 < .001 to what extent do you know about the stages of healing following a surgical incision in the oral cavity? -3.271 1263 0.001 " to what extent do you know about suturing techniques and types of sutures? -6.644 1262 < .001 to which extent do you know about histopathological correlation with clinical presentation in oral cysts? -3.871 1263 < .001 to what extent do you know about the complications and delayed healing after extraction? -3.828 1263 < .001 to which extent do you know about number and distributions of toot before extraction? -4.494 1262 < .001 discussion students from various iraqi colleges participated in this survey, which focused on their professional experience and the role of oral histology in clinical dentistry. these students had completed preclinical training and were actively engaged in clinical practice. clinical-based education is a complex process where students apply the theoretical knowledge acquired during their preclinical studies to real patient cases. since dentistry plays a crucial role in healthcare, improving the quality of clinical dental education is vital for enhancing overall oral and dental health in the population. oral histology forms the basis for clinical dental science including main branches like: orthodontics, operative, oral medicine and oral and maxillofacial branch moreover other dental branches, the histology plays a crucial role in oral diagnosis and treatment planning. however, students often find oral histology challenging and perceive it as less significant for their future dental careers. similar findings were observed by johnson et al. (2015) [18] who compared the learning approaches of dental and medical students. the results of this study declares that in comparing the clinical and basic knowledge between two groups, younger scored higher in understanding structural changes in different studied branches such as the periodontal ligament, bone remodeling during orthodontic treatment and tooth eruption and shedding, enamel structure and cavity design in operative, distinguishing between tumors and cysts, reading pathological tissue slides, and understanding the anatomy and histology of the temporomandibular joint (tmj) in oral medicine, while in oral and maxillofacial branch their responses indicated understanding of surgical interventions such as tooth extractions and dental implants. they also consistently recall histological, anatomical, and physiological fundamentals, linking clinical signs to the cysts and tumors that are seen in the maxillofacial surgery clinic at the college. this is finding supported by a previous study naruishi et al. (2022) [19], when showed younger students often come into dental school with strong basic knowledge due to recent educational experiences. however, they may struggle to apply this knowledge effectively in clinical settings, which can affect their confidence and patient interactions. this foundational knowledge can be advantageous in theoretical assessments and initial learning phases. however, as they transition into clinical training, they may face challenges in applying this knowledge effectively in realworld scenarios. the lack of clinical experience can lead to reduced assessment of oral histology knowledge and its applica8on in clinical orthodon8cs, opera8ve den8stry, oral medicine, and oral surgery vol 12 no 2 (2024) doi 10.5195/d3000.2024.749 10 cooperation and confidence when interacting with patients and performing procedures. younger learners may not yet fully appreciate the clinical relevance of histological details, such as the importance of knowing the vascularization of the periosteum or how bone tissue reacts to extractions or implants. this may lead to a gap in their ability to relate histological knowledge directly to oral surgery. this finding is supported by a previous study [20], that reported that the unprepared for the hands-on aspects of surgical procedures, which can lead to anxiety and decreased self-confidence among young dental student. however, older students demonstrated superior knowledge in areas related to clinical requirement, in orthodontics they showed good understanding of maxillofacial development, orthodontic appliances developmental anomalies requiring orthodontic intervention and mixed dentition. statistical analysis revealed significant differences, indicating that older students generally have stronger clinical knowledge, while younger students excel in certain basic concepts. the analysis of operative question scores shows that older, employed students exhibit a more advanced grasp of complex clinical issues like treating discolored teeth, pulpal responses to caries, and root canal anatomy. on other hand the results showed that there were significant differences in responses scores for distinguishing between tumors and cysts, reading pathological tissue slides, and understanding the anatomy and histology of the temporomandibular joint (tmj) while the older student reported significant differences related to tmj disorder. students working in the healthcare sector excel in the clinical aspects of any surgical intervention in surgical department, as they often perform these procedures in their jobs, which grants them strong clinical skills. older students likely benefit from more clinical exposure, allowing them to observe a wider range of cases, especially those related to maxillofacial development. older students’ extensive clinical exposure helps them grasp the relationship between facial growth, dental development, and orthodontic outcomes. while younger students may excel in basic sciences, older students benefit from their additional clinical experience. this emphasizes the importance of integrating both theoretical knowledge and clinical practice in dental education to ensure a wellrounded learning experience. in iraq, the laws and regulations allow individuals over the age of 18 to enroll in iraqi dental colleges, provided they meet the high academic standards and mental qualifications required for admission, whether the institutions are governmental or private. additionally, the regulations permit individuals up to the age of 28 to join these colleges, often those who are already employed in governmental or private positions. such individuals are generally motivated by a passion for the profession. these roles offer them valuable hands-on experience in clinical procedures, patient care, and the operation of dental equipment. supporting this, agrawal’s et al. (2015) study [21] found that older students accumulate more clinical experience as they progress, which is crucial for applying orthodontic principles effectively. according to previous studies [22,23], most errors in root canal treatments were related to the obturation process. the study showed that assessment of oral histology knowledge and its applica8on in clinical orthodon8cs, opera8ve den8stry, oral medicine, and oral surgery vol 12 no 2 (2024) doi 10.5195/d3000.2024.749 11 dental students were less skilled in performing root canal treatments, with many mistakes being made in contrast to cases managed by more experienced students at higher educational levels. once more, various studies revealed that the change from preclinical to clinical dentistry education can be emotionally upsetting and stressful, which can result in lessthan-optimal learning outcomes [24], diminished stimulus for learning [25] and, in the worst case, signs of anxiety and depression [26]. building and maintaining motivation seems to be essential to averting poor outcomes, the older age students can overcome these situations due to large mentality and easier communication with seniors and patients [27,28]. the result determined that fifth-year students' lack of clinical proficiency was particularly noticeable in the clinical procedures; these issues are currently being resolved. the older group highly engagement with professors and experts tend to have greater interactions with professors and experts in the field, which enhances their understanding and knowledge in addition to clinical experience. in addition to that they may work in some institution that related to health care where they are often immersed in clinical practice and developing their skills in other branches that support the services of the iraqi ministry of health and private clinics. conclusion the results of this investigation demonstrate the various degrees of knowledge and clinical skills between younger and older dental students across various disciplines. younger students tend to excel in basic concepts, in contrast, older students and those with clinical experience demonstrate a deeper understanding of complex clinical scenarios. younger students often possess a strong foundation in oral histology and basic sciences, they may struggle to translate this knowledge into clinical practice, as seen in the challenges faced in operative and surgical procedures. to bridge this gap, dental education programs should aim to integrate both theoretical knowledge and clinical exposure throughout the curriculum. by doing so, students can develop a more comprehensive understanding of how oral histology informs clinical decisionmaking, which is critical for achieving proficiency in various dental specialties. encouraging early clinical involvement while reinforcing basic science concepts can lead to a more well-rounded and confident dental workforce. a deficiency in the clinical competence of fifth-year students was particularly noted in the clinical procedures. conflicts of interest the authors state that there are no conflicts of interest. financial support none. authors’ contributions in this study, salma merza hasan developed the questionnaire sections in partnership with the heads of different branches and distributed it to the majority of fifth-year representatives. concurrently, zainab a. almashhadi gathered the data and performed the statistical analysis, and both contributed to writing the introduction, materials, and methods sections. amjed f. hussein and heider hemeed abbas were responsible for drafting the results and discussion all the results of research . all participants consented to all aspects of the research. assessment of oral histology knowledge and its applica8on in clinical orthodon8cs, opera8ve den8stry, oral medicine, and oral surgery vol 12 no 2 (2024) doi 10.5195/d3000.2024.749 12 references 1. nanci, a. (ed.). (2017). ten cate's oral histology-ebook: ten cate's oral histology-e-book. elsevier health sciences. 2. bennett, j. h., beeley, j. a., anderson, p., belfield, l., brand, h. s., didilescu, a. c., ... & manzanares, m. c. (2020). a core curriculum in the biological and biomedical sciences for dentistry. european journal of dental education, 24(3), 433-441. 3. odell, e. w. (2024). cawson's essentials of oral pathology and oral medicine-e-book. elsevier health sciences. 4. heasman, p. (2008). master dentistry e-book: master dentistry e-book (vol. 2). elsevier health sciences. 5. rajkumar, k., & ramya, r. (2017). textbook of oral anatomy, physiology, histology and tooth morphology. wolters kluwer india pvt ltd. 6. burk, d. t., lee, l. m., & lambert, h. w. (2013). embryology and histology education in north american dental schools: the basic science survey series. journal of dental education, 77(6), 744-756. 7. weyant, r. j. (2019). teaching evidence-based practice: considerations for dental education. dental clinics, 63(1), 97-117. 8. rafai, n., lemos, m., kennes, l. n., hawari, a., gerhardt-szép, s., & classen-linke, i. (2016). anatomy meets dentistry! linking anatomy and clinical practice in the preclinical dental curriculum. bmc medical education, 16, 1-10. 9. matuck, b. f., da silva, l. f. f., warner, b. m., & byrd, k. m. (2023). the need for integrated research autopsies in the era of precision oral medicine. the journal of the american dental association, 154(3), 194205. 10. patel, s., saberi, n., pimental, t., & teng, p. h. (2022). present status and future directions: root resorption. international endodontic journal, 55, 892-921. 11. dʹ apuzzo, f., cappabianca, s., ciavarella, d., monsurrò, a., silvestrini-biavati, a., & perillo, l. (2013). biomarkers of periodontal tissue remodeling during orthodontic tooth movement in mice and men: overview and clinical relevance. the scientific world journal, 2013(1), 105873. 12. ricucci, d., loghin, s., gonçalves, l. s., rôças, i. n., & siqueira jr, j. f. (2018). histobacteriologic conditions of the apical root canal system and periapical tissues in teeth associated with sinus tracts. journal of endodontics, 44(3), 405413. 13. kidd, e. a. m., & fejerskov, o. (2004). what constitutes dental caries? histopathology of carious enamel and dentin related to the action of cariogenic biofilms. journal of dental research, 83(1_suppl), 3538. assessment of oral histology knowledge and its applica8on in clinical orthodon8cs, opera8ve den8stry, oral medicine, and oral surgery vol 12 no 2 (2024) doi 10.5195/d3000.2024.749 13 14. pires, p. m., neves, a. d. a., makeeva, i. m., schwendicke, f., fausmatoses, v., yoshihara, k., ... & sauro, s. (2020). contemporary restorative ion-releasing materials: current status, interfacial properties and operative approaches. british dental journal, 229(7), 450-458. 15. sculean, a., gruber, r., & bosshardt, d. d. (2014). soft tissue wound healing around teeth and dental implants. journal of clinical periodontology, 41. 16. regezi, j. a., sciubba, j., & jordan, r. c. (2016). oral pathology: clinical pathologic correlations. elsevier health sciences. 17. glick, m., greenberg, m. s., lockhart, p. b., & challacombe, s. j. (2021). introduction to oral medicine and oral diagnosis: patient evaluation. burket's oral medicine, 1-18. 18. johnson, s., purkiss, j., holaday, l., selvig, d., & hortsch, m. (2015). learning histology–dental and medical students' study strategies. european journal of dental education, 19(2), 65-73. 19. naruishi, k., wada-mihara, c., oishi, k., & nagata, t. (2022). dental students’ awareness after clinical training between dental treatment and systemic health: a questionnairebased survey. frontiers in dental medicine, 2. https://doi.org/10.3389/fd med.2021.740441. 20. cabbar, f., burdurlu, m. ç., & tomruk, c. ö. (2018). does giving brief information keep patients calm during different oral surgical procedures? quintessence international, 49(10). 21. agarwal, a., wong, s., sarfaty, s., devaiah, a., & hirsch, a. e. (2015). elective courses for medical students during the preclinical curriculum: a systematic review and evaluation. medical education online, 20(1), 26615. 22. alghamdi, n. s., algarni, y. a., ain, t. s., alfaifi, h. m., alqarni, a. a., mashyakhi, j. q., alasmari, s. e., & alshahrani, m. m. (2021). endodontic mishaps during root canal treatment performed by undergraduate dental students: an observational study. medicine, 100(47), e27757. https://doi.org/10.1097/m d.0000000000027757. 23. davey, j., bryant, s. t., & dummer, p. m. h. (2014). the confidence of undergraduate dental students when performing root canal treatment and their perception of the quality of endodontic education. european journal of dental education, 19(4), 229–234. https://doi.org/10.1111/ej e.12130. 24. moore r. psychosocial student functioning in comprehensive dental clinic education: a qualitative study. eur. j. dent. educ. 2018;22:e479– e487. doi: 10.1111/eje.12329. 25. serrano c.m., botelho m.g., wesselink p.r., vervoorn j.m. challenges in the transition to clinical assessment of oral histology knowledge and its applica8on in clinical orthodon8cs, opera8ve den8stry, oral medicine, and oral surgery vol 12 no 2 (2024) doi 10.5195/d3000.2024.749 14 training in dentistry: an adee special interest group initial report. eur. j. dent. educ. 2018;22:e451–e457. doi: 10.1111/eje.12324 26. moore r.m., madsen l.v., trans m. stress sensitivity and signs of anxiety or depression among first year clinical dental and medical students. open j. med. psychol. 2020;9:7–20. doi: 10.4236/ojmp.2020.91 002. 27. moore, r., molsing, s., meyer, n., & schepler, m. (2021). early clinical experience and mentoring of young dental students— a qualitative study. dentistry journal, 9(8), 91. 28. ali, s. n. (2021). clinical competence of undergraduate dental students in pediatric dentistry at a saudi dental school. pesquisa brasileira em odontopediatria e clínica integrada, 21, e0226. 949 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu evaluating the citotoxicity and antibacterial activity of nano zincglyde mixture for intracanal irrigation shammaa anees sahib alansari1, maitha sameer kadhim2, hayder hamed abed3 1college of den*stry, ibn sina university, baghdad, iraq 2al-rafidain university college, baghdad, iraq 3college of den*stry, mustansiriyah university, baghdad, iraq abstract objec8ve: in this research, we evaluated the anhbacterial efficiency and cytotoxicity of glyde including its zinc nps preparahons in three different rahos comparing them with the convenhonal glyde. material and methods: anhbacterial efficiency was evaluated using inhibihon zone method against three common gram-posihve pathogens enterococcus faecali, lactobacillus and streptococcus. three different irrigant preparahons were evaluated, gi: glyde gel alone as a control group, gii: experimental gel (3% zinc nps-glyde), giii: (4% zinc npcs-glyde gel), giv: (5% zinc npcs-glyde gel). we evaluated the cytotoxicity by the mtt test. assessing the cells’ viability as soon as possible a]er 24h, 48h, and 72h. a]er a color change assessment, spectrophotometric analysis with wavelength ranged from 190-780 nm was performed. the spectrum analysis was performed for diluted mixtures in solvent. data were plo^ed and recorded for each wavelength. kruskal–wallis (p < 0.05) and post-hoc bonferroni pairwise (p < 0.05) were used for stahshcal analysis. results: all the groups a reduchon of the three types of bacteria (p > 0.05) was seen, with inhibihon zone increasing with increasing addihon of zinc nps up to 5%. for cytotoxicity, it seems that cell bioavailability remained for 24h, 48h, and declined at 72h. data were not correlated with addihon of zinc nano parhcles, especially within the visible light range. conclusion: the addihon of zinc nanoparhcles has acceptable anhbacterial properhes and cytotoxic features, and glyde gel may be used for root canal disinfechon without remarkable color change. open access cita%on: alansari sas, et al. (2025) evalua%ng the citotoxicity and an%bacterial ac%vity of nano zinc-glyde mixture for instracanal irriga%on. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.949 received: may 24, 2025 accepted: june 26, 2025 published: august 13, 2025 copyright: ©2025 alansari sas, et al. this is an open access ar%cle licensed under a crea%ve commons avribu%on work 4.0 united states license. email: shammaa.alansary@ibnsina.edu.iq introduc)on in most cases of irreversible pulpitis and pulp necrosis, pathogens get into the pulp through mainly caries, periodontal disease or trauma [1,2]. dahlén et al. [3] suggested that this kind of inalammation is often caused by speciaic bacterial strains such as lactobacilli, streptococci, and gram-negative obligate anaerobes. the root canal system is shaped and cleaned chemically and mechanically for the removal of inalamed and/or necrotic pulp tissue, and thus, of established bacteria. chemical cleaning has been recommended for the root canal system [4]. sodium hypochlorite works well at amounts between 0.5 and 5.25% [5-7]. zinc oxide nanoparticle (zno np) is an odorless white powder found in bone, muscle, skin, and teeth. its molecular weight is 81.38 g/mol and the u.s. food and drug administration (fda) recognized it as a safe substance (gras). the use of zinc oxide nanoparticles (zno nps) in biomedicine is safe, show no systemic toxicity, and are cost-effective based on their increased speciaic surface area and enhanced particle surface activity [7, 8]. jowkar et al. found that adding zno to an edta solution for irrigation enhanced the fracture resistance of the roots [9,10]. a study by aguiar et al. discovered that these nps helped with alkalization and acted against e. faecalis when mixed with calcium hydroxide nps and chlorhexidine [11]. material and methods chemicals and reagents glyde gel (dentsply-sirona, switzerland) was used as a base material in this experiment. the nano zinc with high purity of 99.9 %, 35-45 nm, metal base applied in this work was supplied from us research nanomaterial’s, inc. 3302 twig leaf lane, houston, tx77084, usa. glyde-nano zinc mixture was prepared in three different loading percentages 3%, 4 % and 5 %. all preparations were performed under sterile conditions to avoid any samples contaminations. the three experimental mixtures (group ii: iii, and group iv) were prepared by weighting zinc nanoparticles following by vigorous mixing with glyde to prepare exactly the required percentage of evaluahng the citotoxicity and anhbacterial achvity of nano zinc-glyde mixture for intracanal irrigahon vol 13, no 1 (2025) doi 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu 2 mixture of zn nano-glyde. group i represent the glyde material without any additive nano particles to serve as comparison. antibacterial activity test three different human pathogenic bacteria, enterococcus faecali, (a gram-positive, facultative anaerobic coccus), lactobacillus (gram-positive, obligate anaerobes rods) and streptococcus (gram-positive facultative anaerobes cocci) were investigated as tested organisms. a clean cotton swab was used to slide some of the bacterial solution around on the muller-hinton agar medium and make sure it was spread out evenly. after that, the mix was left for 10 minutes. in the layer of agar that came before, 3 wells with a thickness of 5 mm were made. each well got 50 microliters of puriaied and crude eps, and d.w. was put in the middle well as a control. after that, the agar plates were taken out. for 18 hours, the plates were kept at 37 °c. then, the inhibition zones' widths were recorded. the well diffusion experiment was used to see if glyde-zinc nanoparticles could kill pathogens. in each well of the petri plates, three different amounts of nano zinc mix were added: 3%, 4%, and 5%. mtt assay the neutral red (nr) and tetrazolium mtt in vitro cytotoxicity assays were compared for prepared mixtures of glyde – nano zinc mixtures at maximum loading (5%) and for the glyde only on aibroblast cell line as the bio indicator, at three-time intervals (24hr, 48h, and 72h). there was good agreement (r = 0.9052) in how the test agents were ranked based on their median cytotoxicity values (nr50 and mtt50), even though the tests were based on different physiological endpoints. spectroscopic analysis glyde-nano zinc mixtures were scanned using spectrophotometer. the wavelength ranged from 190-780 nm. the expert data were plotted and record in each wavelength. statistical evaluation the three groups were compared using the kruskal–wallis test (p < 0.05): gi (glyde alone), gii (glyde plus 3% zinc), giii (glyde plus 4% zinc), and giv (glyde plus 5% zinc). we used post-hoc bonferroni pairs (p < 0.05) to aind the means that are very different from each other. results table 1 shows the inhibition zones as antibacterial test against lactobacillus (grampositive, obligate anaerobes rods). there was a signiaicant difference depending on the added zinc nanoparticles with different ratios of glyde as intracanal material (p > 0.05). when using post hoc bonferroni, we found a signiaicant difference between gii and giv. in table 2, inhibition zone diameters (mm) of enterococcus faecali, (a gram-positive, facultative anaerobic coccus) by antibacterial action of glyde groups showed a signiaicant difference among tests groups by adding zinc nano particles (p>0.05). post-hoc bonferroni pairwise comparisons showed a signiaicant difference between glyde group alone and group iv (5% zinc). in table 3, there was a signiaicant difference among tests groups (p>0.05) according to the inhibition zones diameters (mm) of streptococcus (gram-positive facultative anaerobes cocci) by the effect of glyde alone and glyde mixed zinc nano particles. using post-hoc bonferroni pairwise comparisons showed a signiaicant difference between glyde group alone and group iv (5% zinc). to evaluate cytotoxic, a human normal aibroblast cell line (bji) was used. the mtt essay was performed at 24h, 48h and 72h. at 24h, the result revealed that glyde alone ic50 was at a dose of 85.6mg/ml. after adding zinc nanoparticles within the same interval, the ic50 was 83.12mg/ml. cell viability was 92.66% at a dose of 50mg/ml, while at the same dose cell viability was 77.16% with glyde. increasing the dose of mtt up to 1000mg/ml decreased cell viability for both glyde and glyde zinc mix (10.94% and 11.15%, respectively) (figures 1 and 2). at 48h the result revealed that glyde alone ic50 was at a dose of 85.6mg/ml. after adding zinc nanoparticles within the same time, the ic50 was 83.12mg/ml. cell viability was 92.66% at a dose of 50mg/ml for glyde only, while at the same dose, cell viability was 77.16% for glyde zinc mix. increasing the dose of mtt up to 1000mg/ml decreased cell viability for both glyde and glyde zinc mix (10.94% and 11.15%, respectively) (figures 3 and 4). at 72h, the result revealed that ic50 was 44.71 mg/ml for glyde alone and 7.71mg/ml for glyde zinc mix. on the other hand, cell viability at a dose of 50mg/ml was 40.12% for glyde alone and 27.17% for glyde zinc mix. when the dose was increased up to 500mg/ml, only 7.25% viability was seen for glyde only and 5.85% viability was seen at a dose of 1000mg/ml (figures 5 and 6). spectroscopic analysis the uv-vis scanning recorded very similar values, without any intense abnormal peaks, especially in visible range. discussion nanoparticles are often used to combat microbial infections because they are less cytotoxic [12,13]. to test the antibiotic effects of added zinc nanoparticles to glyde, enterococcus faecali was picked because it is one of the types most often found in individuals who have had persistent root canal infections. another reason, e. faecalis can live in harsh conditions with few nutrients and for a long time inside canals, even after treatment [14,15]. it can also survive at very hot temperatures or acidic conditions [16]. in our study, selection of streptococcus and lactobacillus species was due to their virulence and involvement in rapid progression of periodontal diseases, endoperio lesions, both acidogenic and aciduric environments, enamel demineralization leading to dental caries, and initiation of bioailm containing streptococcus [17-19]. the antibacterial effect of glyde gel depends on the oxygen bubbling which are liberated from carbamide peroxide through the removing of pulp tissue [20]. as shown in tables 1, 2, and 3 there was a signiaicant difference among tests groups (p>0.05) according to the inhibition zones diameters (mm) for the three bacterial types. results indicated that as we increase the percentage of zinc nanoparticles in the mixtures, the more inhibition zones size appeared in diameter for the three types of bacteria in comparison with the glyde alone or even low zinc percent. these results may relate to electrostatic forces between the positive bacterial cell wall and the positive nps that enhance the antibacterial activity [21]. positively charged nano particles showed higher antibacterial activity against both s. mutans and e. faecalis. while for the airst time zinc nano mixtures was investigated for its antibacterial efaiciency, many other metal nano particles revealed same higher activity against different types of bacteria when added to glyde [22]. or they ailled the canals with nanoparticles as a kind of irrigant [16]. our study looked at the possibility of harmful cytotoxicity of a glyde material (after 24 h, 48 h, and 72 h) with and without 5% zinc nanoparticles as the highest loading percent. to get to a level that is useful in medicine, we used the mtt method to look for deadly activity in the human normal aibroblast cell line (bji). color was used in the mtt test to show how metabolically active cells are and how dangerous they are. this is by turning a yellow color called 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide that dissolves in water into purple formazan crystals that do not dissolve in water [23]. material considers as cytotoxic if it kills more than half of the cell’s lysis, which is a score of ≤ 21. as per iso 10993-5, the test extract was harmful if less than 70% of the cultures survived compared to control cultures that were not handled with it [24,25]. evaluahng the citotoxicity and anhbacterial achvity of nano zinc-glyde mixture for intracanal irrigahon vol 13, no 1 (2025) doi 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu 3 immediately after 24h, the aindings revealed that there was a remarkable difference for adding zinc nano particles to glyde material when ic50 diminished from 220.1 to 56.03 mg/ml but cell viability was still acceptable for both glyde and glyde zinc mix, even when decreased, mostly due to the immediate role of zinc particles. up to 48 h, we saw that both glyde and its zinc additive mixture reached close to ic50 results with cell viability at 50 mg/ml. mtt dose of 92.66473274%, 77.16150079%, which is suitable biocompatibility. while increasing time up to 72h, we noticed cell viability increasing by adding zinc nano particles compared with glyde alone. this could be due to suppression of cytotoxicity by interaction between glyde edta and zinc nano particles [26]. considering the increasing demands for aesthetics, biomaterials should be chromatically stable, present optical properties like dental structures and not exert staining effects to hard dental tissues [27]. biomaterials should be color stable, have visual features like tooth structures, and not damage hard mouth tissues. the zinc mixture used in this study could not change color and zinc, a small metal, seen to cause staining. zinc may even reverse discoloration and greatly reduces the darkening of teeth caused by sdf [28]. conclusions the study results revealed that zinc nano particles, when added to glyde, signiaicantly increased the antibacterial activity against three types of human pathogens: enterococcus faecali, lactobacillus and streptococcus. it also showed it is antibacterial efaiciency is dose dependent. however, this offers a promising material to be used during rotary instrumentation as a lubricant and antibacterial material at the same time. these experimental mixtures revealed favorable cell viability results when tested by mtt assay and they did not induce remarkable color change. references 1. seltzer s, bender ib, ziontz m. the interrelationship of pulp and periodontal disease. oral surg oral med oral pathol. 1963; 16:1474–90. 2. andreasen fm, kahler b. pulpal response after acute dental injury in the permanent dentition: clinical implications—a review. j endod. 2015; 41:299–308. 3. dahlén g. microbiology and treatment of dental abscesses and periodontal—endodontic lesions. periodontol. 2000; 2002(28):206–39. 4. v. gopikrishna. grossman’s endodontic practice, chapter 15, 14th ed.2022 philadelphia. 5. dutner j, mines p, anderson a. irrigation trends among american association of endodontists members: a web—based survey. j endod. 2012; 38:37–40. 6. baek, m., chung, h.-e., yu, j., lee, j.-a., kim, t.-h., oh, j.-m., et al. (2012). int. j. nanomedicine 7, 3081. 7. jiang j, pi j, cai j. the advancing of zinc oxide nanoparticles for biomedical applications. bioinorg chem appl 2018: 106 – 12. 8. sirelkhatim a, mahmud s, seeni a, kaus nhm, ann lc, bakhori skm, et al. review on zinc oxide nanoparticles: antibacterial activity and toxicity mechanism. nanomicro lett. 2015; 7:219–42. 9. jowkar, z., hamidi, s. a., shagiei, f., and ghahramani, y. (2020). the effect of silver, zinc oxide, and titanium dioxide nanoparticles used as ginal irrigation solutions on the fracture resistance of root-gilled teeth. ccide vol. 12, 141–148. doi:10.2147/ccide.s253251. 10. ahmad fliah hassan, suzan mohammed abdul raheem; hani radhi, hayder hamed abed. oral manifestation associated with biochemical and hematological changes in iraqi end-stage renal failure patients. volume 6, issue 6, june 2023, pages 1352-1361. 11. aguiar, a. s., guerreiro-tanomaru, j. m., faria, g., leonardo, r. t., and tanomaru-filho, m. (2015). j. contemp. dent. p. r. 16, 624–629. 12. ashfaq m, verma n, khan s. copper/zinc bimetal nanoparticlesdispersed carbon nanogibers: a novel potential antibiotic material. mater sci eng c mater biol appl. 2016; 59:938–947. 13. ingle a, gade a, pierrat s, sonnichsen c, rai m. mycosynthesis of silver nanoparticles using the fungus fusarium acuminatum and its activity against some human pathogenic bacteria. curr nanosci. 2008; 4(2):141–144. 14. hancock hh, sigurdsson a, trope m, moiseiwitsch j. bacteria isolated after unsuccessful endodontic treatment in a north american population. oral surg oral med oral pathol oral radiol endod 2001; 91:579-86. 15. rocas in, siqueira jf jr, santos kr. association of enterococcus faecalis with different forms of periradicular diseases. j endod 2004; 30:315-20. 16. leila moghadas, mahdi shahmoradi1, tahmineh narimani, antimicrobial activity of a new nanobased endodontic irrigation solution: in vitro study. dental hypotheses journal oct-dec 2012 / vol 3 | issue 4. 17. marsh, philip d. dental plaque as a biogilm and a microbial community–implications for health and disease. bmc oral health. biomed central 2006; 6:1-7. 18. belibasakis gn, maula t, bao k, lindholm m, bostanci n, oscarsson j, ihalin r, johansson a. virulence and pathogenicity properties of aggregatibacter actinomycetemcomitans. pathogens 2019; 8:222. 19. hee-seon kim, woohyung jang, yeong-gwan im, hyun-pil lim. antibacterial effect of zirconia nanoparticles on polyethyl methacrylate resin for provisional crowns. international journal of nanomedicine 2022:17 6551–6560. 20. gernhardt cr, eppendorf k, kozlowski a. toxicity of concentrated sodium hypochlorite used as an endodontic irrigant. int endod j. 2004; 3727280. 21. li, z.; ma, j.; ruan, j.; zhuang, x. using positively charged magnetic nanoparticles to capture bacteria at ultralow concentration. nanoscale res. lett. 2019, 14, 1–8.) (navarre, w.w.; schneewind, o. surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. microbiol. mol. biol. rev. 1999, 63, 174– 229. 22. jamal a. abu al-timan, hussain f. al-huwaizi, hayder h. abed. evaluating the effect of adding chitosan nanoparticles on the disinfection properties of glyde material: an in vitro study. research j. pharm. and tech. 13(1): january 2020. 23. mosmann t. rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. j immunol methods. 1983; 65:55–63. 24. thangaraju p, varthya sb. iso 10993: biological evaluation of medical devices. in: timiri shanmugam ps, thangaraju p, palani n, sampath t, editors. medical device guidelines and regulations handbook. springer, cham. 2022. 25. scudiero pa, shoemaker rh, paull kd, monks a, thierney s, nofziger th, et al. evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. cancer res 1988; 48:4827–33. 26. jan borovanský and patrick a. riley. cytotoxicity of zinc in vitro, chem biol interact. 1989; 69(23):279-91. 27. ioannidis k, beltes p, lambrianidis t, kapagiannidis d, karagiannis v. validation and spectrophotometric analysis of crown discoloration induced by root canal sealers. clin oral inv. 2013 [cited feb 27]. available from: http://dx.doi.org/10.1007/ s00784-012-0850-x. 28. abdullah almulhim, astrid c. valdivia-tapia, guilherme roncari rocha, yan wu, xinyue mao, nora alomeir, danielle benoit, anderson t. hara, tong tong wu, jin xiao, yihong li. effect of zinc on improving silver diamine gluoride-derived tooth discoloration in vitro. bmc oral health (2024) 24:1410. evaluahng the citotoxicity and anhbacterial achvity of nano zinc-glyde mixture for intracanal irrigahon vol 13, no 1 (2025) doi 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu 4 kruskal-wallis test post-hoc bonferroni pairwise comparisons lacto + glyde mixed groups group n median (range) p-value sample 1 + sample 2 test statistics standard error pvalue lacto+ gii 3 21 (1) 0.02 lacto +g ii lacto +gi 0.3 2.9 1.0 lacto+ giii 3 24 (2) lacto+ gii lacto +giii 4.6 2.9 0.6 lacto+ giv 3 28 (1) lacto +gii lacto +giv 7.6 2.9 0.05 lacto+gi 3 22 (2) lacto +gi lacto +giii 4.3 2.9 0.8 lacto +gi lacto +giv 7.3 2.9 0.07 lacto + giii lacto +giv 3.0 2.9 1.0 table 1. inhibition zone diameters in mm of lactobacillus bacteria by glyde alone and mixed groups. evaluahng the citotoxicity and anhbacterial achvity of nano zinc-glyde mixture for intracanal irrigahon vol 13, no 1 (2025) doi 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu 5 table 2. inhibition zone diameters in mm of enterococcus faecali bacteria by glyde alone and mixed groups. enterococcus faecali +glyde mixed groups group n median (range) pvalue sample 1 – sample 2 test statistics standard error p-value entero+ gii 3 20 (2) 0.03 entero + gi entero + gii 2.1 2.8 1.0 entero + giii 3 22 (3) entero + gii entero + giii 1.6 2.9 1.0 entero + giv 3 25 (1) entero + gii entero + giv 5.8 2.9 0.2 entero + gi 3 20 (1) entero + gi entero + giii 3.8 2.9 1.0 entero +gi entero +giv 8.0 2.9 0.02 entero +giii entero +giv 4.1 2.9 0.8 evaluahng the citotoxicity and anhbacterial achvity of nano zinc-glyde mixture for intracanal irrigahon vol 13, no 1 (2025) doi 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu 6 strepto+glyde mixed groups group n median (range) p-value sample 1 – sample 2 test statistics standard error p-value strepto+ gii 3 15 (1) 0.01 entero +gi entero +gii 2.1 2.8 1.0 strepto + giii 3 22 (1) entero +gii entero+ giii 1.6 2.9 1.0 strepto + giv 3 24 (1) entero +gii entero + giv 5.8 2.9 0.2 strepto + gi 3 13 (1) entero + gi entero + giii 3.8 2.9 1.0 entero +gi entero +giv 8.0 2.9 0.02 entero +giii entero + giv 4.1 2.9 0.8 table 3. inhibition zone diameters in mm of streptococcus bacteria by glyde alone and mixed groups. evaluahng the citotoxicity and anhbacterial achvity of nano zinc-glyde mixture for intracanal irrigahon vol 13, no 1 (2025) doi 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu 7 figure 1. cell availability of glyde alone at 24h. figure 2. cell availability of glyde – zinc mix at 24h. figure 3. cell availability of glyde alone at 48h. figure 4. cell availability of glyde-zinc mix at 48h. figure 5. cell availability of glyde alone at 72h. figure 6. cell availability of glyde-zinc mix at 72h. evaluahng the citotoxicity and anhbacterial achvity of nano zinc-glyde mixture for intracanal irrigahon vol 13, no 1 (2025) doi 10.5195/d3000.2025.949 http://dentistry3000.pitt.edu 8 glyde only plus 3% zinc plus 4% zinc plus 5% zinc min in uv 0.106630904 at 364 nm. 0.295807 at 358 nm. 0.177871 at 380 nm. 0.052597 at 372 nm max in uv 4.3346711 at 202 nm. 2.200893 at 192 nm. 4.835236 at 224 nm. 4.492694 at 208 nm. min in visible 0.106996147 at 390 nm. 0.296713 at 424 nm. 0.165798 at 602 nm. 0.0453 at 426 nm, max in visible 0.131435706 at 780 nm. 0.309136 at 772 nm. 0.178722 at 404 nm. 0.0528 at 382 nm. table 4. minimum and maximum values of wavelength absorption according to glyde and its additive mixtures. 959 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.959 http://dentistry3000.pitt.edu enhancing immediate implant success the role of bone subs/tutes and local an/bio/cs in fresh extrac/on sites shefaa h. alnuamy1, ihsan a. kumail2, salam k. rasheed2, hasan skienhe3 1college of den*stry, al esraa university, iraq 2college of den*stry, al ayen iraqi university, iraq 3faculty of dental medicine, lebanese university, beirut, lebanon abstract objec;ve: this study evaluated the effechveness of bone subshtutes and local anhbiohcs in enhancing osseointegrahon during immediate implantahon in fresh extrachon sockets. material and methods: a total of 30 pahents underwent immediate implant placement, divided into three groups: control group receiving no adjuncts, group treated with bone subshtutes, and group treated with bone subshtutes combined with anhbiohcs. ater four months, success rates were assessed through implant stability quohent (isq) measurements. results: the current findings indicated significantly higher success rates in the groups receiving adjunct treatments than in the control, highlighhng the potenhal of bone subshtutes and anhbiohcs in promohng favorable outcomes. conclusion: this study underscores the importance of personalized treatment approaches that consider pahent-specific factors and the use of adjunchve therapies to ophmize implant success. open access cita%on: alnuamy sh, et al. (2025) ehnancing immediate implant success: the role of bone subs%tutes and local an%bio%cs in fresh extrac%on sites. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.959 received: june 10, 2025 accepted: june 26, 2025 published: august 13, 2025 copyright: ©2025 alnuamy sh, et al. this is an open access ar%cle licensed under a crea%ve commons auribu%on work 4.0 united states license. email: shefaa@esraa.edu.iq introduc)on immediate implantation after tooth extraction has gained popularity and recognition in modern dentistry. this shift is driven by possible bene;its, including shorter treatment time, preservation of alveolar bone structure, and increased patient satisfaction [1,2]. however, rapid implant placement faces obstacles, such as decreased bone density and quality, and the possibility of postoperative infections, which may prevent favorable outcomes [3]. researchers and physicians have looked into various complementary strategies to overcome these issues and improve the success of immediate implantation operations. among these, the use of bone substitutes and local antibiotics have emerged as promising techniques for promoting osseointegration while reducing infection risk [4,5]. bone alternatives, which include materials ranging from synthetic grafts to xenografts, are critical in maintaining alveolar ridge dimensions and providing a platform for new bone growth [6]. similarly, the supplementary use of local antibiotics such as chlorhexidine and minocycline has been encouraged to avoid bacterial colonization and infections at the implant site, hence enhancing overall success rates [7,8]. despite increasing evidence supporting the ef;icacy of bone replacements and local antibiotics, more research is needed. this involves choosing appropriate biomaterials, antimicrobial agents, and surgical procedures to improve results and prevent complications [9,10]. furthermore, taking patient-speci;ic aspects into account, such as overall health and anatomical concerns, is critical for personalizing treatment techniques after immediate implantation. also, the risk of necrosis of the bone is reduced due to the decreased surgical trauma. moreover, the natural socket rich in periodontal cells and matrix makes healing faster and more predictable [11]. the current in vivo study seeks to critically assess the effect of bone substitutes and local antibiotics in improving the success rate of immediate implantation in fresh extraction sites and to clarify the effectiveness, safety, and therapeutic consequences of various adjunctive therapies by combining current literature and empirical evidence. furthermore, it intends to give evidence-based recommendations to optimize treatment methods and increase patient outcomes during immediate implantation in fresh extraction sites. material and methods a total of 30 male patients, with a mean age of 35 years (range 25-45 years) were included in this study. all selected patients had three or more unrestorable hopeless enhancing immediate implant success vol 13, no 1 (2025) doi 10.5195/d3000.2025.959 http://dentistry3000.pitt.edu 2 premolar teeth indicated for extraction in the lower arch. ninety neodent implants (grafel;ing, germany) were used in all patients, and three implants were implanted in each patient. each patient who signed an informed consent form was fully educated about the immediate implantation procedures and all the risks involved with this kind of surgery. grouping of samples the patients were divided into 3 study groups (n=30) according to the following protocol used: group a (control): every patient in this group immediately had one tapered implant in the freshly extracted sockets group b: every patient in this group immediately received one implant in the freshly extracted sockets. after implantation, a bone substitute (bioplast-dent) was added to the gap between the implant and socket wall. group c: every patient in this group immediately had one implant in the freshly extracted sockets. after implantation, a bone substitute mixed with an antibiotic (lincocin) was added to the gap between the implant and socket wall. inclusion criteria 1. patient with good health status. 2. patient should be between 25-45 3. reasons for initial tooth extraction (trauma, caries, root resorption, and endodontic failure). 4. presence of adequate gingival architecture with surrounding dentition. 5. good oral hygiene. 6. adequate bone volume. 7. root fracture either vertical/horizontal 8. teeth which are periodontally involved 9. chronic periapical/ periodontal infection exclusion criteria 1. poor oral hygiene. 2. chronic or acute systematic disorders (uncontrolled diabetes, hemorrhagic diathesis, general or auto immunode;iciency). 3. poor interest or cooperation from the patient. 4. existence of non-treated generalized periodontitis. 5. insuf;icient bone volume at the receptor site. 6. pathological changes at the receptor site (cysts, tumors, osteomyelitis 7. patient still growing (child or adolescent). 8. medically allergic and compromised patients. 9. presence of dehiscence or fenestrations. 10. heavy smokers, alcohol or drug abusers. 11. acute periapical/periodontal infections 12. proximity to vital anatomic structures implant size selection radiographic evaluation bone evaluation at the implant placement site was performed, along with the estimation of tooth size to be removed (root length and root width at the cej) and the closeness of the implant site to important anatomical structures using opg, iopa, and computed tomography scans. the implant size used was determined using these characteristics as a reference. the mandibular cone beam computed tomography (cbct) was utilized to assess the diameter and length of sockets dimensions. according to the cbct assessments, the socket diameters were 5.5-7 mm in the crestal direction, 3-4 mm in the apical direction, and 10-13 mm in length. accordingly, tapered self-tapping implants were selected. the implant diameter in groups b and c was carefully chosen to be 2mm less than the crestal socket width, whereas, in group a, it was selected to be the same size as the crestal part of the socket. the length of the implants in all groups was chosen to be 1.5 mm below the crestal bone level (figure 1). surgical procedure atraumatic teeth extraction atraumatic teeth extraction was carried out using periotomes. teeth were extracted with minimal trauma to the alveolus and rotational movement was used to avoid damaging the buccal plate(12). implant insertion immediately after tooth extraction, the implants were placed into the sockets without drilling [12-15]. the implants in group a (control) were inserted into the socket without the addition of any bone substitute. in group b the implants were inserted, and the 2mm gap between the implant and socket walls was ;illed with a bone substitute, however in group c the implants were inserted, and the 2mm gap was ;illed with bone substitute mixed with the antibiotic. after implant insertion for the three groups, a slice of absorbable collagen wound dressing zimmer collagen plug (usa) was placed over the implant, then the buccal and lingual gingiva were sutured using a reverse cut needle 0\4 silk. second stage surgery after four months, the implants of all groups were exposed and assessed for osseointegration using resonance frequency analysis (rfa) using an osstell device (ab integration diagnostics, gothenburg, sweden) [16]. it is expressed as an implant stability quotient (isq) in units ranging from 1 to 100. each transducer is calibrated by the manufacturer, which makes all measurements directly comparable [17-20] (figure 2). the criteria used to de;ine the success or failure of the implants are as follows: 1success: isq value of 60 or more with no mobility. 2failure isq value less than 60 with mobility statistical analysis a chi-square test for independence was conducted to compare the success and failure rates among the three implant groups. this test assesses whether significant differences exist in the distribution of success and failure rates among the three groups. following the chi-square test, pairwise comparisons between the implant groups were performed. these comparisons were conducted using chi-square tests for each pair of groups. the bonferroni correction was applied to control for the family-wise error rate due to multiple comparisons. the adjusted significance level for each pairwise comparison was set at α′=0.05 / 3 ≈ 0.0167. results the frequency of successful and failed implants for each group was [χ2(2, n=90)=11.607, p=0.003]: group a: 19 successful implants, 11 failed implants. group b: 27 successful implants, 3 failed implants. group c: 29 successful implants, 1 failed implant. group a vs group b: χ2(1,n=60)=7.004, p=0.008. group a vs group c: χ2(1,n=60)=9.202, p=0.002. group b vs group c: χ2(1,n=60)=0.352, p=0.553. enhancing immediate implant success vol 13, no 1 (2025) doi 10.5195/d3000.2025.959 http://dentistry3000.pitt.edu 3 discussion the current study showed a signi;icant success rate of immediate implantation procedures done in fresh extraction sites in groups b and c compared to control group a. thus, the null hypothesis regarding using bone substitutes and local antibiotics to enhance osseo-integration was rejected. immediate implantation offered a shortened treatment duration and preservation of alveolar bone structure. both are essential to get the most effective treatment and functional results. however, many challenges such as bone type and post-operative infection can in;luence the treatment outcome [1,2]. one of the most important factors for the success of osseointegration is implant stability which can be classi;ied into primary (mechanical engagement) and secondary stability (osseointegration) [21]. in the present study, the primary stability was achieved by bone substitutes in groups b and c and by socket walls in group a despite not measuring the primary stability. a previous study [22] assessed the stability of a clinically successful implant employing resonance frequency analysis. after a year of loading, the successfully integrated implant had isq values ranging from 57 to 82 isq. results of the present study showed isq levels between 60 and 81 isq with a mean of 70 isq after 4 months of immediate implantation in the mandibular arch. our results agree with those of balleri et al. [22], deng et al. [17], and harirforoush and arzanpour [18] who suggested that the strong interface between the implant and bone is observed in high isq values. however, kittur et al. [23] stated that despite the osstel being a non-invasive and widely used equipment to asses implant stability, his review revealed that there is no single universally accepted method to determine secondary implant stability. the use of bone substitutes in this study could have played a major role in the success rate of group c compared to group a. therefore, bone substitutes could provide support for implants surgically implanted without drilling. this could reveal that the primary stability achieved for successful osseointegration is supposedly related to bone substitutes. this was consistent with several studies that stated that a bone substitute in the ;ixture–socket gap preserved socket volume and supported new bone formation [24,25]. the preservation of the bone structure and minimizing soft tissue trauma was achieved in this study by using atraumatic extraction. so, the gap ;illed by the bone substitute was supported by the alveolar bone and the untraumatized tissue accelerated the healing process and complication. this was in agreement with tarnow et al. [26], who stated that atraumatic extraction preserves the integrity of the socket, and the immediate placement of implants into extraction sockets with an intact buccal wall allows healing and osseointegration. limitations of the study despite the positive results of this study, several limitations need to be considered. the very small sample size and homogeneous patient demographics may restrict the ;indings' application to wider patient populations. future research should look into longer followups to verify the instant implantation methods' long-term safety and effectiveness. additionally, comparative analyses of different antibiotic combinations and bone replacements are required to ascertain the optimal treatment plan based on speci;ic clinical circumstances. conclusions within the limitation of this study, bone substitutes are a potentially effective way to improve the outcome of immediate implantation treatments after tooth extraction. combining local antibiotics with bone substitutes is an effective way to prevent infection. atraumatic extraction as a major factor positively affects the success rate of immediate implantation in fresh extraction socket. conflicts of interest the authors have no con;licts of interest to declare. acknowledgements the authors would like to thank the deanery of the college of dentistry, university of alayen iraqi university for its support to perform the current study. author’s contribu)ons all authors studied conception and design, methodology, statistical analysis and interpretation of results, original draught manuscript preparation, writing, review and editing. all authors reviewed the results and approved the final version of the manuscript to be published. funding no grants or financial support were received from any government or private sector for this study. references 1. araújo, m. g., silva, c. o., misawa, m., & sukekava, f. (2015). alveolar socket healing: what can we learn? periodontology 2000, 68(1), 122– 134. https://doi.org/10.1111/prd.12082 2. mello, c., lemos, c., verri, santos, d. d., goiato, m., & pellizzer, e. (2017). immediate implant placement into fresh extraction sockets versus delayed implants into healed sockets: a systematic review and meta-analysis. international journal of oral and maxillofacial surgery, 46(9), 1162–1177. https://doi.org/10.1016/j.ijom.2017.03.016 3. esposito, m., grusovin, m. g., & worthington, h. v. (2013). interventions for replacing missing teeth: antibiotics at dental implant placement to prevent complications. cochrane library, 2019(10). https://doi.org/10.1002/14651858.cd004152.pu b4 4. mombelli, a., hashim, d., & cionca, n. (2018). what is the impact of titanium particles and biocorrosion on implant survival and complications? a critical review. clinical oral implants research, 29(s18), 37–53. https://doi.org/10.1111/clr.13305 5. schwarz, f., rothamel, d., herten, m., wüstefeld, m., sager, m., ferrari, d., & becker, j. (2008). immunohistochemical characterization of guided bone regeneration at a dehiscence-type defect using different barrier membranes: an experimental study in dogs. clinical oral implants research, 19(4), 402–415. https://doi.org/10.1111/j.16000501.2007.01486.x 6. dam, v. v., trinh, h. a., rokaya, d., & trinh, d. h. (2022). bone augmentation for implant placement: recent advances. international journal of dentistry, 2022, 1–7. https://doi.org/10.1155/2022/8900940 7. anitua, e., alonso, r., girbau, c., aguirre, j. j., muruzabal, f., & orive, g. (2012). antibacterial effect of plasma rich in growth factors (prgf®endoret®) against staphylococcus aureus and staphylococcus epidermidis strains. clinical and experimental dermatology, 37(6), 652–657. https://doi.org/10.1111/j.13652230.2011.04303.x 8. matesanz-pérez, p., garcía-gargallo, m., figuero, e., bascones-martínez, a., sanz, m., & herrera, d. (2013). a systematic review on the effects of local antimicrobials as adjuncts to subgingival debridement, compared with subgingival debridement alone, in the treatment of chronic periodontitis. journal of clinical periodontology, 40(3), 227– 241. https://doi.org/10.1111/jcpe.12026 9. alsaadi, g., quirynen, m., komárek, a., & van steenberghe, d. (2008). impact of local and systemic factors on the incidence of late oral implant loss. clinical oral implants research, 19(7), 670–676. https://doi.org/10.1111/j.16000501.2008.01534.x 10. tarnow dp, chu sj. human histologic verification of osseointegration of an immediate implant placed into a fresh extraction socket with excessive gap distance without primary flap closure, graft, or membrane: a case report. int j periodontics restorative dent. 2011 sepoct;31(5):515-21. pmid: 21845246. 11. hassan ks, alagl as. immediate dental implants and bone grafts. implant dentistry-the most promising discipline of dentistry,2011 prof llserturkyilmaz (ed),isbn:978-953-307-481-8. 12. garber da, salama ma, salama h. immediate total tooth replacement. compend contin educ dent. 2001 mar;22(3):210-6, 218. 35. 13. locante, w. m. (2001). the nonfunctional immediate provisional in immediate extraction sites: a technique to maximize esthetics. implant dentistry, 10(4), 254–258. https://doi.org/10.1097/00008505-20011000000008 enhancing immediate implant success vol 13, no 1 (2025) doi 10.5195/d3000.2025.959 http://dentistry3000.pitt.edu 4 14. lorenzoni, m., pertl, c., zhang, k., wimmer, g., & wegscheider, w. a. (2003). immediate loading of single-tooth implants in the anterior maxilla. preliminary results after one year. clinical oral implants research, 14(2), 180–187. https://doi.org/10.1034/j.16000501.2003.140207.x 15. rosenquist b, grenthe b. immediate placement of implants into extraction sockets: implant survival. int j oral maxillofac implants 1996;112:205–209. 16. alamin y dhahi, salwan y bede, haig khachadourian (2023). reliability of the multipeg™ transducer in measuring dental implant stability by using a resonance frequency analysis device (osstell®): an observational clinical study 17. deng, k. b. c. tan, g. liu, j. geng, and w. yan, ‘‘a new numerical approach for evaluation of dental implant stability using electromagnetic impulse,” j. dent., pp. 1–9, 2008 18. harirforoush, r., & arzanpour, s. (2011). a numerical approach of estimating dental implant healing process using resonance frequency analysis (rfa). volume 2: biomedical and biotechnology engineering; nanoengineering for medicine and biology. https://doi.org/10.1115/imece2011-64415 19. zhou, y., jiang, t., qian, m., zhang, x., wang, j., shi, b., xia, h., cheng, x., & wang, y. (2008). roles of bone scintigraphy and resonance frequency analysis in evaluating osseointegration of endosseous implant. biomaterials, 29(4), 461– 474. https://doi.org/10.1016/j.biomaterials.2007.10.021 20. bhandarkar, s., badgujar, a., rajebahadur, s., & dhatrak, p. (2023). a two-degree of freedom mathematical modelling of a dental implant to estimate frequency and micro-displacement using electromagnetic rfa. e-prime advances in electrical engineering electronics and energy, 6, 100314. https://doi.org/10.1016/j.prime.2023.100314 21. swami, v., vijayaraghavan, v., & swami, v. (2016). current trends to measure implant stability. the journal of indian prosthodontic society, 16(2), 124. https://doi.org/10.4103/09724052.176539 22. balleri, p., cozzolino, a., ghelli, l., momicchioli, g., & varriale, a. (2002). stability measurements of osseointegrated implants using osstell in partially edentulous jaws after 1 year of loading: a pilot study. clinical implant dentistry and related research, 4(3), 128–132. https://doi.org/10.1111/j.17088208.2002.tb00162.x 23. kittur, n., oak, r., dekate, d., jadhav, s., & dhatrak, p. (2021). dental implant stability and its measurements to improve osseointegration at the bone-implant interface: a review. materials today proceedings, 43, 1064–1070. https://doi.org/10.1016/j.matpr.2020.08.243 24. atieh, m. a., alsabeeha, n. h., payne, a. g., duncan, w., faggion, c. m., & esposito, m. (2015a). interventions for replacing missing teeth: alveolar ridge preservation techniques for dental implant site development. cochrane library. https://doi.org/10.1002/14651858.cd010176.pu b2 25. zaki, j., yusuf, n., el-khadem, a., scholten, r. j. p. m., & jenniskens, k. (2021). efficacy of bone-substitute materials use in immediate dental implant placement: a systematic review and metaanalysis. clinical implant dentistry and related research, 23(4), 506–519. https://doi.org/10.1111/cid.13014 26. tarnow, d., chu, s., salama, m., stappert, c., salama, h., garber, d., sarnachiaro, g., sarnachiaro, e., gotta, s., & saito, h. (2014). flapless postextraction socket implant placement in the esthetic zone: part 1. the effect of bone grafting and/or provisional restoration on facial-palatal ridge dimensional change—a retrospective cohort study. the international journal of periodontics & restorative dentistry, 34(3), 323–331. https://doi.org/10.11607/prd.1821 figure 1. diameter and length of the socket before extraction. enhancing immediate implant success vol 13, no 1 (2025) doi 10.5195/d3000.2025.959 http://dentistry3000.pitt.edu 5 figure 2. dental implants 4 months after placement. 1055 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1055 http://dentistry3000.pitt.edu 3d printed cast is more accurate than conventional stone cast for single tooth supported fixed prosthesis alnamel hasanen ali college of den*stry, university of basrah iraq abstract objec0ve: studying the clinical adaptafon of the crown in relafon to the finished line of the prepared abutment and assessing the fit and precision of single unit fixed dental prosthesis made on digitally printed posifve replica casts that created by printers depending on digital intra oral scanning by using filament material of resin material were the goals of this in vitro invesfgafon. materials and methods: aqer acquiring digital virtual casts through intraoral scanning of the prepared teeth using a 3shape trios 4 intraoral scanners, ten digitally printed posifve replica casts were created using a3d printers depending on digital intra oral scanning. the master model was created from filament material of resin material. ten convenfonal type 3stone were used to from convenfonal stone cast (cs), final impression was made from dual viscosity impressions material. every fixed dental prosthesis (fdp) was made using a denfum 5-axis milling machine. a two-way anova was conducted to characterize the whether the deference between groups was significant or not, 3d analysis soqware was used to superimpose the milled fdps' intaglio surface and master model. also post hoc analysis, and tukey honestly significant difference test was employed. results: the internal and marginal root mean square (rms) values of the two groups (three dimensional printed and convenfonal stone cast) were significantly differed, according to a two-way anova. open access cita%on: ali ah. (2025) 3d printed cast is more accurate than on conven%onal stone cast for single tooth supported fixed prosthesis. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1055 received: september 21, 2025 accepted: september 27, 2025 published: november 4, 2025 copyright: ©2025 ali ah. this is an open access ar%cle licensed under a crea%ve commons atribu%on work 4.0 united states license. email: hasanen.muhsen@uobasrah.edu.iq introduc)on accurate prosthesis fabrication requires precise castings. other kinds of casts could be considered as substitutes for conventional type 3 stone cast if dental clinics regularly used digital images acquired with an intraoral scanner. the 3d-printed cast is one such option [1]. the citness of the margin and internal portion of a dental restoration specially cixed dental prosthesis determines its quality. to provide a precise cit, accurate reproduction is necessary. therefore, accuracy is crucial for creating impressions and copies [2,3]. intra oral scan of the teeth and associated intra oral structure by using an intraoral scanner or the traditional technique of (elastomeric impression) can be used to create decinitive dental master casts [4]. for many years, stone casts have been utilized for prosthetics, diagnosis, and treatment. they are vulnerable to damage and fracture and furthermore, because they are heavy, they are challenging to store. these issues can be avoided with digitally printed positive replica casts that are acquired from a digital scan to the intra oral structure. they can be sent digitally and are stored in a digital format [5,6]. after taking an imprint (intra orally), there is no need to create a decinitive cast because the data are taken straight from the intra oral cavity by digital scan [7,8]. decinitive dental casts are still necessary for certain prosthetic treatments [9,10] but by using subtractive or additive manufacturing, 3d digital virtual casts can be used to create decinitive and accurate dental casts. the technique of fabricating prostheses has been made simpler and more accurate by subtractive manufacturing (3d printing dental cast) [11,12]. however, due to the milling machine's restricted axes, it is challenging to replicate complicated geometries and undercuts with this manufacturing method [13]. additionally, due of the milling bur's diameter, this approach produces a lot of waste and may result in inaccuracy (known as drill compensation) [14,15]. additive manufacturing, also referred to as 3d printing, on the other hand, develops the intended shapes layer by layer after converting the planned cad ciles into slice data [16,17]. features like undercuts and intricate interior forms can be produced with little material waste [17,18]. 3d printed cast is more accurate than convenfonal stone cast for single tooth supported fixed prosthesis vol 13, no 1 (2025) doi 10.5195/d3000.2025.1055 http://dentistry3000.pitt.edu 2 additionally, multiple goods can be produced at the same time. due to these benecits, 3d printers are becoming more and more popular in the prosthodontic cield. furthermore, 3d printers are now less expensive because the original patent for the concept has expired [19]. the photopolymerization process is used in digital printing. on the whole platform, the 3d printer printing a single image as a layer concurrently. because all layers are exposed simultaneously, dlp can thereby shorten printing times [20]. using high quality three diamantine printer affects accuracy of parenting [21]. furthermore, it has been noted that the dlp technique produces diagnosis casts with greater accuracy than other 3d printer kinds [22]. currently, the primary applications of 3d printers are the creation of surgical guides and orthodontic casts for dental implant procedures. they are anticipated to be utilized more frequently, nevertheless, to produce decinitive castings for prosthodontics as well as prosthetics. the precision of 3d-printed orthodontic diagnostic castings has been assessed in studies. nevertheless, there aren't many studies on digitally printed positive replica casts were created using a3d printers depending on digital intra oral scanning (fdp). thus, the objective of this in vitro investigation was to assess the accuracy of 3d-printed casts in comparison to a traditional stone cast (cs) and the marginal and internal cit of fdps made on a 3dp. materials and methods a single unit fdp was abutted by the maxillary right cirst molars utilizing a cilament resin typodont (ag-3 zpvk; frasaco gmbh). the standard abutment preparation type was full crown preparation (360 degree, 1mm with margins of chamfer type). the created printed cast as a master model made of cilament epoxy resin duplicated ither from intra oral scan or by scan to the conventional cast made by conventional impression by using an optical scanner (3shape trios4 4 advance wireless intraoral scanner) with a 6mm accuracy. metal rim look stock tray (mendcy, italy) were used with heavy body silicon (zemach, germany) for primary impression making, after the study cast fabrication, it was layered by 1 mm baseplate wax as spacer and acrylic non perforated special tray was fabricated, cinished and painted with adhesive material to provide mechanical attachment to the impression material. dual viscosity impressions were used (10 minutes according to the manufacturer's instruction to ensure for full polymerization) the tray impression was moved with the snap out removed to fabricate ten master model by purring the cinal impression and cast creating according to the manufacturer's specicications [22], it was then stored in an incubator for 8 hours at 23°c to control water evaporation and prevent dimensional changes. type iii dental stone (fuji rock; gc) was poured into the tray, and the stone cast separated from the impression after 45 minutes to ensure full polymerization. following a 8-hour storage period, the stone castings were digitalized using a reference scanner and saved as a standard tessellation language (stl) cile (cs group). a trained clinician created ten three dimensional printed casts (3dp group) by scanning the master model with an intraoral scanner. using the pertinent exported stl ciles (digital virtual casts), three dimensional printed casts were produced using a three-dimensional dental model printer (3dent; envisiontec gmbh). in around three hours, the 3d printer created ten castings at a 50 mm resolution. these cast then scanned using the digital intra oral scanner, and the analytic software (stl cile) was stored. the single-unit fdps were built using cad-cam software (dentium milling) throe an stl cile, the crowns were fabricated to the collected data include the scanning of the conventional cast data and intra oral scan. using a 5axis milling machine (dwx-50; roland dg corp), the single-unit fdps were produced from a polyurethane block (innoblanc model; innoblanc gmbh). following milling, the intaglio of the cinished single-unit fdps was captured by a reference scanner and stored as an stl cile. using 3d analysis tools, all stl ciles were reduced to the region of interest, eliminating defects and artifacts for precise superimposition (geomagic verify 2015; geomagic gmbh). the single unit fdps intaglio scan data and the master model data were automatically aligned before being overlaid. for precise alignment, the best-cit alignment the acceptance varieties were then strongminded in this way: the maximum value/the minimum value (±10 mm); and the overall deviation was computed using standards from a dye chart. root mean squares (rmss) were used to quantify the dimensional differences between the digitalized single unit fdps' intaglio surface data and the master model [23]. rmss were computed using the subsequent formula: root mean squares (rmss) where xi is the measurement argument of the master model and n is the quantity of the measured points. in this study, an exact 3d match was indicated by a low rms score. to measure the 3d data, the castings were also separated into the mesial and distal sides using a line that split each abutment's mesiodistal region in half, and between the marginal and interior sections using a line that curved sharply towards the axial wall (figures 1 and 2). the statistical analysis was carried out using statistical software (ibm spss statistics, v22.0; ibm corp.). to cind out if the kind of group and side, as well as how they interacted, affected the rms values, a 2-way anova was employed. to cind signicicant differences between the groups, the post hoc tukey hsd honestly signicicant difference (hsd) test (a=.05) was used. results the mean rms and standard deviation values for internal and marginal disagreement are displayed in table 1. the internal (entire) rms values in cs and 3dp were 25.55 mm and 53.77 mm, respectively, whereas the marginal (entire) rms values were 34.88 mm and 43.56 mm. furthermore, it was shown that the side accuracy between 3dp type and cast group had a signicicant interaction (p=0.009). signicicant differences were found between groups (p<0.001) and between side (p=0.007) internal rms values, as shown in table 2. nevertheless, the interaction effect between the groups and sides type (p=0.571) and the marginal rms values between on the sides (p=0.762) were not signicicant. as seen in table 3, the marginal and internal rms values for 3pd were signicicantly higher than those for cs (p<0.001) according to the post hoc tukey hsd test. color difference maps (cigure 3) showed the variations between the fdps' intaglio surface and the master model. the inside part of 3dp is shown in light blue on the vertical slopes (negative discrepancies) and dark yellow on the occlusal surfaces (positive discrepancies). discussion since disparities in the marginal and internal cits between the sides (mesial side and distal side) and groups (cs and 3dp) were found, the study's cindings supported the rejection of the null hypothesis. one signicicant development for dental ofcices is the use of intraoral scanners. stone cast alternatives, including 3d-printed casts, will be needed as this technique gains traction. in this work, fdps created on stone casts and fdps made on 3d-printed castings were compared for internal and marginal cit. the entire digital process—from getting the scan to making the prosthesis—was contrasted with the conventional process in this study. since the precision of the casts was compared in earlier studies, mistakes in the prosthesis fabrication process were eliminated [8,11,23]. as a result, the complete 3d printed cast is more accurate than convenfonal stone cast for single tooth supported fixed prosthesis vol 13, no 1 (2025) doi 10.5195/d3000.2025.1055 http://dentistry3000.pitt.edu 3 fabrication process was not evaluated. because the current investigation was carried out in a standardized environment, the overall number of manufacturing errors at every stage of the workclow was reduced. both 2d and 3d approaches can be used to measure the prosthesis' internal and marginal cit. the replica, direct view, and crosssectioning techniques are all part of the 2d measurement strategy. one these techniques quantify the amount of variation in pictures captured with a stereomicroscope or similar instruments. the incapacity of 2d measures to assess discrepancy at multiple sites is their primary drawback. in contrast, the 3d analysis measures the thickness of the inside and outside of virtual space numerous times. its use of a color map to visually represent the entire discrepancy is advantageous, as no information is lost that might arise from measuring just one area [24]. the temperature differential between the oral cavity and the surrounding air causes thermal contraction in silicone impression materials [24]. because the imprint was created at ambient temperature, these errors were not replicated in our investigation. consequently, a clinical impression was less accurate than a standard impression control. according to mously et al., the internal gap increased as the spacer thickness setting increased. additionally, several studies have demonstrated that the spacer thickness setting affects how well the prosthesis cits [2,3,25]. to get an exact rms result in the current experiment, the spacer thickness was set to 0. both the internal and marginal cits are incluenced by the prosthesis's material [25]. in this investigation, a polyurethane block was utilized as the prosthetic material. because this block is machined to the same size as the desired form, it has the advantage of reducing the issue of expansion and shrinkage during milling [18,25]. furthermore, because it does not reclect light like metal, zirconia, or ceramic do, it is perfect for scanning and appropriate for 3d evaluation. a prosthesis made using cad-cam technology has had its marginal accuracy assessed [4,26]. the range of marginal discrepancies that are clinically important is unknown, and there are differences in the ranges of marginal cit that are clinically acceptable. in a 5-year clinical study involving 1000 restorations, mclean and von fraunhofer determined that the maximum permissible marginal gap was 120 mm. furthermore, the decinition of clinically adequate internal cit values, which have been reported using a range of techniques, is still up for debate [25,26]. the present study also revealed that the rms value of 3dp (53.77 mm) was higher than that of cs (25.55 mm) in the interior (entire) area, as indicated in table 1 and depicted in figure 3. according to anadioti et al., a prosthesis built using the 3d printing method of stereolithography (sla) die had a substantially better internal cit than one made using a stone die. these numerical values and results are comparable to those of the current investigation, even though the prosthesis material, measurement techniques, and 3d printing technology utilized to create the cast varied. table 1 indicates that the current study found that in the marginal (entire) area, the rms value of 3dp was greater than that of cs. this is consistent with anadioti et al.'s cindings [19,26], who concluded that a prosthesis manufactured on a stone die had a somewhat better cit than one manufactured on a sla die. numerous factors, such as postprocessing, materials, manufacturer setting parameters, and 3d printing procedures, affect the resolution and accuracy of 3d printers. for example, the sla process is affected by horizontal resolution, which is determined by the diameter of the laser beam, and vertical resolution, which is dependent on layer thickness [27]. moreover, changing the setup parameters leads to varying machining accuracy and build timeframes, which may result in residual internal tension during the post-curing phase and perhaps distort the prosthesis. when compared to the cs group, the 3dp group's greater rms values in the internal and marginal areas are caused by the 3d printer's formative processes. in a 3d printer, materials are deposited toward the axis layer by layer. this layer-by-layer technique creates a stair-step impression on the object's surface [27,28]. as a result, the product may have dimensional errors or uneven surfaces [28]. furthermore, because the dlp technique utilized in this study is based on a bottom-up projection in which the build platform moves higher, a polymerized layer is sandwiched between the resin vat and the layer that came before. when the build platform is removed from the vat during the construction process, the coagulated material may attach cirmly to the resin and deform the item. [29]. the current study evaluated cast discrepancies by splitting the internal and marginal cit into mesial and distal sides in order to separate the data and appropriately measure the castings. the mesial and distal side rms values in the marginal and internal areas were not statistically different, except for the 3dp group's internal rms values (figure 4). this result appears to be due to the qualities of the material used in the 3d printer. a photopolymerization approach is required for resin, one of the materials used in the dlp process. photopolymerization-induced material shrinkage can result in residual tension, skewing, or distortion in the cinal product. the rms values on the distal side appear to be higher because the strain is directed toward the mesial side (figure 4). according to an analysis of study results, prostheses made from stone casts cit better than those made from 3d-printed castings. the difference was barely noticeable as all cindings fell below the clinically acceptable range (<120 mm) [30]. these cindings strengthen the case for using 3d-printed casts for longterm prosthesis. however, because various factors in the mouth cavity incluence the digitalization and impression processes, clinical trials should be conducted to assess 3d printing. conclusion the following deductions were made considering the results of this in vitro investigation: 1) the 3dp group's internal and marginal rms values were noticeably greater than the cs groups. 2) nonetheless, the internal and marginal f values for both groups were within the clinically acceptable range (<120 mm). 3d printer accuracy needs to be further improved. references 1. kuhn k, ostertag s, ostertag m, walter mh, luthardt rg, rudolphh. comparison of an analog and digital quantitative and qualitative analysis for the hit of dental copings. compute biol med 2015; 57:32-41. 2. mously ha, finkelman m, zandparsa r, hirayama h. marginal and internal adaptation of ceramic crown restorations fabricated with cad/cam technology and the heat-press technique. j prosthetic dent 2014; 112:249-56. 3. kale e, seker e, yilmaz b, o] zcelik tb. effect of cement space on the marginal hit of cad-cam-fabricated monolithic zirconia crowns. j prosthetic dent 2016; 116:890-5. 4. chochlidakis km, papaspyridakos p, geminiani a, chen cj, feng ij, ercoli c. digital versus conventional impressions for hixed prosthodontics: a systematic review and meta-analysis. j prosthet dent 2016; 116:184-90. 5. anadioti e, aquilino sa, gratton dg, holloway ja, denry il, thomas gw, et al. 3d and 2d marginal hit of pressed and cad/cam lithium disilicate crowns made from digital and conventional impressions. j prosthodontic2014; 23:610-7. 6. patzelt sb, bishti s, stampf s, att w. accuracy of computer-aided design/ computer-aided manufacturing generated dental casts based on intraoral scanner data. j am dent assoc 2014; 145:1133-40. 7. patzelt sb, lamprinos c, stampf s, att w. the time efhiciency of intraoral scanners: an in vitro comparative study. j am dent assoc 2014; 145:542-51. 3d printed cast is more accurate than convenfonal stone cast for single tooth supported fixed prosthesis vol 13, no 1 (2025) doi 10.5195/d3000.2025.1055 http://dentistry3000.pitt.edu 4 8. lee sj, gallucci go. digital vs. conventional implant impressions: efhiciency outcomes. clinical oral implants res 2013; 24:111-5. 9. shim js, lee js, lee jy, choi yj, shin sw, ryu jj. effect of software version and parameter settings on the marginal and internal adaptation of crowns fabricated with the cad/cam system. j appl oral sci 2015; 23:515-22. 10. pfeiffer j. dental cad/cam technologies: the optical impression (i). int j compute dent 1998; 1:29. 11. dawood a, marti bm, sauret-jackson v, dawood a. 3d printing in dentistry. br dent j 2015; 216:521-9. 12. van noort r. the future of dental devices is digital. dent mater 2012; 28:3-12. 13. mclean jw, von fraunhofer ja. the estimation of cement hilm thickness by an in vitro technique. br dent j 1971; 131:107-11. 14. persson as, andersson m, odén a, sandburgenglund g. computer aided analysis of digitized dental stone replicas by dental cad/cam technology. dent mater 2008; 24:1123-30. 15. ender a, mehl a. accuracy of complete-arch dental impressions: a new method of measuring trueness and precision. j prosthet dent 2013; 109:121-8. 16. cho sh, schaefer o, thompson ga, guentsch a. comparison of accuracy and reproducibility of casts made by digital and conventional methods. j prosthet dent 2015; 113:310-5. 17. güth jf, keul c, stimmelmayr m, beuer f, edelhoff d. accuracy of digital models obtained by direct and indirect data capturing. clin oral investing 2013; 17:1201-8. 18. kim cm, kim sr, kim jh, kim hy, kim wc. trueness of milled prostheses according to number of ball-end mill burs. j prosthet dent 2016; 115:624-9. 19. anadioti e, aquilino sa, gratton dg, holloway ja, denry il, thomas gw, et al. internal hit of pressed and computer-aided design/computeraided manufacturing ceramic crowns made from digital and conventional impressions. j prosthet dent 2015; 113:304-9. 20. liu q, leu mc, schmitt sm. rapid prototyping in dentistry: technology and application. int j adv manuf technol 2006; 29:317-35. 21. o] rtorp a, jönsson d, mouhsen a, von steyern pv. the hit of cobalt chromium three-unit hixed dental prostheses fabricated with four different techniques: a comparative in vitro study. dent mater 2011;27: 356-63. 22. hoang ln, thompson ga, cho sh, berzins dw, ahn kw. die spacer thickness reproduction for central incisor crown fabrication with combined computer-aided design and 3d printing technology: an in vitro study. j prosthet dent 2015; 113:398-404. 23. zhou jg, herscovici d, chen cc. parametric process optimization to improve the accuracy of rapid prototyped stereolithography parts. int j mach tool manufact 2000; 40:363-79. 24. zhou c, chen y, yang zg, khoshnevis b. digital material fabrication using mask-image-projection-based stereolithography. rapid prototype j 2013;19: 153-65. 25. chae mp, rozen wm, mcmenamin pg, findlay mw, spychal rt, hunter smith dj. emerging applications of bedside 3d printing in plastic surgery. front surg 2015; 2:25. 26. murugesan k, anandapandian pa, sharma sk, kumar mv. comparative evaluation of dimension and surface detail accuracy of models produced by three different rapid prototype techniques. j indian prosthodont soc 2012;12: 16-20. 27. hazeveld a, huddleston slater jjr, ren y. accuracy and reproducibility of dental replica models reconstructed by different rapid prototyping techniques. am j ortho dentofacial orthop 2014; 145:108-15. 28. wadhwani cp, johnson gh, lepe x, raigrodski aj. accuracy of newly formulated fast-setting elastomeric impression materials. j prosthet dent 2005; 93:530-9. 29. schaefer o, kuepper h, thompson ga, cachovan g, hefti af, guentsch a. effect of cncmilling on the marginal and internal hit of dental ceramics: a pilot study. dent mater 2013; 29:8518. 30. chew cl, chee ww, donovan te. the inhluence of temperature on the dimensional stability of poly (vinyl siloxane) impression materials. int j prosthodont 1993; 6:528-32. figure 1. scanning image analysis for single unit cixed dental prosthesis. 3d printed cast is more accurate than convenfonal stone cast for single tooth supported fixed prosthesis vol 13, no 1 (2025) doi 10.5195/d3000.2025.1055 http://dentistry3000.pitt.edu 5 figure 2. single unit fdps intaglio scan data subdivisions. table 1. rms values for cixed partial dentures' internal (complete) gap and marginal (complete) discrepancy (mm). the area groups n (rms) (micro m) mean /standard deviation p-value discrepancy of the marginal cs 10 34.88/3.4 3dp 10 43.56/2.6 inferior gap cs 10 25.55/2.1 3dp 10 53.77/2.5 0.001 table 2. mean and ±sd between groups and between sides (2-way anova). groups n mesial mean and standard deviation distal mean and standard deviation p-value conventional cast three d printed 10 27.66/2.4 28.77/2.4 10 52.67/4.3 57.88/4.5 0.001 3d printed cast is more accurate than convenfonal stone cast for single tooth supported fixed prosthesis vol 13, no 1 (2025) doi 10.5195/d3000.2025.1055 http://dentistry3000.pitt.edu 6 table 3. mean and ±sd between groups and between sides (post-hoc tukey hsd test). groups n mesial mean and standard deviation distal mean and standard deviation p-value conventional cast three d printed 10 39.66/2.4 39.77/2.4 10 54.67/4.3 54.88/4.5 0.001 figure 3. maps showing differences in color. a, cs internal cit. b, 3dp's internal cit. c, cs's marginal cit. d, 3dp's marginal cit. 3dp stands for 3d-printed cast; cs stands for traditional stone cast. a b c d 3d printed cast is more accurate than convenfonal stone cast for single tooth supported fixed prosthesis vol 13, no 1 (2025) doi 10.5195/d3000.2025.1055 http://dentistry3000.pitt.edu 7 figure 4. color difference maps of inconsistencies. a: the mesial side of the cs's interior region. b: distal side in 3dp's interior area. c: mesial side in cs's interior area. d represents the distal side of the 3dp's internal area. 3dp stands for 3d-printed cast, while cs is for conventional stone cast. a b c d 943 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.943 http://dentistry3000.pitt.edu gaps in medical emergency preparedness among iraqi dentists: a nationwide cross-sectional analysis awf sh. mahmood1, meena muneeb2 1college of den*stry, al-iraqia university, baghdad, iraq 2dijlah university college, baghdad, iraq abstract objec5ve: to evaluate the preparedness of iraqi dental practitioners in managing medical emergencies by assessing self-reported confidence, theoretical knowledge, prior clinical exposure, and formal training in basic life support (bls) and emergency management protocols. material and methods: a nationwide cross-sectional study was conducted from november 2024 to march 2025 using a validated, self-administered electronic questionnaire distributed to licensed iraqi dentists. the survey assessed demographic characteristics, knowledge of emergency protocols, prior exposure to emergencies, and participation in bls or related training. data were analyzed using spss version 22 to explore associations between preparedness and professional or demographic variables. results: a total of 531 dentists participated; 58.9% were female, and 86% were general practitioners. only 47.2% reported confidence in managing medical emergencies. furthermore, 86.4% demonstrated moderate to low knowledge, and most had limited clinical exposure. formal bls/cpr training was reported by 35.2%, and 31.7% had attended structured emergency management courses. prior training was significantly associated with greater confidence and preparedness (p < 0.05). conclusion: substantial deficiencies were identified in the emergency preparedness of iraqi dentists, particularly in clinical exposure and formal training. these findings highlight the urgent need to incorporate mandatory bls certification, simulation-based instruction, and continuous professional development into dental education and licensure frameworks, aligning national practice with international standards and enhancing patient safety. open access cita%on: mahmood as, et al. (2025) gaps in medical emergency preparedness among iraqi den%sts: a na%onwide cross-sec%onal analysis. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.943 received: may 16, 2025 accepted: july 6, 2025 published: august 11, 2025 copyright: ©2025 mahmood as, et al. this is an open access ar%cle licensed under a crea%ve commons auribu%on work 4.0 united states license. email: awf.sh.mahmood@aliraqia.edu.iq introduc)on although medical emergencies are relatively rare in dental settings, their occurrence can be life-threatening and demand immediate, competent intervention. beyond performing routine procedures, dental practitioners are responsible for recognizing and managing acute medical events that may arise due to patients' underlying systemic conditions, anxiety, procedural stress, or adverse drug interactions [1-3]. common emergencies encountered in dental clinics include vasovagal syncope, angina, hypoglycemia, seizures, airway obstruction, asthma exacerbations, anaphylaxis, and, in severe cases, cardiac arrest [4-6]. with the growing prevalence of chronic diseases, the likelihood of encountering such scenarios is increasing, underscoring the need for preparedness to mitigate preventable morbidity, mortality, and medicolegal consequences. despite the existence of international guidelines emphasizing the importance of emergency readiness, evidence suggests that many dental practitioners lack adequate training in medical emergency management [1,7]. competence in this area requires formal instruction in basic life support (bls), advanced cardiovascular life support (acls), familiarity with emergency pharmacology, and proficiency in the use of emergency equipment. nevertheless, global studies continue to reveal deficiencies in preparedness, often attributed to limited training opportunities, insufficient regulatory mandates, and variability in emergency protocols across clinical settings [8-10]. furthermore, many dental professionals report low confidence in managing emergencies, reflecting a discrepancy between theoretical knowledge and clinical performance [11]. compounding this issue, the availability of emergency medications and essential equipment in dental clinics is frequently inadequate, particularly in private practice settings. such deficiencies can delay life-saving interventions, especially in cases where gaps in medical emergency preparedness among iraqi den`sts: a na`onwide cross-sec`onal analysis vol 13, no 1 (2025) doi 10.5195/d3000/2025.943 http://dentistry3000.pitt.edu 2 emergency medical services are delayed or unavailable [6,12]. in iraq, despite the critical nature of this issue, there is a notable lack of data on dental practitioners' preparedness for medical emergencies. as such, this study aims to evaluate the knowledge, confidence, prior exposure, and training of iraqi dentists regarding emergency management. by identifying existing gaps, this research seeks to provide an evidence-based foundation for educational reform and policy development within a developing healthcare system. material and methods study design this descriptive cross-sectional study was conducted between december 2024 and march 2025 to assess the preparedness of iraqi dental practitioners in managing medical emergencies. the study adhered to the strobe guidelines for cross-sectional research. survey design & development of questionnaire a structured, self-developed questionnaire was designed by the authors based on a comprehensive review of the literature on emergency management in dental settings. content validity was established through evaluation by five independent experts in oral and maxillofacial surgery and oral medicine, who assessed each item for clarity, relevance, and completeness. a pilot study involving 30 licensed practicing dentists was conducted to evaluate validity and internal consistency, with refinements made accordingly before [inal distribution. the final questionnaire consisted of three core domains: 1. demographic and professional profile, including gender, academic qualification, specialty, and years of clinical experience. 2. knowledge assessment, comprising 14 multiple-choice items focused on emergency recognition, pharmacologic protocols, and clinical response. each correct response was awarded one point. total knowledge scores were categorized as high (11–14), moderate (7–10), or low (0–6). 3. training and preparedness, capturing self-reported confidence in handling medical emergencies, prior exposure to such events, and previous participation in basic life support (bls), cardiopulmonary resuscitation (cpr), and advanced cardiovascular life support (acls) training courses. the complete questionnaire is available in table 1 to support transparency and reproducibility. data collection the questionnaire was distributed electronically through official mailing lists of dental associations, academic platforms, and professional social media groups. eligible participants were licensed iraqi general dentists and dental specialists actively engaged in clinical practice. dental students and nonpracticing individuals were excluded to ensure the accuracy and relevance of the sample. a convenience sampling approach was used. sample size determination the required sample size was calculated using slovin's formula, assuming a target population of 19,000 iraqi dentists, a 95% confidence level, and a 5% margin of error. the minimum calculated sample was 392. a total of 531 valid responses were collected, exceeding the requirement and enhancing the generalizability of the results. ethical considerations ethical approval was obtained from the faculty of dentistry, dijlah university (ref no.29 on 16 october 2024). all participants were informed of the voluntary and anonymous nature of the study, and electronic informed consent was obtained prior to participation and completion of the survey. statical analysis data was analyzed using spss version 25 (ibm corp., armonk, ny, usa). descriptive statistics, including frequencies and percentages, were used to summarize categorical and continuous variables. inferential statistics were applied to examine associations between knowledge levels and participant characteristics. pearson chi-square tests and post hoc analyses were used to evaluate group differences. the contingency coefficient (c.c.) was calculated to determine the strength of associations among categorical variables. a p-value of 0.05 or less was considered statistically significant, and outcomes were classified as non-significant, significant, or highly significant. no subgroup or sensitivity analyses were conducted beyond the descriptive and comparative statistics reported, as the primary objective was to assess preparedness levels and associated factors within the overall study sample consistent with the cross-sectional design. results a total of 531 licensed dental practitioners from across iraq participated in this study. of these, 313 (58.9%) were female and 218 (41.1%) were male. most respondents (81.7%) reported less than five years of clinical experience, while 7.7% had practiced for 5–10 years, 4.9% for 11–19 years, and 5.6% for 20 years or more. regarding qualifications, general practitioners represented 86% of the sample (n = 457), while specialists comprised 14% (n = 74). the sociodemographic characteristics of the participants are summarized in table 2. emergency preparedness is detailed in table 3. notably, 47.3% of respondents (n = 251) reported confidence in managing medical emergencies, whereas 52.7% (n = 280) did not; this difference was not statistically significant (p = 0.557). a history of encountering a medical emergency was reported by 45.0% (n = 239), compared to 55.0% (n = 292) who had not (p = 0.275). in contrast, only 35.2% (n = 187) had received formal basic life support (bls) or cardiopulmonary resuscitation (cpr) training, a statistically significant difference compared to the 64.8% (n = 344) who had not (p = 0.001). furthermore, 31.7% of participants (n = 168) attended structured emergency management courses, while 68.3% (n = 363) had not—a highly significant finding (p < 0.001). knowledge was assessed using a 14-point scale covering core emergency management concepts. overall, 13.3% of respondents demonstrated high knowledge (mean score: 11.79), 47.1% had moderate knowledge (mean score: 8.55), and 39.4% exhibited low knowledge (mean score: 4.63), as demonstrated in table 4. statistical analysis revealed several significant associations. emergency exposure was significantly associated with gender [correlation coefficient (cc) = 0.179, p = 0.03] and professional qualification (cc = 0.239, p = 0.013). confidence in managing emergencies was strongly associated with prior exposure (cc = 0.242, p = 0.003) and significantly related to attendance at emergency management courses (cc = 0.201, p = 0.014). prior exposure to emergencies was also highly associated with having received bls training (cc = 0.270, p = 0.001) and with participation in emergency courses (cc = 0.256, p = 0.002). these correlations are detailed in table 5. discussion this study reveals significant deficiencies in the medical emergency preparedness of iraqi dental practitioners in terms of clinical gaps in medical emergency preparedness among iraqi den`sts: a na`onwide cross-sec`onal analysis vol 13, no 1 (2025) doi 10.5195/d3000/2025.943 http://dentistry3000.pitt.edu 3 exposure, confidence, and formal training. only 47.2% of respondents reported confidence in managing medical emergencies. this alarming figure aligns with international literature that links limited practical exposure and inadequate training to diminished self-efficacy in emergency response. these findings highlight a compelling need to integrate structured, simulation-based emergency training within both undergraduate and postgraduate dental education in iraq to ensure baseline competence and enhance clinical responsiveness. the lack of clinical exposure emerged as a significant concern, with 55% of participants indicating they had never encountered a medical emergency in practice. additionally, only 35.2% had received formal basic life support (bls) or cardiopulmonary resuscitation (cpr) training, and just 31.7% had attended structured emergency management courses—both statistically significant gaps (p = 0.001 and p < 0.001, respectively). these deficiencies directly threaten patient safety and hinder effective emergency management. prior research, including those by wu et al. and manton et al, has consistently demonstrated that repeated exposure to simulation-based scenarios significantly enhances readiness and decision-making in acute clinical situations. [13,14]. similarly, anwar et al. emphasized the importance of systematic, curriculum-based emergency instruction in bridging the knowledge-practice gap among dental professionals [15]. comparable inadequacies have been documented in regional studies. a national survey by alotaibi et al. on bls certi[ication rates among saudi dentists, while ilyas et al. and tar binti midzi et al. found that the absence of formal emergency education in malaysia and saudi arabia similarly compromised preparedness [16-18]. in contrast, regulatory frameworks in the united states and the united kingdom mandate bls/cpr certification for licensure, with periodic recertification ensuring sustained competency. iraq currently lacks such regulatory enforcement, exacerbating the systemic vulnerability in emergency management. substantial international evidence affirms that mandatory training and revalidation policies significantly improve preparedness and clinical outcomes [19]. notably, years of clinical experience were not significantly associated with preparedness (p > 0.05), a finding consistent with bhagat et al. [20]. this suggests that clinical tenure alone does not ensure competence reinforces the necessity of structured, recurrent training. however, male practitioners and dental specialists were significantly more likely to have encountered medical emergencies (p = 0.030 and p = 0.013, respectively), likely due to greater clinical exposure levels and practice environments. these patterns are consistent with observations by varoni et al., who identified clinical settings and patient caseload as critical determinants of preparedness [21]. further multivariate analysis revealed that confidence in managing emergencies was significantly associated with previous clinical exposure (p = 0.003), participation in emergency training courses (p = 0.014), and receipt of bls/cpr instruction (p = 0.002). these associations confirm that structured training improves theoretical knowledge, practical confidence, and operational agility. accordingly, integrating emergency preparedness training into continuing professional development (cpd) frameworks and licensure requirements is essential for a resilient and competent dental workforce. the overall preparedness score in this study reflects a moderate to poor level of readiness, echoing findings from jaber et al. and other regional studies. given the well-documented decline in emergency skills, periodic re-evaluation and refresher training are imperative. sustainable preparedness hinges on initial educational interventions and longitudinal reinforcement through validated assessments and mandatory workshops [6,22]. conclusions the findings reveal a substantial deficiency in medical emergency preparedness among iraqi dental practitioners. bridging the gap necessitates the urgent need for comprehensive, well-structured practical training programs. incorporating mandatory bls/cpr certification, simulation-based training, and continuous professional development (cpd) into dental education and licensure requirements is essential. these interventions will reinforce practitioner competence, enhance patient safety, and align emergency response protocols in iraq with international standards of care. conflicts of interest the authors state that there are no con[licts of interest. financial support this research was not supported by any grants or external funding sources. references 1. fernandes al, malik jb, ansari sr, murali s, thirupathii j. knowledge, attitude, and practice of dentists in the management of medical emergencies in india: a cross-sectional study. j oral biol craniofac res. 2023 nov;13(6):758–63. 2. abraham s, badgujar m, nagmode p, lokhande n. common medical emergencies in dentistry: a review. world j adv res rev. 2022;15(3):443–9. 3. pius l, brady n, overby m, zhu j, ferraro n. emergency protocol in the dental clinic: assessing medical emergency training requirements and guidelines for dentists. j am dent assoc. 2023 apr;154(4):301–10. 4. müller mp, hänsel m, stehr sn, weber s, koch t. a state-wide survey of medical emergency management in dental practices: incidence of emergencies and training experience. emerg med j. 2008 may;25(5):296–300. 5. reed kl. basic management of medical emergencies: recognizing a patient's distress. j am dent assoc. 2010 may;141 suppl 1:s20–4. 6. choufani a, moussallem m, dib jb, asmar mk, yeretzian js. lebanese dentists’ preparedness to deal with medical emergencies in their clinics: a national survey. int dent j. 2025 feb;75(1):324–32. 7. solanki c, geisinger ml, luepke pg, al-bitar k, palomo l, lee w, et al. assessing readiness to manage medical emergencies among dental students at four dental schools. j dent educ. 2021 sep;85(9):1462–70. 8. cheng a, magid dj, auerbach m, bhanji f, bigham bl, blewer al, et al. part 6: resuscitation education science: 2020 american heart association guidelines for cardiopulmonary resuscitation and emergency cardiovascular care. circulation. 2020 oct 20;142(16 suppl 2):s551–79. 9. vaughan m, park a, sholapurkar a, esterman a. medical emergencies in dental practice– management requirements and international practitioner proficiency: a scoping review. aust dent j. 2018 dec;63(4):455–66. 10. cardoso mb, brusca mi, verdu sd, jewtuchowicz v. medical emergencies in the dental office: challenges and solutions. odontol (montevideo). 2023;(1):15. 11. kishimoto n, sanuki t, liu y, tran sd, seo k. simulation training for medical emergencies of dental patients: a review of the dental literature. jpn dent sci rev. 2023 dec;59:104– 13. 12. nemati s, hamedani s. emergency medications and equipment indispensable for dental offices and clinics in iran. j dent (shiraz). 2024 dec;25(4):374. 13. wu jh, lin pc, lee kt, liu hl, lu py, lee cy. situational simulation teaching effectively improves dental students’ non-operational clinical competency and objective structured clinical examination performance. bmc med educ. 2024 may 14;24(1):533. 14. manton jw, kennedy ks, lipps ja, pfeil sa, cornelius bw. medical emergency management in the dental office (memdo): a pilot study assessing a simulation-based training gaps in medical emergency preparedness among iraqi den`sts: a na`onwide cross-sec`onal analysis vol 13, no 1 (2025) doi 10.5195/d3000/2025.943 http://dentistry3000.pitt.edu 4 curriculum for dentists. anesth prog. 2021 jun 29;68(2):76. 15. anwar ma, rehman iu, khalil f, saeed t, shakeel s, arif k, kushef h. knowledge, skills, and perceived competency in handling medical emergencies among dental house officers and general dentists. pak j health sci. 2024 oct 31:158–63. 16. alotaibi o, alamri f, almufleh l, alsougi w. basic life support: knowledge and attitude among dental students and staff in the college of dentistry, king saud university. saudi j dent res. 2016 jan;7(1):51–6. 17. ilyas m, ul ma, saleem a, shaikh g, tariq a. knowledge and attitude of dental professionals towards basic life support. saudi j oral dent res. 2020;5(6):291–4. 18. tarmidzi na, ramii nb, amran n, norazmi hn, arifin ns. knowledge, attitude and perception of private dental practitioners towards medical emergencies in klang valley, malaysia. j int dent med res. 2022 may;15(2):649–55. 19. shivakumar s, doddawad vg, shetty sk, shivanagappa m, narayanaswamy cs, shetty a, bhat hk. effectiveness of basic life support training course intervention among dental practitioners and students: an experimental study. natl j maxillofac surg. 2024 jan;15(1):87–92. 20. bhagat t, shrestha a, agrawal sk, gautam u, mishal r. medical emergencies preparedness in dental clinic among postgraduate residents in nepal. j chitwan med coll. 2024 mar 30;14(1):14–9. 21. jaber l, al-qarni f, alsaati m, al-nefaiee m, shetty ac, shaban s, et al. perspectives of dental practitioners regarding their readiness for medical emergencies: a study in saudi arabia. open dent j. 2021 dec 31;15(1):72–8. 22. varoni em, rigoni m, lodi g, sardella a, muti p, vitello a, et al. medical emergencies in dental practice: a nationwide web-based survey of italian dentists. heliyon. 2023 mar 1;9(3):e13910. table 1. the questionnaire of the study part (1): sociodemographic part q1 gender □ male □ female q2 years of experience □ less than 5 years □ 5–10 years □ 10–19 years □ more than 20 years q3 qualification □ general practitioner □ specialist part (2): knowledge-based assessment q4 what is the most frequently encountered medical emergency in dental practice? a. syncope b. hypoglycemia c. allergic reaction d. seizure q5 a 25-year-old asthmatic patient begins wheezing and has difficulty breathing during a dental procedure. what should be done initially? a. administer oxygen b. administer an epinephrine im injection c. administer albuterol d. call emergency medical assistance q6 a diabetic patient, during a dental procedure, feels sweaty, tired, disoriented, and has a headache. the condition is most likely: a. hyperglycemia b. hypoglycemia c. angina d. anaphylaxis q7 during a dental procedure, a young adult patient loses consciousness, followed by generalized tonic-clonic movements; after 2 minutes, the seizure subsides, but the patient remains unconscious. what is the next immediate step in managing this condition? a. administer 5 mg midazolam b. activate emergency medical services c. place the patient in a recovery position and maintain the airway d. monitor vital signs and wait for the patient to regain consciousness q8 an adult male patient feels dizzy and sweaty and experiences crushing pain radiating down the left arm, neck, and mandible, associated with nausea and vomiting. the condition is most likely: a. angina b. insulin shock c. asthmatic attack d. myocardial infarction q9 when do most medical emergencies occur in dental practice? a. during treatment b. immediately after treatment c. 2–3 hours after treatment d. during or shortly after local anesthesia injection q10 what is the primary cause of syncope in the dental office? a. anxiety b. hypertension c. overexertion d. prolonged treatment q11 what is the immediate action when a patient experiences syncope during dental treatment? a. place the patient in the trendelenburg position b. ask the patient to sit in an upright position c. recline the patient and give oral glucose d. place the patient on their side q12 a conscious adult patient complains of difficulty breathing and speaking, holding their neck with one hand. the probable diagnosis is: a. asthma – give albuterol b. angina – administer oxygen c. aspiration of a foreign body – heimlich maneuver d. respiratory arrest – back blows gaps in medical emergency preparedness among iraqi den`sts: a na`onwide cross-sec`onal analysis vol 13, no 1 (2025) doi 10.5195/d3000/2025.943 http://dentistry3000.pitt.edu 5 q13 a diabetic patient shows symptoms of rapid pulse and pale, cold, and clammy skin. what is the first line of treatment? a. oral glucose b. glucagon 1 mg injection c. sublingual nitroglycerine d. oxygen q14 a young adult patient suddenly becomes pale with a flushed face and develops shortness of breath and gasping. the best action is: a. epinephrine 1 mg/ml im b. gtn 400 micrograms c. salbutamol inhaler 100 micrograms d. dispersible aspirin 300 mg q15 a young adult patient suddenly becomes pale with a flushed face and develops shortness of breath and gasping. the best action is: a. epinephrine 1 mg/ml im b. gtn 400 micrograms c. salbutamol inhaler 100 micrograms d. dispersible aspirin 300 mg q16 what is the best position for a conscious patient who has developed a myocardial infarction, and which drug would you choose for such a condition? a. comfortable position and aspirin b. supine position and aspirin c. reclined position and oxygen d. supine position and oxygen q17 what is the best treatment for a patient who has crushing pain extending to the neck and jaw? a. aspirin b. sublingual glyceryl trinitrate c. iv saline infusion d. insulin sc answer key – questions 4 to 17 4. a | 5. c | 6. b | 7. c | 8. d | 9. d | 10. a | 11. a | 12. c | 13. a | 14. a | 15. a | 16. a | 17. b part (3): training and preparedness assessment q18 do you feel confident in handling medical emergencies during dental procedures? □ yes □ no q19 have you ever encountered a medical emergency during a dental procedure? □ yes □ no q20 did you receive practical basic life support (bls) or cardiopulmonary resuscitation (cpr) training? □ yes □ no q21 have you attended any courses in the management of medical emergencies? □ yes □ no table 2. sociodemographic characteristics (sdc) of the participants. sdc groups no. % gender male 218 41.1 female 313 58.9 years of experience < 5 yrs. 434 81.7 5 10 yrs. 41 7.7 11 19 yrs. 26 4.9 ≥ 20 yrs. 30 5.6 qualification gp 457 86 specialists 74 14 gaps in medical emergency preparedness among iraqi den`sts: a na`onwide cross-sec`onal analysis vol 13, no 1 (2025) doi 10.5195/d3000/2025.943 http://dentistry3000.pitt.edu 6 table 3. confidence, exposure, and trainingrelated to medical emergency preparedness. table 4. knowledge level stratification of participants based on a 14-point assessment scale. knowledge score no. of participants % of sample average score minimum score high knowledge (11–14 points) 71 13.3 11.79 11 moderate knowledge (7–10 points) 251 47.1 8.55 7 low knowledge (0–6 points) 209 39.4 4.63 0 note: scores reflect participant responses on a 14-point knowledge assessment scale. questions response n % p-value confident in handling medical emergencies? yes 251 47.3% 0.557 no 280 52.7% have you encountered a medical emergency previously? yes 239 45.0% 0.275 no 292 55.0% have you received bls/cpr training? yes 187 35.2% 0.001 no 344 64.8% have you attended emergency management courses? yes 168 31.7% <0.001 no 363 68.3% gaps in medical emergency preparedness among iraqi den`sts: a na`onwide cross-sec`onal analysis vol 13, no 1 (2025) doi 10.5195/d3000/2025.943 http://dentistry3000.pitt.edu 7 table 5. correlation analysis of exposure, confidence, training, gender, and professional qualification. variables analyzed correlation coefjicient (cc) p-value gender and emergency exposure 0.179 0.03 quali[ication and emergency exposure 0.239 0.013 con[idence and prior exposure 0.242 0.003 con[idence and emergency course attendance 0.201 0.014 encountered emergency and bls training 0.270 0.001 encountered emergency and emergency course attendance 0.256 0.002 cc: correlation coef[icient; hs: highly signi[icant (p ≤ 0.01); s: signi[icant (p ≤ 0.05) 1058 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1058 http://dentistry3000.pitt.edu analyzing the effects of polident and thyme extract oil on denture base hawraa khalid aziz, maha kareem jabbar, najwah yousuf hameed, sara abdulbasit turki college of health and medical techniques, middle technical university, baghdad, iraq abstract objec@ve: dentures are the most common remedy for edentulous human populaion. natural products and essenial oils provide promising therapeuic agents for the treatment of oral infecions. the increasing understanding of the diverse applicaions of natural resources has made them a favored subsitute for manufactured materials. therefore, the goal of this study was to find out how different immersion soluions [thyme oil, polident denture clean] changed the roughness, hardness, and transverse strength of 3d-printed pmma denture base. materials and methods: the current study produced ninety 3d-printed resin samples, each measuring [65mm ×10mm×2.5mm] and classified them into three groups including 30 samples, each group were submerged into three groups depending on immersion soluions [control, thyme essenial oil, and polident denture cleaner] for a duraion of 15 days. awer that the samples were tested for surface roughness, hardness, and transverse strength. results: the polident immersion group had the highest surface roughness values followed by thyme essenial oil, whereas the control group was demonstrated the lowest mean values. likewise, the surface hardness test findings demonstrated that the polident immersion group exhibited the greatest mean values, while the thyme immersion group had the lowest mean values but staisically was not significant. furthermore, the transverse strength test indicated the lowest average transverse strength for the thyme essenial oil group. conclusion: thyme essenial oil as a denture cleaner be\er preserves surface roughness but has a lesser influence on the hardness and transverse strength of the 3d print denture base compared to polident denture cleanser. open access cita%on: aziz hk, et al. (2025) analyzing the effects of polident and thyme extract oil on denture base. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1058 received: september 30, 2025 accepted: october 2, 2025 published: november 4, 2025 copyright: ©2025 aziz hk, et al. this is an open access ar%cle licensed under a crea%ve commons awribu%on work 4.0 united states license. email: hawraa.khalid.azizaziz@mtu.edu.iq introduc)on an increasing number of older people are regularly using dentures as a result of the rising prevalence of edentulism and aging [1,2]. people who wear dentures for a long time have more mutans, streptococci, lactobacilli, staphylococci, microorganisms, and yeasts in their mouths than people who do not wear them [3,4]. throughout the denture placement appointment, home care instructions are essential to help maintain healthy oral mucosa, especially for elderly patients with disease, dementia, and poor dexterity of who cannot brush their dentures adequately [5,6]. alqanas et al. have proposed denture cleansers for denture cleaning and maintenance protocols, emphasizing the importance of keeping excellent denture hygiene for healthy oral mucosa. denture cleaners should get rid of biojilm without changing the properties of the denture base material. long-term use can change the color, hardness, and surface roughness of denture base resins [7]. an ideal denture cleaning should be biocompatible, bactericidal, fungicidal, non-harmful to the denture structure, effective in removing both organic and inorganic deposits, and user-friendly [8]. almost all cleaners’ dentures contain oxidizing agents, reducing agents, effervescent compounds, detergents, enzymes, and disinfectants [9]. the most frequently used alkaline peroxides, but denture cleansers are breaking down the physical characteristics of denture base resins and pathogenic microorganisms were becoming more resistant to these chemicals. this has encouraged people to look at plant extracts as novel antibacterial and antifungal drugs. natural products and essential oils give excellent therapeutic agents for oral infections [10,11]. research comparing plant extracts indicates that thyme essential oil is an effective antibacterial agent against candida albicans, rendering it appropriate for denture cleaning [12]. despite its recognized antibacterial capabilities, there is limited research on how thyme essential oil affects the physical qualities of analyzing the effects of polident and thyme extract oil on denture base vol 13, no 1 (2025) doi 10.5195/d3000.2025.1058 http://dentistry3000.pitt.edu 2 denture base materials when used as a denture cleaning agent. the hypothesis of present study is that the solution of thyme essential oil and polident will not change the properties of the 3d printed resin. so, this study oriented to assess the effects of two distinct denture cleansers on surface roughness, hardness, and transverse strength of three-dimensional printed denture base. materials and methods fabrication of 3d printing design cad specimens the specimens were constructed of 3d printed resin denture base (flexo denture base, v2, senertek, turkey) by using 3d printing cad device (phrozen, thailand), before printing the specimens designed with bar-shaped dimensions (65mm, 10mm, and 2.5mm) for length, width, and thickness, respectively using chitobox software according to ada no. 12, 1999. the sample plan was saved as an stl file, which was then loaded into 3d printing software for the planned denture base [13]. they exported the 3d printer software at a 90 degree angle [14]. then added an appropriate quantity of resin to the print tank. following the printing process by three-dimensional printing with a specified 50 µm layer thickness , removed the denture base specimen from the platform and trimmed off any excess materials, after that immersed them in to isopropyl ethyl alcohol in two steps: first for 2 minutes in one container, followed by another 3 minutes in a second container using an ultrasonic instrument (bella gusto, china) [15]. the uv light post-curing process for 3d printed specimens the samples were subsequently subjected to uv light at an intensity of 405 nm for polymerization, which took place over a duration of 10 minutes [16]. the support structures were then separated by exerting light pressure at the junction where the support was attached to the printed component. after the removal of the support structures, finished and polished the external surface only of the denture bases specimens using the same tools and techniques used for finishing and polishing conventional heat cured denture base [15]. specimen grouping the study used ninety specimens made of 3d printed denture base resin. three tests, which included the surface roughness, surface hardness, and transverse strength tests. each one involved thirty specimens. the immersion procedure divided the 30 specimens into three groups, each including 10 specimens, as shown below. group i: the specimens immersed in 100ml of distilled water, which was considered a control. group ii: the specimens were immersed in 100ml of thyme essential oil solution for denture cleaning. group iii: the specimens were immersed in 100ml of polident denture cleanser tablet. prepare a denture cleanser solution preparation of thyme essential oil to create 1000 ml of a diluted mixture for immersion treatments, blend 5ml of thyme essential oil, 5ml of 0.5% tween, 80ml of ethanol, and 910 ml of distilled water [11]. preparation of polident denture cleanser to prepare the denture cleanser, dissolve one polydent tablets[stafford-miller, ireland] in 100ml of distilled water. after manufacturing denture cleanser solutions, perform the immersion process by immersing the specimens in each corresponding solution for a continuous duration of 15 days [17]. cleansing method specimens experienced daily washing by dipping 10specimens from each group. among the three different solutions, continuously for period 15 days, which replicated three years of usage based on their respective subgroups at room temperature. the determination of these immersion durations was executed using the subsequent procedure. one hour comprised three immersions of 20 minutes each, while a 24-hour period equated to 72 immersions of 20 minutes daily. consequently, to fulfill a three-year immersion simulation [1095 days], fifty days were necessary [18]. testing the specimens surface roughness test the profilometer device (time group inc., tr220, china) uses a sharp, sensitive diamond-crafted needle (stylus) as a surface analyzer to outline the profile of surface imperfections. the test was conducted according to the instructions provided by the profilometer. then divided each sample into three equal portions, and the stylus contacted these three standardized portions to obtain three readings for each one. the stylus makes contact at 5mm in each area of the sample, and positioned the sample on a firm and stable surface, allowed the stylus to contact the first region and the digital scale automatically displayed the reading. the mean of the three measurements was regarded as the roughness value (δra), represented by µm. surface hardness test the shore d durometer was employed to assess the hardness of 3d printed acrylic samples. the testing value was determined by calculating the mean of three distinct readings from the durometer scale. after established a twenty-millimeter distance between the surface of each sample and the indenter in three time (right, center, and left)] for the hardness test, and recorded the average values for each specimen. transverse strength test the universal instron testing equipment (jianqiao testing equipment, china) was used for the transverse strength test. the three-point bending technique was used to examine the transverse strength of acrylic samples. the prepared thirty samples were positioned them on a bending setup, which featured two parallel supporting arms spaced about 50mm apart. a maximum load of 50 kg was applied at a cross-head speed of 1 mm/min via a rod positioned at the center of the sample until fracture initiation occurred. subsequently, calculated the transverse strength values by employing the following equation: t=3pl /2bd2 t represents transverse strength (n/mm2). p denotes the greatest load exerted on samples (newton). l indicates the space between the supporting arms(mm). the ada specification defines b as the width and d as the depth of samples (mm) (ada specification no.12, 1999). statistical analysis the data study computerized by using spss (statistical package for the social sciences). to compare the mean values of the different groups, one-way anova and tukey-hsd test post hoc test were utilized. all statistical calculations were conducted at a 0.05 significance level. results surface roughness test table 1 showed the mean and standard deviation for all the groups of 3 d-polymerized resins that were tested after being soaked in different denture cleaners. the polident cleaner group iii had the highest surface roughness (δra) of the 3 d-polymerized resins, measuring 1.4348 µm. the thyme essential oil group ii came next, with a measurement of 1.0961 µm, whereas the control group i had the lowest roughness (δra). an anova-test was applied to evaluate the data, indicating a significance difference of statistics among these groups (p<0.05). as seen in table 2, the tukey-test revealed, after all examined groups, a significant difference in surface roughness between each two groups. surface hardness test table 3 compares the mean values for surface hardness test after soaking in various denture cleaning methods. the surface hardness findings of the groups indicate that the analyzing the effects of polident and thyme extract oil on denture base vol 13, no 1 (2025) doi 10.5195/d3000.2025.1058 http://dentistry3000.pitt.edu 3 polident immersion group iii exhibits the greatest mean values, while the thyme immersion group ii displays the lowest mean values. however, the anova-test result revealed no differences statistically among all groups. transverse strength test the data were evaluated utilizing descriptive statistics. the specimens in group iii that were immersed in polident solution had the highest mean transverse strength (17.14250 n/mm²). the specimens in control group had the lowest mean value (14.22690 n/mm²). further analysis was need for comparison among the three groups by the anova test that was revealed extremely signijicant differences (table 4). the tukey hsd-test was utilized for multiple comparisons among all groups (table 5). the tukey test jindings demonstrated a highly signijicant difference within the groups. discussion physical cleaning procedures frequently combine with chemical denture cleansing techniques to ensure the cleanliness and hygiene of dentures. older patients with severe diseases, such as disabling illnesses, diminished manual dexterity, and neuromuscular dysfunction, sometimes are unable to clean dentures. so that allows for the growth of bacterial and candida, leading to severe resources for disseminating infection [19]. some research indicates that denture cleanser usage greatly reduces the amount of bacteria on dentures, particularly in older adults [20]. traditional chemical denture cleaners are good at killing microbes, but natural plant extracts will soon be used instead because microbes are becoming more resistant to them. mouth rinses, toothpastes, and similar products utilize these plant extracts for their effective antibacterial and antifungal activities [8]. gutierrez et al. [21] had proved that thyme essential oil was selected as the botanical extract for the denture cleaning since it completely inhibits microorganisms. gonçalves et al. [19] described that thyme essential oil was the most useful for killing streptococcus mutans. several studies have investigated the beneficial properties of thyme essential oil, but less research has focused on its impact on denture base resin. researchers like peracini et al. [5] and sharma et al. [22] found that polident was better than other commercially available denture cleaners, especially when compared to hypochlorite and alkaline peroxides. it also didn't change the strength or roughness properties of denture base resins in a big way. thus, it was utilized for comparison with the denture cleaner derived from the thyme vulgaris plant extract. in this study, the null hypothesis was thrown out due to significant differences seen between the chemical cleanser [polident] and the essential oil cleanser (thyme extract) regarding the roughness, hardness, and flexural strengths of all examined material. the surface roughness test evaluates the imperfections of denture surfaces, especially the fitting surface, which might harbor microorganisms and facilitate persistent reinfection of the palate. several factors affect the surface texture of acrylic dentures. these include the amount of residual methyl methacrylate monomer, the polymerization process and cycle, the amount of time the dentures are stored in water, and how often they are washed [23]. research indicates that the quantity of microbes on smooth surfaces is comparatively lower than on rough surfaces and the removal of biofilm becomes more difficult as the surface roughness increases [24]. when exposed to denture cleaners, the texture of denture foundation acrylic resin undergoes extensive surface morphological alterations [6]. surface roughness, characterized by ra values, is an acknowledged means of analyzing surface textures in research [25]. the results presented in table 2 indicate that the polident denture cleanser group had a greater mean roughness value (1.4348 µm) compared to the thymes essential oil group (1.0961 µm) and the control group (0.3321 µm). this led to a rise in the roughness of the acrylic specimens. these results may be due to the perborate used in polident denture cleaning, which might cause surface degradation, resulting in enhanced surface roughness. the present study's results align with those of mossa et al. [26]. also, these findings were similar to those of namala and hedge [8], they concluded that the surface roughness in the thyme oil group increased less than that of the polident denture cleanser group. alqanas et al. [6], they demonstrated that soaking 3d-printed acrylic in polident denture cleanser increased surface roughness. the findings of this study agreed with our observations. the findings of panariello et al. [27], were different from this study’s result because the surface roughness did not get better in the polident denture cleaning group; but it stayed the same as in the untreated group. khandelwal et al., showed that the thymes oil group had a lower average value than both the untreated group and the polident denture cleanser group. they explained their results by stating that the variation in surface roughness is attributable to their composition and the immersion solution. thymol, which (2-isopropyl-5-methylphenol) and carvacrol (5-isopropyl-2-methylphenol) have the principal antibacterial constituents found in thyme oil. thyme essential oil was utilized as an extract from plants as a denture cleanser due to its low minimum inhibitory concentration (mic) values and its antifungal and antibacterial properties [28]. conversely, the outcomes of the surface hardness test showing no significant variances within these groups. following 15 days of immersion, table 3 indicates that the thymes oil group exhibited a lower mean surface hardness value (39.4000 mpa) related to the control group (41.1000 mpa), but the polident group recorded a value of (41.7000 mpa). these findings agreed with those reported by hatim et al. [29], they concluded that when thyme oil and nigella oil were added at concentrations of 1%, 1.5%, and 2%, the denture base became harder, but there were no big differences between the concentrations. the interaction between the unreacted monomer and the oil-coated polymer explains this alteration. panariello et al. [27], demonstrated that the application of this protocol had no impact on hardness, which was partially accepted. similar results to this study were found by neppelenbroek et al. [30], who also discovered that denture base resins that were treated with sodium hypochlorite and perborate mixture exhibited a significant decrease in average hardness values. the explanation of this result by stating that the thymol oil exerts its solvent effect on the surface of acrylic and other thermoplastic resins, the saturated salt solution results in increased water absorption within the acrylic, as sodium chloride, an ionic compound, forms an infinite repeating lattice of ions when dissolved in water. the polar nature of resin molecules makes it easier for water to dissolve due to electrostatic interactions. this makes the material's surface less hard. this investigation aligns with the conclusions of the current research. moussa et al. [26], contest our results, saying that the mean surface hardness of the polident denture cleaner group is lower than that of the control group. the hardness values significantly diminished among all groups, contradicting the findings of alqanace et al. [6]. a study by khandelwal et al. [28], they found that the control group had a harder surface than both the thyme essential oil group and the polident denture cleanser group. this varies from our studies; these differences may be attributed to several reasons, like immersion duration, the polymerization method of pmma, and polishing denture procedures. the current study demonstrates that the transverse strength of the pmma denture analyzing the effects of polident and thyme extract oil on denture base vol 13, no 1 (2025) doi 10.5195/d3000.2025.1058 http://dentistry3000.pitt.edu 4 base resin affects the longevity of the prosthesis. inadequate flexural strength leads to a higher frequency of denture fractures, occurring both intraorally and extraorally. this study revealed that the thyme essential oil group had a superior mean transverse strength value compared to the control group following 15 days of immersion. the results were similarly to those of anjum et al. [11], who discovered that following 30 days of immersion. the thyme essential oil group had better results than the control group. this study's results contradicted those of namala and hedge[8], who reported that the transverse strength mean value of the control group exceeded that of the thyme oil group. in this study, the results showed that the mean transverse strength of the polident denture cleaner group exceeded that of both the control and polident groups. namala and hedge [8] also found that the mean transverse strength of polident denture cleaner was above than that of the un treated control group. they found that the polident denture cleaner group had the same flexural strength as sharma et al. and anjum et al. [11,31]. the mean transverse strength value of the polident denture cleaner exceeded that of the control group. mohammed et al.'s research [17], they revealed that the thyme essential oil group had a greater mean transverse strength value compared to the control group. the difference between the polident denture cleanser group and the control group was significant, as the control group had a greater mean value of transverse strength than the polident denture cleanser group. thyme extract did not negatively affect the jlexural strengths of the investigated material. the results were the same as those of sidhant et al. [20] reported that thyme essential oil exhibited superior jlexural strength compared to the chemical combination approach and may be utilized successfully as a denture cleanser. conclusion the present investigation demonstrated that the denture cleaner polident and essential oil of thyme injluence surface roughness. however, they can be safely used to clean denture base material without affecting its surface hardness or transverse strength. conflict of interest none. references [1] a. f. al-fouzan, l. a. al-mejrad, and a. m. albarrag, “adherence of candida to complete denture surfaces in vitro: a comparison of convendonal and cad/cam complete dentures,” j. adv. prosthodont., vol. 9, no. 5, p. 402, 2017. doi:10.4047/jap.2017.9.5.402 [2] m. k. jabbar and s. f. dulaimi, “effect of the combined zirconium dioxide surface treatment on the shear bond strength of a veneering ceramic to zirconium dioxide,” dent. med. probl., vol. 57, no. 2, pp. 177–183, 2020. doi: 10.17219/dmp/116409 [3] s. sato, m. r. s. cavalcante, i. a. orsi, h. de f. o. paranhos, and o. zaniquelli, “assessment of flexural strength and color alteradon of heat-polymerized acrylic resins aaer simulated use of denture cleansers,” braz. dent. j., vol. 16, pp. 124–128, 2005. hbps://doi.org/10.1590/s010364402005000200007 [4] n. y. hameed, a. m. alnaser, and s. f. dulaimi, “the effect of contact dme between alginate impression material and type iii dental stone on the surface properdes of stone casts”. doi: hbps://doi.org/10.26477/jbcd.v33i3.2949 [5] a. peracini, l. r. davi, n. de queiroz ribeiro, r. f. de souza, c. h. l. da silva, and h. de f. o. paranhos, “effect of denture cleansers on physical properdes of heat-polymerized acrylic resin,” j. prosthodont. res., vol. 54, no. 2, pp. 78–83, 2010. hbps://doi.org/10.1016/j.jpor.2009.11.004 [6] s. s. alqanas, r. a. alfuhaid, s. f. alghamdi, f. d. al-qarni, and m. m. gad, “effect of denture cleansers on the surface properdes and color stability of 3d printed denture base materials,” j. dent., vol. 120, p. 104089, 2022. hbps://doi.org/10.1016/j.jdent.2022.104089 [7] h. de f. o. paranhos, l. r. davi, a. peracini, r. b. soares, c. h. da s. lovato, and r. f. de souza, “comparison of physical and mechanical properdes of microwave-polymerized acrylic resin aaer disinfecdon in sodium hypochlorite soludons,” braz. dent. j., vol. 20, pp. 331–335, 2009. hbps://doi.org/10.1590/s010364402009000400012 [8] b. b. namala and v. hegde, “comparadve evaluadon of the effect of plant extract, thymus vulgaris and commercially available denture cleanser on the flexural strength and surface roughness of denture base resin,” j. indian prosthodont. soc., vol. 19, no. 3, pp. 261–265, 2019. hbps://doi.org/10.4103/jips.jips_141_19 [9] s. gajwani-jain, d. magdum, a. karagir, and p. pharane, “denture cleansers: a review,” iosr j. dent. med. sci., vol. 1, no. 14, pp. 94–96, 2015. doi: 10.9790/0853-14249496 [10] d. srinivasan, s. nathan, t. suresh, and p. l. perumalsamy, “andmicrobial acdvity of certain indian medicinal plants used in folkloric medicine,” j. ethnopharmacol., vol. 74, no. 3, pp. 217–220, 2001. hbps://doi.org/10.1016/s0378-8741[00]00345-7 [11] r. anjum, s. v dhaded, s. joshi, c. s. sajjan, p. konin, and y. reddy, “effect of plant extract denture cleansing on heat-cured acrylic denture base resin: an: in vitro: study,” j. indian prosthodont. soc., vol. 17, no. 4, pp. 401–405, 2017. hbps://doi.org/10.4103/jips.jips_97_17 [12] k. a. hammer, c. f. carson, and t. v riley, “andmicrobial acdvity of essendal oils and other plant extracts,” j. appl. microbiol., vol. 86, no. 6, pp. 985–990, 1999. hbps://doi.org/10.1046/j.13652672.1999.00780 [13] e. bayarsaikhan et al., “effects of postcuring temperature on the mechanical properdes and biocompadbility of three-dimensional printed dental resin material,” polymers (basel)., vol. 13, no. 8, p. 1180, 2021. hbps://doi.org/10.3390/polym13081180 [14] a. a. alshaikh et al., “3d-printed nanocomposite denture-base resins: effect of zro2 nanopardcles on the mechanical and surface properdes in vitro,” nanomaterials, vol. 12, no. 14, p. 2451, 2022 hbps://doi.org/10.3390/nano12142451. [15] m. i. el-nogoomi, n. m. awaad, and n. nader, “surface roughness of 3d printed maxillary denture bases versus convendonally fabricated ones: in-vitro study,” adv. dent. j., vol. 5, no. 4, pp. 871– 882, 2023. hbps://doi.org/10.21608/adjc.2023.219290.1352 [16] c.-h. lin, y.-m. lin, y.-l. lai, and s.-y. lee, “mechanical properdes, accuracy, and cytotoxicity of uv-polymerized 3d prindng resins composed of bisema, udma, and tegdma,” j. prosthet. dent., vol. 123, no. 2, pp. 349–354, 2020. hbps://doi.org/10.1016/j.prosdent.2019.05.002 [17] s. mohamed, e. elddamony, and e. mohamed, “flexural strength of three different denture base materials aaer immersion in a thyme extract and a chemical cleanser: an in vitro comparadve study,” egypt. dent. j., vol. 69, no. 1, pp. 505–513, 2023. hbp://dx.doi.org/10.21608/edj.2022.160262.2239 [18] m. x. pisani, c. h. l. da silva, h. de f. o. paranhos, r. f. souza, and a. p. macedo, “the effect of experimental denture cleanser soludon ricinus communis on acrylic resin properdes,” mater. res., vol. 13, pp. 369–373, 2010. hbps://doi.org/10.1590/s151614392010000300015 [19] d. felton et al., “evidence-based guidelines for the care and maintenance of complete dentures: a publicadon of the american college of prosthodondsts,” j. prosthodont. implant. esthet. reconstr. dent., vol. 20, pp. s1–s12, 2011. hbps://doi.org/10.1111/j.1532-849x.2010.00683.x [20] s. sudan, p. verma, p. bhagat, s. kohli, u. parmar, and a. sahu, “flexural strength of heat cure acrylic denture base resin by denture cleansing and plant extract immersion: an in-vitro study,” group, vol. 1, pp. 82–83, 2020. hbps://www.oraljournal.com/archives/2020/6/2/e/6-2-42 [21] s.-o. lee, g.-j. choi, k.-s. jang, h.-k. lim, k.-y. cho, and j.-c. kim, “andfungal acdvity of five plant essendal oils as fumigant against postharvest and soilborne plant pathogenic fungi,” plant pathol. j., vol. 23, no. 2, pp. 97–102, 2007. doi: hbps://doi.org/10.5423/ppj.2007.23.2.097 [22] p. sharma, s. garg, and n. m. kalra, “effect of denture cleansers on surface roughness and flexural strength of heat cure denture base resin-an in vitro study,” j. clin. diagnosnc res. jcdr, vol. 11, no. 8, p. zc94, 2017. hbps://doi.org/10.7860/jcdr/2017/27307.10483 [23] s. singh, j. n. palaskar, and s. mibal, “comparadve evaluadon of surface porosides in convendonal heat polymerized acrylic resin cured by water bath and microwave energy with microwavable acrylic resin cured by microwave energy,” contemp. clin. dent., vol. 4, no. 2, pp. 147–151, 2013. hbps://doi.org/10.4103/0976-237x.114844 [24] m. quirynen and c. m. l. bollen, “the influence of surface roughness and surface-free energy on supra-and subgingival plaque formadon in man: a review of the literature,” j. clin. periodontol., vol. 22, no. 1, pp. 1–14, 1995. hbps://doi.org/10.1111/j.1600051x.1995.tb01765.x analyzing the effects of polident and thyme extract oil on denture base vol 13, no 1 (2025) doi 10.5195/d3000.2025.1058 http://dentistry3000.pitt.edu 5 [25] k. degirmenci, m. hayad atala, and c. sabak, “effect of different denture base cleansers on surface roughness of heat polymerised acrylic materials with different curing process. odovtos [online]. 2020, vol. 22, n. 3,” issn. hbp://dx.doi.org/10.15517/ijds.2020.41900 [26] a. ramadan moussa, w. mohamed dehis, a. nabil elboraey, and h. samir elgabry, “a comparadve clinical study of the effect of denture cleansing on the surface roughness and hardness of two denture base materials,” 2016. hbps://doi.org/10.3889/oamjms.2016.089 [27] b. h. panariello, f. e. izumida, e. b. moffa, a. c. pavarina, j. h. jorge, and e. t. giampaolo, “effects of short-term immersion and brushing with different denture cleansers on the roughness, hardness, and color of two types of acrylic resin,” am j dent, vol. 28, no. 3, pp. 150–156, 2015. . hbps://pubmed.ncbi.nlm.nih.gov/26201226/ [28] v. punia, m. khandelwal, c. sharda, a. porwal, and a. sethia, “a comparadve evaluadon of effect of denture cleansers on color stability, surface roughness and hardness of polyamide denture base material,” j. cancer res. ther., vol. 19, no. 7, pp. 2031–2035, 2023. hbps://doi.org/10.4103/jcrt.jcrt_945_22 [29] n. a. hadm, a. a. taqa, w. abbas, and a. m. shuker, “the effect of thyme and nigella oil on some properdes of acrylic resin denture base,” alrafidain dent. j., vol. 10, no. 2, pp. 205–213, 2010. . hbp://dx.doi.org/10.33899/rden.2010.9032 [30] k. h. neppelenbroek, a. c. pavarina, c. e. vergani, and e. t. giampaolo, “hardness of heat-polymerized acrylic resins aaer disinfecdon and longterm water immersion,” j. prosthet. dent., vol. 93, no. 2, pp. 171–176, 2005. hbps://doi.org/10.1016/j.prosdent.2004.10.020 [31] a. sharma and h. s. shashidhara, “a review: flexible removable pardal dentures,” j dent med sci, vol. 13, no. 12, pp. 58–62, 2014. hbp://dx.doi.org/10.9790/0853-131265862 table 1. descriptive of data and anova analysis for surface roughness assessments among all immersion groups. n mean standard deviation standard error minimum value maximum value anova test pvalue group i 10 0.3321 0.06723 0.02126 0.25 0.47 0.000 group ii 10 1.0961 0.09803 0.031 0.92 0.18 group iii 10 1.4348 0.09063 0.02866 0.34 0.58 table 2. comparative analysis tukey hsd analysis of surface roughness test. groups mean difference standard error p-value 95% conjidence interval lower bound upper bound group i group ii 0.764 0.0386 0.000 0.8597 0.6683 group iii 1.1027 0.0386 0.000 1.1984 1.007 group ii group iii 0.3387 0.0386 0.000 0.243 0.4344 table 3. the descriptive statistics and anova test results for the surface hardness test among all immersion groups. table 4. descriptive statistics and the anova test for the transverse strength among all immersion groups. table 5. tukey hsd analysis of transverse strength tests for all immersion groups. groups mean difference standard error p-value 95% conjidence interval lower bound upper bound group i group ii 0.8037 0.228885 0.004 1.3712 0.2362 group iii 2.9156 0.228885 0.000 3.4831 2.3481 group ii group iii 2.1119 0.228885 0.000 1.5444 2.6794 groups n mean standard deviation standard error minimum value maximum value anova test p-value group i 10 41.1 1.59513 0.50442 39 44 0.071 group ii 10 39.4 2.75681 0.87178 32 41 group iii 10 41.7 2.11082 0.6675 37 45 groups n mean standard deviation standard error minimum value maximum value anova test pvalue group i 10 14.2269 0.220758 0.06981 13.944 14.532 0.000 group ii 10 15.0306 0.786233 0.248629 14.314 16.945 group iii 10 17.1425 0.344856 0.109053 16.827 17.88 939 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.939 http://dentistry3000.pitt.edu effect of curing technique and emax type on the polymerization of dual cure resin cement amani abduljabar altaie, emad farhan alkhalidi university of mosul, mosul, iraq abstract objec6ve: the objeccve of this study was to evaluate the effect of different types of curing techniques and composicon of two different types of emax materials on degree of conversion and polymerizacon of dual cure resin cement ater 24 hours. material and methods: resin cement discs (90 total) were made by polymerizacon through two types of emax discs: ivoclar ips e.max (n=30) and gc lisi press (n=30). 30 samples without an emax disc were used as controls. for each group, three subgroups according to the type of curing technique were determined: 10 samples were made by a concnuous curing technique, 10 samples were made with a sot start curing technique, 10 samples were made by an intermi^ent curing technique. each of 90 resin discs were evaluated with the degree of conversion test by fourier transform infrared spectroscopy (ftir) ater 24 hours. results: gc lisi press had a higher polymerizacon rate than ivoclar ips e.max. the intermi^ent curing technique sub-group had the highest polymerizacon rate compared to the other techniques independent of the emax type. significant differences between each type of emax and curing procedure type were found using a one-way anova test (p<0.05). conclusion: gc lisi press allowed for more light curing in comparison to ivoclar ips e.max, and the intermi^ent curing technique increased polymerizacon rate of dual cure resin cement. the degree of conversion for dual cure resin cement was directly influenced by the type of emax veneer and the curing method. open access cita%on: altaie aa, et al. (2025) effect of curing technique and emax type on the polymeriza%on of dual cure resin cement. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.939 received: may 7, 2025 accepted: june 26, 2025 published: august 6, 2025 copyright: ©2025 altaie aa, et al. this is an open access ar%cle licensed under a crea%ve commons atribu%on work 4.0 united states license. email: amani.abduljabar@uomosul.edu.iq introduc)on in recent years, the -ield of dentistry has used an increasing number of cosmetic materials; this could be because of the need to satisfy patient needs and new fabrication developments methods [1]. all-ceramic restorations (those lacking a metal substrate) can be used in the aesthetic area because of their increased translucency [2,3]. lithium disilicate glass ceramics are the most widely used ceramic system because of its good strength (350–450 mpa), ideal adhesion to tooth structures, easy production and superior aesthetic qualities [4]. to improve the appearance of an anterior tooth, the laminate is a conservative substitute for full covering. one of the most common restorations in aesthetic dentistry during the past few decades is laminate veneers [5]. the current method of restoration fabricating technologies ensures tooth-colored restorations is computer-aided design and computer-aided manufacturing (cad/cam) system [6] which is simple, time ef-icient and quick using resin and ceramic materials in a timely way [7,8]. for many years, resin luting cements have been utilized in dental restorations such as crowns, bridges, inlays, and onlays. glass -illers and polymerizable methacrylates are the main component of resin cement, and they can be cured by light-curing, self-curing, or both methods [9]. for indirect restorations, the methacrylate-based resin material must chemically bond to the surfaces, which is often accomplished in two or three steps. micromechanical retention for ceramic restorations incorporating silica is achieved by etching the surface with hydro-luoric acid (hf) and then priming it to become hydrophobic and resin friendly. additional hydroxyl groups on the ceramic substrates are chemically activated by etching [10]. dual-cure materials, which combine the advantages of light-curing with chemical polymerization, have gained a lot of popularity [11]. however, it was stated that exposure to light is required for the resin cements to achieve the highest possible degree of conversion rate [12]. this is signi-icant as the lack of polymerization for the self-curing form of also produces reduced mechanical characteristics and increased solubility of resin cements with two curing times [13]. hardness testing is frequently employed as a straightforward and trustworthy technique to assess appropriate resin cement polymerization [14,15]. effect of curing technique and emax type on the polymerizacon of dual cure resin cement vol 13, no 1 (2025) doi 10.5195/d3000/2025.939 http://dentistry3000.pitt.edu 2 degree of conversion (doc) is the percentage of c=c double bonds that are changed into c-c single bonds or the amount that monomers react to produce polymers [16]. several studies in the literature seem to support the idea that different curing techniques improve the polymerization of direct resin materials [17]. these techniques are based on the knowledge that a slower conversion rate improves material -low, which lowers the resin's shrinkage stress and so helps avoid gap development [18]. the longevity of resin-bonded ceramic restorations is largely dependent on adequate polymerization. color change may result from incomplete resin cement polymerization, postoperative sensitivity, toxicity from leftover monomer, and reduced bond strength, which raises the risk of cavities and microleakage [19]. to the best of our knowledge, no research has assessed how the emax type and curing technique affect the degree of conversion of dual-cured resin cement. this study aimed to assess how the degree of conversion of dualcure resin cement is affected by emax type and curing technique. material and methods we used a dual cure resin cement (fgm, joinville, brazil and a2 shade) and two types of lithium disilicate (ivoclar ips e.max press ingots and gc initial lisi press shade a2) (table 1). the thickness of discs that were fabricated was 1.2mm. since color shade and thickness have direct effect on the light transmission, for this study we kept the color shade and thickness of emax constant. figure 1 summarizes the design of the study. each group was subdivided into three subgroups according to the type of curing technique: continuous mode with 700mw/cm² intensity, soft start mode with 150mw/cm2 followed by 700mw/cm2 intensity and intermittent curing technique with 700mw/cm2 [22]. a total of 90 dual resin cement samples were put within an elastomeric mold that was 1 mm thick. each sample's upper surface was covered with individual mylar strips to separate them from the emax disk [18]. after that an emax disc sample was put on top of the resin cement and loaded with 200 g for 60 seconds, an led-curing light was then used to cure the resin cement in three curing techniques at zero distance from emax discs [19]. all samples of resin cement disc were stored in a dry and black container for 24 hours at room temperature before polymerization. following a 24-hour storage period, an atr ftir spectroscopy equipment was used to evaluate the degree of conversion (doc). each specimen of resin cement was placed on the surface of the zinc selenide pellet (specac) of atr-ftir to obtain the absorption spectra of the non-polymerized (c=c) and polymerized (c-c) forms of the specimens. 64 pictures at a resolution of 4 cm were obtained from the 1500–1800 cm region. this region contains the reference peak of the aromatic component (discovered at 1608 cm) and the strength of the aliphatic carbon double-bond (c=c) absorption peak [23,24]. both peaks are present in materials having aromatic vinyl bonds of bisphenol and aliphatic bonds of the methacrylate functional group. the following formulas were then used to calculate the doc of each distinct resin cement specimen [25,26]: doc (%) = 100 [1 – (r polymerized/r nonpolymerized)] r = the ratio of the absorbance peaks at 1608 cm and 1638 cm results the highest mean value of (doc) was observed among controls. mean value of gc emax was higher than ivoclar’s. the intermittent technique of curing had the highest mean value of doc compared to the rest. to determine whether there were signi-icant differences between the subgroups within each group, the analysis of variance (one way anova) was performed. figure 2 shows the three scenarios had the same patterns of response with differences in the absolute value (p<0.005). figures 3, 4, and 5 show the chart of entire ftir/atr spectra for zero cure (unset) dual cure resin cement material for controls, ivoclair and gc. discussion this study assessed how various porcelain veneer types (ivoclar and gc emax veneer) and different curing techniques (continuous, soft start and intermittent) affected the polymerization doc of dual cure resin cement. the null hypothesis of this research was rejected because different types of emax veneer showed direct effect on doc. the highest mean value of doc was obtained in the controls. ivoclar emax disk had lower doc than gc emax disk likely because of the difference in composition of the two materials. ips e.max press (ivoclar, vivadent) creates pressable ingots of lithium disilicate glass ceramic, a synthetic glass-based porcelain. ips, the microstructure of e.max press is composed of approximately 70% glass-matriculated lithium disilicate (li2 si2 o5) crystals, which range in length from 3 to 6 µm [27]. while initial lisi press (gc emax) consist of lithium disilicate micro-crystals equally dispersed in a glass matrix [18], so microcrystalline containing material showed lower scattering of light and more light transmission through it [28]. in each group we found that intermittent curing technique had the highest mean value of polymerization. this method uses a dark gap between each exposure. the polymerization reaction proceeds slowly at this phase. three to -ive minutes of delay results in the largest reduction in shrinkage [29]. additionally, the polymerization process may allow for stress release because to the lengthy curing process that allows resin cement to -low [30,31]. many authors attribute these results to elongation of pre-gel stage that would permit the material to -low to get the volumetric reduction [32,33]. other writers contended that chain relaxation, rather than viscous -low, would be a more acceptable explanation for the observed stress alleviation [34,35]. from the results of this research we observed that soft start curing technique had the lowest mean value of doc. soft-start polymerization uses a low irradiance at -irst, then a high irradiance cure toward the end. better material -low is made possible by a slower rate of conversion. before curing, the material retains its -lexibility for a long time, which also reduces contraction stresses and improves marginal adaption [19,36]. starting the light-activation process with a low light intensity allowed for a low degree of convergence and prolonged maintenance of a higher level of molecular mobility inside the polymer matrix. goracci et al. [37] introduced the "slow and gradual polymerization" in 1992 for that reason. this technique entails polymerizing the initial composite layer with progressively increasing light intensity over the course of four minutes to extend the phase of the early setting of the composite. this allows polymerization shrinkage to occur while the molecules still can change their direction to compensate for the internal stress, reducing the contraction stress that develops during the -inal setting and making it resistant to the bonding's adhesive forces [37]. a light source with a consistent intensity is applied to the resin cement for an established period in continuous curing [38], mean value of doc of this type of curing technique was more than soft start and less than intermittent curing technique in the result of this study. effect of curing technique and emax type on the polymerizacon of dual cure resin cement vol 13, no 1 (2025) doi 10.5195/d3000/2025.939 http://dentistry3000.pitt.edu 3 conclusions composition of emax veneer had direct effect on the polymerization of resin cement, initial lisi press gc with microcrystalline structure transported more light from light cure unit to resin cement in comparison to the ips e.max press ivoclar vivadent. curing technique had direct effect on polymerization of resin cement, intermittent curing technique displayed more degree of conversion for resin cement than continuous and soft start curing technique. effect of continuous curing technique was increase in (doc) of resin cement more than soft start curing technique. references [1] in7luence of different surface pretreatments on shear bond strength of an adhesive resin cement to various zirconia ceramics. colombo m, gallo s, padovan s, chiesa m, poggio c, scribante a. materials. 2020;13(3):652. [2] effect of resin cement color on the final color of lithium disilicate all-ceramic restorations. vafaee f, heidari b, khoshhal m, hooshyarfard a, izadi m, shahbazi a, moghimbeigi a. j dent (tehran). 2018;15(3):143-150. [3] surface roughness assessment of the sandblasted and acid-etched zirconia ceramic. abdulateef mr. nayif mm. al–ra7idain. dent j. 2021;21(1):146-157. [4] strength, fracture toughness and microstructure of a selection of all-ceramic materials. part i. pressable and alumina glass-in7iltrated ceramics. guazzato m, albaky m, ringer sp, swain mv. dent mater. 2004. june;20(5):441–8. [5] evaluation of cements curing techniques regarding microleakage during cementation of emax laminate veneers. alqahtani w, galal rm, hamza g. open access maced j med sci. 2021; 9(4):155-159. [6] effect of different storage temperatures on degree of polymerization of tetric evoceram bulk fill composite resin (an in vitro study). ahmed d. and abd al-jabar m. al-ra7idain dental journal. 2022,22(1); 194-202. [7] effect of plasma treatment on tensile bond strength of (5) yttrium zirconia coping fixed on titanium implant abutment. altaie aa, alkhalidi ef. pharmacog j. 2024;16(1):1-7. [8] cad-cam technology: a literature review. abdulla ma., ali h kh., jamel rs. al–ra7idain dent j. 2020;20(1):95-113. [9] effectiveness of a silane coupling agent in a resin luting cement. erick, y., qiang, m. and byoung s. international journal of adhesion and adhesives;134(2024): 103-808. [10] aspects of silane coupling agents and surface conditioning in dentistry: an overview. lung, c.y. and matinlinna, j.p., dental materials;2012,28(5):467-477. [11] effect of curing technique on the polymerization characteristics of dual-cured resin cement systems. arrais cag, rueggeberg fa, waller jl, de goes mf, giannini m. j dent 2008; 36: 418-426. [12] mechanical properties of resin cements with different activation techniques. braga rr, cesar pf, gonzaga cc. j oral rehabil 2002; 29: 257-262. [13] the effect of light exposure on water sorption and solubility of self-adhesive resin cements. aguiar tr, andré cb, ambrosano gmb, giannini m. int sch res not 2014; 2014: 1-6. [14] in7luence of the feldspathic ceramic thickness and shade on the microhardness of dual resin cement. soares cj, da silva nr, fonseca rb. oper dent 2006 may-jun;31(3):384-389. [15] effect of different thicknesses of pressable ceramic veneers on polymerization of lightcured and dual-cured resin cements. cho s-h, lopez a, berzins dw, prasad s, ahn kw. j contemp dent pract 2015;16(5):347-352. [16] degree of conversion of two dual-cured resin cements light-irradiated through zirconia ceramic disks. kim m, kim k, kim y, kwon t. j adv prosthodont 2013 nov;5(4):464-470. [17] effect of curing methods and base materials on the stress generated by the polymerization shrinkage of a resin composite. cunha lg, alonso rcb, sinhoreti mac, goes mf, correr-sobrinho l. dtc¦"l"qtcn"uek, 2004; 3: 609-614. [18] clinical criteria for the successful curing of composite materials. mahn, e. rev. clin. periodoncia implantol. rehabil. oral. 2013, 6(3); 148-153. [19] effect of different thicknesses of pressable ceramic veneers on polymerization of lightcured and dual-cured resin cements. cho s-h, lopez a, berzins dw, prasad s, ahn kw. j contemp dent pract 2015;16(5):347-352. [20] effect of plasma treatment on tensile bond strength of (5) yttrium zirconia coping fixed on titanium implant abutment. altaie aa, alkhalidi ef. pharmacog j. 2024;16(1): 1-7. [21] effect of light intensity, light-curing unit exposure time, and porcelain thickness of ips e.max press and vintage ld press on the hardness of resin cement. naliani s, elias s, tjandrawinata r. sci dent j 2020;4:21-5. [22] effect of different initial light intensity by the soft-start photoactivation on the bond strength and knoop hardness of a dental composite. dall'magro e; sinhoreti m; américo, correr a; sobrinho l; consani s; rontani r. braz. dent j.(2007) 18 (2): 15-22. [23] orthodontic composite in static magnetic field. al qassar s., taqa a. and mohiaalden h. journal of international dental and medical research. 2021,14(1):67-73. [24] calibration of ftir conversion analysis of contemporary dental resin composites. rueggeberg f, hashinger d, fairhurst c. dent mater 1990 oct;6(4):241-249. [25] a pilot trial on lithium disilicate partial crowns using a novel prosthodontic functional index for teeth (fit). cagidiaco, e.f.; grandini, s.; goracci, c.; joda, t. bmc oral health 2019, 19, 276. [26] in7luence of the curing technique on the degree of conversion of a dual-cured self-adhesive resin luting cement beneath ceramic. souza e, borges b, oliveira d, brandt w, hirata r, silva e, sinhoreti m. acta odontol scand 2013;71(3-4):444448. [27] scienti7ic documentation ips e.max®press. bühler zemp p, völkel t, fischer k. . 2011. pp: 140. [28] the in7luence of microcrystalline structure and crystalline size on visible light transmission of polyvinyl alcohol optical 7ilms. ding, c., wenzhong ma and zhong, j., optical materials,147,2024, pp:114-627. [29] comparison of variation in the light curing cycle with a time gap and its effect on polymerization shrinkage, degree of conversion and microhardness of a nanohybrid composite. subbiya a, mary ns, suresh m, vivekanandhan p, dhakshinamoorthy m, sukumaran vg. j conserv dent: jcd. 2015 mar;18(2):154. [30] in7luence of light energy density on effectiveness of composite cure. yap, a. u. and c. senevirante.. operative dentistry 2001, 26 5:460– 466. [31] comparison of composite curing parameters: effects of light source and curing mode on conversion, temperature rise and polymerization shrinkage. z. tarle; a. knezevic; n. demoli; a. meniga; j. sutalo; g. unterbrink; m. ristic; g. pichler. oper dent (2006) 31 (2): 219–226. [32] shrinkage stress and microhardness of composites photoactivated by intermittent-delay curing. pfeifer c., braga r. & ferracane j: operative dentistry, 2006, 31-5, 610-615. [33] reduction of polymerization contraction stress for dental composites by two-step light-activation. lim bs, ferracane jl, sakaguchi rl & condon jr (2002) dental materials, 18(6):436444. [34] towards the elucidation of shrinkage stress development and relaxation in dental composites. lu h, stansbury jw & bowman cn (2004) dental materials 20(10) 979-986. [35] effect of stepped light intensity on polymerization force and conversion in a photoactivated composite. bouschlicher mr, rueggeberg fa & boyer db (2000) journal of esthetic dentistry 12(1) 23-32. [36] in7luence of light intensity on polymerization shrinkage and integrity of restoration cavity interface. feilzer aj, dooren lh, de gee aj, davidson cl. eur j oral sci 1995;103:322326. [37] curing light intensity and marginal leakage of resin composite restorations. oracci g, mori g, martinis lc. quint int 1996;27: 355-362. [38] effect of different modes of light curing and resin composites on microleakage of class ii restorations part ii. s.t. hardan l.s., amm e.w., ghayad a., ghosn c. and khraisat a. t.d.j. 2009, 32: 1229. effect of curing technique and emax type on the polymerizacon of dual cure resin cement vol 13, no 1 (2025) doi 10.5195/d3000/2025.939 http://dentistry3000.pitt.edu 4 figure 1. study design schematics. table 1. materials used in this study. materials composition manufacture color allcem dual resin cement bis-ema/bisgma/tegdma, -silicate glass -barium aluminum -silicon dioxide 62% weight [20] fgm, brazil a2 initial lisi press equivalently distributed lithium disilicate microcrystals in a glass matrix dispersed in a glass matrix [21]. gc a2 ips e.max press about 70% of li2si2o5 and lithium disilicate crystals are immersed in a glassy matrix [21]. ivoclar vivadent a2 no emax disc cover resin cement (n=30) • continous curing mode technique (n=10) • soft start curing technique (n=10) • intermittent curing technique (n=10) ivoclar emax disc cover resin cement (n=30) • continous curing mode technique (n=10) • soft start curing technique (n=10) • intermittent curing technique (n=10) gc emax cover resin cement (n=30) • continous curing mode technique (n=10) • soft start curing technique (n=10) • intermittent curing technique (n=10) 90 resin cement samples for a whole day, all resin cement discs were kept at room temperature in a dry, black container for every resin cement disc, the degree of conversion was assessed using ftir effect of curing technique and emax type on the polymerizacon of dual cure resin cement vol 13, no 1 (2025) doi 10.5195/d3000/2025.939 http://dentistry3000.pitt.edu 5 figure 2. mean values of doc for all groups and subgroups. ivoclar (to the left), gc (in the middle), and controls (to the right). blue is continuous polymerization, red is soft start, and green and intermittent. : figure 3. diagram of full spectra of unset (zero cure) resin cement. 0 10 20 30 40 50 11.5 24 32.5 6.25 14.25 21.75 25.5 33.75 44.25 a1 a2 a3 effect of curing technique and emax type on the polymerizacon of dual cure resin cement vol 13, no 1 (2025) doi 10.5195/d3000/2025.939 http://dentistry3000.pitt.edu 6 figure 4. diagram of full spectra of polymerized controls. figure 5. diagram of full spectra of polymerized gc emax. 981 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.981 http://dentistry3000.pitt.edu impact of smoking on halitosis and oral bacterial infections doaa raad alghaffari1, mays wael sulaiman al alousi2, raya alani3, dina ahmed mahdi4 1boston univers,y school of public health, boston, usa 2private prac,ce, zionsville, indiana, usa 3private prac,ce, boynton beach, florida, usa 4college of den,stry, albayan university, baghdad, iraq abstract objec<ve: the present study sought to inveshgate the frequency of halitosis in a sample of smokers and non-smokers and the associahon of smoking and perceived oral bacterial infechons. material and methods: a queshonnaire was administered to 301 respondents. the structured queshonnaire included demographics, oral hygiene behaviors, medical and dental history, lifestyle behaviors, awareness of halitosis and its social consequences in others. stahshcal analyses were performed with chi-square tests to idenhfy any associahons of halitosis and possible risk factors. results: among respondents, 22.9% self-reported using tobacco products while 63.4% self-reported experiencing halitosis somehmes or frequently. smoking, periodontal disease, dental caries, poor clinical hygiene (not brushing, not flossing or using mouthwash) and some systemic diseases were related to self-reporhng halitosis (p < 0.01). only 9.6% of respondents accessed treatment. most respondents (86%) realized their halitosis themselves rather than it being diagnosed professionally. conclusion: this study provides insight into the relahonship between smoking and self-reported halitosis and the need for greater awareness and tailored public health inihahves. open access cita%on: alghaffari dr, et al. (2025) impact of smoking on halitosis and oral bacterial infec%ons. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.981 received: july 5, 2025 accepted: july 6, 2025 published: august 21, 2025 copyright: ©2025 alghaffari dr, et al. this is an open access ar%cle licensed under a crea%ve commons auribu%on work 4.0 united states license. email: duaaraad@bu.edu introduc)on halitosis, also known as bad breath, is a common problem for many people all around the world. it can result from many local or systemic factors – almost 90% of halitosis cases are due to oral factors. among these oral reasons, oral malodor comes from an imbalance in the oral ecosystem, so that microbial degradation of food debris, epithelial cells and blood products in the mouth cause volatile sulfur compounds (vscs), including hydrogen sulgide and methyl mercaptan, which are the principal odor components of oral malodor [1]. it is well known that one of the greatest risk factors for halitosis is smoking, due among other factors to multiple effects on the oral ecosystem. for example, smoking worsens plaque accumulations, decreases salivary glow, changes the oral glora, and increases pathogenic anaerobic bacteria producing vscs [2]. additionally, smokers are more likely to have periodontal diseases and dental caries, both of which are strongly related to halitosis and bacterial overgrowth [3]. aside from its physiological effects, halitosis has psychosocial outcomes, such as embarrassment, poor self-esteem, and social isolation among individuals when they do not know they have it or want to treat it [4]. several studies have examined the relationship between smoking and oral ailment, but focused investigations that assess the combined effect of smoking on halitosis and oral bacterial infections in a population-based research design are still necessary. the surveybased studies provide an insight into the respondents' personal habits and hygiene behavior, as well as their perceptions of themselves regarding oral health. consequently, this study, sought to establish the prevalence of halitosis and its relationship with the infection caused from oral bacteria in smokers. the outcomes of this study should help establish the behavioral and clinical aspects of both problems of halitosis and support the development of public health strategies to address the overall oral health issue of smokers [5]. materials and methods the study included 301 participants from all age groups, backgrounds and levels of education. participants provided the study with social diversity through convenience sampling. also, participants under 10 years of age were excluded, as well as individuals unwilling to participate. initially, participants completed verbal informed consent from the impact of smoking on halitosis and oral bacterial infechons vol 13, no 1 (2025) doi 10.5195/d3000.2025.981 http://dentistry3000.pitt.edu 2 study crew. the study consisted of a fully voluntary written questionnaire. the questionnaire had both closed-ended and multiple-choice questions. the questionnaire asked basic participant demographical information and was constructed in 5 areas of interest: • demographical information: age, gender, and highest educational level completed. • dental hygiene practices: frequency of brushing teeth, use of mouthwash, tongue cleaning, glossing, brushing technique, and how the participant brushes. • dental and health history: history of periodontal disease/periodontal disease, history of decay, malocclusion of teeth, and systemic diseases. • lifestyle: history of smoking (length of time and quantity of smoking), history of using vape products, consumption of alcohol, and food choices. • halitosis: participant self-reported frequency of halitosis, indication of when participants report awareness of halitosis awareness, treatment history, and psychological or social problems associated with halitosis. in-person administration of the questionnaire was utilized with the researcher clarifying any item that may not have been fully understood to adequately avoid any misinterpretation by the participants. given that all responses were anonymous, the participants were excited to reflect their thinking honestly and demonstrate their skills to their peers. the data were statistically analyzed using ibm spss statistics (version 26), (2019). descriptive statistics were used to summarize frequencies and percentages. chi-square tests (χ²) were utilized to determine the associations between halitosis and other categorical variables such as smoking status, oral hygiene behaviors, sickness and health conditions. the statistical significance was examined at two levels: • p ≤ 0.05: statistically significant • p ≤ 0.01: highly statistically significant • ns: not significance (p > 0.05) this analysis provided signigicant associations between smoking and halitosis and other oral health indicators. results a total of 301 participants were included in this study; 218 (72.4%) were male, and 83 (27.6%) were female. the age group 21-31 years had the highest representation of participants (56.8%), followed by the 10-20 years group (20.9%). most participants were educated at the university level (78.4%), with no participants having attained postgraduate education (10.6%) or had less than high school education. oral hygiene practices most (94.4%) of the participants reported brushing their teeth, with 46.2% responding they brushed their teeth twice a day and 14.3% brushing three or more times. approximately 32.2% of individuals used mouthwash, while 52.8% cleaned their tongue. nonetheless, a considerable number of participants (67.8%) did not use mouthwash consistently. with respect to the frequency of visiting the dentist, half of the participants (51.5%) visited the dentist once per year and just 14.3% reported having more than three visits a year. smoking and halitosis of the participants, 69 (22.9%) reported smoking. most of them had been smoking for more than 3 years (10%); 6% and 4.3% reported smoking for 3 and 2 years respectively. a small number of participants reported daily smoking of 1-20 cigarettes (9.3%), and 12.6% reported vaping. self-reported halitosis was prevalent: • 59.1% said they sometimes had it, • 4.3% often had it, • and 36.5% said they rarely or never had it. just 3% admitted to suffering from bad breath and 12% said they had bad breath sometimes. in fact, 86% of the respondents said they realized they had bad breath by having self-realization and only 10.3% were notigied of having bad breath. oral and systemic health • 32.2% of respondents had periodontal disease, • 62.1% had dental caries, • 35.9% reported malocclusion. • 11.6% had systemic disease, with the most common being paranasal sinus conditions (27.9%), allergies (12.6%) and respiratory issues (7%). there was a significant association for halitosis and smoking status (p < 0.0001), dental caries (p < 0.0001), and periodontal disease (p < 0.0001). lifestyle factors including not cleaning the tongue, irregular brushing habits and infrequent floss or mouthwash use were also statistically associated with bad breath. social impact of halitosis only 9.6% of participants indicated that they had received any treatment for halitosis, with 17.3% used techniques associated with oral hygiene (like cleaning the mouth). most (59.9%) did not have a method for dealing with halitosis. some social effects were also related, such as 8% indicated they had social problems due to bad breath and 40.9% were concerned about how others perceived their breath. statistical summary using the chi-square test, statistically significant relationships (p ≤ 0.01) were found between halitosis and the following factors, see table 1: • smoking habits • technique and frequency of tooth brushing • use of floss and rinses • periodontal and dental disease • pattern of breathing • the presence of systemic illness discussion the results of this study shown a marked association between smoking and the prevalence of halitosis and oral microbial infections for the participants surveyed. the majority of participants reporting halitosis were smokers, indicating a strong connection between tobacco use and the presence of oral malodor. this agrees with other studies that demonstrated smoking predisposes individuals to pathogenic dental plaque and periodontal pathogens, and smoking is known to reduce salivary flow [3,6]. the high percentage of individuals reporting occasional halitosis (59.1%) shows how common this condition is. however, the much lower percentage of individuals seeking care for this condition (9.6%) speaks to the public's lack of awareness. halitosis is a socially sensitive, stigmatizing condition, including data from this study that indicated that most participants (86%) reported noticing their own halitosis and did not have a medical professional document their apparent halitosis [1]. this may delay or ignore appropriate management. poor oral hygiene practices were significantly related to halitosis and are consistent with previous literature. irregular brushing habits, no flossing habits, and limited use of mouthwash and tongue cleaning had statistically significant associations with bad breath. these behaviors provide areas for retention of food debris and bacterial plaque and are compounded in smokers who have an already altered oral microbiome [2]. the amount of mouthwash use (32.2%) is impact of smoking on halitosis and oral bacterial infechons vol 13, no 1 (2025) doi 10.5195/d3000.2025.981 http://dentistry3000.pitt.edu 3 important because it is known to reduce anaerobic bacteria loads and vscs when utilized regularly [7] and highlights the need for educating the population on the importance of preventive care. this study also confirmed the strong association of halitosis and oral diseases, especially periodontal disease (32.2%) and dental caries (62.1%). these two diseases have been established in previous literature to promote oral malodor due to the colonization of anaerobic bacteria in periodontal pockets and carious lesions [8]. furthermore, systemic illnesses such as rhinosinusitis, allergies, and respiratory diseases also showed signification correlation with halitosis and lends itself to some consideration of the notion that it should be recognized that extraoral sources must be determined as part of diagnosis and treatment [4]. the psychosocial burden of halitosis was evident with 40.9% of respondents concerned with how others perceived their breath and 8% of participants reported social problems. these findings corroborate previous research demonstrating a psychological burden related to halitosis including embarrassment, social anxiety and reduction in self-esteem [9]. the gap between symptom perception and action supports the need for public health materials around halitosis, as well as provide some access to dental services. overall, our findings highlight the multifactorial etiology of halitosis, where smoking is an important modifiable risk factor. addressing smoking cessation while supporting oral hygiene practices and regular visits to the dentist, may lessen the burden of halitosis and infections. conclusions this study showed a strong relationship between tobacco use and the incidence of halitosis and oral bacterial disease. smokers were more likely to report symptoms of bad breath, and had higher prevalence of periodontal disease and dental caries, both of which are major contributors to oral malodor. inadequate oral hygiene such as sporadic brushing habits, lack of tongue cleaning, and less frequent mouthwash and flossing habits were also strongly associated with halitosis. these results suggest that halitosis is not just a social or aesthetic issue; it may be a clinical reflection of serious oral health issues, especially when observed in an individual with poor hygiene behaviors or tobacco habits. finally, the observed low levels of treatment-seeking behavior and awareness of halitosis, highlights the need for more public education and the need for preventive strategies. in conclusion, halitosis can be limited by addressing risk factors that are potentially modigiable (e.g. tobacco habits, oral hygiene practices), and implementing better access to dental care, and/or awareness programs will potentially reduce the burden of halitosis or related oral bacterial infections among non-clinical populations. references 1.porter, s. r., & scully, c. (2006). oral malodor (halitosis). bmj, 333(7569), 632–635. https://doi.org/10.1136/bmj.38954.631968.ae 2.lee, s. s., zhang, w., & li, y. (2007). the antimicrobial potential of 14 natural herbal dentifrices: a comparative study using an in vitro biofilm model. journal of the american dental association, 138(3), 351–357. 3.aylıkcı, b. u., & çolak, h. (2013). halitosis: from diagnosis to management. journal of natural science, biology, and medicine, 4(1), 14–23. https://doi.org/10.4103/0976-9668.107255 4.miyazaki, h., sakao, s., katoh, y., takehara, t. (1995). correlation between volatile sulfur compounds and certain oral health measurements in the general population. journal of periodontology, 66(8), 679–684. 5.alkhafaji, s. h. a., al-sudani, s. f. k., & alsunbuli, m. m. b. (2025). smoking and the hidden consequences: bad breath and oral infection of bacteria. microbial biosystems, 10(4), [in press]. 6.scully, c., & greenman, j. (2012). halitosis (breath odor). periodontology 2000, 48(1), 66–75. https://doi.org/10.1111/j.16000757.2008.00267.x 7.young, a., jonski, g., & rölla, g. (2003). a study of triclosan and zinc citrate as combined agents in mouthwash: the effect on volatile sulfur compounds. journal of clinical periodontology, 30(4), 300–306. 8.rosenberg, m., & mcculloch, c. a. g. (1992). measurement of oral malodor: current methods and future prospects. journal of periodontology, 63(9), 776–782. 9.kumar, s., tadakamadla, j., & johnson, n. w. (2016). effect of self-perceived halitosis on social interactions: a systematic review. journal of oral rehabilitation, 43(10), 875–883. https://doi.org/10.1111/joor.12438 impact of smoking on halitosis and oral bacterial infechons vol 13, no 1 (2025) doi 10.5195/d3000.2025.981 http://dentistry3000.pitt.edu 4 table 1. associations between halitosis and the different factors. questionnaire number of respondents percentage (%) χ2 (p-value) gender males 218 72.4% 60.548 ** (0.0001) females 83 27.6% total 301 100% - age 10-20 63 20.9% 72.605 ** (0.0001) 21-31 171 56.8% 32-43 47 15.6% 44-55 14 4.7% 56-66 5 1.7% more than 66 1 0.3% total 301 100% - education level primary school 8 2.7% 648.11 ** (0.0001) junior high school 8 2.7% high school 17 5.6% university 236 78.4% master degree 32 10.6% total 301 100% frequency of dental visit per year 1 155 51.5% 114.82 ** (0.0001) 2 59 19.6% impact of smoking on halitosis and oral bacterial infechons vol 13, no 1 (2025) doi 10.5195/d3000.2025.981 http://dentistry3000.pitt.edu 5 3 44 14.6% 3< 43 14.3% total 301 100% do you brush your teeth? yes 284 94.4% 236.84 ** (0.0001) no 17 5.6% total 301 100% frequency of toothbrush per day 1 116 38.5% 159.26 ** (0.0001) 2 139 46.2% 3 43 14.3% 3< 3 1% total 301 100% do you use mouthwash? yes 97 32.2% 38.04 ** (0.0001) no 204 67.8% total 301 100% tongue cleaning yes 159 52.8% 0.960 ns (0.3272) no 142 47.2% total 301 100% suffering from halitosis never/rarely 110 36.5% 122.87 ** impact of smoking on halitosis and oral bacterial infechons vol 13, no 1 (2025) doi 10.5195/d3000.2025.981 http://dentistry3000.pitt.edu 6 sometimes 178 59.1% (0.0001) frequently 13 4.3% total 301 100% frequency of tooth brushing after every meal 41 13.6% 107.67 ** (0.0001) twice/day 136 45.2% once/day 100 33.2% some times 24 8% total 301 100% timing of tooth brushing before bed 55 18.3% 86.920 ** (0.0001) morning 46 15.2% morning and before bed 145 48.2% no gixed time 55 18.3% total 301 100% way of tooth brushing vertical stroke 63 20.9% 11.758 ** (0.0083) horizontal stroke 61 20.3% semi-circular 79 26.2% gum to incisal edge 98 32.6% total 301 100% carbohydrate foods yes 240 79.7% 106.44 ** (0.0001) impact of smoking on halitosis and oral bacterial infechons vol 13, no 1 (2025) doi 10.5195/d3000.2025.981 http://dentistry3000.pitt.edu 7 no 61 20.3% total 301 100% breathing pattern through the mouth 29 9.6% 78.160 ** (0.0001) through the nose 144 47.8% both 128 42.5% total 301 100% smoking history yes 69 22.9% 157.89 ** (0.0001) no 232 77.1% total 301 100% systemic disease no 266 88.4% 177.28 ** (0.0001) yes 35 11.6% total 301 100% periodontal disease yes 97 32.2% 38.036 ** (0.0001) no 204 67.8% total 301 100% dental caries yes 187 62.1% 17.704 ** (0.0001) no 114 37.9% total 301 100% impact of smoking on halitosis and oral bacterial infechons vol 13, no 1 (2025) doi 10.5195/d3000.2025.981 http://dentistry3000.pitt.edu 8 malocclusion yes 108 35.9% 24.003 ** (0.0001) no 193 64.1% total 301 100% medical history regarding diseases of the blood, heart, respiratory system, intestines, liver, kidneys, and known allergies heart diseases 21 7% 52.702 ** (0.0001) respiratory diseases 21 7% liver diseases 12 4% renal diseases 18 5.9% allergies 38 12.6% paranasal sinuses 84 27.9% other than that 61 20.3% don’t have any diseases 46 15.3% total 301 100% a history of diabetes was recorded yes 13 4.3% 251.24 ** (0.0001) no 288 95.7% total 301 100% do you use sloss yes 53 17.6% 37.266 ** (0.0001) sometimes 137 45.5% never 111 36.9% total 301 100% impact of smoking on halitosis and oral bacterial infechons vol 13, no 1 (2025) doi 10.5195/d3000.2025.981 http://dentistry3000.pitt.edu 9 do you use any mouth rinses yes 58 19.3% 33.948 ** (0.0001) sometimes 103 34.2% never 140 46.5% total 301 100% consumption of alcoholic beverages never 272 90.4% 690.61 ** (0.0001) rarely 25 8.3% dilly 3 1% several times per day 1 0.3% total 301 100% smoking: the exact number of cigarettes smoked daily 1-20 28 9.3% 163.85 ** (0.0001) 20-40 4 1.3% 40-60 2 0.7% never 229 76.1% vape 38 12.6% total 301 100% how long have you been smoking? 1 year 9 3% 207.44 ** (0.0001) 2 year 13 4.3% 3 year 18 6% more than 3 year 30 10% impact of smoking on halitosis and oral bacterial infechons vol 13, no 1 (2025) doi 10.5195/d3000.2025.981 http://dentistry3000.pitt.edu 10 never 231 76.7% total 301 100% do you suffer from bad breath odor ? yes 9 3% 369.63 ** (0.0001) sometimes 36 12% never 256 85% total 301 100% how did you know that you suffer from bad breath odor someone told me 31 10.3% 218.91 ** (0.0001) people act funnily around me 0 0% i just know 259 86% my dentist 11 3.7% total 301 100% have you had any treatments for bad breath odor yes 29 9.6% 196.17 ** (0.0001) no 272 90.4% total 301 100% if you got it treated, what measurement did you use drugs 9 2.9% 328.74 ** (0.0001) don’t suffering from halitosis 10 3.3% cleaning mouth 52 17.3% don’t use any things 180 59.9% other than that 50 16.6% impact of smoking on halitosis and oral bacterial infechons vol 13, no 1 (2025) doi 10.5195/d3000.2025.981 http://dentistry3000.pitt.edu 11 total 301 100% do you have any social problems because of your bad breath odor yes 24 8% 212.65 ** (0.0001) no 277 92% total 301 100% are you concerned about other people's behavior toward yourself on account of your breath odor yes 123 40.9% 10.049 ** (0.0015) no 178 59.1% total 301 100% * (p≤0.05), ** (p≤0.01), ns: non-signigicant. impact of smoking on halitosis and oral bacterial infechons vol 13, no 1 (2025) doi 10.5195/d3000.2025.981 http://dentistry3000.pitt.edu 12 figure 1. the bar graph illustrates the inhibition zones (mm) for staphylococcus aureus isolates at three-time intervals (t0 (a), t1 (b), t2 (c)), when exposed to six different antibiotics: amoxicillin, vancomycin, ampicillin, erythromycin, azithromycin, and clindamycin. statistical comparisons between groups are indicated with asterisks (* for p < 0.05, ** for p < 0.01, *** for p < 0.001) and "ns" for non-signigicant differences. impact of smoking on halitosis and oral bacterial infechons vol 13, no 1 (2025) doi 10.5195/d3000.2025.981 http://dentistry3000.pitt.edu 13 figure 2. the bar graph illustrates the inhibition zones (mm) for candida albicans isolates at three-time intervals (t0 (a), t1 (b), t2 (c)), when exposed to six antifungal agents: clotrimazole, fluconazole, nystatin, 5-flucytosine, caspofungin, and amphotericin b. statistical comparisons between groups are annotated with *** for p < 0.001 and “ns” for non-signigicant differences. 861 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.861 http://dentistry3000.pitt.edu topical impact of nanosimvastatin on bone fracture luay h. jalil college of den*stry, al-farahidi university, baghdad, iraq abstract bone healing is a vert complex process. fracture healing is a major challenge in orthopaedic prac<ce and research. simvasta<n is an encouraging novel bone regenera<on inducer due to its safety, low cost, and clinical convenience. however, the specific mechanism of simvasta<n's effect on bone growth, as well as the best dose, are unknown. simvasta<n is a biodegradable polymer that promotes bone growth. a total number of 16 adult albino rabbits were divided into two groups (8 rabbits /each). a defect was done in bone and filled with gel only for controls and treatment specimens were filled with nanoliposomal simvasta<n. the results showed that the treatment group with nanosimvasta<n gel had a significant improvement in bone healing compared to the control group. open access cita%on: jalil lh, et al. (2025) topical impact of nanosimvasta%n on bone fracture. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.861 received: february 24, 2025 accepted: march 16, 2025 published: june 26, 2025 copyright: ©2025 jalil lh. this is an open access ar%cle licensed under a crea%ve commons avribu%on work 4.0 united states license. email: luay.h.jalil@@uofarahidi.edu.iq introduc)on bone healing is a very complex process that relies on the coordinated action of numerous cell lineages on a series of biological processes. fracture healing assessment is a major challenge in orthopedic practice and research [1]. bone healing requires the regeneration of a functional bone structure [2]. traditional therapies, such as bone grafts, and different types of synthetic bone substitutes are still challenging either because of their plain side effects or because they are cinancially. tissue-engineering technique, in which osteogenic stem cells and/or bone growth factors are multiplied or loaded in extracellular matrices in vitro before being transplanted in vivo to repair bone defects, is the most recent approach. the growth factors stimulate bone formation, while the matrices operate as a scaffold that incrementally dissolves as bone ingrowth progresses. this should give the new bone sufcicient structure and functionality. however, due to the absence of a solid technique for amplifying and guided differentiation, the use of stem cells is still in the experimental stage [3]. simvastatin is a white, crystalline powder with a low water solubility. it possesses antihypercholesterolemia properties. it works by blocking the reductase of 3-hydroxy-3-methylglutaryl coenzyme a (hmg co-a). this enzyme catalysis cholesterol production. pravastatin was originally reported to prevent heart transplant rejection in 1990, indicating that statins had an immunosuppressive impact [4]. simvastatin, a cholesterol-lowering medicine, was found to improve bone production by boosting the expression of the bone morphogenetic protein2 (bmp-2) gene in bone cells in a previous investigation of osteoporosis. simvastatin has also been linked to enhanced bone density and a lower risk of osteoporotic fracture in more recent clinical investigations. simvastatin has been gaining traction as a promising novel bone regeneration inducer due to its safety, low cost, and clinical availability. however, the specicic mechanism of simvastatin's effect on bone growth, as well as the best dose, are unknown. the success of bone repair is aided by a variety of cytokines and growth factors [5]. the purpose of this study was to determine a better bone regeneration inducer. materials and methods we applied the prepared gel to a surgical damage to the left rabbit femur with followup of the healing process. we used simvastatin in a vehicle gel that included cholesterol and lecithin and simvastatin phosphate buffer (ph 7.4). the chloroform cilm approach was used to standardize a variety of liposomal solutions. cholesterol and lecithin were combined and topical impact of nanosimvasta1n on bone fracture vol 13, no 1 (2025) doi 10.5195/d3000/2025.861 http://dentistry3000.pitt.edu 2 dissolved in a 9 ml organic combination of chloroform and methanol (2:1), respectively. a thin coating of lipids was deposited inside the round bottom clask of the device after the organic solvent was evaporated using a rotary evaporator. with vigorous shaking, sixty ml of 0.2 m phosphate saline buffer ph 7.4 containing 1mg s simvastatin [6] and 1 mm edta was added to the deposited cilm, and liposome suspension formed instantly [7]. a total of sixteen adult albino rabbits were used in current study (2.2-3 kg). animals were kept in individual cages and were kept in similar conditions (22-24 c0) with light and dark cycle, as well as unlimited pellet meal and water ad libitum. before the surgical procedure, a veterinary professional performed an assessment to examine the animal's overall health and condition [8]. food was stopped 9-10 hours before anesthesia was administered. animal hair was shaved off the left femur, and the region was disinfected with a povidone iodide solution [9]. food was not eaten for eight to ten hours before anaesthesia was administered. each animal was given 0.2 mg of vagal tonus to reduce intramuscular atropine sulphate dose of mg/kg injection. anesthesia was used to put the animals to sleep. ketamine (50 mg/kg) was injected intramuscularly. intramuscular injection of (weight in kilograms) and intramuscular injection of (weight in kilograms) diazepam (5.0–10.0 mg/kg body weight). antimicrobial prophylaxes (preoperative antimicrobial prophylaxes) consisting of ceftrioxone was injected at a rate of 50 mg/kg. the defect that done in bone was cilled with nanoliposomal simvastatin for treatment groups and cilled with gel only for control groups; sectioned femurs were cixed in 10% formalin in phosphate buffer saline pbs 0.1 m for 48 hrs. two concentrations were excluded from the study (2% and 0.5%). sectioned femurs were cixed in 10% formalin in phosphate buffer saline pbs 0.1 m for 48 hrs.: at the end of the experimental periods after 6th weeks. the areas of connective tissue and bone were measured in relation to the overall measurement area in histomorphometric evaluations. to avoid any potential bias, the middle section of each core was chosen. i used the image j analysis tool to investigate. 𝐏𝐞𝐫𝐜𝐞𝐧𝐭𝐚𝐠𝐞 𝐨𝐟 𝐜𝐡𝐚𝐧𝐠𝐞 % = 𝐝𝐞𝐟𝐞𝐜𝐭 𝐨𝐟 𝐛𝐨𝐧𝐞(𝐀) − 𝐜𝐨𝐧𝐭𝐫𝐚𝐬𝐭 𝐚𝐫𝐞𝐚(𝐁) 𝐦𝐞𝐚𝐧 𝐨𝐟 𝐢𝐧𝐭𝐞𝐫𝐜𝐨𝐫𝐭𝐢𝐜𝐚𝐥𝐛𝐨𝐧𝐞 𝐚𝐫𝐞𝐚 (𝐂) − 𝐜𝐨𝐧𝐭𝐫𝐚𝐬𝐭 𝐚𝐫𝐞𝐚(𝐁) ∗ 100 volume percentages (percent) of new bone were estimated using histomorphometric measurements of the samples. after identifying the external and internal surfaces of the original femur, the total area was calculated, and the cavity and soft tissue (non-bone region) were circled and decined(10)determined the following histomorphometric parameter. area new bone %= [(1 – (non-bone area / total area)] × 100 (%) results 1. figure 1 shows surgical procedure. at the end of the study, no wound infection was observed. (figure 2). nano simvastatin showed mineralization early and complete bridging of defect was seen at end 6th week after treatment group, in contrast to control group that reported less evidence of healing with significant differences (p-value 0.03, table 1). in figure 3, an example of the image j analysis taken for rabbit femur. the area of the defect was replacement with low density bone of granulation tissue in the controls (figure 4). for the treated animals, there was signicicant increase of cellular component of the newly formed soft callus at the site of defect compared with 6th week sections appeared as clusters of osteoblasts surrounded by variable amount of eosinophilic pink osteoid partially mineralized to appear slightly grayish pink at some foci, generally osteoid precipitation is more diffused compared with 6th week sections (control) (figures 5 and 6). at the end of 6th week of treatment, bone formation in the treatment group was signicicantly higher than control group (p= 0.000) (table 2). discussion numerous experimental research has investigated the use of statins in bone tissues in combination with various vehicle systems [10]. simvastatin works by increasing the activity of osteoblasts while inhibiting the activity of osteoclasts. this is accomplished by bone morphogenetic proteins (bmps) driving osteoblastic cell development (through tnf-a-to-ras/rho/mitogen-activated protein kinase competition) [11]. it was reported that the outcome of radiological investigation in all treatment periods revealed an increase in radiopacity and has come to agreement with following gel production and application in the rabbit femur, after taking the right dose [12]. biological complexity of bone healing of fractures is still an open cield for research [13]. tumors, bone infection, atrophy, and surgical procedures can all cause bone defects in the maxillofacial region. rabbits were used as models because they acquire skeletal maturity at around [6] months of age. in comparison to other animals, the rabbit has a rapid bone turnover [14]. when compared to rats and mice, the rabbit model has the benecit of being easier to manage and set aside for greater bone abnormalities. the usage of males was limited to avoid possible hormonal changes in females, which could have incluenced the results. the cycle of rabbit bone repair takes approximately 42 days, according to consensus. allows for the investigation of the beginning, intermediate, and cinal stages of bone repair evaluations conducted over 7, 21, and 42 days . because it contains red bone marrow and a considerable number of spongy bone and osteoprogenitor cells, the femoral epiphysis is a good candidate for cavity preparation. the femoral epiphysis also allows for the creation of a defect [14]. the goal of this study is to see how simvastatin affects different pathways of bone growth. the effect of simvastatin on entochondrostosis of long tubular bones and intra-membranous ossicication of clat bones was investigated using a rabbit radial defect and a monkey calvaria defect model, respectively. the rabbit radial defect is the traditional model for studying bone formation, with a 20 mm long defect regarded the upper limit for self-repair [15]. simvastatin's ability to increase bone formation and decrease bone loss was cirst demonstrated in rats after stomach administration and subcutaneous tissue injection [15]. conclusion the current study showed that nano-simvastatin gel as a treatment for fractures and bone deformities accelerated their recovery. references 1. hoveizi e, naddaf h. effects of graphene quantum dots on the osteogenic differentiation of stem cells from human endometrial. armaghane danesh. 2020;25(4):423–37. 2. dang l, zhu j, song c. the effect of topical administration of simvastatin on entochondrostosis and intramembranous ossirication: an animal experiment. j orthop transl. 2021;28:1–9. 3. chiadika sm, shobayo fo, naqvi sh, saraykar ss, ambrose cg, rianon nj. lower femoral neck bone mineral density (bmd) in elderly women not on statins. women health. topical impact of nanosimvasta1n on bone fracture vol 13, no 1 (2025) doi 10.5195/d3000/2025.861 http://dentistry3000.pitt.edu 3 2019;59(8):845–53. 4. chauhan as, maria a, managutti a. efricacy of simvastatin in bone regeneration after surgical removal of mandibular third molars: a clinical pilot study. j maxillofac oral surg. 2015;14(3):578–85. 5. bach tj. hydroxymethylglutaryl-coa reductase, a key enzyme in phytosterol synthesis? lipids. 1986;21(1):82–8. 6. le ad, basi dl, abubaker ao. wound healing: rindings of the 2005 aaoms research summit. j oral maxillofac surg. 2005;63(10):1426–35. 7. kheirallah m, almeshaly h. simvastatin, dosage and delivery system for supporting bone regeneration, an update review. j oral maxillofac surgery, med pathol [internet]. 2016;28(3):205–9. available from: http://dx.doi.org/10.1016/j.ajoms.2015.10.0 05 8. marie nk, habeeb ad. preparation and evaluation of salbutamol liposomal suspension using chloroform rilm method. 2012;11(2):39–44. 9. bostan hb, mehri s, hosseinzadeh h. toxicology effects of saffron and its constituents: a review. iran j basic med sci. 2017;20(2):110. 10. aljumaily ma, alnuaimy h, al-allaf h. correlation between computerized tomography densimetry and histomorphometry in assessment of bone healing. j fac med baghdad. 2010;52(4):388– 91. 11. chen c-h, lee fc, liang c-w, chien c-c, chen s-y. histomorphometric and radiographic study of bone healing in critical size defects in rabbit tibia. part ii-study of biomaterials. 輔仁 醫學期刊. 2013;11(4):285–95. 12. yaylacı a, karaarslan es, hatırlı h. evaluation of the radiopacity of restorative materials with different structures and thicknesses using a digital radiography system. imaging sci dent. 2021;51(3):261. 13. almeida m, laurent mr, dubois v, claessens f, o’brien ca, bouillon r, et al. estrogens and androgens in skeletal physiology and pathophysiology. physiol rev. 2017;97(1):135–87. 14. saraei p, asadi i, kakar ma, moradi-kor n. the benericial effects of metformin on cancer prevention and therapy: a comprehensive review of recent advances. cancer manag res. 2019;11:3295–313. 15. venkatesan n, liyanage adt, castro-núñez j, asafo-adjei t, cunningham ll, dziubla td, et al. biodegradable polymerized simvastatin stimulates bone formation. acta biomater. 2019;93:192–199. figure 3. rabbit femur for test (a) and control animal (b). figure 1. surgical procedure. figure 2. no infection was observed. b a topical impact of nanosimvasta1n on bone fracture vol 13, no 1 (2025) doi 10.5195/d3000/2025.861 http://dentistry3000.pitt.edu 4 figure 4. (a) photomicrograph of rabbit left femoral bone at site of defect at control group after 2 weeks of defect induction showing the original mature bone (a) and the soft callus tissue formed at site of defect composed of mesh like collagen bundles (b), a nidus or foci of osteoid precipitations (c), and newly formed capillaries (d). magnicication 165 x, staining h&e. (b) the process if ossicication amount of maturation and mineralization of osteiod is much more progressed at peripherally than it is in center of newly formed callus. (c) few remnants of cibrin casts still apparent, no sign of inclammatory reaction, few newly formed capillaries can be noted, few multinucleated osteoclasts observed at noticed sections. topical impact of nanosimvasta1n on bone fracture vol 13, no 1 (2025) doi 10.5195/d3000/2025.861 http://dentistry3000.pitt.edu 5 group statistics group n mean standard deviation standard.error mean p-value control group 8 46.0000 10.74709 4.80625 0.030* treatment group 8 64.4000 11.37102 5.08527 independent t test signicicant difference-p value ≤0.05* figure 5. the areolar tissue bridges between those partially ossicied segments still recognizable, vascularization and capillaries are more prominent. photomicrograph of rabbit, femoral bone at site of defect at treated group after 6 weeks of defect induction showing areolar tissue at the center of the callus demonstrating mature capillaries and foci of proliferating osteoblasts. staining h&e. figure 6. photomicrograph of rabbit femoral bone at site of defect at treated group after 6 weeks of defect induction showing a magnicied view of previous h&e staining. topical impact of nanosimvasta1n on bone fracture vol 13, no 1 (2025) doi 10.5195/d3000/2025.861 http://dentistry3000.pitt.edu 6 table 2. new bone after created defects in rabbit femur (n=8). group statistics group n mean standard deviation standard error mean p-value control group 8 0.5720 0.04970 0.02223 0.000 treatment group 8 0.9020 0.07190 0.03216 independent t test signicicant difference-p value ≤0.05 860 final effect of menthe and green tea vola3le oil extract on streptococcus mutans, a bacterium species isolated from dental caries vol 13 no 1 (2025) doi 10.5195/d3000.2025.860 h#p://den*stry3000.pi#.edu effect of menthe and green tea volatile oil extract on streptococcus mutans, a bacterium species isolated from dental caries mohamed abdulmunem abdulafeet, sura a. jaber, anes a. alshamaa, sohaib mohameed awad college of dentistry, university of al maarif, anbar, iraq abstract objec&ves: to iden(fy phytochemical components, evaluate the yield or vola(le oil extrac(on, and assess the impact of vola(le oil extract on a strain of streptococcus mutans that was isolated from a dental caries. materials and methods: menthe spicata (menthe) and camellia sinensis (green tea) were harvested fresh during the flowering period of 2022, and their vola(le oils were extracted for about 24 hours using a sox let apparatus. green tea and menthe were prepared at four different concentra(ons (50, 75, 100, and 150 mg/ml). streptococcus mutans growth isolates were inhibited, and the results were compared with extracts from both kinds of plants. results: menthe produced the most vola(le oil extract (3%), while green tea produced the least (2%). alkaloid, phenol, glycoside, and flavonoid showed posi(ve results in the phytochemical screening of m. spicata vola(le oil extract, but saponin showed nega(ve results. the results also showed that the vola(le oil of green tea and menthol oils (100 and 150 mm/ml) were more inhibitory than those of 50 and 75 mm/ml. overall, given these results, vola(le oil of menthe has greater effect on dental caries compared to vola(le oil of green tea. conclusions: the vola(le oil extract on menthe inhibitor is more effec(ve on the s. mutans bacteria compared to vola(le oil of the green tea. keywords: dental caries; m. spicata; streptococcus mutans; vola(le oils; green tea. cita:on: abdulafeet ma, et al. (2025) effect of menthe and green tea vola:le oil extract on streptococcus mutans, a bacterium species isolated from dental caries. den:stry 3000. 1:a001 doi:10.5195/d3000.2025.860 received: february 24, 2025 accepted: march 14, 2025 published: april 25, 2025 copyright: ©2025 abdulafeet ma, et al. this is an open access ar:cle licensed under a crea:ve commons ayribu:on work 4.0 united states license. email: mohamadjasem@yahoo.com introduction one of the most prevalent oral conditions affecting people worldwide is dental caries. it is a chronic infectious disease caused by series microbial activities and chemical reactions linked to form a biofilm on dental plaque. thus, minerals will be removed from the calcified tissues of teeth and cause decomposition of organic ingredients [1]. the presence of bacteria, consuming carbohydrates and producing acids by bacteria are the main causes of dental caries [2]. there are many different types of bacteria in the mouth, but only few types can cause dental caries [2,3]. one of the most well-known biofilm-forming bacteria, streptococcus mutans, is a member of the oral micro flora and is thought to be the main cause of dental caries (or tooth decay) [3,4]. it is quite concerning that infectious diseases are on the rise and that harmful bacteria and fungi are rapidly developing medication resistance. the mortality and morbidity related to microbial infections remain high despite improving the knowledge about effect of menthe and green tea vola3le oil extract on streptococcus mutans, a bacterium species isolated from dental caries vol 13 no 1 (2025) doi 10.5195/d3000.2025.860 h#p://den*stry3000.pi#.edu microbial pathophysiology and the use of different methods of treatments [5]. as a result, scientists in this sector had to come up with alternatives, including plant extracts or therapeutic herbs, as the primary sources of pharmaceuticals to treat a variety of bacterial illnesses, including gingivitis [6]. the m. spicata (menthe) plant is common in tropical and subtropical climates areas which consider as a source of terpene rich essential oils [7]. m. spicata are members of the lamiaceae family and defined as the key sources of physiologically active chemicals [8]. the dried leaves are used commonly for herbal tea and may use as a medication, whereas the fresh leaves are used as a flavoring herb or raw vegetable [9]. several biological effects of mint have been related to them, including antiinflammatory, antioxidant, anticancer, and antibacterial properties [9]. different esters are present in the volatile oil in different ratios; phenol reaches 5%, while menthol 5% and ketones 3% [9,10]. in this study, green tea (camellia sinensis), which is made from a tree whose leaves grow in southeast asia and has been consumed by the chinese for thousands of years, was chosen [11]. it contains phenolic compounds, also known as flavonoids, which are biologically effective compounds as antioxidants and anti-bacterial. these compounds bind free radicals, which are produced when food is burned for energy and whose presence works in cell membranes to stress the cell and make it vulnerable to dna damage, causing the cell to become stressed [12]. material and methods plant collection the plants menthe and green tea from karbalaa city in hindia, iraq were collected during the flowering stage. the m. spicata were dried at oven degree 45 cº and then were powdered and grinded by using mechanical grinder [13,14]. plant extraction 80 gram of plant was placed in thimble with 200 ml of ether 75% in flask round volume 1000 ml for 24 hours. then, a rotary evaporation apparatus was used to evaporate the extract at 45 cº [15,16]. the percentage is evaluated using the equation listed below [17-19]. 𝒀𝒊𝒆𝒍𝒅 (𝑾𝒕.%) = 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒐𝒊𝒍 𝒑𝒓𝒐𝒅𝒖𝒄𝒆𝒅 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒔𝒆𝒆𝒅 𝒑𝒐𝒘𝒅𝒆𝒓 𝒖𝒔𝒆𝒅𝒙𝟏𝟎𝟎 reagents were prepared according to previous work [20-24]. we examined the impact of various doses on the development of specific pathogenic microorganisms [22]. statistical analysis the experiments were conducted and analyzed as factorial experiments with three replications using a completely randomized design (crd) with two or three factor tested by least effect of menthe and green tea vola3le oil extract on streptococcus mutans, a bacterium species isolated from dental caries vol 13 no 1 (2025) doi 10.5195/d3000.2025.860 h#p://den*stry3000.pi#.edu significant difference (l.s.d) at probability of 1% (p ≤ 0.01) [25]. results percentage yield of volatile oils extracted results in table 1 showed the percentage yield of volatile oils offered the height percentage of the mentha volatile oils extract (3%), and green tea has a variety of volatile oil components that are naturally produced and accumulate in plants (2%). table 1. percentage yield of volatile oils extracted. percentage yield of oils extracted oils extracted 3/100g *100 = 3% m. spicata 2/100 g*100= 2% green tea secondary metabolism screen study table 2 displays the findings of the secondary metabolism screen, which was conducted to identify the active chemicals extracted from m. spicata. the results showed positive results for alkaloids, phenols, glycosides, and flavonoids but negative results for saponin. while tests on green tea's alkaloid, phenol, glycoside, flavonoid, and saponin yielded a favorable result. table 2. secondary metabolism screen of m. spicata. green tea menthe compounds + + alkaloid + + phenol + + glycoside + + flavonoid + saponin effect of menthe and green tea vola3le oil extract on streptococcus mutans, a bacterium species isolated from dental caries vol 13 no 1 (2025) doi 10.5195/d3000.2025.860 h#p://den*stry3000.pi#.edu effect of menthe and green tea volatile oil extract on s. mutans the goal of the study was to determine how the volatile menthol and green tea oils affected the development of the s. mutans bacteria by measuring the widths of the inhibition zones 48 hours after incubation, and then the findings were determined. the results showed that the effect of 100 and 150 mm/ml of green tea volatile oil on the dental caries were more inhibitory than those of 50 and 75 mm/ml as explained in table 3. table 3. inhibitory activity of the green tea volatile oil extract of the s. mutans. volatile oil concentration inhibitor zone control 0 50 % 18 75% 23 100% 28 150% 30 l.s.d (0.01) 1.52 the results in table 4 listed the effect of menthe volatile oil on dental caries which showed that the inhibitory effect of treatment volatile oil (100 and 150 mm/ml) was greater than that of (50 and 75 mm/ml). table 4. inhibitory activity of the menthe volatile oil extract of the s. mutans. volatile oil concentration inhibitor zone control 0 50 % 19 75% 25 100% 31 150% 32 l.s.d (0.01) 2.13 discussion the application of herbal treatments to treat dental caries is of potential interest [26]. the inhibition process in bacteria is directly proportional to the rise in concentration. the effect of the active extract on the bacterial species increases with the concentration of the extract [27]. the extract's potency is a result of the presence of active ingredients, which enhance its ability to stop the growth of microorganisms by blocking the function of the cell wall and its effects on biological processes [28]. for example, the extract's capacity to form hydrogen bonds with proteins results in the destruction of protein effect of menthe and green tea vola3le oil extract on streptococcus mutans, a bacterium species isolated from dental caries vol 13 no 1 (2025) doi 10.5195/d3000.2025.860 h#p://den*stry3000.pi#.edu synthesis in bacterial cells, which stops the growth of bacteria. it also has an impact on the genetic makeup of these bacteria [28]. previous work [29] showed that an inhibition diameter of 30 mm was caused by the green tea extract, compared to 28 mm for hot water extracts and 26 mm for cold water extracts. the inhibitory activity was due to the nature of the substances present in the plant, such as the presence of alkaloid compounds, anti-substances include phenols, tannins, flavonoids, saponins, and resins. for bacteria, tatins work to restrict the growth of bacteria by interfering with dna and having a demonstrable impact on the bacterial cell. the cell membrane's carrier enzymes are present, and phenols are distinguished by their capacity to combine with other molecules complexes between extracellular proteins and cells [30]. the findings of current study were consistent with previous research [31], which demonstrated that green tea can be used as mouthwash to prevent cavities and is an inhibitor of s. mutans isolates. the outcomes concurred with those of a team of researchers [32] who discovered that green tea has the power to suppress the s. mutans bacteria that causes caries in teeth by stopping their attachment to teeth surfaces and thus reducing the production of essential membranes. the results in this study were also agreed with a previous study [33] where they concluded that green tea extracts possess an inhibitory activity against s. mutans. the inhibitory activity is due to the nature of the substances contained in the plant, such as the presence of alkaloid compounds phenols, tannins, flavonoids, saponins, and resins which considered as antisubstances for bacteria. they have a clear effect in inhibiting the growth of bacteria to the bacterial cell and interfere with the dna the carrier enzymes present in the cell membrane, while phenols are characterized by their ability to form a complex with extracellular proteins complex with the cell wall and lead to disruption of the cell membrane of bacteria. conclusion the volatile oil extract of menthe inhibitor is more effective on the s. mutans compared to volatile oil of the green tea. overall, using 100 and 150 mm/ml of volatile oil (wither menthe or green tea) are more effective in decreasing dental carries compared to lower concentrations. acknowledgements the facilities at university of almaarif in al-anbar, are acknowledged by the writers, who are also appreciative of the faculty of dentistry staff's assistance in practice. ethical approval because this research was conducted using samples unrelated to humans or animals, ethical approval was not necessary. effect of menthe and green tea vola3le oil extract on streptococcus mutans, a bacterium species isolated from dental caries vol 13 no 1 (2025) doi 10.5195/d3000.2025.860 h#p://den*stry3000.pi#.edu conflicts of interest authors have no conflicts of interest to declare. funding authors were self-funded in this research. references 1. pitts, n.b., twetman, s., fisher, j. and marsh, p.d., 2021. understanding dental caries as a non-communicable disease. british dental journal, 231(12), pp.749753. 2. cheng, l., zhang, l., yue, l., ling, j., fan, m., yang, d., huang, z., niu, y., liu, j., zhao, j. and li, y., 2022. expert consensus on dental caries management. international journal of oral science, 14(1), p.17. 3. yu, o.y., lam, w.y.h., wong, a.w.y., duangthip, d. and chu, c.h., 2021. nonrestorative management of dental caries. dentistry journal, 9(10), p.121. 4. prados-privado, m., garcía villalón, j., martínez-martínez, c.h., ivorra, c. and prados-frutos, j.c., 2020. dental caries diagnosis and detection using neural networks: a systematic review. journal of clinical medicine, 9(11), p.3579. 5. safini, i.n.m., zakaria, n.f.s., saad, m.i.h., yahya, m.f.z. and jamil, n.m., 2024. understanding bacterial persistence under antibiotic pressure: a review. science letters, 18(2), pp.56-69. 6. jassim, n.n. and hassan, r.h., 2019. the inhibitory activity of plant extracts (mentha citrato l. and citrus aurantium l.) towards some bacteria strains isolated from inflamed gums. tikrit journal of pure science, 24(7), pp.40-44. 7. ayaz, e. and memon, a., 2021. development of the aromatic medicinal plants, mentha x piperita l. and mentha pulegium l. through in vitro callus induction and micropropagation. 8. chrysargyris, a., koutsoumpeli, e., xylia, p., fytrou, a., konstantopoulou, m. and tzortzakis, n., 2021. organic cultivation and deficit irrigation practices to improve chemical and biological activity of mentha spicata plants. agronomy, 11(3), p.599. 9. tang, k.s., konczak, i. and zhao, j., 2016. identification and quantification of phenolics in australian native mint (mentha australis r. br.). food chemistry, 192, pp.698-705. 10. hatami, t., johner, j.c., zabot, g.l. and meireles, m.a.a., 2019. supercritical fluid extraction assisted by cold pressing from clove buds: extraction performance, volatile oil composition, and economic evaluation. the journal of supercritical fluids, 144, pp.39-47. 11. zhao, t., li, c., wang, s. and song, x., 2022. green tea (camellia sinensis): a review of its phytochemistry, pharmacology, and toxicology. molecules, 27(12), p.3909. 12. jelic, m.d., mandic, a.d., maricic, s.m. and srdjenovic, b.u., 2021. oxidative stress and its role in cancer. journal of cancer research and therapeutics, 17(1), pp.22-28. 13. nibras, a.i. and hasan, r.m., 2019. identification of artemisinin compound in artemisia herba alba belong to the asteracea by hplc and gc/ms. al-kufa university journal for biology, 11(2), p.32. 912 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu histopathological assessment of wound healing in mice treated with helianthus tuberosus powder ali ismiel yawir, layla sabri yas college of den*stry, university of baghdad, baghdad, iraq abstract objec5ve: several medicinal plants have been given significant a?en5on in the medical field due to their effect on wound healing. this study aims to iden5fy the effec5veness of tuberosus helianthus powder in skin wound healing in mice. material and methods: tuberosis helianthus powder was purchased from a commercial supplier (medicinal korean herbs, prince herb). the powder was stored in a dry, air5ght container at room temperature un5l use. it was applied directly to the incisional wounds at a specific concentra5on and frequency (50 mg twice daily). thirty-six male mice were randomly assigned to three 5me points (3, 7, and 14 days), with 12 mice per 5me point. each mouse received two incisional wounds: one on the lev side, serving as the control, and another on the right side, designated as the experimental wound. at each 5me point, 12 mice were euthanized, yielding 24 wound samples (12 control and 12 experimental) per 5me point. in total, 36 control wounds and 36 experimental wounds were analyzed. three physical parameters were taken, including the number of inflammatory cells, blood vessels, and epidermis thickness. all these parameters were on the dorsal aspect of the thoracolumbar region, into which tuberous helianthus powder was typically done in the treated day dura5on. meanwhile, the day dura5ons for control mice were lev without any treatment. the mice were euthanized on days 3, 7, and 14 aver wound healing for histopathological study. results: the histopathological analysis using hematoxylin and eosin (h&e) staining revealed significant differences between the control and treatment groups. on day 3, the control group exhibited pronounced inflammatory responses with extensive inflammatory cell infiltra5on, necro5c 5ssue, and fibrin clot forma5on. in contrast, the treatment group showed early signs of wound healing, such as mild inflammatory response and reepithelializa5on with kera5nized stra5fied squamous epithelium. throughout the study, the treatment group demonstrated faster wound contrac5on, enhanced 5ssue remodeling, and reduced inflammatory cells compared to the control. by day 14, treated mice displayed mature epidermis and dermal granula5on 5ssue with significant angiogenesis, sugges5ng accelerated wound healing. these findings support the poten5al of helianthus tuberosus powder in promo5ng wound healing. conclusion: tuberosus helianthus powder plays an important role in the healing pathway in wound healing. open access cita%on: yawir ai, et al. (2025) histopathological assessment of wound healing in mice treated with helianthus tuberous powder. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.912 received: april 18, 2025 accepted: may 14, 2025 published: june 27, 2025 copyright: ©2025 yawir ai, et al. this is an open access ar%cle licensed under a crea%ve commons auribu%on work 4.0 united states license. email: ali.yamir2206@codental.uobaghdad.edu.iq histopathological assessment of wound healing in mice treated with helianthus tuberosus powder vol 13, no 1 (2025) doi 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 2 introduc)on the healing of skin wounds is a problematic biological procedure by which skin repairs itself after a wound [1,2]. wound healing is a natural process that returns the skin or mucosa to its normal state [3]. this procedure comprises three highly combined stages: inclammation, tissue proliferation, and remodeling [4]. it includes the activation of inclammatory cells, endothelial cells, keratinocytes, and cibroblasts [5], along with the mixture and liberation of active mediators (cytokines and development parameters) to adjust tissue repair and recreation [2,6]. these stages and their physiological parts might happen in the suitable series and at certain times to realize the ideal function and the clexible strength of the wounded position of the source skin [6]. different methods are utilized to support skin wound healing, such as managing local or systemic antibiotics or using antiseptic results [7]. the increasing of opposing pathogenic against presently utilized simulated medications managers like antifungal [8], antiviral products and antibiotics has encouraged interest among several investigators in the determination of novel antibacterial combinations created from plants [9], plant depend on phytochemicals as potential alternatives to antibiotics that are still utilized in modern and classical schemes of drug for the disease treatment [10]. the existence of multiple life-sustaining constituents in plants has encouraged researchers to concentrate on plants for their extensive healing wounds potential [11] . currently, plants or chemical combinations derived from plants have been utilized as pharmaceutical product managers for treating skin wounds [12]. medicinal plants show a vital key in wound healing via several different mechanisms like reducing the bacterial amount, improving angiogenesis, rushing the deposition of collagen, and rising cibroblast proliferation [13]. the biological features of medicinal plants affect key actions in mammalian wound healing, displaying a vital character in regulating the wound-healing pathway [14]. the knowledge of the medicinal value of plants and other substances and their use goes back to the earliest settlers [15]. they are hence regarded as a basis of several macromolecules like fatty acids, carbohydrates, and proteins [16], which can be utilized as biomaterials for emerging new candidate medications to support wound healing [17]. around 500,000 plant types exist worldwide, and only 1% have been investigated [18]. tuberosus helianthus is a sunclower that appeared in eastern north america and is extensively distributed in the middle east, particularly iraq [19]. this plant is (2–3 mm) long with supercicial leaves and plump tubers [20]. as the plant was civilized by native americans cirst, they denoted the plant as sun roots. while the plant was presented to other portions of the world [21], varied standard terms were attributed in several languages [22]. it has been recognized that this type shows several medical activities, such as diuretic, purgative, and bowel tonic impacts [23]. it has also been utilized as a folk medicine for treating bone fractures and cutaneous wounds, and even for releasing pain [24]. studies have also shown that tuberous helianthus combinations have antiinclammatory, antimicrobial, antioxidant, antifungal, anticancer, antipyretic, and analgesic impacts [25]. tuberosus helianthus is rich in fatty acids like plasmatic acid that resemble those current in the sebaceous glands in the skin [26]. these fatty acids have been recorded to keep strong antimicrobial–disinfectant features in the skin [27]. this plant has been utilized generally as a dietary supplement for pain management, to decrease swelling [23], to increase the immune system, and to treat skin wounds in general medicine. it is an abundant source of bioactive compounds like phenolic acids, coumarins [28], and clavonoids that are recognized to use pharmacological actions, comprising anti-inclammatory, antimicrobial, and antioxidant features [29]. this study aims to identify the effectiveness of tuberosus helianthus powder on skin wound healing in mice. material and methods this study was cross-sectional and approved by baghdad university, baghdad dental college, iraq—thirty-six adult male mice from april 2024 to july 2024. the mice used in the experiment protocol were from baghdad university baghdad dental college. the samples were treated at the laboratories of the animal house center in the college of baghdad medicine, baghdad university. thirtysix adult male mice will be subjected to the induced wound on the right dorsal region of each mouse. aincisional wound on the left side of the dorsal region will be treated daily with normal saline for three different durations (3, 7 and 14 days). b-experimental group: 36 mice will be subjected to the induced incisional wound on the right side of the dorsal region of the mice and will be treated daily with tuberosus helianthus powder for three different day durations (3, 7 and 14 days). a full thickness wound1cm×1 cm in size was established on the right side of the dorsal aspect of the animal, into which tuberosus helianthus powder was topically applied in the treated group. in contrast, the control group was left without any treatment. all samples were excised and cixed in 10% formalin. after cixation, they were processed; block and slides were prepared and then stained in hematoxylin / eosin for histological studies [30]. preparation of tuberous helianthus powder: tuberosus helianthus powder was purchased from a commercial supplier (medicinal korean herbs, prince herb). the powder was stored in a dry, airtight container at room temperature until use [31]. it was applied directly to the incisional wounds at a specicic concentration and frequency (50 mg twice daily). using helianthus tuberosus in powder form instead of liquid or other preparations can have several advantages in wound healing, like stability and shelf life, controlled dosage, sustained release, direct application and enhanced absorption and coverage [32]. 36 adult male mice were classicied into experimental and control groups (one incision on the left side -the control and one on the right side experimental), with three different day durations (3, 7 and 14 days). three physical parameters were taken, like number of inclammatory cells, blood vessels, and epidermis thickness. all these parameters were on the dorsal aspect of the thoracolumbar region, into which tuberous helianthus powder was topically done in the treated day duration. the day durations for the control incision were left without any treatment. the mice were euthanized on days 3, 7, and 14 after wound healing for histopathological study. animals: thirty-six male young adults (7-8 weeks old) kunming mice weighing 20-25 g were purchased from the animal house at the college of veterinary medicine, baghdad university. the environment in the cages was kept at room temperature of 25 ± 2°c [33], humidity 55-60%, with a 12:12 h light/dark cycle. the animals were fed standard pellets and tap water. we used male mice in this study to reduce the contribution of hormonal variation associated with the oestrous cycle of female mice, which can affect inclammatory responses and tissue remodeling in wound healing [34]. wound creation: general anaesthesia was performed with a mixture of 90 mg/kg ketamine (alfasan, holland) and 10 mg/kg xylazine (rugby-virbac laboratories, france) intraperitoneally. the dorsal part of the thoracolumbar area on both sides was surgically prepared, and full-thickness 1 cm×1 cm wounds were created with a surgical scalpel (blade no.11) on all the animals. surgery was performed under a sterile condition [35]. the lesions on the left side were left undressed overnight to the environment to grow, microbial histopathological assessment of wound healing in mice treated with helianthus tuberosus powder vol 13, no 1 (2025) doi 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 3 colonization and proliferation. in contrast, the right side was treated with the powder, and 50 mg of h. tuberosus powder was topically applied twice daily for the injury. the incisional wound was made because it enables precise evaluation of inflammation, fibroblast activity, and collagen remodeling. it is a reliable and reproducible method of measuring wound closure over time. also, it simulates surgical wounds, which makes it pertinent for possible helianthus tuberosus clinical applications, such as post-surgical healing [36]. sample size calculations: the sample size calculation was done utilizing statistical analyses achieved using the statistical package for social sciences (spss) ver. 29 with microsoft excel 2021. results of the complete number of mice 36 adult male mice from april 2024 to july 2024. the α error was 0.05, and the power was 0.90 for sample size calculation. frequency distribution for nominated variables describing the detailed mice with samples was done. the entire test was commonly distributed, as variables are presented by different statistical parameters like mean, standard deviation (sd), and standard error mean. a one-way anova test was used to compare the experimental and control groups for three days, shown at a signicicance level of 0.05. results this study was cross-sectional and approved by baghdad university, baghdad dental college, iraq. thirty-six adult male mice from april 2024 to july 2024. all mice will be subjected to a traumatic incision of 1^1cm, with a surgical blade (no 11) on the right side of the dorsum for each mouse with the application of tuberous helianthus powder on the wound. the mice were euthanized on days 3, 7, and 14 days after the wound, making for a histopathological study. using imagej software, the histopathological analysis will be achieved on hematoxylin and eosin-stained slides, including the inclammatory cells, epithelial thickness, and blood vessel numbers. macroscopic assessment of wound healing: wound contraction over time was macroscopically evaluated as the healing process of the incisional wounds. digital photographs of these wounds were acquired at multiple time points (d3, d7, and d14) for both groups as visual estimates of wound closure. in addition, , wound contraction was quanticied by a vernier caliper to evaluate the decreasing surface area of wounds with time. wound healing was noticeably enhanced in helianthus tuberosus powder-treated animals compared to untreated animals. dressed wounds showed earlier signs of closure with lower levels of inflammation and higher reductions in wound size over time. in contrast, the control group displayed impaired healing with an increased wound size and prolonged inflammation. these results concluded that helianthus tuberosus powder could improve wounds' healing response, leading to faster contraction and lower inflammatory responses in the early days. this observation is further supported by representative images at various time points of wound closure in both groups (figure 1). microscopic assessment of wound healing-histopathological result (hematoxylin & eosin) stain: figures a through n summarize microscopic assessements. effect of the number of indlammatory cells results on all day durations: a oneway anova test is used to analyze the effect of the number of inclammatory cells on allday durations. a p-value >0.05 indicates no signicicant differences between the number of inclammatory cells results and all-day durations. at the same time, p-value < 0.05 represents the signicicant differences between the number of inclammatory cells and all-day durations. there was a highly signicicant difference in the number of inclammatory cells results among all day durations according to p-value=0.000. it can be observed that the highest mean value (98.00) of 3 days of control is attributed to the number of inclammatory cells at this duration being very high compared with other day durations. the lowest standard deviation value (0.707) of 14 days (control negative) was due to a slight difference in values among all the inclammatory cells at this duration, as displayed in table 1. effect of the number of blood vessels results on all day durations: a one-way anova test is used to analyze the effect of the number of blood vessel results according to all-day durations. there was a highly signicicant difference between the numbers of blood vessel results and all-day durations concerning p-value=0.000, as displayed in table 2. it can be observed that the maximum mean value (8.20) of 7 days post-treatment is attributed to the number of blood vessels at this duration, which is very high compared to other day durations. the minimum standard deviation value (0.447) of 14day durations post-treatment is due to a slight difference in values among the number of blood vessels at this duration. effect of the thickness of epidermis results on all day durations: a one-way anova test was used to analyze the effect of the thickness of epidermis results according to all-day durations. there was a highly signicicant difference between the thickness of epidermis results and all-day durations concerning p-value=0.000, as exhibited in table 3. it can be clearly shown that the maximum mean value (82.1164) of 3-day durations post-treatment is attributed to the thickness of the epidermis at this duration being very high compared with other day durations. the minimum standard deviation value (0.5254) of 14-day durations (control negative) is due to a slight value difference among all the epidermis thicknesses at this duration. furthermore, there was no formation around the tissue at this duration. discussion this study evaluated the histopathological aspects of the treatment of wound healing, emphasizing inclammatory cell inciltration, vascularization, epidermal thickness and maturity of the connective tissue. the results concirmed signicicant differences between experimental groups at different time points using one-way anova followed by post hoc tests, emphasizing the efcicacy of the tested treatment in enhancing the wound healing process [37]. however, on day 3, the treated group had signicicantly fewer inclammatory cells (mean = 32.40), indicating that the treatment helped reduce the early inclammatory response. controlled inclammation is essential for wound healing; however, it should not be excessive, since it leads to delayed wound healing [38]. additionally, the reductions in inclammatory cell counts observed in the post-treatment groups persisted with time. on day 14, the treated group had the least inclammatory cells (mean = 1.80), suggesting a reduced inclammatory response. p < 0.05, highlighting the promise of treatment to drive the less inclammatory environment required for wound healing [39]. vascularization: angiogenesis—forming new blood vessels—is a key component of wound healing. the data were analysed statistically, and a marked increase in the number of blood vessels in the treated groups vs the control groups was seen (o/l). the treated group had a mean of 8.20 blood vessels per hpf (p<0.05) compared to the 6.40 in the control on day 7 post-treatment. indeed, the treatment-generated difference (p < 0.05) suggests that angiogenesis was encouraged and allows for the delivery of oxygen and nutrients to the regenerating tissue, as well as transitioning the healing process from inclammatory to proliferative [38]. these results also validated the favorable effect of the treatment on vascularization, which would be shown by improvements in the vascular content of the treated groups over the other groups at later time points (days 7 and 14). histopathological assessment of wound healing in mice treated with helianthus tuberosus powder vol 13, no 1 (2025) doi 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 4 epidermal thickness: epidermal thickness is a critical indicator of epithelialization, and our results show a signicicant increase in epidermal thickness in the post-treatment groups. on day 3 post-treatment, the treated group had an average epidermal thickness of 82.12 µm, signicicantly more signicicant than the control group, which showed a cibrin clot and necrotic tissue in the epidermal layer. the above difference (p<0.05) concirms that the treatment resulted in a faster reepithelization and a thicker epidermal sheet with stronger morphology(40). the treated group still had epithelial messaging by day 14, emphasizing how egf treatment accelerated wound healing and induced epidermal integrity. maturation of connective tissue: another very important factor of wound healing is the maturation of connective tissue. connective tissue type analysis showed signicicant differences in inclammatory cell numbers about each type. the control group at day 3 showed necrotic tissue with many inclammatory cells (mean = 98.00), while the treated groups exhibited more mature granulation tissue intestinal types. statistical analysis suggested that necrotic connective tissue was signicicantly associated with more signicicant inclammatory cell counts than other tissue types, including immature granulation tissue and mature granulation tissue. these had the combined effect of enhancing the shift from necrotic tissue to more granulation tissue, which is crucial in the remodeling phase of wound healing [41]. conclusion wound healing is a complex process that involves inclammation, granulation and tissue remodeling. interactions of different cells, extracellular matrix proteins and their receptors are involved in wound healing and are mediated by cytokines and growth factors [42]. the statistical analysis of the histopathological data corroborates the fact that the treatment dramatically accelerates the healing process. the decrease in inclammatory cells, increased vascularity, thickened epidermal layer, and collagen maturation suggest that treatment acts on multiple parts of the wound healing cascade. these data corroborate the literature on non-invasive therapeutic strategies for regenerative enhancement of cutaneous wound healing [38]. overall, since the treatment reduced inclammation, promoted angiogenesis, and accelerated healing of the epithelial and connective tissues, it may be an effective therapeutic strategy in wound healing. references 1. tottoli em, dorati r, genta i, chiesa e, pisani s, conti b. skin wound healing process and new emerging technologies for skin wound care and regeneration. pharmaceutics. 2020;12(8):735. 2. al-zubaidy ey, ghani ba, ibrahim nr. local application of opuntia ficus-indica/punica granatum oils on cutaneous wound healing: a histochemical study. journal of baghdad college of dentistry. 2023;35(4):28-34. 3. al zamely zr, kadhim ef. evaluation of the effect of local exogenous application of osteopontin on wound healing in rats. journal of baghdad college of dentistry. 2018;30(2):23-8. 4. mancini a, gentile mt, pentimalli f, cortellino s, grieco m, giordano a. multiple aspects of matrix stiffness in cancer progression. frontiers in oncology. 2024;14:1406644. 5. stunova a, vistejnova l. dermal fibroblasts—a heterogeneous population with regulatory function in wound healing. cytokine & growth factor reviews. 2018;39:137-50. 6. lasocka i, jastrzębska e, szulcdąbrowska l, skibniewski m, pasternak i, kalbacova mh, et al. the effects of graphene and mesenchymal stem cells in cutaneous wound healing and their putative action mechanism. international journal of nanomedicine. 2019:2281-99. 7. kramer a, dissemond j, kim s, willy c, mayer d, papke r, et al. consensus on wound antisepsis: update 2018. skin pharmacology and physiology. 2018;31(1):28-58. 8. nicola am, albuquerque p, paes hc, fernandes l, costa ff, kioshima es, et al. antifungal drugs: new insights in research & development. pharmacology & therapeutics. 2019;195:21-38. 9. denaro m, smeriglio a, barreca d, de francesco c, occhiuto c, milano g, et al. antiviral activity of plants and their isolated bioactive compounds: an update. phytotherapy research. 2020;34(4):742-68. 10. haque ma, jantan i, bukhari sna. tinospora species: an overview of their modulating effects on the immune system. journal of ethnopharmacology. 2017;207:67-85. 11. alwan mh, ghani ba. histological evaluation of local application of flavonoid extract of capparis spinosa on wound healing in alloxaninduced diabetic rats. diyala journal of medicine. 2020;18(1):1-13. 12. sharma a, khanna s, kaur g, singh i. medicinal plants and their components for wound healing applications. future journal of pharmaceutical sciences. 2021;7(1):1-13. 13. ghosal k, chakraborty d, roychowdhury v, ghosh s, dutta s. recent advancement of functional hydrogels toward diabetic wound management. acs omega. 2022;7(48):43364-80. 14. biswal a, purohit ss, swain sk. chitosan based composite scaffolds in skin wound repair: a review. journal of drug delivery science and technology. 2023;84:104549. 15. khalil am, fadhil e. histological and immunohistochemical study of osteocalcin to evaluate the effect of local application of lepidium sativum oil on bone healing of rats. biochemical & cellular archives. 2020;20(2). 16. su l, feng y, wei k, xu x, liu r, chen g. carbohydrate-based macromolecular biomaterials. chemical reviews. 2021;121(18):10950-1029. 17. chouhan d, dey n, bhardwaj n, mandal bb. emerging and innovative approaches for wound healing and skin regeneration: current status and advances. biomaterials. 2019;216:119267. 18. lavoie c. biological collections in an ever changing world: herbaria as tools for biogeographical and environmental studies. perspectives in plant ecology, evolution and systematics. 2013;15(1):68-76. 19. kaya y, balalic i, milic v. eastern europe perspectives on sunflower production and processing. sunflower: elsevier; 2015. p. 575-637. 20. atiyah ag, al-falahi nhr. the role of helianthus tuberosus powder in healing of fullthickness wounds in mice. veterinary world. 2021;14(5):1290. 21. sawicka b, skiba d, umachandran k, dickson a. alternative and new plants. preparation of phytopharmaceuticals for the management of disorders: elsevier; 2021. p. 491537. 22. seiler gj, qi ll, marek lf. utilization of sunflower crop wild relatives for cultivated sunflower improvement. crop science. 2017;57(3):1083-101. 23. sawicka b, skiba d, pszczóå p, aslan i, sharifi j, krochmal-marczak b. jerusalem artichoke (helianthus tuberosus l.) as a medicinal plant and its natural products. cellular and molecular biology. 2020;66(4):160-77. 24. de araujo taa, locatelli fr, da silvalópez re, do carmo fa. formulations with active plant molecules and additional therapies in wound healing. revista brasileira de farmacognosia. 2024:1-22. 25. chen f-j, long x-h, li e-z. evaluation of antifungal phenolics from helianthus tuberosus l. leaves against phytophthora capsici leonian by chemometric analysis. molecules. 2019;24(23):4300. 26. pal d, lal p, hiremath d. sunflower and passiflora seeds and their roles in cancer with special reference to secondary metabolites present within them. seeds: anti-proliferative storehouse for bioactive secondary metabolites: springer; 2024. p. 243-83. 27. jackman ja, yoon bk, li d, cho n-j. nanotechnology formulations for antibacterial free fatty acids and monoglycerides. molecules. 2016;21(3):305. 28. sharma sk, alam a. biological prospecting of the "hidden diversity" of medicinal plants (asteraceae) in south-eastern rajasthan. geophytology. 2022;51(1-2):143-8. 29. kaszás l, alshaal t, el-ramady h, kovács z, koroknai j, elhawat n, et al. identification of bioactive phytochemicals in leaf protein concentrate of jerusalem artichoke (helianthus tuberosus l.). plants. 2020;9(7):889. 30. gathwan kh, albir aa. quantitative and histological study of the effect of cadmium oxide on both body and kidney of mice. indian journal of forensic medicine & toxicology. 2020;14(3):1186-9. 31. catană l, catană m, iorga e, lazăr a, lazăr m, teodorescu r, et al. valorification of jerusalem artichoke tubers (helianthus tuberosus) for achieving of functional ingredient with high nutritional value. sciendo. 2018;1:27683. histopathological assessment of wound healing in mice treated with helianthus tuberosus powder vol 13, no 1 (2025) doi 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 5 32. lee k-d, sun h-j, lee m, chun j, shin ts, choi ks, et al. ethanolic extract of pancake mixture powder supplemented with helianthus tuberosus enhances antidiabetic effects via inhibiting inflammatory mediator no production. 2022. 33. albir aa, al-kaisy z. histological investigation of the hepatic tissue in mice induced by soft laser with different doses. biomedical and pharmacology journal. 2019;12(2):989-92. 34. sheridan jf, padgett da, avitsur r, marucha pt. experimental models of stress and wound healing. world journal of surgery. 2004;28:327-30. 35. dawood as, al-ghaban n. histological and immunohistochemical study of the effect of local application of moringa oliefera/marine collagen on bone healing in rats. baghdad university. mcs, thesis; 2021. 36. ansell dm, campbell l, thomason ha, brass a, hardman mj. a statistical analysis of murine incisional and excisional acute wound models. wound repair and regeneration. 2014;22(2):281-7. 37. wynn ta. common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. the journal of clinical investigation. 2007;117(3):524-9. 38. gurtner gc, werner s, barrandon y, longaker mt. wound repair and regeneration. nature. 2008;453(7193):314-21. 39. takeo m, lee w, ito m. wound healing and skin regeneration. cold spring harbor perspectives in medicine. 2015;5(1):a023267. 40. madison kc. barrier function of the skin:“la raison d'etre” of the epidermis. journal of investigative dermatology. 2003;121(2):231-41. 41. clark ra. wound repair. the molecular and cellular biology of wound repair. 1996;2:3-35. 42. muhammad nh, ghani ba. histological evaluation of the effect of topical application of curcumin powder and essential oil on skin wound healing. journal of baghdad college of dentistry. 2015;27(3):58-63. histopathological assessment of wound healing in mice treated with helianthus tuberosus powder vol 13, no 1 (2025) doi 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 6 figure 1. macroscopic images of the wound contraction in the treated and control groups. (a) day 3 control. (b) day 3 treated. (c) day 7 control. (d) day 7 treated. (e) day 14 control. (f) day 14 treated. histopathological assessment of wound healing in mice treated with helianthus tuberosus powder vol 13, no 1 (2025) doi 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 7 3 days (control negative) the histopathological cigures of the skin (control negative) showed marked dermatitis that characterized by marked loss of the epidermis that replaced with thick layer of cibrin clot followed by thick line of necrotic tissue with marked inciltration of inclammatory cells (mncs) (a and b), the dermis revealed dermatitis with severe degeneration and necrosis of dermal collagen cibers, severe inciltration of leukocytes and edema (c). a b c 3 days (posttreatment) the histopathological cigures of the skin dermis revealed marked reepithelization (proliferation of epithelial cells) and formation of keratinized straticied squamous epithelium (d). there were thin line of necrotic tissue with mild inciltration of mono nuclear leukocytes (mncs) mainly lymphocytes with little hemorrhage between dermis and epidermis (d and e). the dermis comprised of immature cibrous connective tissue with furthermore cibroblasts and no hair follicles or sebaceous glands (f). d e f histopathological assessment of wound healing in mice treated with helianthus tuberosus powder vol 13, no 1 (2025) doi 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 8 7 days (control negative) the histopathological cigures of the skin (control negative) showed proliferating epithelial cells of epidermis (straticied squamous epithelium (g). the dermis revealed immature cibrous connective tissue with marked meshwork of angiogenesis (newly formed blood vessel) and moderate inciltration of leukocytes, the cigures revealed no sebaceous glands or hair follicles (h). g h 7 days post treatment the histopathological cigures of the skin revealed marked epidermal hyperplasia that characterized by formation of epidermal papilla (i). the dermis comprised of slightly mature granulation tissue with little inciltration of leukocytes and marked angiogenesis. the cigures revealed nor hair follicles and neither sebaceous glands (j). i j 14 days (control negative) the histopathological cigures of the skin (control negative) showed mature epithelial cells of epidermis (straticied squamous epithelium) (k). the dermis revealed slightly mature granulation tissue with many newly formed blood vessels, there was no inciltration of leukocytes and the cigures revealed no sebaceous glands or hair follicles (l). k l histopathological assessment of wound healing in mice treated with helianthus tuberosus powder vol 13, no 1 (2025) doi 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 9 table 1. the relative frequency of the number of inclammatory cells in each duration using the one-way anova test. no. of indlammatory cells day durations n mean std. deviation std. error p-value 3 days-control 5 98.00 5.385 2.408 0.000 3 days post-treatment 5 32.40 6.229 2.786 7 days-control 5 19.60 3.362 1.503 7 days-post treatment 5 11.60 1.517 .678 14 days-control 5 3.00 .707 .316 14 days-post treatment 5 1.80 .837 .374 total 30 27.73 33.825 6.176 table 2. the relative frequency of the number of blood vessels results in each duration using the one-way anova test. no. of blood vessels day durations n mean standard deviation standard error p-value 3 days-control 5 1.00 1.000 .447 0.000 3 days post-treatment 5 4.00 1.225 .548 7 days-control 5 6.40 .894 .400 7 days-post treatment 5 8.20 1.095 .490 14 days-control 5 1.80 .837 .374 14 days-post treatment 5 3.20 .447 .200 total 30 4.10 2.695 .492 14 days post treatment the histopathological cigures of the skin revealed mature epidermis epithelial cells (m). the dermis comprised of mature granulation tissue with marked angiogenesis (numerous blood vessels). the cigures revealed no hair follicles and neither sebaceous glands and there is normal adipose tissue of hypodermis (n). m n histopathological assessment of wound healing in mice treated with helianthus tuberosus powder vol 13, no 1 (2025) doi 10.5195/d3000/2025.912 http://dentistry3000.pitt.edu 10 table 3. the relative frequency of the thickness of the epidermis results in each duration using the one-way anova test. thickness of epidermis day durations n mean standard deviation standard error p-value 3 days-control 5 .00000 .0000 .0000 0.000 3 days post-treatment 5 82.1164 3.1397 1.4041 7 days-control 5 43.8006 2.4992 1.1177 7 days post-treatment 5 34.6008 1.9686 .8803 14 days-control 5 25.8884 .5254 .2350 14 days post-treatment 5 26.5034 1.9070 .8528 total 30 35.4849 25.2418 4.6085 1068 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1068 http://dentistry3000.pitt.edu acrylic denture basis’ long-term colour stability shahrazad fouad karkosh1, amer hussian makki2 1middle technical university, technical medical ins4tute, baghdah, iraq 2uruk university, college of health and medical technologies, baghdad, iraq abstract objec:ve: this study invesegated how staining solueons affected the acrylic denture bases' colour stability. materials and methods: fioy-six samples of acrylic denture base (hot and cold) were submerged in diselled water for control and three staining solueons. a spectrophotometer was uelized to measure colour before immersion, every sample's inieal colour value was noted. following a 24-hour, one-week, and two-week immersion period, colour change values were calculated. results: hot coffee was the most preferred beverage, showing strong significance over both eme periods (p = 0.002 for 24 hours, p = 0.013 for 1 week). this indicates a consistent preference for hot coffee. karak and hibiscus demonstrated increasing preference over eme, especially in their hot forms (p = 0.001 for hot karak, p = 0.006 for hot hibiscus). this suggests that these drinks become more favored as habits develop. comparison between 24-hour and 1-week data showed that coffee remained stable in preference, while karak and hibiscus became more preferred over eme. conclusion: the denture base's discoloraeon rose in direct proporeon to the immersion eme, and the instant coffee solueon was discovered to be the most chromogenic staining solueon. to maintain the color stability of acrylic dentures, users should adopt good oral hygiene praceces and minimize prolonged exposure to these beverages. open access cita%on: karkosh sf, et al. (2025) acrylic denture basis’ long-term colour stability. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1068 received: october 8, 2025 accepted: october 15, 2025 published: november 10, 2025 copyright: ©2025 karkosh sf, et al. this is an open access ar%cle licensed under a crea%ve commons auribu%on work 4.0 united states license. email: shahrazad.almunjm@mtu.edu.iq introduc)on one of the most popular materials for creating a denture base is acrylic, also known as polymethyl methacrylate [1]. due to its superior mechanical and physical characteristics. its drawbacks, however, include brittleness and liquid absorption with extended usage, which could damage its mechanical qualities [2]. the polar characteristics of resin molecules are the main cause of liquid absorption, which makes pmma denture base materials more susceptible to food consumption, liquid exposure, and staining from denture cleaners [3]. the colour match and the material's colour stability over time are the primary factors that determine whether an aesthetic restorative material is successful or not. one crucial characteristic of many materials used in dentistry is colour stability, or the capacity of the material to maintain its original colour over time in a particular environment [4]. over time, a number of internal and external factors that impact the mechanical and physical characteristics of acrylic resin can cause colour change [5]. beverages, such as black tea, coffee, cola drinks, and fruit juices, have been shown to discolor denture surfaces. these beverages may also impact the surface minish of the dentures [6]. an annual herbaceous plant, hibiscus grows vertically and can reach a height of roughly two meters. the plant's branches have a reddish-green colour. the simple leaves have serrated edges and long petioles. reddish-green leaves in the style of palms [7]. one of the most valuable plants for the culinary and pharmaceutical industries is hibiscus sabdariffa, which is used as a pleasant beverage, particularly after being sweetened with sugar. after concentration, the extract is regarded as a colourful and enhanced material for the unique mlavour [8]. karak tea, a popular beverage in the middle east, is known for its robust mlavor from black tea, sugar, milk, and a mixture of spices, including cloves, cardamom, cinnamon, and ginger. this aromatic concoction offers several health benemits, primarily attributed to its individual components. karak tea combines the health-promoting properties of black tea and various spices, offering antioxidant, digestive, anti-inmlammatory, and metabolic benemits. however, mindful preparation and consumption are essential to maximize its advantages while minimizing potential drawbacks [9]. acrylic denture basis’ long-term colour stability vol 13, no 1 (2025) doi 10.5195/d3000.2025.1068 http://dentistry3000.pitt.edu 2 one of the most consumed drinks in the world, coffee is prized for its deep mlavour and invigorating properties.it is made from roasted coffee beans, which come from the coffee plant. the drink contains caffeine, a natural stimulant that helps boost energy and alertness. coffee is consumed in various forms, including espresso, cappuccino, and instant coffee, and can be served hot or cold. in many cultures, coffee is not just a drink but also a social tradition, often enjoyed with friends, family, or during work breaks [10]. materials and methods this study aimed to evaluate the colour stability of acrylic denture bases. that were heat-cured and cold-cured using three widely consumed beverages as staining agents and distilled water as a control. fiftysix (56) specimens were prepared with wax, separated into two primary classes based on the type of denture base material after being conventionally converted to acrylic. 28 specimens were prepared with heat cured acrylic resin and 28 specimens with cold cure acrylic resin. each group was split up into four smaller groups, each with seven samples, based on the kind of staining solution that was submerged in it. in total, 7 specimens immersion in coffee, 7 specimens immersion in karak tea, 7 specimens immersion in hibiscus tea and 7 specimens immersion in distilled water only as control for 24 hours, one week and two weeks. mold preparation for this study, brass matrices were manufactured to standardize the dimensions of the specimens (50 mm length, 20 mm wide, 2 mm thick). the brass matrixes were invested in a denture mlask with type ii stone cast, to obtain molds, which were used to manufacture the standardized specimens. sample preparation preparation of the heat cured acrylic samples the mold was prepared using the traditional mlasking method for full dentures. following the manufacturer's directions, all materials were combined and worked in each step, from packing and curing to minishing, polishing, and conditioning. preparation of the cold cured acrylic samples following the manufacturer's instructions, a 3:1 by volume mixture of polymer and monomer was used to create the necessary amount of self-cure acrylic resin (italy). preparing staining solutions according to the guidelines provided by the manufacturer, 250 ml of water was mixed with 5 g of coffee (jordan), karak tea (jordan), and hibiscus tea (saudi arabia). the mixture was then put over a low heat and taken off before boiling. a paper milter was then used to milter the mixture. every day, new solutions were made. each time, the samples were carefully cleaned with soft toothbrush and water for 30 seconds to get rid of each debris from immersion [11]. spectrophotometer measurements following polishing and minishing, the specimens were submerged in staining solutions and washed with room-temperature distilled water. as a control, untreated group specimens were employed. the colour change was measured with a spectrophotometer, after 24 hours, 1 week, and 2 weeks [12]. results (tables 1-6 and figures 1-3) there was a statistically signimicant difference between cold and hot distilled water (p = 0.041). the difference between hot and cold coffee was statistically signimicant (p = 0.002). a difference between cold and hot hibiscus was observed (p = 0.008). there was no statistically signimicant difference between cold and hot karak (p = 0.216). for distilled water, cold versus hot did not signimicantly affect consumption. for coffee, karak, and hibiscus, the temperature did have a statistically significant effect, with cold drinks being preferred for karak and hibiscus, and hot coffee being preferred over cold. no significant difference was found between cold and hot variants for distilled water. hot coffee significantly differed from cold coffee, with hot coffee having a higher mean. hot karak significantly differed from cold karak, with hot karak having a higher mean. hot hibiscus significantly differed from cold hibiscus, with hot hibiscus having a higher mean. hot beverages generally showed significant differences, except for distilled water (hot). for cold beverages, distilled water showed the only significant difference for cold. karak and hibiscus showed differences for hot conditions. only cold coffee (cold), and hot and cold hibiscus showed statistically significant differences implying that time (24 hours versus 1 week) affected their consumption in meaningful ways. other beverages like distilled water and karak did not show significant differences in either hot or cold conditions between 24 hours and 1 week. there was a significant difference between cold and hot coffee, and hot karak. hibiscus also showed signimicant differences for both cold and hot. discussion since colour is regarded as a crucial component of any denture material, its stability during the course of the material's survival is seen as a key determinant of the success or failure of the prosthesis . all denture base materials were found to undergo colour changes, and these changes grew for every group during the immersion period. denture base materials may change colour for a variety of reasons, which can be separated into intrinsic and extrinsic factors. these include the chemical makeup of the material, any remaining monomers, water absorption, the breakdown of intrinsic pigments, component leaching, stain accumulation, and surface roughness. nevertheless, the types of food consumed are also important factors that contribute to colour stability over an extended length of time, in addition to polishing ability and material structure [13]. the base material used to make dentures is exposed to a variety of environments with mluctuating oral temperatures, salivary ph levels, and their constituents. it also has to come into contact with a number of foods, beverages, and drinks consumed at different temperatures, making it vulnerable to changes in its physical composition and appearance as a result of the absorption of various contaminants [14]. colour stability is the ability of a material to hold onto its colour over time in a specimic environment. it is thought to be an essential physical property of dental materials [15]. by forming micro cracks associated with absorption and hydrolytic degradation of the polymer, water molecules that are absorbed by acrylic resin function as plasticizers to weaken the material's mechanical resistance, causing linkage cleavage and the slow deterioration of its infrastructure [16]. because coffee is typically utilized in vitro research, it was chosen as the staining material for this investigation. because coffee is yellow-brown, tannic acid has been identimied as the main staining agent [17]. the mindings demonstrated that all denture material types utilized in this study had color changes after immersion in staining solution, depending on the length of time. this is likely since denture base materials collect deposits and stains in the same way as natural teeth, but that hard deposits and stains, like those from coffee, karak, and hibiscus, are much harder to remove. the material's surface has a certain amount of porosity and surface roughness, as well as an organic mucin and inorganic. the mindings that demonstrated the surface characteristic, porosity that allows particles to accumulate and cause noticeable discoloration. this clarimied the mindings that acrylic denture basis’ long-term colour stability vol 13, no 1 (2025) doi 10.5195/d3000.2025.1068 http://dentistry3000.pitt.edu 3 indicated notable distinctions between heatcured and cold-cured acrylic resin. the impact of solutions on the colour variations of denture base materials was found to be comparable in the current investigation. coffee and hibiscus had the biggest colour change, whereas karak showed the least. compared to cold methyl methacrylate resin provisional materials, hot methyl methacrylate resin provisional materials were more colour stable [18]. conclusion the following results were reached within the constraints of this investigation: 1. the denture base's discoloration rose in direct proportion to the immersion duration. 2. the instant coffee solution was determined to be the most chromogenic staining solution. references 1. mikael j, al-samaraie s, ikram f. evaluation of some properties of acrylic resin denture base reinforced with calcium carbonate nanoparticles. erbil dental journal (edj). 2018 jun 6;1(1):41-7. 2. alqutaibi ay, baik a, almuzaini sa, farghal ae, alnazzawi aa, borzangy s, et al. polymeric denture base materials: a review. vol. 15, polymers. multidisciplinary digital publishing institute (mdpi); 2023;15:3258. 3. keyf f, etikan i. evaluation of gloss changes of two denture acrylic resin materials in four different beverages. dental materials. 2004;20:244-51. 4. madhyastha, prashanthi s., et al. effect of staining solutions on color stability of silorane & methacrylate restorative material. international journal of biomedical science: ijbs, 2015, 11.1: 29. 5. hong g, murata h, li y, sadamori s, hamada t. inaluence of denture cleansers on the color stability of three types of denture base acrylic resin. j prosthet dent 2009;101:205-13. 6. sham ask, chu fcs, chai j, chow tw. color stability of provisional prosthodontic materials. journal of prosthetic dentistry. 2004;91:447-52. 7. al-kahtani n, almurayh a, subbarayalu av, sebastian t, alkahtani h, aljabri d. sustaining blended and online learning during the normal and new normal conditions in a saudi higher education institution: health science students' perspectives. heliyon. 2022 oct 1;8. 8. shruthi vh, ramachandra ct, udaykumar nidoni un, sharanagouda hiregoudar sh, nagaraj naik nn, kurubar ar. roselle (hibiscus sabdariffa l.) as a source of natural colour: a review. plant archives 2016;16(2):515-522. 9. ahmed s. the drink of dearborn. kennesaw state university 2024. english 1101 showcase 37. 10. 10-myhrvold, nathan. coffee encyclopedia britannica, 18 mar 2025. 11. valizadeh s, asiaie z, kiomarsi n, kharazifard mj. color stability of self-adhering composite resins in different solutions. dental and medical problems. 2020 jan 1;57(1):31-8. 12. prasad dk, alva h, shetty m. evaluation of colour stability of provisional restorative materials exposed to different mouth rinses at varying time intervals: an in vitro study. the journal of indian prosthodontic society. 2014 mar;14(1):8592. 13. hollis s, eisenbeisz e, versluis a. color stability of denture resins after staining and exposure to cleansing agents. the journal of prosthetic dentistry. 2015 nov 1;114(5):709-14. 14. assunção wg, barão va, pita ms, goiato mc (2009). effect of polymerization methods and thermal cycling on color stability of acrylic resin denture teeth. j prosthet dent, 102, 385-392 15. goiato mc, nóbrega as, santos dm, andreotti am, moreno a. effect of different solutions on color stability of acrylic resin-based dentures. brazilian oral research. 2013 dec 17;28:1-7. 16. imirzalioglu p, karacaer o, yilmaz b, ozmen msc i. color stability of denture acrylic resins and a soft lining material against tea, coffee, and nicotine. j prosthodont. 2010; 19 (2):118-24. 17. hollis s, eisenbeisz e, versluis a. color stability of denture resins after staining and exposure to cleansing agents j prosthet dent. 2015;114: 709–14. 18. bayindir f, kürklü d, yanikoğlu nd. the effect of staining solutions on the color stability of provisional prosthodontic materials. journal of dentistry. 2012 dec 1;40:e41-6. table 1. results after 24 h. n mean standard deviation p-value distilled water cold 5 49.7840 4.48322 0.041 hot 5 38.3400 3.08086 coffee cold 5 26.7760 6.21864 0.002 hot 5 56.7080 6.38243 karak cold 5 24.9640 3.65085 0.216 hot 5 26.1900 2.36219 hibiscus cold 5 29.1780 2.02280 0.008 hot 5 31.9700 1.41317 acrylic denture basis’ long-term colour stability vol 13, no 1 (2025) doi 10.5195/d3000.2025.1068 http://dentistry3000.pitt.edu 4 table 2. results after 1 week. n mean standard deviation p-value distilled water cold 5 38.526 3.284 0.176 hot 5 40.126 3.65267 coffee cold 5 32.75 2.64833 0.023 hot 5 38.352 3.27629 karak cold 5 19.112 3.60012 0.048 hot 5 14.662 3.1038 hibiscus cold 5 15.022 1.76379 0.047 hot 5 21.25 4.64618 0 10 20 30 40 50 60 cold hot cold hot cold hot cold hot distal water coffee karak hibscus 49.784 38.34 26.776 56.708 24.964 26.19 29.178 31.97 figure 1. average ae values that were acquired from samples that were immersed in various solutions for 24 h. acrylic denture basis’ long-term colour stability vol 13, no 1 (2025) doi 10.5195/d3000.2025.1068 http://dentistry3000.pitt.edu 5 table 3. results after 2 weeks. n mean standard deviation p-value distilled water cold 5 26.622 36.48096 0.324 hot 5 27.282 37.3602 coffee cold 5 41.726 3.43239 0.000 hot 5 57.896 3.49028 karak cold 5 21.95 1.98813 0.045 hot 5 24.384 2.0034 hibiscus cold 5 36.706 5.66449 0.005 hot 5 55.08 3.71653 0 5 10 15 20 25 30 35 40 45 cold hot cold hot cold hot cold hot distal water coffee karak hibscus 38.526 40.126 32.75 38.352 19.112 14.662 15.022 21.25 figure 2. average ae values derived from samples that were immersed in various solutions one week. acrylic denture basis’ long-term colour stability vol 13, no 1 (2025) doi 10.5195/d3000.2025.1068 http://dentistry3000.pitt.edu 6 figure 3. mean ae values obtained from the samples soaked in different solution for two weeks. table 4. comparison between 24 hours and 1 week. t p-value distilled water 24hr-1 week (cold) 2.335 0.047 24hr-1 week (hot) -1.589 0.187 coffee 24hr-1 week (cold) -2.342 0.049 24hr-1 week (hot) 4.306 0.013 karak 24hr-1 week (cold) 2.077 0.106 24hr-1 week (hot) 7.775 0.001 hibiscus 24hr-1 week(cold) 9.572 0.001 24hr-1 week (hot) 5.344 0.006 .0000 10.0000 20.0000 30.0000 40.0000 50.0000 60.0000 cold hot cold hot cold hot cold hot distal water coffee karak hibscus 26.6220 27.2820 41.7260 57.8960 21.9500 24.3840 36.7060 55.0800 acrylic denture basis’ long-term colour stability vol 13, no 1 (2025) doi 10.5195/d3000.2025.1068 http://dentistry3000.pitt.edu 7 table 5. comparison between 24 hours and 2 weeks. t p-value distilled water 24hr -2 weeks (cold) 1.568 0.192 24hr -2 weeks (hot) 0.894 0.422 coffee 24hr-2 weeks (cold) -4.283 0.013 24hr-2 weeks (hot) -0.327 0.76 karak 24hr-2 weeks (cold) 1.582 0.189 24hr-2 weeks (hot) 1.49 0.21 hibiscus 24hr-2 weeks(cold) -2.36 0.048 24hr-1 week (hot) -14.31 0.000 . table 6. comparison between 1 week and 2 weeks. t p-value distilled water 1 week-2 weeks (cold) 0.903 0.418 1 week-2 weeks (hot) 0.956 0.393 coffee 1 week-2 weeks (cold) -8.282 0.001 1 week-2 weeks (hot) -10.443 0.000 karak 1 week-2 weeks (cold) -1.225 0.288 1 week-2 weeks (hot) -6.905 0.002 hibiscus 1 week-2 weeks (cold) -10.852 0.000 1 week-2 weeks (hot) -9.541 0.001 962 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.962 http://dentistry3000.pitt.edu radiographic quality of root canal therapy accomplished clinically by undergraduate dental students thulficar ghali hameed al-khafaji1, azal al-masoody2, qasim abdulkareem mohammed1, zainab mahmood al-jammali1, mays ali azeez alanz3 1college of den*stry, university of babylon, hilla, iraq 2college of den*stry, university of alkafeel, najaf, iraq 3iraqi ministry of health, babylon, iraq abstract objecbve: to analyze the technical quality of root canal therapy done by dental students while in clinical training. material and methods: the frequency of common technical errors was assessed using dental periapical radiographs. 870 charts (n= 870) were evaluated which involved 870. in each chart, 3 periapical radiographs were used for evaluation of the incidence rate and types of errors happening within the root canal procedure. the 3 periapical radiographs were diagnostic (pre-treatment) radiograph, working length determination radiograph, and gutta-percha and sealer (obturation) radiograph. results: it was found that phase 3 (obturation phase) shows the highest frequency of errors 54.37%, followed by phase 1 (access opening) 11.84%, and jinally phase 2 (root canal preparation) shows the least frequency 4.01%. conclusion: this study idendfied that the least rate of errors in endodondc therapy happened during access opening and instrumentadon when compared to obturadon. open access cita%on: al-khafaji tgh, et al. (2025) radiographic quality of root canal therapy accomplished clinically by undergraduate dental students. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.962 received: june 14, 2025 accepted: june 26, 2025 published: august 13, 2025 copyright: ©2025 al-khafaji tgh, et al. this is an open access ar%cle licensed under a crea%ve commons auribu%on work 4.0 united states license. email: dent.thulficar.ghali@uobabylon.edu.iq introduc)on in the school of dentistry, one of the important requirements that should be done by the undergraduate students is the root canal therapy (rct) [1]. this treatments involves cleaning, shaping and appropriate filling of the radicular space [2]. the main aim of the rct is to allow healing of the periapical tissues surrounding the root of the tooth and prevent the re-infection of the radicular space [3]. the outcome of the endodontic treatment mainly rests on the quality of the procedural technique of the root canal filling (rcf) [4,5]. the common method that has been implemented to judge the quality of the root canal fillings is periapical radiographs during the different phases of the root canal therapy [6-8]. the successful root canal therapy (rct) prevents tooth pain. it should also reduce the risk of tooth loss and/or developing apical periodontitis [9]. dentists should remember that in each phase of the treatment, any operative mistake may have negative effect on prognosis, as these mistakes increase the risk to failure of the entire treatment [9]. according to previous research, errors during rct can be divided into three groups according to the treatment phase as follows [10-14]: phase 1 (access opening errors): • gouging • coronal perforation • over preparation phase 2 (cleaning and preparation errors): • ledge • transportation "zipping" • strip perforation • broken instruments • apical perforation phase 3 (root canal obturation errors): • void • over-filling • under-jilling usually, the evaluation of the rct technical quality is based on careful radiographic examination [6,15-17]. therefore, the current study assessed radiographically the quality of endodontic therapy done by undergraduate students through these 3 phases of treatment, while in their clinical training. the frequency of common technical errors was evaluated by multiple dental periapical radiographs. material and methods this study was done at the dental school, university of babylon, iraq. it was done in radiographic quality of root canal therapy accomplished clinically by undergraduate dental students vol 13, no 1 (2025) doi 10.5195/d3000.2025.962 http://dentistry3000.pitt.edu 2 agreement with the 1964 declaration of helsinki and its succeeding revisions. the root canal therapy was performed by under-graduate students in their jifth year of study in the clinics of the dental school under the supervision of qualijied supervisors (endodontists). the rct was performed on single-root canal treated teeth on upper and lower incisors, canines, and premolars, during 6-year period from october 2014 to october 2020. the single-root canal teeth were endodontically treated, jirstly access opening was done into the pulp space of the tooth and the dental pulp was extirpated, and then the root canal system was instrumented by k-jiles (dentsply, maillefer, switzerland) with a 0.02 taper. the conventional step-back technique was applied. canals irrigation was performed using normal saline and an endodontic gauge 27 syringe. obturation of the canals was then performed by cold lateral compaction technique using standardized gutta-percha cones (dentsply maillefer, switzerland) and a zoe-based endodontic sealer (endojill, dentsply, brazil). in this study, 870 charts (n= 870) were evaluated which involved 870. in each chart, 3 periapical radiographs were used for the evaluation of the executed endodontic procedures in terms of types and incidence of errors. the 3 periapical radiographs were diagnostic (pre-treatment) radiograph, working length determination radiograph, and gutta-percha and sealer (obturation) radiograph. two qualijied dentists (two endodontists) assessed these radiographs on a light box x-ray viewer in dark environment [11]. a comparative evaluation between these radiographs was used to verify any errors that could have occurred during the phases of the root canal therapy. the applied parameters in this study were: no-fault cases; where there was no detected procedural mistakes and gutta percha is shorter than the radiographic apex by less than 2 mm, coronal-perforation, over preparation, gouging, strip perforation, apical perforation, broken instruments, ledging, transportation, void in the jilling material, overjilling, and under jilling of guttapercha [10,11]. before conducting this study, intraand inter-examiner agreements were verijied by values calculation of cohen's kappa (k) using 35 randomly chosen periapical radiographs that had been used in this study [18]. the results were 0.88 and 0.81, respectively, so both values indicated excellent intraand inter-examiner agreements. finally, spss software (version 20.0, spss inc., chicago, il, usa) was implemented for the processing of data. pearson’s chi-square test was applied to analyze the data at p<0.05. results table 1 shows that 70.33% of cases have various types of errors, but 29.77% of them have no error (normal). the various types of errors in phase 1 are 4.48% gouging and 7.36% over preparation, in phase 2 are 1.26% ledge, 0.8% strip perforation, 0.69% broken instruments and 1.26% apical perforation, and in phase 3 are 12.99% void, 4.83% overjilling and 36.55% underjilling. it seems that the dominant type of error is the underjilling which shows the highest frequency, followed by void, over preparation, overjilling then gouging. while, ledge, apical perforation, strip perforation and broken instruments show tiny frequencies. however, the difference between all types of were signijicantly different, p<0.05, table 2. it also seems that phase 3 (obturation phase) shows the highest frequency of errors 54.37%, followed by phase 1 (access opening) 11.84%, and jinally phase 2 (root canal preparation) shows the least frequency 4.01% (table 1). discussion the accurate chemo-mechanical cleaning and shaping of the dental root canal could result in successful rcf. any error happens during the 3 phases of the root canal therapy could affect the prognosis. in dental schools, the undergraduate students should be taught about these errors to avoid any technical error that could happen during the rct and to improve the prognosis of the rct. this study paid close attention to the assessment of the quality of the rct whether the treatment was without error (normal) or with error during all phases of the rct. the periapical radiographs were utilized to evaluate the success of root canal therapy in patients operated by the 5th year under-graduate students at the dental school clinics of university of babylon. several studies have evaluated the errors, such as gouging, ledges, apical perforation, broken instruments, underjilling, overjilling and voids which could happen during rct treatment [10,11,16,19]. in this study, errors occurred during all the phases of the rct, but the most frequent errors were during the third phase (obturation phase). this was followed by phase 1 (access opening), whereas phase 2 (root canal preparation) recorded the least frequency of errors. in phase 3, the underjilling error showed the highest incidence. this could be attributed to the debris accumulating at the apical third of the root canal due to instrumentation without enough irrigation of the canal which could result in shortening of the working length and jinally results in underjilling. the other possible reason for the underjilling, is the non-complete instrumentation of the root canal which could happen from the wrong measurement of the root canal length [20]. again, in phase 3, voids error was the next higher incidence, in which spaces could form between the gutta-percha cones. these voids might harbor pathological microorganisms. these voids could be due to not-enough condensation of the gutta-percha cones into the radicular canals [21]. in addition, the nonhomogeneous mixing of the endodontic sealer could also result in theses voids [22]. finally in phase 3, the overjilling was with the least frequent error. it could have been the result of over-estimation of working length of root canal, which could end in loss of the apical stop. generally, it has been agreed that root canal jilling is preferred to be within 2 mm distance away from the radiographic apex [6,23]. higher success rates of rcf were associated in teeth jilled with root canal jilling ≤ 2 mm short from the radiographic apex with decreased occurrence of apical periodontitis in comparison with teeth jilled > 2 mm away from the radiographic apex [24]. according to this study, the quality of the endodontic therapy that is offered for the patient by undergraduate students is not good enough. this result is supported by the results of three previous studies [10,16,25]. improving knowledge, training and skills of the undergraduate students is very important. this can be done by improvement of several factors such as increasing the ratio of supervisors to students and improvement of the training aids of the undergraduate students, in addition to introduction of different methods for evaluation the work of the students. conclusions this study identijied that the access opening phase and instrumentation phase in root canal therapy have less incidence of errors when compared with the obturation phase. whereas, in obturation phase, the errors which recorded the highest incidence rate were underjilling, voids and then overjilling. consequently, it is advised to enhance the practical training sessions of the rct, especially in the last phase (obturation phase). ethical approval this study was reviewed and approved by the research ethical approval committee of the dental school of the university of babylon (babylon, iraq), with reference number (27) on (29/5/2024). radiographic quality of root canal therapy accomplished clinically by undergraduate dental students vol 13, no 1 (2025) doi 10.5195/d3000.2025.962 http://dentistry3000.pitt.edu 3 references 1. de moor r, hülsmann m, kirkevang ll, et al. undergraduate curriculum guidelines for endodontology. interna@onal endodon@c journal. 2013;46(12):1105-14. 2. general dental councilthe first five years (interim). 3rd edi@on ed2008. 3. ng y-l, mann v, rahbaran s, et al. outcome of primary root canal treatment: systema@c review of the literature – part 2. influence of clinical factors. interna@onal endodon@c journal. 2008;41(1):6-31. 4. peak j, hayes s, bryant s, et al. endodon@cs: the outcome of root canal treatment. a retrospec@ve study within the armed forces (royal air force). bri@sh dental journal. 2001;190:140-4. 5. smith cs, setchell dj, harty fj. factors influencing the success of conven@onal root canal therapy--a five-year retrospec@ve study. int endod j. 1993;26(6):321-33. 6. er o, sagsen b, maden m, et al. radiographic technical quality of root fillings performed by dental students in turkey. int endod j. 2006;39(11):867-72. 7. eleeheriadis gi, lambrianidis tp. technical quality of root canal treatment and detec@on of iatrogenic errors in an undergraduate dental clinic. int endod j. 2005;38(10):725-34. 8. tsuneishi m, yamamoto t, yamanaka r, et al. radiographic evalua@on of periapical status and prevalence of endodon@c treatment in an adult japanese popula@on. oral surg oral med oral pathol oral radiol endod. 2005;100(5):631-5. 9. estrela c, pécora j, estrela c, et al. common opera@ve procedural errors and clinical factors associated with root canal treatment. brazilian dental journal. 2017;28:179-90. 10. haji-hassani n, bakhshi m, shahabi s. frequency of iatrogenic errors through root canal treatment procedure in 1335 charts of dental pa@ents. j int oral health. 2015;7:14-7. 11. yavari h, samiei m, shahi s, et al. radiographic evalua@on of root canal fillings accomplished by undergraduate dental students. iran endod j. 2015;10(2):127-30. 12. ibrahim ma, elgendy aae, elsewify tm. compara@ve evalua@on of shaping abili@es of two different rotary files (an in vitro study). ain shams dental journal. 2021;22(2):28-38. 13. bialy mf, el shazli ah, osama m, et al. compara@ve evalua@on of different techniques and devices for removal of intra canal separated instruments with different lengths (an in vitro study). ain shams dental journal. 2024;36(4):12-9. 14. othman ahmed s, fahmy s, el faramawy mt. comparison of the shaping ability and cujng efficiency of three different rotary nickel-titanium systems. (an in vitro study). ain shams dental journal. 2024;34(2):141-51. 15. khabbaz mg, protogerou e, douka e. radiographic quality of root fillings performed by undergraduate students. int endod j. 2010;43(6):499-508. 16. ghali t, hameed a, alanz a, et al. errors during root canal treatment performed by dental students. interna@onal medical journal. 2020;25:1499-504. 17. saber s, kataia e, darwish k. evalua@on of the quality of root canal obtura@on using resin and silicon-based sealers: a randomized clinical trial. ain shams dental journal. 2024;35(3):85-94. 18. mukhaimer r, hussein e, orafi i. prevalence of apical periodon@@s and quality of root canal treatment in an adult pales@nian subpopula@on. the saudi dental journal. 2012;24(3):149-55. 19. roman-richon s, faus-matoses v, alegredomingo t, et al. radiographic technical quality of root canal treatment performed ex vivo by dental students at valencia university medical and dental school, spain. med oral patol oral cir bucal. 2014;19(1):e93-7. 20. lin lm, rosenberg pa, lin j. do procedural errors cause endodon@c treatment failure? the journal of the american dental associa@on. 2005;136(2):187-93. 21. jafarzadeh m, saatchi m, jafarnejadi p, et al. digital radiographic evalua@on of the quality of different root canal obtura@on techniques in deciduous mandibular molars aeer prepara@on with rotary technique. j dent. 2019;20(3):152-8. 22. heran j, khalid s, albaaj f, et al. the single cone obtura@on technique with a modified warm filler. journal of den@stry. 2019;89:103181. 23. barrieshi-nusair km, al-omari ma, alhiyasat as. radiographic technical quality of root canal treatment performed by dental students at the dental teaching center in jordan. j dent. 2004;32(4):301-7. 24. sjogren u, hagglund b fau sundqvist g, sundqvist g fau wing k, et al. factors affec@ng the long-term results of endodon@c treatment. j endod. 1990;16(10):498-504. 25. polyzos nk, sarris kg, pita ai, et al. quality of root canal fillings performed by undergraduate students and the related factors on the treatment outcome: a 2to 5-year follow-up. eur endod j. 2018;3(3):179-85. table1. incidence of errors throughout root canal therapy. type of error number (%) no error (normal) 259 (29.77%) phase 1 (access opening) gouging 39 (4.48%) 11.84% 70.33% over preparation 64 (7.36%) phase 2 (preparation) ledge 11 (1.26%) 4.01% strip perforation 7 (0.80%) broken instruments 6 (0.69%) apical perforation 11 (1.26%) phase 3 (obturation) void 113 (12.99%) 54.37 over jilling 42 (4.83%) under jilling 318 (36.55%) total 870 (100%) 1044 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1044 http://dentistry3000.pitt.edu sodium fluoride varnish versus silver diamine fluoride effectiveness to stop dental decay in primary molars lubna abdulelah abdulrahman almallah1, mohammed qays mahmoud fahmi2, wurood khairallah al-lehaibi1 1dijah university college, iraq 2college of den7stry, musransiriyah university, iraq abstract objec<ve: the aim of this study was to determine how well 38% sdf and 5% naf varnish inhibited primary molar decay. materials and methods: 84 children between the ages of 6 and 9 who had caries without pulpal involvement in their primary teeth were included in the randomized controlled experiment. children were divided between two groups at random. 5% naf varnish was applied to group 1 (n = 42), and 38% sdf was applied to group 2 (n = 42). in both groups, the second applicavon was completed six months later. children were examined for caries arrest and recalled at 6and 12-month. results: axer 6-month intervals, the rate of caries arrest was in group 1(naf varnish – 40.4%) while in group 2 (sdf – 66.6%) and axer 12-month intervals the rate of caries arrest was (naf varnish – 47.6%) for group 1 and (sdf88%) for group 2 (p<0.05). conclusion: the sdf group was shown to have higher caries arresvng potenval than the naf varnish group. open access cita%on: almallah laa, et al. (2025) sodium fluoride varnish versus silver diamine fluoride effec%veness to stop dental decay in primary molars. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1044 received: september 14, 2025 accepted: september 25, 2025 published: october 15, 2025 copyright: ©2025 almallah laa, et al. this is an open access ar%cle licensed under a crea%ve commons asribu%on work 4.0 united states license. email: iubna.abd@duc.edu.iq introduc)on dental caries begins with 0irst lesions, which are dynamic and resulting from an imbalance between the dental surface's demineralization and remineralization [1]. if remineralization is greater than demineralization, these initial lesions may be reversed or stopped. on the other side, if demineralization is greater than remineralization, they may become cavitations [2,3]. active early caries on smooth or dry occlusal surfaces can be easily detected clinically which appears as chalky white lesions [4]. conversely, proximal surfaces have consistently shown dif0iculties for early detection; the inability to self-clean, limited salivary access, and clinical diagnosis in the contact point area have all contributed to this challenge regarding the early diagnose and treatment [5]. the treatment modalities for these lesions include conventional restorations, caries in0iltration, proximal sealants, increased 0luoride usage and maintaining dental hygiene [6]. many studies have been demonstrated that using silver 0luoride (sf) products to treat carious lesions in both adults and children is an effective minimally invasive treatment option [7]. on the other hand, sodium 0luoride varnish (naf) has been shown to be useful in reducing the progression of dental cavities by promoting remineralization and inhibiting demineralization. according to these studies, using 0luoride varnishes can prevent caries in young children and reduce cavities by 25% to 45% [8-10]. by utilizing caries arresting agents and materials with cariostatic properties that reduce the bacterial load, such as arginine, sealants, 0luoride varnish, nanosilver 0luorides, and silver diamine 0luoride (sdf), the concept of cavity control by restoration has been replaced by prevention with minimal intervention [8]. in 2014, the united states food and drug administration authorized sdf. since then, demand for using it "off-label" to treat carious lesions in children has grown [11]. sdf offers an extensive list of advantages in managing caries. it is an effective in stopping cavitated carious lesions [12-14]. in addition, it is inexpensive and does not produce any aerosol and does not need any dental equipment. nevertheless, discoloration is its primary drawback [14]. in dentistry, topical 0luorides, including 0luoride varnishes, are commonly utilized to remineralize enamel in the earliest lesions sodium fluoride varnish versus silver diamine fluoride effecvveness to stop dental decay in primary molars vol 13, no 1 (2025) doi 10.5195/d3000.2025.1044 http://dentistry3000.pitt.edu 2 without causing tooth discoloration. as it is necessary to assess these topical 0luorides' ability to prevent cavitated lesions, for this, the current study was carried to assess and compare the potential of semiannually application of both 38% sdf and 5% naf varnish to primary molars in stopping dental cavities. materials and methods the total number of participating children was 92 randomly chose and their parents from the department of pediatric and preventive dentistry of dijlah university during the academic year 2023–2024 only 84 were responded. the dijlah university faculty of dentistry's ethical committee created and authorized the informed ethical consent form. following screening, the children who satis0ied the study's inclusion requirements were given permission to participate. during the questionnaire interview, each participant was requested to sign a formal ethical consent form. children were chosen following the cpitn probe evaluation of the carious lesion and the intraoral periapical radiograph, which con0irmed the depth of the lesion in teeth with radiolucency restricted to the outer third of the dentin. a total of 84 child was randomized into two groups: group 1 consisting of 5% naf varnish and group 2 consisting of 38% sdf. children whose parents are strongly against 0luoride staining with silver diamine, children whose medical conditions are too dif0icult to treat in clinic, children who are sensitive to silver products, were excluded. children between the ages of 6 and 9 who were cooperative and willing to take part in the study were those with occlusal carries in primary molar teeth without any pulpal involvement. although the socioeconomic status of the families’ children whose participate was quite close to one another, the researcher matched the two groups using the socioeconomic status (ses) questionnaire that was already in use (28) (one of the parents completed the questionnaire) to minimize the inaccuracy of the results. in this study, oral health behaviors like cleaning one's teeth and consuming sugar were matched by age, gender, and ses status using the group matching approach. after using a rubber cup and no 0luoridated pumice powder to clean the chosen teeth, they were rinsed and allowed to dry for 0ive seconds. cotton rollers were used for isolation. at the initial visit and six months later, sdf and naf varnish were applied to every carious primary molar a single operator performed every clinical operation. a tiny applicator tip was used to apply group 1 topical naf varnish (embrace varnish, pulpdent, usa), which was then given a minute to dry. the patient was instructed not to eat or drink anything for thirty minutes in addition to receiving information on good oral hygiene. the 5% naf varnish was re-applied after 6 and 12-month intervals from baseline. to prevent sdf discoloration, vaseline was administered around the tooth and gingiva in group 2, then using a tiny applicator tip, sdf (riva star, sdi inc., australia) was applied to the lesion with cavitation, for approximately 2 minutes were allowed to pass. a gentle air0low was used to accelerate the drying process. if possible, the lesion was kept isolated for three minutes. to reduce systemic absorption, excess sdf was eliminated using a cotton pellet [11]. on the other hand, potential lesions were cleaned and dried by a piece of cotton before examination. assessing lesion activity was done by visual inspection and tactile detection using a 0.5 mm ball-ended community periodontal index (cpi) periodontal probe and a disposable dental mirror. lesions were assessed at baseline and after 6 months and after 12 months, if the cavity wall or 0loor could be readily pierced with minimal force using the cpitn probe, active caries was detected during the recall visits. hard-surfaced cavities were categorized as stopped caries, whenever an active tooth caries lesion detected at baseline became inactive during follow-up, stopped caries was reported [5]. the statistical package for social sciences (spss) (version 25) was used to analyze the data. the chi-square test was used to assess the collected data. statistical signi0icance was determined by considering a probability value of < 0.05. results in this study, children’s participation is described in the consort 0low diagram (figure 1). the total sample 84 child, the research was done during the academic year 2023–2024 were randomized into two groups: group 1 consisting of 5% naf varnish and group 2 consisting of 38% sdf. the potential of caries arresting for both materials at 6 and 12-month intervals as intragroup comparison, this difference was found to be statistically signi0icant in the sdf (p <0.05) while not signi0icant for naf varnish (p >0.05) groups (table 1). the potential of caries arresting was found to be greater for sdf (66.6%) at 6-month intervals on intergroup comparison than for naf varnish (40.4%), and the differences were determined to be statistically signi0icant (p < 0.05). likewise, at 12-month intervals, there was a statistically signi0icant difference (p < 0.05) in the performance of sdf (88%) and naf varnish (47.6%) (table 2). discussion two approaches for treating active carious lesions are indicated. the two types of cavity control are nonrestorative (nrcc) and restorative (rcc) [15,16]. since nrcc doesn't include drilling or anesthetic and tries to keep primary teeth functioning until they are exfoliated, it is regarded as a kid-friendly procedure [16]. it has an advantage over conventional restorative therapy since it does away with the anxiety and tension that intrusive restorative therapy frequently causes. a squamous layer of silver-protein conjugate forms when sdf is applied to a decaying surface, strengthening the tooth's resistance to enzymatic digestion and acid breakdown [17,18]. as the depth of the lesion diminishes, the mineral density and hardness of the treated lesion increase [13]. silver ions destroy the germs causing dental cavities [14,15] by rupturing the cell membrane, denaturing proteins, and preventing dna replication. when the sdf’s 0luoride ingredient combines with calcium phosphate and hydroxyapatite to generate 0luorapatite and calcium 0luoride, it strengthens the oral hard tissues' resistance to acid attack [13,17]. sdf used in the control group as it is a topical mediation that has been shown to be effective in stopping cavitated caries lesions. sdf is currently receiving a lot of interest from researchers and dentists. sdf differs from other caries-preventive agents in that it has the capacity to regulate the caries process. because 0luoride varnishes are so simple to use on pre-cooperative children as well as those with special needs, they have become extremely popular in the pediatric dental community. according to virupaxi et al. [18], 0luoride varnish can reduce dental cavities by 25% to 45% and helps in preventing caries in young children. since it has been determined from the research that children in the age range of 6 to 9 years old have a high frequency of untreated carious lesions, these children were taken into consideration for the study. to prevent dentinal caries, we investigated at an application of semiannual regimen for sdf and naf varnish. according to fung et al. [19], 38% sdf applied semiannually (75.7%) is more successful in preventing dentinal caries than sdf applied annually (66.9%). according to the sodium fluoride varnish versus silver diamine fluoride effecvveness to stop dental decay in primary molars vol 13, no 1 (2025) doi 10.5195/d3000.2025.1044 http://dentistry3000.pitt.edu 3 american dental association council on scienti0ic affairs, high-risk people can successfully lower their caries prevalence by administering 0luoride varnish every six months [20]. in the current study, there was no attempt to excavate carious tissue, facilitating an easier and faster application process. compared to proximal lesions, occlusal cavitated lesions are more easily accessible for hygiene management. the darkening of the lesions treated with sdf typically ranges from gray to black. in primary molars, sdf was found to have a greater caries stopping capacity than naf varnish at 6and 12-months periods in the present investigation. this may be because 38% sdf has an alkaline nature and a high concentration of 0luoride (44,800 ppm) and silver (253,870 ppm). it essentially prevents bacterial growth and then promotes dentin remineralization. on the other hand, the 0luoride concentration of 5% naf varnish is lower—22,600 ppm. these 0indings are corresponded to a 12month follow-up randomized controlled study on young children carried out by mabangkhru et al. [21]. they concluded that, when applied semiannually, 38% sdf had a stronger caries arresting potential than 5% naf varnish. in addition, duangthip et al. found that sdf had a 66% greater ef0icacy than naf varnish (41%), in reducing carious lesions [22]. similarly, 38% of sdf has 65%–91% caries arresting potential in the primary dentition, while 0luoride varnish has 38%–44% caries arresting potential, according to an umbrella review by seifo et al [23]. fluoride varnish showed a 42% caries arresting potential in the current investigation at 12-month intervals. fluoride varnish use was found to minimize caries in the primary dentition with a 37% preventive percentage in a cochrane comprehensive study [19]. the primary mechanism by which 0luoride varnish regulates the advancement of caries is one. it raises the hard tissues' ability to resist demineralization in the event of subsequent ph dips and restores the mineral content that was lost while the caries process was active [6]. fluoride varnish showed a 42% caries arresting potential in the current investigation at 12-month intervals. fluoride varnish use was found to minimize caries in the primary dentition with a 37% preventive percentage in a cochrane comprehensive study [24]. the primary mechanism by which 0luoride varnish regulates the advancement of caries is a single. in the instance of additional ph drops, it strengthens the hard tissues' resistance to demineralization and brings back the mineral content lost during the active caries process [13]. in the current investigation, the sdf showed a higher rate of caries arrest than naf varnish groups at 6 and 12-month intervals. this might be because of the second application separated six months apart. in a similar vein, zhi et al. [25] found that when sdf treatment frequency was raised to every six months, the percentage of active dentin that had been arrested increased. according to mabangkhru et al. [21], sdf and naf varnish had greater caries arrest rates at the 12month time periods than they did at the 6month time periods. this might be brought on by maintaining proper dental hygiene and using topical 0luoride on a regular application. children who are uncooperative with conventional caries therapy can safely use sdf. it takes less time and requires little equipment, making it easy to become involved in a communal environment. this facilitates better dental care accessibility in rural locations. most of the time, using sdf has more bene0its than disadvantages, such as the staining of dentin lesions black, beside it can stop dental cavities. naf varnish is not as effective as sdf. therefore, sdf can be regularly applied to cavitated primary molars as a technique of interim therapy. the study's therapeutic implications include evidence that 38% of sdf successfully stops caries lesions with cavitation. therefore, this can be applied in underprivileged areas where early primary tooth loss and untreated dental caries are common. conclusion sdf demonstrated more caries arresting potential than naf varnish when applied semiannually to cavitated primary molars. conflict of interest none. acknowledgements the authors would like to thank dijlah university, baghdad, iraq, for their support in the present work. financial support and sponsorship none. references 1. marsh, p. in sickness and in health—what does the oral microbiome mean to us? an ecological perspective. adv. dent. res.2018; 29: 60–65. 2. kachuie, m.; khoroushi, m. prevention and treatment of white spot lesions in orthodontic patients. contemp. clin. dent.2017; 8: 11–19. 3. rechmann, p.; kinsel, r.; featherstone, j.d.b. integrating caries management by risk assessment (cambra) and prevention strategies into the contemporary dental practice. compend. contin. educ. dent. 2018; 39: 226. 4. agarwal, d.; machale, p.s.; hegde-shetiya, s. the incipient caries. j. contemp. dent. 2013; 3:20–24. 5. mejàre, i.; källestål, c.; stenlund, h. incidence and progression of approximal caries from 11 to 22 years of age in sweden: a prospective radiographic study. caries res. 1999; 33: 93–100. 6. splieth, c.; kanzow, p.; wiegand, a.; schmoeckel, j.; jablonski-momeni, a. how to intervene in the caries process: proximal caries in adolescents and adults—a systematic review and meta-analysis. clin. oral investig. 2020; 24:1623–1636. 7. contreras, v.; toro, m.j.; elıás-boneta, a.r.; encarnación-burgos, a. effectiveness of silver diamine iluoride in caries prevention and arrest: a systematic literature review. gen. dent. 2017; 65: 22–28. 8. chu ch, lo e. uses of sodium iluoride varnish in dental practice. ann r australasian coll dent surg 2008; 19:58–61. 9. autio-gold jt, courts f. assessing the effect of iluoride varnish on early enamel carious lesions in the primary dentition. j am dent assoc 2001; 132:1247–53. 10. weintraub ja, ramos-gomez f, jue b, shain s, hoover ci, featherstone jd, et al. fluoride varnish efiicacy in preventing early childhood caries. j dent res 2006; 85:172–6. 11. seifo n, cassie h, radford j, innes n. “it's really no more difiicult than putting on iluoride varnish”: a qualitative exploration of dental professionals’ views of silver diamine iluoride for the management of carious lesions in children. bmc oral health 2020; 20:257. 12. lo ec, chu ch, lin hc. a community-based caries control program for pre-school children using topical iluorides:18-month results. j dent res 2001; 80:2071–4. 13. chibinski ac, wambier lm, feltrin j, loguercio ad, wambier ds, reis a. silver diamine iluoride has efiicacy in controlling caries progression in primary teeth: a systematic review and meta-analysis. caries res 2017; 51:527–41. 14. hiraishi n., yiu c.k., king n.m., tagami j., tay f.r. antimicrobial efiicacy of 3.8% silver diamine iluoride and its effect on root dentin. j. endod. 2010; 36:1026–1029. 15. tan h.p., lo e.c. risk indicators for root caries in institutionalized elders. community dent. oral epidemiol. 2014;42:435–440. 16. van strijp g, van loveren c. no removal and inactivation of carious tissue: non-restorative cavity control. monogr oral sci 2018;27:124–36. 17. oliveira bh, rajendra a, veitz-keenan a, niederman r. the effect of silver diamine iluoride in preventing caries in the primary dentition: a systematic review and meta-analysis. caries res 2019;53:24–32. 18. virupaxi sg, roshan nm, poornima p, nagaveni nb, neena ie, bharath kp. comparative evaluation of longevity of iluoride release from three different iluoride varnishes-an in vitro study. j clin diagn res 2016;10:c33–6. 19. fung mh, duangthip d, wong mc, lo ec, chu ch. randomized clinical trial of 12% and 38% sodium fluoride varnish versus silver diamine fluoride effecvveness to stop dental decay in primary molars vol 13, no 1 (2025) doi 10.5195/d3000.2025.1044 http://dentistry3000.pitt.edu 4 silver diamine iluoride treatment. j dent res 2018;97:171–8. 20. american dental association council on scientiiic affairs. professionally applied topical iluoride:evidence-based clinical recommendations. j am dent assoc 2006;137:1151–9. 21. mabangkhru s, duangthip d, chu ch, phonghanyudh a, jirarattanasopha v. a randomized clinical trial to arrest dentin caries in young children using silver diamine iluoride. j dent 2020;99:103375. 22. duangthip d, jiang m, chu ch, lo ec. non-surgical treatment of dentin caries in preschool children-systematic review. bmc oral health 2015;15:44. 23. seifo n, cassie h, radford jr, innes np. silver diamine iluoride for managing carious lesions: an umbrella review. bmc oral health 2019;19:145. 24. marinho vc, worthington hv, walsh t, clarkson je. fluoride varnishes for preventing dental caries in children and adolescents. cochrane database syst rev 2013 (7):cd002279. 25. zhi qh, lo ec, lin hc. randomized clinical trial on effectiveness of silver diamine iluoride and glass ionomer in arresting dentine caries in preschool children. j dent 2012;40:962–7 table 1. comparison of effectiveness between silver diamine 0luoride and sodium 0luoride varnish at two different time. table 2. comparison of the effectiveness of sodium 0luoride varnish and silver diamine 0luoride in arresting caries lesion at two distinct times. group time interval caries activity x2 p<0.05 caries arrested caries active sdf 6 28 (66.6%) 14 (28.5%) 5.509 df = 1 critical value = 3.84 12 37 (88%) 5 (11.9%) naf varnish 6 17 (40.4%) 25 (59.5%) 0.434 12 20 (47.6%) 22 (52.3%) time interval group caries activity x2 p<0.05 caries arrested caries active 6 sdf 28 (66.6%) 14 (28.5%) 5.791 df = 1 critical value = 3.84 naf varnish 17 (40.4%) 25 (59.5%) 12 sdf 37 (88%) 5 (11.9%) 15.773 naf varnish 20 (47.6%) 22 (52.3%) sodium fluoride varnish versus silver diamine fluoride effecvveness to stop dental decay in primary molars vol 13, no 1 (2025) doi 10.5195/d3000.2025.1044 http://dentistry3000.pitt.edu 5 figure 1. the 0low graphic shows how children participate in the study. 6 months follow up 12 months follow up randomize (n=84 children) group i, 5%naf (n=42) follow up17 (40.4%) follow up20 (47.6%) group ii, 38%sdf (n=42) follow up28 (66.6%) follow up 37 (88%) 1020 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1020 http://dentistry3000.pitt.edu serum b12 and folic acid level among a sample of iraqi patients with geographic tongue noor s. mohammed ali college of den*stry, university of baghdad, baghdad, iraq abstract objec4ve: to assess the serum concentragons of vitamin b12 and folic acid in individuals suffering from geographic tongue. it also aims to invesggate whether deficiencies in these hemagnic nutrients are significantly linked to the occurrence of this condigon. subjects and methods: the data for this retrospecgve analysis were obtained from the medical records of individuals with oral lesions who required laboratory tesgng for serum b12 and folic acid between october 2024 and may 2025 due to suspected anemia. 30 pagents had geographic tongue who examined by oral medicine specialist at college of dengstry/university of baghdad and 30 healthy subjects, according to the inclusion and exclusion criteria. stagsgcal analysis was performed to compare mean levels between groups and to evaluate potengal correlagons. results: the average serum levels of vitamin b12 (p < 0.001) and folic acid (p < 0.05) were significantly decreased in pagents with geographic tongue compared to healthy controls. a considerable proporgon of these pagents had values falling below the normal reference range. these findings suggest a possible involvement of hemagnic deficiencies in the underlying pathogenesis of geographic tongue. conclusion: the study's findings indicate that deficiencies in vitamin b12 and folic acid may contribute to the development of geographic tongue. evaluagng and addressing these nutrigonal gaps could be beneficial in the clinical approach to managing gt, pargcularly in pagents presengng with symptoms. addigonal longitudinal research is warranted to be]er understand the causal relagonship and the potengal benefits of targeted supplementagon. open access cita%on: ali nsm. (2025) serum b12 and folic acid level among a sample of iraqi pa%ents with geographic tongue. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1020 received: august 18, 2025 accepted: august 28, 2025 published: september 10, 2025 copyright: ©2025 ali nsm. this is an open access ar%cle licensed under a crea%ve commons atribu%on work 4.0 united states license. email: noorsaad2011@codental.uobaghdad.edu.iq introduc)on geographic tongue (gt) is a common and benign inflammatory disorder of the oral mucosa, generally manifesting as annular lesions marked by the absence of filiform papillae. these depapillated areas appear red and atrophic, often bordered by a distinct yellowish-white margin. the dorsal surface of the tongue is the most frequently affected site, although the lateral borders may also be involved in some cases [1]; it was first described in 1931 [2]. among the many names for geographic tongue are migratory glossitis, annulus migrans, and erythema migrans as well. on the tongue, the disease manifests as irregular red spots with white borders that are somewhat elevated. these lesions frequently have a map-like appearance, which is the basis for the term "geographic" [3], the lesions associated with geographic tongue may persist for several days to months before resolving spontaneously, often reappearing at different sites on the tongue. notably, no scarring is observed during remission [4]. gt is predominantly localized to the dorsal surface of the tongue [5]. it impacts approximately 1-3% of the whole population and is present in the males and females [6], other studies indicate a slightly elevated prevalence in females, demonstrating a female-to-male ratio of approximately 2:1 [7]. this could be linked to gt's correlation with female hormones [8]. geographic tongue primarily occurs in adolescents and adults, with instances documented in persons ranging from 5 to 84 years of age. among adults the clinical signs are generally more apparent than in children [9-11]. although the exact etiology of gt remains unknown, numerous contributing factors have been proposed. these include genetic predisposition, psoriasis, allergic conditions, diabetes mellitus, hormonal imbalances, nutritional deficiencies, psychological stress, and the use of certain medications such as oral contraceptives. additionally, this condition has been documented in conjunction with serum b12 and folic acid level among a sample of iraqi pagents with geographic tongue vol 13, no 1 (2025) doi 10.5195/d3000.2025.1020 http://dentistry3000.pitt.edu 2 systemic disorders like reiter's syndrome, down syndrome, and lichen planus [12,13]. hematologic deficiencies, especially those related to vitamin b12 and folic acid, have garnered significant attention as potential contributing factors. these vitamins are crucial for dna production, cellular division, and the preservation of epithelium integrity. deficiencies can result in various mucosal changes, including glossitis and other oral lesions [14]. several studies have suggested a potential association between geographic tongue and low serum levels of these nutrients [14,15]. the present study aims to examine serum concentrations of vitamin b12 and folic acid in persons diagnosed with geographical tongue to healthy controls, and to evaluate the possible link between these de\iciencies and the presence of the condition, with the goal of informing appropriate clinical management strategies. subjects and methods this retrospective analysis utilized data obtained from the medical records of patients with oral lesions submitted for laboratory assessment of serum vitamin b12 and folic acid since there was clinical suspicion of anemia. the data collection period extended from october 2024 to may 2025. a total of 30 patients diagnosed with geographic tongue, examined by an oral medicine specialist at the college of dentistry, university of baghdad, were included in the study group. the control group comprised 30 healthy individuals, chosen based on identical eligibility and exclusion requirements as patients visiting the same center. all participants reported that they had not taken any vitamin supplements in the eight weeks prior to the study. individuals with systemic diseases, such as chronic renal disorders, psoriasis, reiter’s syndrome, dermatitis, insulin-dependent diabetes, and bronchitis, as well as pregnant women, were excluded from the study [16]. control subjects were con\irmed to be free of geographic tongue and the designated systemic disorders and had not utilized any drugs. in this investigation, blood b12 vitamin levels of ≤ 220 pg/ml and folic acid levels of < 4 ng/ml are considered insuf\icient (17,18). spss software, version 18.0 (spss inc., chicago, il), was used for statistical analysis. welch's t-test was used to determine statistical signi\icance because the data was quantitative. statistical signi\icance was de\ined as a p-value of less than 0.05, while extremely signi\icant was de\ined as a p-value of less than 0.001. results thirty people with geographic tongue and thirty healthy controls were included in the \inal analysis. age and gender were used to match the gt group with the control group. every participant was sourced from the university of baghdad's college of dentistry's outpatient oral medicine clinic. the average age of the 19 female and 11 male members of the gt group was 34.83 ± 8.66 years. the mean age of the 14 male and 16 female members of the control group was 32.03 ± 8.71 years. the demographic characteristics of both groups are shown in table 1. table 1. demographic data of the gt patients and healthy controls. welch's t-test was used in this study to compare the serum vitamin b12 levels of patients with geographic tongue (gt) and healthy controls. for those with low serum vitamin b12 levels, the gt group's mean value was 143.8 ± 36.5 pg/ml (n = 18), while the control group was 202.6 ± 11.5 pg/ml (n = 8). a highly signi\icant difference between the two groups was indicated by the statistically signi\icant difference (t ≈ -6.18, df ≈ 23, and p < 0.00001). the gt group had a mean of 396.15 ± 124.3 pg/ml (n = 12) for persons with normal serum vitamin b12 levels, whereas the control group had a mean of 519.5 ± 138.7 pg/ml (n = 22). additionally, there was statistical signi\icance in this difference (t = -2.65, df ≈ 27, p ≈ 0.012). the mean serum b12 levels for gt patients were 244.74 ± 82.94 pg/ml (n = 30), whereas the mean for healthy controls was signi\icantly higher at 434.99 ± 120.2 pg/ml (n = 30). there was a signi\icant difference (t = -7.13, df = 58, p < 0.001). twelve gt patients and fourteen controls were included to examine serum folic acid levels among those with low folic acid levels. statistical signi\icance was indicated by the mean values of 1.4 ± 0.6 ng/ml and 2.3 ± 1.2 ng/ml, respectively, with t ≈ -2.36, df = 24, and p ≈ 0.027. the gt group had a mean of 10.5 ± 3.2 ng/ml (n = 18) for people with normal folic acid levels, while the control group had a mean of 15.32 ± 4.3 ng/ml (n = 16). there was a signi\icant difference (t ≈ -3.74, df = 32, p ≈ 0.0007). overall, the gt group's mean folic acid level was 6.86 ± 2.52 ng/ml, compared to 9.24 ± 3.25 ng/ml for the control group. a statistically signi\icant difference was discovered (t ≈ -3.17, df = 58, p ≈ 0.0024). table 2 provides a summary of all the data. discussion the serum levels of folic acid and vitamin b12 in people with geographic tongue (gt), a benign but occasionally symptomatic oral disorder of unknown cause, were investigated in this study. when compared to healthy controls, the results showed that vitamin b12 and folic acid deficits were far more common in gt patients. previous research indicates that nutritional deficiencies, especially haematinic factors like b 12 and folate, may play a role in the pathogenesis of gt. these vitamins are essential for dna synthesis and epithelial cell regeneration. insufficient levels of these nutrients may hinder the regeneration of the oral mucosa, resulting in epithelial atrophy and desquamative lesions hallmark of gt [18,19]. our results are in line with those reported by alikhani et al., who found significantly lower serum levels of vitamin b12 and folate in gt patients compared to healthy individuals [20]. similarly, jahanbani et al. observed an association between hematinic deficiencies and gt, underscoring the importance of evaluating systemic nutritional status in affected individuals [21]. it is well-established that vitamin b12 deficiency may lead to glossitis, mucosal atrophy, and burning sensations in the oral cavity—clinical features that can mimic or overlap with those seen in gt [22,23]. likewise, folic acid deficiency has been linked to increased mucosal fragility and susceptibility to inflammatory changes on the tongue [24]. however, not all gt patients in our study demonstrated deficiencies in these vitamins, suggesting that nutritional factors, while important, are not the sole contributors to gt. the etiology of gt is likely multifactorial, involving a combination of genetic predisposition, immunological mechanisms, psychological stress, hormonal influences, and possibly allergic or atopic conditions [25]. nutritional status itself is a complex indicator influenced by socio-economic factors, food availability, and individual metabolic needs. the world health organization views nutritional status as both a determinant and a consequence of overall development and health [26]. optimal nutrition remains crucial for the maintenance of oral and dental tissue integrity [27]. the serum levels of folic acid and vitamin b12 in people with geographic tongue (gt), a benign but occasionally symptomatic oral disorder of unknown cause, were investigated in this study. when compared to healthy controls, the results showed that vitamin b12 and folic acid deficits were far more common in gt patients. previous research indicates that nutritional deficiencies, especially haematinic factors like b 12 and folate, may play a role in the pathogenesis of gt. these vitamins are essential for dna gt patients healthy control no. of subjects 30 30 age 34.83 ± 8.66 32.03 ± 8.71 male:female 11:19 14:16 serum b12 and folic acid level among a sample of iraqi pagents with geographic tongue vol 13, no 1 (2025) doi 10.5195/d3000.2025.1020 http://dentistry3000.pitt.edu 3 synthesis and epithelial cell regeneration. insufficient levels of these nutrients may hinder the regeneration of the oral mucosa, resulting in epithelial atrophy and desquamative lesions hallmark of gt [18,19]. our results are in line with those reported by alikhani et al., who found significantly lower serum levels of vitamin b12 and folate in gt patients compared to healthy individuals [20]. similarly, jahanbani et al. observed an association between hematinic deficiencies and gt, underscoring the importance of evaluating systemic nutritional status in affected individuals [21]. it is well-established that vitamin b12 deficiency may lead to glossitis, mucosal atrophy, and burning sensations in the oral cavity—clinical features that can mimic or overlap with those seen in gt [22,23]. likewise, folic acid deficiency has been linked to increased mucosal fragility and susceptibility to inflammatory changes on the tongue [24]. however, not all gt patients in our study demonstrated deficiencies in these vitamins, suggesting that nutritional factors, while important, are not the sole contributors to gt. the etiology of gt is likely multifactorial, involving a combination of genetic predisposition, immunological mechanisms, psychological stress, hormonal influences, and possibly allergic or atopic conditions [25]. nutritional status itself is a complex indicator influenced by socio-economic factors, food availability, and individual metabolic needs. the world health organization views nutritional status as both a determinant and a consequence of overall development and health [26]. optimal nutrition remains crucial for the maintenance of oral and dental tissue integrity [27]. despite being frequently asymptomatic and discovered incidentally, gt can cause discomfort in some patients. in such symptomatic cases especially those reporting burning sensations it is advisable to assess serum levels of hematinic nutrients. although treatment with vitamin supplementation has led to symptomatic relief in some individuals, the supporting evidence remains limited and largely anecdotal [28]. conclusion the results of this study confirm that the existence of geographic tongue is significantly correlated with low serum levels of folic acid and vitamin b12. even though not every patient had these deficits, they seem to have played a significant role in the pathophysiology and clinical presentation of gt. as a result, when evaluating gt patients clinically, especially those who exhibit symptoms like burning or mucosal irritation, hematinic status assessment should be considered. to investigate the therapeutic potential of vitamin supplementation in the management of gt, more prospective research is necessary. conflict of interest none. references 1. hashemipoor m, rad m, dastboos a. prevalence, clinical features of geographic tongue. journal of dentistry.2015: 1558; 8:93–81. 2. campana f, vigarios e, fricain jc, sibaud v (2019) geographic stomatitis with palate involvement. ann bras dermatol 94(4): 449-451. 3. picciani b, santos vc, teixeira st, izahias lm, curty a, et al. (2017) investigation of the clinical features of geographic tongue: unveiling its relationship with oral psoriasis. int j dermatol 56(4): 421-427. 4. ogueta ci, ramirez pm, jimenez oc, cifuentes mm (2019) geographic tongue: what a dermatologist should know. actas dermosifiliogr (engl ed) 110(5): 341346. 5. shahjahan s, ettefagh l (2023) geographic tongue. in: stat pearls. treasure island (fl): stat pearls publishing. 6. farah c, balasubramaniam r, mccullough mj. contemporary oral medicine. springer nature; 2019. doi:10.1007/978-3-319-72303-7 7. scully c, bagan j, pedro diz dios am. oral medicine and pathology at a glance. 1st ed. willeyblackwell; 2016. 8. teixeira-souza t, carneiro s, moore d, et al. geographic tongue and atopy: is there an association? brazilian j dent. 2018:1–7. doi:10.18363/rbo. v75.2018.e1186 9. ghom ag, ghom sa. textbook of oral medicine. 3th ed. jaypee brothers medical publishers; 2014. doi:10.5005/jp/books/12631_48 10. ongole r, p bn. textbook of oral medicine, oral diagnosis and oral radiology. 2nd ed. elsevier; 2013. 11. scully c. oral and maxillofacial medicine. elsevier; 2013. doi:10.1111/j.16010825.2008.01461.x 12. picciani bl, santos lr, amin tn, et al. applicability of the geographic tongue area and severity index among healthcare professionals: a crosssectional clinical validation of a newly developed geographic tongue scoring system. j clin med. 2021; 10(23):5493. doi:10.3390/ jcm10235493 13. gonz l, garc j, garc m. geographic tongue: predisposing factors, diagnosis and treatment: a systematic review. rev clíínica española. 2018. doi:10.1016/j.rceng.2018.05.009. 14. wu yh, yu-fong chang j, wang yp, wu yc, chen hm, sun a. hemoglobin, iron, vitamin b12, and folic acid deficiencies and hyperhomocysteinemia in behcet’s disease patients with atrophic glossitis. journal of the formosan medical association 2018; 117(7):559e65. 15. chiang cp, chang jy, wang yp, wu yc, wu yh, sun a. significantly higher frequencies of anemia, hematinic deficiencies, hyperhomocysteinemia, and serum gastric parietal cell antibody positivity in atrophic glossitis patients.j formos med assoc. 2018 dec;117(12):1065-1071. 16. mina khayamzadeh, shamsoulmolouk najafi, parastoo sadrolodabaei, faranak vakili, mohammad javad kharrazi fard. determining salivary and serum levels of iron, zinc and vitamin b12 in patients with geographic tongue. j dent res dent clin dent prospect 2019; 13(3):221-226. 17. farkhanda ghafoor, ayyaz a khan. association of vitamin b12, serum ferritin and folate levels with recurrent oral ulceration. pak j med res vol. 51, no. 4, 2012. 18. conclusions of a who technical consultation on folate and vitamin b12 deficiencies. food and nutrition bulletin, 2008, 29:s238–244. 19. porter sr, scully c. oral manifestations of systemic disease. dent update. 2000; 27(4):194–202. 20. alikhani m, rasti m, jafari m. evaluation of serum vitamin b12, folate, and iron levels in patients with geographic tongue. j res med sci. 2015; 20(7):707–710. 21. jahanbani j, shajari a, ranjbar r, et al. geographic tongue and hematinic deficiencies. j contemp dent pract. 2009; 10(2):e041–e048. 22. aya k. taher, nada jafer mh radhi. salivary irisin in relation to recurrent aphthous ulcer and weight status in diyala city/iraq. j. bagh. coll. dent. vol. 36, no. 1. 2024 23. field ea, allan rb. oral mucosal disease: the role of b-group vitamins in oral mucosal disease. br j oral maxillofac surg. 2003; 41(5):327–333. 24. zegarelli dj. glossitis and its relationship to vitamin b-complex deficiency. oral surg oral med oral pathol. 1967; 23(3):282–289. 25. assimakopoulos d, patrikakos g, fotika c, elisaf m. benign migratory glossitis or geographic tongue: an enigmatic oral lesion. am j med. 2002; 113(9):751–755. 26. tagreed altaei. treatment of recurrent aphthous ulceration by mastic orabase. j bagh coll dentistry 2017; 29(3):45-53 27. mohamed taha elfezary, shaimaa eldeeb, mohamed elsayed moteea, mohammad said abu samadah, ahmed safaa waliy. impact of nutrient deficiencies on early childhood caries: a case-control study on vitamin d, calcium, and ferritin serum levels. j. bagh. coll. dent. vol. 36, no. 4. 2024 28. mignogna md, fortuna g, leuci s, adamo d. symptomatic benign migratory glossitis (geographic tongue): successful treatment with topical tacrolimus. clin oral investig. 2011; 15(6):991– 998. serum b12 and folic acid level among a sample of iraqi pagents with geographic tongue vol 13, no 1 (2025) doi 10.5195/d3000.2025.1020 http://dentistry3000.pitt.edu 4 table 2. comparison of serum vitamin b12 and folic acid levels in patients with gt and healthy controls. variable gt patients healthy control no. mean ± sd no. mean ± sd vitamin b12 (pg/ml) low 18 143.8 ± 36.5 8 202.6 ± 11.5** normal 12 396.15 ± 124.3 22 519.5 ± 138.7* total 30 244.74 ± 82.94 30 434.99 ± 120.2 ** folic acid (ng/ml) low 12 1.4 ± 0.6 14 2.3± 1.2* normal 18 10.5 ± 3.2 16 15.32± 4.3** total 30 6.86 ± 2.52 30 9.24 ± 3.25* * p < o.o5 ** p < o.oo1 955 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.955 http://dentistry3000.pitt.edu efficacy of two diode laser wavelenghts in the management of masticatory disorders and myofascial pain anes adnan alshamaa1, hani mohammed al-kufi2, anmar sadaa al-halbosi3, sohaib mohammed awad1 1college of den*stry, university of almaarif, ramadi, iraq 2college of den*stry, the islamic university, najaf, iraq 3habbaniyah primary health care sector, ministry of health, anbar, iraq abstract objec;ve: this study aimed to evaluate the effect of photobiomodulalon on restricted mouth opening and pain relief in painful temporomandibular palents by using two wavelengths of diode laser. material and methods: in this study, thirty-six parlcipants—ages 18 to 45 yeas—who suffered from restricted mouth opening, pain in the temporomandibular joint area and tenderness in the temporal and masseter muscles were divided into two groups: group a (n = 15) received 980 nm laser therapy, and group b (n = 15) received 635 nm laser therapy. six parlcipants were excluded. over the course of two weeks, each group had six therapy sessions. the maximal mouth opening was measured by digital caliper and the pain severity was evaluated using the visual analog scale. the mouth opening and pain scale of the groups were then evaluated stalslcally using mullple wilcoxon sign rank adjusted by dunn-bonferroni method, and tukey’s hsd tests. the significance level was set to 0.05. results: the outcomes demonstrated that there were notable improvements in mouth opening and discomfort reduclons in both groups. the 980 nm treatment displayed a consistent reduclon from baseline, with stalslcally significant differences between all-lme points. the 635 nm treatment showed a more pronounced reduclon, especially between baseline and six weeks or one month, but no significant difference between six weeks and one month. conclusion: this study demonstrated the significant impact of the therapeulc efficacy of diode laser wavelengths on the management of maslcatory disorders and myofascial pain. open access cita%on: alshamaa aa, et al. (2025) efficacy of two diode laser wavelenghts in the management of mas%catory disorders and myofascial pain. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.955 received: june 2, 2025 accepted: june 26, 2025 published: august 13, 2025 copyright: ©2025 alshamaa aa, et al. this is an open access ar%cle licensed under a crea%ve commons asribu%on work 4.0 united states license. email: anes.adnan@uoa.edu.iq introduc)on temporomandibular disorder (tmd) tmd refers to conditions affecting musculoskeletal system structures, such as the joints (tmj), masticatory muscles and other related structures. tenderness in the joints and/or muscles, sounds from the joints, and restricted jaw movement and limitation of mouth opening are the common conditions [1-3]. tmd occur due to injury due, neuropsychological stress, malocclusion, and stroke-related parafunctional activity [4]. intense pain felt in chewing muscles is quite common in temporomandibular disorders. this pain can often be debilitating. problems with chewing can arise for a variety of causes that affect the muscles of the jaw, which is felt in the pre-auricular area and masticatory muscles [5,6]. pbm and arndtschulz law photobiomodulation is an application that relies on the use of certain wavelengths in low and sub-ablative power settings that produce minimal and accepted thermal effects on tissues to stimulate cellular responses. many experiments show high compliance with pbm, with no invasiveness, atraumaticity, and no collateral effects [7,8]. the wavelengths shorter than 600 nm tend to be absorbed excessively by pigmental proteins of the cell, while water serves as an effective absorber of the bands longer than 1150 nm [9]. the therapeutic window is the range of suitable wavelengths appropriated for photobiomodulation. this range lies between 600 and 1150 nm [10]. the phrase "arndt-schulz law" refers to a law of treatment effect by medication. it guides the optimal dosing of a therapeutic stimulus. the arndt-schulz curve expresses the optimum response of a living organism between 0.1 to 10 joules/ cm2 [11]. the curve suggests that: 1. low doses (low energy densities) < 2 joule/cm2 insufficient for effect or may be weak stimulatory, promoting cellular function and healing. efficacy of two diode laser wavelenghts in the management of maslcatory disorders and myofascial pain vol 13, no 1 (2025) doi 10.5195/d3000.2025.955 http://dentistry3000.pitt.edu 2 2. moderate doses (110 joules/cm2) lead to an optimal therapeutic effect. 3. at 10 joules/ cm2 indicate the analgesic effect 4. high doses (high energy densities) > 10 joules / cm2 can be inhibitory or even harmful. pain relief and muscle relaxing low level lasers can improve and restore muscle function and alleviate pain threshold, ultimately leading to the restoration of normal cell and tissue functions. as inhibiting of the pain signals that nerve cells send to the brain, laser treatment affects these cells. the brain and adrenal gland produce large amounts of painkillers including endorphins and encephalin, which is a representation of the pain blocking mechanism [12, 13]. using a non-invasive approach, laser therapy targets muscle trigger points to relieve musculoskeletal discomfort and accelerating angiogenesis, which results in transient vasodilatation, provides an additional advantage [14]. this study aimed to evaluate the effect of photobiomodulation on districted mouth opening and pain relief on painful temporomandibular patients by use two wavelengths of diode laser. material and methods participants the experimental in vivo trials of this study were approved by research ethics committee at faculty of dentistry, university of almaarif, ramadi, iraq. (ref. no. 2, 2024). in this study, thirty-six participants—ages 18 to 45 years—who suffered from restricted mouth opening, pain in the temporomandibular joint area and tenderness in the temporal and masseter muscles presented to the dental clinic at the university of almaarif. the established guidelines were used to conduct a clinical evaluation of the relevant subjects. a digital measuring instrument was used to measure the participants’ mouth opening, and the visual analog scale was employed to gauge the patients' subjective pain level [15]. participants were chosen according to predefined inclusion criteria. to ensure excluding unwanted instances and maintain other dental and surgical aspects of the research unaware of it, an mri was taken. inclusion participants have been divided into two groups, with group b receiving laser therapy at 635 nm and group a receiving laser therapy at 980 nm. diagnosis extraoral and intraoral examinations are achieved in the counselling dental clinic: palpation, percussion, xray, mri, and the history case of each patient had taken. 2.1 inclusion and exclusion subjects the study included patients with pain in the temporomandibular joint region, tenderness in the masticatory muscles, and reduced mouth opening. the excluded cases include complicated conditions such as dislocated joint capsules, trauma, tumors, dental pain, and oral lesions, vascular diseases, arthritis, any known neuropsychological or hormonal problems [16]. six participants were excluded. laser equipment 1. the laser system used was a medency triplo laser device, production date 08/2024. 2. therapy handpiece (directo hat-top). directo is a special collimated handpiece developed to increase therapy’s reliability with no energy dispersion. laser parameters each patient received irradiance per session as the following equations: [17] power density = power / area of laser beam energy density = e /area of laser beam peak power = e/ duration of each treatment session in seconds. a. 980 nm wavelength power 0.8 w, beam spot diameter 2.2 cm, power density 0.2105 w/cm2 exposure time 30 seconds per trigger points in continuous mode, energy density per session 6.32 j/cm2 b. 635 nm wavelength power 0.5 w, beam spot diameter 2.2 cm, power density per point 0.1315 w/cm2 exposure time 30 seconds per trigger points in continuous mode. energy density of each exposure 3.95 j/cm2 treatment approach participants in the treatment groups received laser therapy sessions, during which the directo hat-top handpiece was placed over the trigger points, with each exposure time for 30 seconds. each participant underwent a total of six treatment sessions, which included three laser therapy sessions per week over a duration of two weeks. subjects in group a were treated with a 980 nm wavelength at a power setting of 800 mw and an energy density of 6.32 j/cm2. meanwhile, group b was administered 635 nm laser therapy at a power setting of 300 mw and an energy density of 3.95 j/cm2 [18]. for every participant, the maximal mouth opening and baseline vas pain scores were documented. a mean value of pain and mo during function was to be recorded by the patients over two days in between each treatment session. the laser beam was applied in a contact delivery with continuous mode. the contributors monitored patients pain intensity using the visual analog scale before, during, and after the therapy. mouth opening was measured using a digital caliper. ibm spss software version 22 was used for the statistical analysis (spss inc., chicago, il, usa). for the categorical and nominal variables, descriptive statistics included to characterize the study variables, while means and standard deviations were used to present the continuous variables. results after completion of laser treatment, the drop in pain intensity and development of mouth opening has been recorded for one month (tables 1 through 7 and figures 1 and 2). discussion the values of laser parameters and usable settings were included in therapeutic range of photobiomodulation curve. laser therapy is a treatment that is effective in alleviating pain and discomfort. it is also useful for relaxing muscle spasms and increasing circulation to speed up inflammation healing. when the measurements were obtained after treatment period, there was a significant drop in the levels of pain. those receiving treatment from the 635 nm laser had better results when it comes to mouth opening compared to those receiving treatment from the 980 nm laser. it was helpful that laser light's anti-inflammatory and painrelieving impact took place in the target tissues. the differing results of 980 and 635 nm laser treatments were possibly due to the varying depth of penetration and biological effects of these two laser wavelengths. normality test of maximal mouth opening among periods and groups. pbm transfers energy to intracellular lightabsorbing molecules. chromophores are present as porphyrins and the mitochondrial etransport chain. the result is a metabolic energy through the respiratory chain direct to redox change of photons and production of adenosine triphosphate (atp), and release of nitric oxide (no) may act as a potent extracellular vasodilator [19,20]. photoreceptors are present as two complementary groups inside mitochondria. the respiratory chain enzymes at the mitochondrial level act as the main photoreceiver for photobiomodulation outcomes within wavelengths of the visible band. ion channels embedded on the cell membrane act as the primary photoreceiver for pbm outcomes at infrared wavelength bands [21-23]. laser-tissue interaction determines the absorption and scattering nature of light, especially the wavelength and its chromophores inside the tissues both laser treatments show significant reductions in maximal mouth opening over time. the 980nm treatment displays a consistent reduction from baseline, with statistically significant differences between all efficacy of two diode laser wavelenghts in the management of maslcatory disorders and myofascial pain vol 13, no 1 (2025) doi 10.5195/d3000.2025.955 http://dentistry3000.pitt.edu 3 time points. the 635 nm treatment shows a more pronounced reduction, especially between baseline and six weeks the study's kindings concur with those of other researchers, such as mortazavi and martins-júnior. the most prevalent age for the development of this condition is between 20 and 40 years old, according to another research conducted by lipton and glass & glaros [24, 25]. this study supports minghella's kindings that 68% of patients with these diseases were young adults [26]. the present study contradicts emshoff and de abreu venancio, who found no alleviation of tmj pain with lllt treatment [27]. according to petrucci, lllt is insufkicient for easing tmj pain [28]. numerous research concurs with this one since they found that lllt application is a useful treatment for tmd sufferers. according to mazzetto, çetiner, and venezian's investigations, patients were monitored for up to 30 days following their kinal laser application sessions. according to çetiner and bisson, the pain decrease remained statistically signikicant during this time [29-31]. mazzetto stated that despite these outcomes, the last laser application session eliminated the minimal sensitivity to palpation. for a year, lassemi monitored the patients and recorded pertinent results in terms of pain relief [32]. according to their research, arani, myer used lasers and trigger points to treat the afklicted area. the study reported a signikicant reduction in pain in all the patients with a laser beam at a wavelength of 980 nm and 635 nm. the corresponding outcomes of pbm with 980 nm were documented in previous studies [33-35]. the mean vas between the two groups reached a signikicant difference (p < 0.05) after the fourth session which continued till the 6th session and one month follow-up period. according to turky index, the procedure is considered successful if there is 50 % reduction in the vas. it is recorded as 75 % reduction in 635 nm group. the mean mmo between the two groups reached a signikicant difference (p < 0.05) after the sixth session which continued till month follow-up period. the result was in accordance with the study by khalighi et al., in which results demonstrated 33.6% increase in mouth opening in the laser group which started from the eighth session [36]. the application of photobiomodulation lasers with different wavelengths become popular for pain reduction and healing acceleration after surgeries. infrared lasers are the best because of their deeper penetration, according to the kindings of last research and the laser's wavelength ranged from 635 to 980 nm [37-38]. conclusions this study has demonstrated the signikicant impact of the therapeutic efkicacy of diode laser wavelengths on management of masticatory disorders and myofascial pain. by examining two diode laser wavelengths 980 nm, 635 nm, the research highlights relaxing muscle spasms and increasing circulation to speed up inklammation healing. when the measurements were obtained after treatment period, there was a signikicant drop in the levels of pain to use a 635 nm laser therapy as a biostimulaton aids with myogenic tmd. while the study determines key aspects of diode laser effect on soft tissue, further investigation is needed to explore as applying other laser wavelengths. however, since there were no further side effects of two wavelengths observed after laser session. it is possible to recommend the use of a 635 nm as a photo-biomodulation second option in treatment of masticatory disorders and myofascial pain patients. acknowledgements this research has the academic and kinancial support by university of almaarif and deanship college of dentistry. the authors thank the staff of the maxillofacial surgery department at al-ramadi teaching hospital. references [1] tecco, s, nota, a, caruso, s, primozic, j, marzo, g, baldini, a, et al. temporomandibular clinical exploration in italian adolescents. cranio. (2019) 37:77–84. doi: 10.1080/08869634.2017.1391963 [2]. christidis, n, lindstrom ndanshau, e, sandberg, a, and tsilingaridis, g. prevalence and treatment strategies regarding temporomandibular disorders in children and adolescents-a systematic review. j oral rehabil. (2019) 46:291–301. doi: 10.1111/joor.12759 [3] köhler, aa, helkimo, an, magnusson, t, and hugoson, a. prevalence of symptoms and signs indicative of temporomandibular disorders in children and adolescents. a cross-sectional epidemiological investigation covering two decades. eur arch paediatr dent. (2009) 10:16–25. doi: 10.1007/bf03262695 [4] al-kufi, h.m., al-bayati, a.m. efficacy of photobiomodulation therapy (pbmt) in management of temporomandibular disorder (tmd). laser dent sci 7, 77–83 (2023). [5] fricton j. myogenous temporomandibular disorders: diagnostic and management considerations. dent clin north am. 2007 jan;51(1):6183, vi. doi: 10.1016/j.cden.2006.10.002. [6] badel, tomislav, andreja carek, dijana podoreški, ivana savić pavičin and sandra kocijan lovko. “temporomandibular joint disorder in a patient with multiple sclerosis--review of literature with a clinical report.” collegium antropologicum 34 3 (2010): 1155-9. [7] almasri, m.; mandal, n.; kulkarni, p.; raj, a.; zeya, a.; mann, n. low-level laser therapy role in surgical extractions:an original research. j pharm biol sci. 2022,14,245. [8] john, s.s.; mohanty, s.; chaudhary, z.; sharma, p.; kumari, s.; verma, a. comparative evaluation of low level laser therapy and cryotherapy in pain control and wound healing following orthodontic tooth extraction: a double blind study. j cranio maxillofac.surg.2020,48,251–260 [9] hamblin, m.r.; liebert, a. photobiomodulation therapy mechanisms beyond cytochrome c oxidase. photobiomodul. photomed. laser surg. 2022, 40, 75–77. [10] hamblin, m.r.; liebert, a. photobiomodulation therapy mechanisms beyond cytochrome c oxidase. photobiomodul. photomed. laser surg. 2022, 40, 75–77. [11] debenedittis, m.; petruzzi, m.; pastore, l.; inchingolo, f.; serpico, r. nd:yag laser for gingivectomy in sturge-weber syndrome. j. oral maxillofac. surg. 2007, 65, 314–316. [12] huang,y.-y.; sharma, s.k.; carroll, j.; hamblin, m.r. biphasic dose response in low level light therapy—an update. dose response 2011, 9, 602–618. [13] debenedittis, m.; petruzzi, m.; pastore, l.; inchingolo, f.; serpico, r. nd:yag laser for gingivectomy in sturge-weber syndrome. j oral maxillofac surg. 2007, 65, 314–316. [14] bjordal jm, johnson mi, iversen v: ''low-level laser therapy in acute pain: a systematic review of possible mechanisms of action and clinical effects in randomized placebo-controlled trials''. photomed and laser surg 2006; 24:158168. [15] feeling the heat: evolutionary and microbial basis for the analgesic mechanisms of photobiomodulation therapy scientioic figure on researchgate. available from: https://www.researchgate.net/oigure/the-pbm-dose-regimensrepresent-a-multi-phasic-biological-responseand-emphasize-the_oig3_334618194 [accessed 11 may 2025] [16] delgado da, lambert bs, boutris n, mcculloch pc, robbins ab, moreno mr, harris jd. validation of digital visual analog scale pain scoring with a traditional paper-based visual analog scale in adults. journal of the american academy of orthopaedic surgeons. global research & reviews. 2018 mar;2(3). [17] mohamed, r. k., elsayed, n. m., mahmoud, s. a., gaweesh, y. y. (2024). photobiomodulation versus corticosteroid in the management of erosive oral lichen planus: a randomized controlled clinical trial. bmc oral health, 24(1). [18] kumar, s. (2017). the emerging role of botulinum toxin in the treatment of orofacial disorders: literature update. asian journal of pharmaceutical and clinical research, 10(9), 21. [19] shirani am, gutknecht n, taghizadeh m, mir m. low-level laser therapy and myofacial pain dysfunction syndrome: a randomized controlled clinical trial. lasers med sci. 2009 sep;24(5):715-20. doi: 10.1007/s10103-008-0624-5. [20] mester, e.; mester, a.f.; mester, a. the biomedical effects of laser application. laserssurg.med. 1985,5,31–39. [21] inchingolo, a.m.; malcangi, g.; ferrara, i.; viapiano, f.; netti, a.; buongiorno, s.; latini, g.; azzollini, d.; deleonardis, n.; deruvo, e.; et al. laser surgical approach of upper labial frenulum: a systematic review. int j environ res public health 2023,20,1302. [22] rapone, b.; ferrara, e.; santacroce, l.; topi, s.; gnoni, a.; dipalma, g.; mancini, a.; di domenico, m.; tartaglia, g.m.; scarano, a.; et al. the gaseous efficacy of two diode laser wavelenghts in the management of maslcatory disorders and myofascial pain vol 13, no 1 (2025) doi 10.5195/d3000.2025.955 http://dentistry3000.pitt.edu 4 ozone therapy as a promising antiseptic adjuvant of periodontal treatment: a randomized controlled clinical trial. int. j. environ. res. public health 2022, 19, 985. [23] inchingolo f, tatullo m, abenavoli fm, marrelli m, inchingolo ad, inchingolo am, dipalma g. comparison between traditional surgery, co2 and nd:yag laser treatment for generalized gingival hyperplasia in sturge-weber syndrome: a retrospective study. j investig clin dent. 2010 nov;1(2):85-9. [24] martins-júnior rl, palma aj, et al. ''temporomandibular disorders: a report of 124 patients''. j contemp dent pract. 2010; 14:071–078. [25] mortazavi h, javadzadeh a, et al. ''myofascial pain dysfunction syndrome''. iranian j otorhinolaryngol.2010; 22:61. [26] minghelli b, et al. ''prevalence of temporomandibular disorder in children and adolescents from public schools in southern portugal''. n am j med sci. 2014; 6(3):126–132. [27] emshoff r, bosch r, et al. ''low level laser therapy for treatment of temporomandibular joint pain: a double-blind and placebo-controlled trial''. oral surg oral med oral pathol oral radiol endod 2008; 105(4); 452-6. [28] a. petrucci, f. sgolastra, r. gatto, a. mattei, and a. monaco, “effectiveness of low-level laser therapy in temporomandibular disorders: a systematic review and meta-analysis,” journal of orofacial pain, vol. 25, no. 4, pp. 298–307, 2011. [29] venezian gc, da silva ma, mazzetto rg, et al. ''low level laser effects on pain to palpation and electromyographic activity in tmd patients: a double blind, randomized, placebo controlled study''. cranio. apr 2010; 28(2):84-91. [30] m. o. mazzetto, t. h. hotta, and r. c. d. a. pizzo, “measurements of jaw movements and tmj pain intensity in patients treated with gaalas laser,” brazilian dental journal, 2010, vol. 21, no. 4, pp. 356–360. [31] bissoon ak, whaites e. ''evaluation of common operator errors in panoramic radiography in trinidad and tobago: a comparison of formally vs informally trained operators''. west indian med j. 2012; 61:733–738 [32] e. lassemi, et al. “lowlevel laser therapy in the management of temporomandibular joint disorder,” the journal of oral laser applications, vol. 8, pp. 83–86, 2008. [33] khiavi ha, ebrahimi h, najaoi s, nakisa m, habibzadeh s, khayamzadeh m, efoicacy of lowlevel laser, hard occlusal appliance and conventional pharmacotherapy in the management of myofascial pain dysfunction syndrome; j lasers med sci 2020;11:37–44. [34] ashour em, khalifa fa, khalifa ga. low level laser therapy versus pharmacotherapy and inter occlusal splint therapy in improving myofascial pain disorder syndrome al-azhar dent j girls 2021;8:91–7. [35] mahmoud nr, fayed hm. efoicacy of low-level laser therapy in the management of myofascial pain dysfunction syndrome. comparative controlled clinical trial egypt dent j 2023;69:1799– 809. [36] khalighi hr, mortazavi h, mojahedi sm, azari-marhabi s, parvaie p, anbari f. the efoicacy of low-level diode laser versus laser acupuncture for the treatment of myofascial pain dysfunction syndrome (mpds) j dent anesthe pain med 2022;22:19–27. [37] arany, p.r. photobiomodulation-activated latent transforming growth factor-b1: a critical clinical therapeutic pathway and an endogenous optogenetic tool for discovery. photobiomodul. photomed. laser surg. 2022, 40, 136–147. [38] l. mayer et al., “histologic and resonance frequency analysis of peri-implant bone healing after low-level laser therapy: an in vivo study,” int. j. oral maxillofac implants 30(5), 1028–1035 (2015). table 1. pain relief 1-10. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 980 nm pre 7 7 8 6 7 8 8 9 9 10 9 7 8 9 6 post 1st 5 3 5 2 5 4 5 5 6 7 7 7 5 6 2 post 6th 1 2 2 0 2 0 2 1 2 2 4 5 3 2 0 1 month 1 2 1 0 2 2 3 4 2 5 7 7 5 2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 635 nm pre 8 6 7 7 9 7 6 7 6 9 8 8 7 10 8 post 1st 5 4 3 3 6 5 3 4 4 6 5 6 3 6 5 post 6th 2 0 1 0 2 1 1 2 0 4 2 2 1 2 2 1 month 0 0 1 0 2 2 0 4 0 4 0 0 0 2 2 efficacy of two diode laser wavelenghts in the management of maslcatory disorders and myofascial pain vol 13, no 1 (2025) doi 10.5195/d3000.2025.955 http://dentistry3000.pitt.edu 5 figure 1. mean pain relief over time. table 2. maximum mouth opening (mm). no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 980 nm pre 25 27 24 31 27 24 23 21 21 21 25 27 25 24 30 post 1st 37 39 34 37 41 40 36 32 36 32 38 34 36 36 44 post 6th 42 44 40 41 42 49 46 41 48 40 45 40 44 40 52 1 month 37 40 40 41 40 44 46 37 42 40 40 36 44 42 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 635 nm pre 22 32 23 25 20 26 30 25 33 22 24 22 27 20 22 post 1st 26 36 30 31 28 36 40 35 41 28 34 30 37 30 31 post 6th 40 52 42 42 40 51 55 52 53 41 47 43 54 41 44 1 month 42 50 44 43 41 54 55 50 50 40 45 43 52 40 40 efficacy of two diode laser wavelenghts in the management of maslcatory disorders and myofascial pain vol 13, no 1 (2025) doi 10.5195/d3000.2025.955 http://dentistry3000.pitt.edu 6 figure 2. mean mouth opening over time. table 3. maximum mouth opening comparison using shapiro wilk test at p>0.05. period groups 980nm 635nm statistic df p value statistic df p value baseline 0.930 15 0.274 0.898 15 0.090 one 0.956 15 0.631 0.947 15 0.475 six 0.885 15 0.054 0.886 15 0.055 1month 0.928 15 0.257 0.884 15 0.054 table 4. mean maximum mouth opening comparisons. period groups t test p-value 980nm 635nm mean ±sd mean ±sd baseline 25.000 3.024 24.867 4.086 0.102 0.920 one 36.800 3.278 32.867 4.518 2.729 0.011 six 41.267 3.654 45.933 5.351 2.789 0.010 1 month 43.600 3.757 46.467 5.705 1.625 0.117 f 89.200 239.857 p-value 0.000 0.000 effect size 0.957 0.984 efficacy of two diode laser wavelenghts in the management of maslcatory disorders and myofascial pain vol 13, no 1 (2025) doi 10.5195/d3000.2025.955 http://dentistry3000.pitt.edu 7 table 5. multiple pairwise comparison of maximum mouth opening. groups period mean difference p value 980nm baseline one -11.800 0.000 six -16.267 0.000 1 month -18.600 0.000 one six -4.467 0.000 1 month -6.800 0.000 six 1 month -2.333 0.018 635nm baseline one -8.000 0.000 six -21.067 0.000 1 month -21.600 0.000 one six -13.067 0.000 1 month -13.600 0.000 six 1 month -0.533 1.000 table 6. mean pain scores comparisons. period groups wsr p-value 980nm 635nm median mean rank1 mean rank 2 median mean rank1 mean rank 2 baseline 8 16.77 3.93 7 14.23 4.00 0.812 0.436 one 5 16.83 2.93 5 14.17 2.97 0.852 0.412 six 2 16.77 1.30 2 14.23 1.60 0.839 0.436 1 month 2 19.13 1.83 0 11.87 1.43 2.339 0.023 friedman 40.664 42.277 p-value 0.000 0.000 effect size 2.03 2.11 wsr=wilcoxon sum rank, mean rank 1=between groups, mean rank 2 between periods. table 7. multiple pairwise comparisons of pain (multiple wilcoxon sign rank adjusted by the dunn-bonferroni method). groups sample 1-sample 2 wilcoxon sign rank p-value 980nm pain1-pain0 2.121 0.034 pain6-pain0 5.586 0.000 pain1month-pain0 4.455 0.000 pain6-pain1 3.465 0.001 pain1month-pain1 2.333 0.020 pain6-pain1month 1.131 0.258 635nm pain1-pain0 2.192 0.028 pain6-pain0 5.091 0.000 pain1month-pain0 5.445 0.000 pain6-pain1 2.899 0.004 pain1month-pain1 3.253 0.001 pain1month-pain6 0.354 0.724 1069 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1069 http://dentistry3000.pitt.edu effect of nanoparticles on the mechanical properties of polymethyl methacrylate denture base material omar m. faruq abdlrahman1, farhad wahid rasool2, fahd sudad ikram3 1khanzad teaching dental center, erbil, iraq 2soran health center, soran, iraq 3college of den;stry, hawler medical university, erbil, iraq abstract polymethylmethacrylate (pmma) has been widely used as a denture base material due to its favorable propernes but exhibits limitanons in mechanical strength. this study invesngated the effects of incorporanng three different nanoparncles zirconium dioxide (zro₂), silicon dioxide (sio₂), and diamond nanoparncles (dnps) at varying concentranons (0.5%, 1.0%, 2.5%, and 5.0%) on the mechanical propernes of pmma denture base material. the incorporanon of nanoparncles demonstrated concentranon-dependent effects on pmma propernes. zro₂ nanoparncles at 1.0% concentranon showed opnmal results with improved hardness (21.4 ± 0.8 vhn) while maintaining acceptable surface roughness. higher concentranons (2.5% and 5.0%) led to increased surface roughness and decreased hardness across all nanoparncle types. diamond nanoparncles exhibited the highest surface roughness (0.146 ± 0.017 μm) at 5.0% concentranon, while sio₂ showed moderate improvements in mechanical propernes at lower concentranons. surface morphology analysis revealed excellent parncle dispersion at 0.5-1.0% concentranons, with significant agglomeranon observed at higher concentranons. the study determines that 1.0% concentranon is the opnmum level to unlize for the addinon of nanoparncles into pmma denture base materials, wherein zro₂ nanoparncles show the opnmum balance of enhanced material propernes and preserved surface features. the findings show that precise control of parncle concentranon is required to achieve enhanced material propernes without compromising surface integrity that will in turn translate to enhanced clinical performance of dental prostheses. open access cita%on: abdlrahman omf, et al. (2025) effect of nanopar%cles on the mechanical proper%es of polymethyl methacrylate denture base material. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1069 received: october 8, 2025 accepted: october 15, 2025 published: november 10, 2025 copyright: ©2025 abdlrahman omf, et al. this is an open access ar%cle licensed under a crea%ve commons atribu%on work 4.0 united states license. email: zeenaadwal.a@gmail.com introduc)on polymethylmethacrylate (pmma) has remained the preferred material in the process of making denture bases since the 1930s based on its advantageous traits of biocompatibility, esthetics of look, ease of processability, and cost-effectiveness. notwithstanding these, though, there are some disadvantages of pmma to a larger extent on the aspect of mechanics that could lead to breakage along with failure of the denture base in use in clinical practice [1,2]. mechanical shortcomings of conventional pmma have evoked investigations of new methods of reinforcement. promising leads that originate from advances in the realm of nanotechnology open new opportunities for prosthodontic material enhancement. incorporation of nanoparticles in pmma has emerged as an important method of its physical and mechanical enhancement. due to the small diameter size and high surface area to volume ratio, the nanoparticles can improve the material's functionality if properly distributed in the polymer matrix [3]. some of the nanoparticles that have been investigated for reinforcement of pmma denture bases include metal oxides zirconium oxide (zro2), titanium dioxide (tio2), and silicon dioxide (sio2). nanoparticles found some level of success in improving qlexural strength, impact resistance, and surface hardness. reinforcement with nanoparticles is based on several parameters like particle size, concentration, dispersion within polymer matrix, and particle-matrix interface [4]. zirconium oxide nanoparticles have particularly captured interest since they are highly biocompatible, very strong, and white in nature that will never undermine the esthetics of the denture base. it has been established by research that zro2 nanoparticles can effect of nanoparncles on the mechanical propernes of polymethyl methacrylate denture base material vol 13, no 1 (2025) doi 10.5195/d3000.2025.1069 http://dentistry3000.pitt.edu 2 signiqicantly improve the mechanical strength of pmma when used in appropriate concentrations and along with proper surface modiqication [5]. titanium dioxide nanoparticles incorporation is also proving to be beneqicial in modifying the property of pmma. besides enhancing the material's mechanical property, tio2 nanoparticles possess some level of antimicrobial activity that can be useful in avoiding infection of the denture. the level of tio2 nanoparticles incorporation should be controlled in such a way that there is optimal performance without sacriqicing other material's properties [6]. silicon dioxide nanoparticles have also been found to improve the mechanical performance of pmma without degrading its transparency. the addition of sio2 nanoparticles has been found to improve the wear resistance as well as tensile strength of the material but will be particle concentrationand surface treatment-sensitive [7]. nanoparticle reinforcement is highly reliant on proper dispersion within the pmma matrix as well as between polymer and nanoparticles with good bonding. nanoparticles are surface-functionalized using silanization or with some coupling agents to improve particle dispersion as well as inorganic nanoparticles-polymer matrix interface. the changes are of utmost signiqicance in regulating the qinal property of the resulting nanoparticle-reinforced pmma denture base material [2]. addition of nanoparticles in polymethyl methacrylate has increased its mechanical characteristics. it has been proved by research that addition of nanoparticles of sio2, tio2, al2o3, zro2, and diamond increases the impact resistance, transverse resistance, hardness, and young's modulus of polymethyl methacrylate [8,9,2]. yet, the performance is based on the type and concentration of nanoparticles added. low concentrations (less than 1% wt.) are generally recommended for minimizing agglomeration impacts and achieving optimum beneqits [10]. the surface roughness can signiqicantly increase at concentrations above 0.5%, while hardness is increased at various concentrations [2]. a silane coupling agent can resolve agglomeration problems [10]. pmma's mechanical properties can generally be enhanced by incorporating nanoparticles, but the optimum type and quantity need to be established. materials and methods zirconium dioxide nanoparticles (znps) with an average particle size of 14 nm and 99.9% purity (aritech chemazone pvt. ltd., iraq) were selected and silanized with 97% γ-mps (shanghai richem international co., ltd.) surface treatment process was carried out according to standard procedures to optimize the interfacial adhesion between nanoparticles and the polymer matrix. silicon dioxide nanoparticles (snps) (aerosil r812; evonik degussa) having particle size 12 nm, speciqic surface area 150-550 m²/g, and intensity 2.2 g/cm³ were purchased. the snps were processed through a salinization process performed according to previously reported methods to improve their compatibility with the acrylic resin matrix. diamond nanoparticles (dnps) of about 19 nm in size (shanghai richem international co. ltd) were procured and processed according to routine surface modiqication procedures. surface treatment of dnps was performed to improve their dispersion behavior in the polymer matrix and prevent agglomeration. all nanoparticles were weighed using a highprecision electronic balance (s-234; denver instrument gmbh, göttingen, germany) to prepare concentrations of 0.5%, 1.0%, 2.5%, and 5.0% by weight of the acrylic powder. the weighed nanoparticles were then combined with the acrylic powder to form different mixtures of pmma/nanoparticles. these mixtures were mechanically stirred for 30 minutes using an electric mixer operating at 400 rpm at room temperature to ensure uniform distribution of the nanoparticles throughout the acrylic powder. the prepared nanoparticle-pmma mixtures were stored in sealed containers in a dry environment until further processing to prevent moisture contamination and maintain the integrity of the surface treatment. the handling and preparation of all nanoparticles were conducted in accordance with material safety guidelines and manufacturer recommendations. heat-polymerized acrylic resin powder (major base 20 resin; prodotti dentari spa) was selected as the primary denture base material for this study. the material was supplied as a two-component system consisting of pre-polymerized polymethyl methacrylate powder and methyl methacrylate monomer liquid. the powder and liquid components were stored at room temperature (23 ± 1°c) in their original sealed containers until use, following the manufacturer's storage recommendations. the ratio of the polymer powder to monomer liquid employed for blending was carefully maintained at the suggested 2.5:1 weight ratio by the manufacturer. this was ascertained with a calibrated electronic balance with the potential to achieve an accuracy of 0.001g. room temperature (23 ± 1°c) and controlled humidity (50 ± 10%) were employed in a clean ceramic mixing bowl to blend the contents. prior to the inclusion of the nanoparticles, the base material was quality-checked and proven for uniformity. the monomer was inspected for transparency and lack of premature polymerization, while the powder was examined for signs of moisture contamination or agglomeration. all materials were handled in accordance with the manufacturer's guidelines and material safety protocols. the conventional mixing technique was employed where the polymer powder was gradually incorporated into the monomer liquid. the mixture was stirred gently with a clean, dry spatula to ensure complete wetting of the powder particles. the material was then allowed to reach a dough-like consistency suitable for packing, as per the manufacturer's working time speciqications. this standardized preparation process was maintained throughout the study to ensure consistency in the base material properties before nanoparticle incorporation. znps were surface treated using 97% γ-mps silane coupling agent following established protocols. snps were silanized according to previously documented procedures, while dnps were treated following standardized surface modiqication methods. all surface treatments were performed under controlled laboratory conditions to enhance the interfacial bonding between the nanoparticles and the pmma matrix. the treated nanoparticles were mechanically mixed with the acrylic powder using an electric mixer operating at 400 rpm for 30 minutes at room temperature to ensure homogeneous distribution. a total of 260 specimens were fabricated and divided into thirteen groups (n=10/group). the control group was prepared using pure pmma without any nanoparticle reinforcement. for the experimental groups, three different types of nanoparticles (znps, snps, and dnps) were incorporated into the pmma powder at four different concentrations (0.5%, 1.0%, 2.5%, and 5.0% by weight). the nanoparticle-modiqied powder was then mixed with the monomer following the manufacturer's recommended powderto-liquid ratio. disk-shaped specimens measuring 15 mm in diameter and 2 mm in thickness were fabricated using standardized metal molds. the molds were qirst isolated using a separating medium and allowed to dry. the mixed pmma dough was packed into the molds at the dough stage, following conventional denture processing techniques. the molds were then compressed under hydraulic pressure of 3000 psi and maintained under pressure during the initial setting period. the packed specimens were heat-polymerized following a standardized curing cycle. the curing process was initiated at 74°c for 90 minutes, followed by a terminal boiling at effect of nanoparncles on the mechanical propernes of polymethyl methacrylate denture base material vol 13, no 1 (2025) doi 10.5195/d3000.2025.1069 http://dentistry3000.pitt.edu 3 100°c for 30 minutes. after polymerization, the qlasks were allowed to cool slowly to room temperature for 30 minutes on the bench and then for 15 minutes under running water to minimize residual monomer content and prevent warpage. ten specimens were fabricated for each concentration of each type of nanoparticle, resulting in a total of 120 experimental specimens (40 specimens per nanoparticle type) and 10 control specimens (table 1). after demolding, all specimens were qinished using tungsten carbide burs to remove excess material and qlash. the specimens were then stored in distilled water at 37°c for 48 hours before testing to ensure complete polymerization and to simulate oral conditions. following polymerization, all specimens were subjected to a standardized qinishing and polishing protocol. initial qinishing was performed using tungsten carbide burs (hm79gx-040-hp; meisinger, centennial, co) operating at 18,000 rpm to remove surface irregularities. this was followed by progressive polishing using a series of rubber polishing tips (finopol polishers, 64830, laboshop gmbh, germany) to achieve a uniform surface texture. surface standardization was accomplished using a mechanical polisher (metaserve 250 grinder-polisher, buehler) equipped with a polishing cloth disc (texmet c10in, 42-3210, buehler gmbh). the specimens were polished at 100 rpm under wet conditions for 5 minutes to ensure consistent surface qinish. after polishing, all specimens were stored in distilled water maintained at 37°c for 7 days before testing to simulate oral conditions and ensure dimensional stability. surface roughness measurements were conducted using a noncontact optical proqilometer (contour gt-k1 optical proqiler; bruker nano, inc., tucson, az) with a resolution of 0.01 mm. five areas were randomly selected and scanned on each specimen using a standard camera at 20× magniqication. the acquired images were analyzed using specialized software (vision64, bruker nano) to determine the average roughness (ra) values. five readings were recorded for each specimen, and the mean ra value was calculated to represent the surface roughness. the surface hardness of the specimens was evaluated using a vickers hardness tester (tukon 1102, wilson hardness, itw test & measurement, shanghai, china). a diamond pyramid indenter was applied to put a 300g load for 15 seconds at a right angle to the surface of the test piece. five indentations were created at different locations in each test piece with sufqicient spacing between them not to interfere with each other. the qive values' average was considered to obtain the qinal hardness reading for each test piece. scanning electron microscopy (sem) (fei, inspect s50) was used to study the topography and surface features of the specimens. the specimens were gold-coated in a dedicated coating unit (quorum, q150r es, uk) prior to sem observation to enhance the surface conductivity and image quality. sem imaging was performed at various magniqications ranging from 500× to 10,000× to observe the surface features, nanoparticles' distribution, and probable agglomeration patterns. several areas in each sample were examined to ensure comprehensive surface analysis and documentation of characteristic surface details. results the roughness of the pmma samples' surface was evaluated following the inclusion of various nanoparticles at varying concentrations. control samples that were not provided with any inclusion of nanoparticles were determined to possess a surface roughness of 0.113 ± 0.012 μm, and it was taken as the reference for comparison. when zro₂ nanoparticles were incorporated, no signiqicant changes in surface roughness were observed at concentrations of 0.5% and 1.0% (p>0.05). however, significant increases in surface roughness were detected at higher concentrations, with measurements of 0.125 ± 0.015 μm at 2.5% (p=0.028) and 0.138 ± 0.018 μm at 5.0% (p=0.008) being recorded as shown in table 2. in specimens modiqied with sio₂ nanoparticles, similar trends were observed. the roughness of surface was not signiqicantly affected at lower concentrations of 0.5% and 1.0%. signiqicant increases were noted at 2.5% concentration, where the roughness was measured at 0.127 ± 0.012 μm (p=0.023), and at 5.0% concentration, where it was measured at 0.135 ± 0.016 μm (p=0.014). the incorporation of diamond nanoparticles produced the most pronounced effects on surface roughness at higher concentrations. while no signiqicant changes were detected at 0.5% and 1.0% concentrations, substantial increases were measured at 2.5% (0.133 ± 0.014 μm, p=0.013) and 5.0% (0.146 ± 0.017 μm, p=0.002). the highest surface roughness among all tested specimens was recorded in the 5.0% diamond nanoparticle group. it was concluded that all three types of nanoparticles demonstrated a concentration-dependent effect on surface roughness, with signiqicant increases being observed at concentrations of 2.5% and above. lower concentrations (0.5% and 1.0%) were found to maintain surface roughness values comparable to the control specimens, regardless of the nanoparticle type used (figure 1). vickers hardness of pmma specimens was evaluated following the incorporation of different nanoparticles at varying concentrations. the control specimens, which contained no nanoparticle additions, were measured to have a baseline hardness value of 18.3 ± 0.9 vhn. for zro₂ nanoparticlemodiqied specimens, signiqicant improvements in hardness were observed at speciqic concentrations. while the 0.5% concentration showed a marginal increase (19.7 ± 0.7 vhn, p=0.051), signiqicant enhancements were recorded at 1.0% (21.4 ± 0.8 vhn, p=0.001) and 2.5% (20.9 ± 0.9 vhn, p=0.003) concentrations. however, at 5.0% concentration, the hardness was found to decrease to 19.4 ± 1.0 vhn (p=0.07), showing no signiqicant difference from the control as shown in table 3. in specimens modiqied with sio₂ nanoparticles, moderate improvements in hardness were detected. the 0.5% concentration showed no signiqicant change (19.0 ± 0.8 vhn, p=0.09), while signiqicant increases were measured at 1.0% (20.1 ± 0.9 vhn, p=0.02) and 2.5% (19.6 ± 0.8 vhn, p=0.032) concentrations. like zro₂, the hardness at 5.0% concentration (18.7 ± 0.9 vhn, p=0.42) was not signiqicantly different from the control. the addition of diamond nanoparticles was found to have minimal impact on the hardness of pmma specimens. no statistically signiqicant differences were observed across all concentrations tested (0.5% to 5.0%), with values ranging from 18.4 to 19.2 vhn (all p-values >0.05). it was concluded that zro₂ nanoparticles demonstrated the most effective enhancement of pmma hardness, particularly at 1.0% and 2.5% concentrations. sio₂ nanoparticles showed moderate improvement at similar concentrations, while diamond nanoparticles were found to have no signiqicant effect on hardness. higher concentrations (5.0%) of all nanoparticle types were observed to result in decreased hardness values compared to their optimal concentrations. the surface morphology and nanoparticle dispersion characteristics of pmma specimens were analyzed using scanning electron microscopy (sem). the control specimens were observed to exhibit a smooth, continuous matrix with a homogeneous surface structure and no inclusions, receiving the highest dispersion quality rating of 5. in zro₂ nanoparticle-modiqied specimens, excellent dispersion was observed at lower concentrations (0.5% and 1.0%), with uniform particle-matrix interfaces and optimal integration being noted. however, at 2.5% effect of nanoparncles on the mechanical propernes of polymethyl methacrylate denture base material vol 13, no 1 (2025) doi 10.5195/d3000.2025.1069 http://dentistry3000.pitt.edu 4 concentration, mild agglomeration and slightly rougher surfaces with clustered areas were detected. the highest concentration (5.0%) was characterized by visible agglomeration, uneven surfaces, and the presence of microvoids as shown in table 4. for sio₂ nanoparticle specimens, good dispersion quality was documented at 0.5% and 1.0% concentrations, with largely smooth surfaces and qine nanoparticle distribution being observed. as concentration increased to 2.5%, noticeable clusters were detected on the surface topography. at 5.0% concentration, irregular surfaces with numerous agglomerates were identiqied, leading to a disrupted matrix with cluster artifacts. the incorporation of diamond nanoparticles was found to result in varying surface characteristics across concentrations. lower concentrations (0.5% and 1.0%) showed smooth surfaces with slight undulation and rare cluster formation. however, signiqicant deterioration in surface quality was observed at higher concentrations, with 2.5% showing uneven surfaces and moderate clustering. the most severe effects were noted at 5.0% concentration, where discontinuous surfaces with high agglomeration, large clusters, numerous voids, and matrix gaps were observed. it was concluded that optimal nanoparticle dispersion was achieved at lower concentrations (0.5-1.0%) for all types of nanoparticles, with zro₂ showing the most favorable integration characteristics (figure 2). surface quality and dispersion were found to deteriorate signiqicantly at higher concentrations (2.5-5.0%), with diamond nanoparticles showing the most pronounced negative effects at elevated concentrations. a comparative analysis was conducted to evaluate the effects of different nanoparticles on the mechanical properties of pmma denture base material (table 5). the surface roughness, surface hardness, and dispersion quality were assessed, and signiqicant variations were observed across different nanoparticle groups. for surface roughness measurements, dnp-5.0 (5% diamond nanoparticles) was found to exhibit the highest value, while znp-1.0 (1% zirconia nanoparticles) demonstrated the lowest surface roughness. statistically signiqicant differences (p < 0.001) were observed between the control group and several modiqied groups, including znp-2.5, znp-5.0, snp-2.5, snp-5.0, dnp2.5, and dnp-5.0. in terms of surface hardness, the highest values were recorded in the znp-1.0 group (1% zirconia nanoparticles), while the lowest values were observed in both the control group and dnp-5.0 group. signiqicant improvements in hardness (p < 0.001) were noted in znp-1.0, znp-2.5, snp-1.0, and snp-2.5 groups compared to the control. the dispersion quality evaluation revealed that both the control group and znp-1.0 demonstrated the highest quality of particle dispersion, while dnp-5.0 showed the poorest dispersion characteristics. signiqicantly lower dispersion quality (p < 0.001) was observed in znp-2.5, znp-5.0, snp-5.0, dnp-2.5, and dnp-5.0 groups compared to the control. overall, these results indicated that the type and concentration of nanoparticles signiqicantly inqluenced the mechanical properties of pmma denture base material, with each nanoparticle type showing distinct effects on different parameters. the concentration-dependent effects of different nanoparticles on pmma denture base material were evaluated (table 6 and figure 3), revealing distinct patterns of inqluence on surface roughness and hardness properties. for zro₂ nanoparticles (znps), values comparable to the control were observed at 0.5% concentration. at 1.0% concentration, which was identiqied as optimal, a decrease in surface roughness and a signiqicant increase in hardness were documented. when the concentration was increased to 2.5%, an increase in surface roughness was noted along with maintained improved hardness. however, at 5.0% concentration, a substantial increase in surface roughness was observed while hardness values were found to decrease. in specimens modiqied with sio₂ nanoparticles (snps), control-like values were maintained at 0.5% concentration. the 1.0% concentration resulted in a slight decrease in surface roughness and improved hardness. at 2.5% concentration, increased surface roughness was noted while hardness improvements were maintained. the highest concentration of 5.0% led to a substantial increase in surface roughness, while hardness values returned to levels like the control. for diamond nanoparticles (dnps), the 0.5% concentration showed values like the control group. at 1.0% concentration, a slight decrease in surface roughness was observed while hardness remained unchanged. the 2.5% concentration resulted in increased surface roughness with no signiqicant change in hardness. at the highest concentration of 5.0%, a substantial increase in surface roughness was recorded along with a decrease in hardness values. it was concluded that the optimal concentration for all nanoparticle types was found to be 1.0%, with zro₂ nanoparticles showing the most favorable combination of properties. higher concentrations were generally associated with deteriorating mechanical properties, particularly at 5.0% concentration across all nanoparticle types. discussion incorporation of nanoparticles into polymethylmethacrylate (pmma) denture base material has yielded varied effects on its properties. incorporation of zirconium oxide (zro₂), silicon dioxide (sio₂), and diamond nanoparticles at concentrations of up to 1% had no signiqicant effect on surface roughness, whereas 2.5% and higher concentrations led to higher roughness [2]. similarly, titanium oxide nanoparticles made the surface roughness increase in comparison to zro₂ nanoparticles [11]. nanodiamond addition (0.1-0.5%) improved elastic modulus, qlexural strength, and surface hardness and conferred fungal resistance [4]. zro₂ nanoparticle addition improved hardness but decreased transverse strength of pmma, the water sorption and solubility rising with increasing concentration [3]. the above qindings suggest that the addition of nanoparticles can improve certain properties of pmma but that care should be taken in selecting the concentration and kind of nanoparticles to achieve the best result. the addition of nanoparticles to polyethyl methacrylate (pmma) denture base material has had various impacts on its mechanical performance. zro₂ nanoparticles were found to enhance the hardest by the highest amount at 1.0% and 2.5% concentrations [2]. also, the incorporation of zro₂, tio₂, and glass qlakes micronized enhanced fracture toughness and hardness of pmma [7]. there was signiqicant enhancement in impact strength, transverse strength, and radio-opacity with 5% concentration of nano-modiqied zro₂ in pmma [5]. however, higher nano-zro₂ concentrations (10% and 20%) decreased transverse strength but improved water sorption and solubility [3]. surface roughness was also improved with nanoparticle addition but within the acceptable clinical practice limits [2,3]. from the qindings, there are optimum amounts of nanoparticles that improve some mechanical characteristics of pmma denture base materials. this summary integrates results of four studies on nanoparticle-modiqied polymethylmethacrylate (pmma) denture base materials. nanoparticle incorporation generally enhanced mechanical properties with optimum results at low concentrations. zro₂ nanoparticles enhanced hardness but adversely affected surface roughness above 0.5% [2]. incorporation of nanodiamond (0.1-0.5 wt%) signiqicantly enhanced qlexural strength, elastic modulus, and surface hardness and prevented fungal adhesion (mangal et al., 2019). zro₂ nanoparticles improved hardness at 510% concentrations but decreased transverse strength and increased water sorption/solubility at higher concentrations [3]. silica nanoparticles improved effect of nanoparncles on the mechanical propernes of polymethyl methacrylate denture base material vol 13, no 1 (2025) doi 10.5195/d3000.2025.1069 http://dentistry3000.pitt.edu 5 microhardness and fracture toughness at as low as 0.023% concentration, while higher concentrations led to agglomeration and lesser effect [1]. these tests altogether suggest that nanoparticles can be incorporated to improve pmma but its optimal concentration is also required to cause desired improvement without impacting other properties. the addition of different nanoparticles to pmma denture base materials showed contrasting effects on mechanical properties, wherein the optimal concentration was the limiting factor in the desired outcome. it was observed that the addition of 1.0% concentration of zirconia nanoparticles (zro2) showed the optimal balance of properties, with enhanced hardness and still clinically acceptable values of surface roughness. however, higher concentrations of all three tested nanoparticles (zro2, sio2, and dnps) tended to impair mechanical properties at particularly 5.0% concentration [2]. this is in line with other studies whereby high nanoparticle loading has led to aggregation and loss of material properties [4]. the test for surface roughness also indicated that loadings over 0.5% had signiqicantly increased ra values compared to the clinical maximum of 0.2 μm [3]. interestingly, although the diamond nanoparticles exhibited mechanical improvement properties, they also tended to agglomerate at higher concentrations, thus presenting higher surface roughness and lower hardness values [12]. the qindings illustrate the need to preserve the optimal concentration of the nanoparticles, around 1.0%, to achieve better mechanical properties while not compromising the clinical performance of the material. conclusion the incorporation of nanoparticles into pmma denture base material exhibited signiqicant concentration-dependent alterations on mechanical and surface properties. the study revealed that lower concentration levels (0.5-1.0%) of all the tested nanoparticles-retained surface properties comparable to those of control specimens, while 2.5% or greater led to signiqicant alterations in material properties. this trend was always observed for all kinds of tested nanoparticles, although to different degrees of effect. the best modiqier was found to be the zro₂ nanoparticles, particularly at 1.0% concentration, where these maintained the optimal compromise between enhanced hardness (21.4 ± 0.8 vhn) and retained smoothness of the surface. the excellent performance of zro₂ was also complemented by excellent particle dispersion and uniform integration with the pmma matrix at this concentration. sio₂ nanoparticles exhibited mild improvements in mechanical properties, whereas diamond nanoparticles, to the contrary, exhibited mild beneqits with a high drawback at higher concentration levels. surface morphology analysis revealed a threshold concentration of nanoparticles, beyond which material properties began to deteriorate. the 5.0% concentration always resulted in adverse effects in all forms of nanoparticles, with characteristics of higher surface roughness, lower hardness, and poor particle dispersion. this was particularly true for diamond nanoparticle samples, which exhibited the highest surface roughness (0.146 ± 0.017 μm) and poorest agglomeration at this concentration. these qindings strongly suggest 1.0% concentration as the optimal level of nanoparticle addition to pmma denture base materials, with zro₂ nanoparticles providing the most favorable balance of enhanced mechanical properties and maintained surface qualities. the results also emphasize the necessity of strict concentration control of nanoparticle-modiqied dental materials, as concentrations higher than the optimum can lead to property degradation instead of improvement. systematic exploration of surface morphology, hardness and roughness across different types and concentrations of nanoparticles provides valuable information for the development of improved dental materials. this work demonstrates that it is possible to improve material properties by selective pmma modiqication with some nanoparticles at precisely controlled concentrations while maintaining important surface properties, potentially leading to improved clinical performance of dental prostheses. there are many recommendations for research: 1) maintain 1.0% levels of nanoparticles for optimal performance as higher concentration levels provide poor properties. this is very crucial in the case of zro₂ nanoparticles, whose performance was optimal when concentration was at this level. 2) utilize zro₂ nanoparticles instead of sio₂ and diamond nanoparticles for pmma modiqication, as they exhibited better mechanical properties and integration with the matrix. implement strong quality checks during the incorporation of nanoparticles to determine uniform dispersion and prevent agglomeration to maintain the best possible surface properties as well as mechanical properties. references 1. baloš, s., pilic b., markovic d., pavlicevic j., and luzanin o. “poly(methyl-methacrylate) nanocomposites with low silica addition.” *the journal of prosthetic dentistry*, vol. 111, no. 4, 2014, pp. 327–34. https://doi.org/10.1016/j.prosdent.2013.06.021. 2. gad, m. m., al-thobity, a. m., rahoma, a., arrejaie, a. s., and al-harbi, f. a. “inqluence of addition of different nanoparticles on the surface properties of poly(methylmethacrylate) denture base material.” *journal of prosthodontics*, vol. 29, no. 5, 2020, pp. 422–28. https://doi.org/10.1111/jopr.13168. 3. ergun, g., sahin, z., and ataol, a. s. “the effects of adding various ratios of zirconium oxide nanoparticles to poly(methyl methacrylate) on physical and mechanical properties.” *journal of oral science,* vol. 60, no. 2, 2018, pp. 304–15. https://doi.org/10.2334/josnusd.17-0206. 4. mangal, u., kim, j., and yoo, g. “novel poly(methyl methacrylate) containing nanodiamond to improve the mechanical properties and fungal resistance.” *materials,* vol. 12, no. 20, 2019, article no. 3438. https://doi.org/10.3390/ma12203438. 5. moudhaffar, m., and ns. i. n. s. “evaluation of the effect of modiqied nano-fillers addition on some properties of heat cured acrylic denture base material.” *journal of baghdad college of dentistry,* vol. 23, no. 1, 2011, pp. 23–29. 6. kaurani, a., tomar, p., and kaurani, m. “effect of addition of titanium oxide and zirconium oxide nanoparticles on the surface roughness of heat cured denture base resins: an in -vitro study.” manuscript, 2021. 7. nejatian, t., johnson, a., and van noort, r. “reinforcement of denture base resin.” *advances in science and technology,* vol. 49, 2006, pp. 124–130. https://doi.org/10.4028/www.scientibic.net/ast.49.124. 8. salman, a. d., jani, g. h., and fatalla, a. a. “comparative study of the effect of incorporating sio₂ nano-particles on properties of poly methyl methacrylate denture bases.” *biomedical & pharmacology journal,* vol. 10, no. 3, 2017, pp. 1499–1506. https://doi.org/10.13005/bpj/1262. 9. rashahmadi, s., mosalman, s., and hasanzadeh, r. “improving the mechanical properties of poly methyl methacrylate nanocomposites for dentistry applications reinforced with different nanoparticles.” *polymer-plastics technology and engineering,* vol. 56, no. 16, 2017, pp. 1730–40. https://doi.org/10.1080/03602559.2017.12894 02. effect of nanoparncles on the mechanical propernes of polymethyl methacrylate denture base material vol 13, no 1 (2025) doi 10.5195/d3000.2025.1069 http://dentistry3000.pitt.edu 6 10. naguib, ghada h., et al. “inqluence of inorganic nanoparticles on dental materials’ mechanical properties. a narrative review.” *bmc oral health,* vol. 23, 2023, article no. 3652. https://doi.org/10.1186/s12903-02303652-1. 11. kaurani, a., tomar, p., and kaurani, m. “effect of addition of titanium oxide and zirconium oxide nanoparticles on the surface roughness of heat cured denture base resins: an in vitro study.” manuscript, 2021. 12. zidan, s., silikas, n., alhotan, a., haider, j., and yates, j. “investigating the mechanical properties of zro₂-impregnated pmma nanocomposite for denture-based applications.” *materials,* vol. 12, no. 8, 2019, article no. 1344. https://doi.org/10.3390/ma12081344. table 1. experimental grouping and specimen distribution. group no. of specimens (n) nanoparticle type concentration (% wt) no. of specimens (n) key notes control 10 none 0% 10 pure pmma baseline znp-0.5 10 zro2 0.5% 10 znp, silanized znp-1.0 10 zro2 1.0% 10 snp, silanized znp-2.5 10 zro2 2.5% 10 znp-5.0 10 zro2 5.0% 10 snp-0.5 10 sio2 0.5% 10 snp-1.0 10 sio2 1.0% 10 snp-2.5 10 sio2 2.5% 10 snp-5.0 10 sio2 5.0% 10 dnp-0.5 10 diamond 0.5% 10 dnp, surface-modiqied dnp-1.0 10 diamond 1.0% 10 dnp-2.5 10 diamond 2.5% 10 dnp-5.0 10 diamond 5.0% 10 total 130 130 x2 for testing/duplicate sets table 2. surface roughness (ra, μm) of pmma specimens with various nanoparticle additions. group nanoparticle type concentration (% wt) surface roughness mean (ra, μm) ± sd signiqicance vs control (pvalue) signiqicant difference (yes/no) control none 0 0.113 ± 0.012 na na znp-0.5 zro₂ 0.5 0.112 ± 0.01 0.87 no znp-1.0 zro₂ 1.0 0.108 ± 0.011 0.59 no znp-2.5 zro₂ 2.5 0.125 ± 0.015 0.028 yes znp-5.0 zro₂ 5.0 0.138 ± 0.018 0.008 yes snp-0.5 sio₂ 0.5 0.115 ± 0.011 0.94 no snp-1.0 sio₂ 1.0 0.114 ± 0.009 0.88 no snp-2.5 sio₂ 2.5 0.127 ± 0.012 0.023 yes snp-5.0 sio₂ 5.0 0.135 ± 0.016 0.014 yes dnp-0.5 diamond 0.5 0.114 ± 0.010 0.91 no dnp-1.0 diamond 1.0 0.116 ± 0.011 0.80 no dnp-2.5 diamond 2.5 0.133 ± 0.014 0.013 yes dnp-5.0 diamond 5.0 0.146 ± 0.017 0.002 yes effect of nanoparncles on the mechanical propernes of polymethyl methacrylate denture base material vol 13, no 1 (2025) doi 10.5195/d3000.2025.1069 http://dentistry3000.pitt.edu 7 figure 1. surface roughness (ra) evaluation of pmma specimens reinforced with various nanoparticles and concentrations. table 3. vickers hardness values of pmma specimens with different nanoparticle additions. group nanoparticle type concentration (% wt) vickers hardness mean ± sd signiqicance vs control (pvalue) signiqicant difference (yes/no) control none 0 18.3 ± 0.9 na na znp-0.5 zro₂ 0.5 19.7 ± 0.7 0.051 no znp-1.0 zro₂ 1.0 21.4 ± 0.8 0.001 yes znp-2.5 zro₂ 2.5 20.9 ± 0.9 0.003 yes znp-5.0 zro₂ 5.0 19.4 ± 1.0 0.07 no snp-0.5 sio₂ 0.5 19.0 ± 0.8 0.09 no snp-1.0 sio₂ 1.0 20.1 ± 0.9 0.02 yes snp-2.5 sio₂ 2.5 19.6 ± 0.8 0.032 yes snp-5.0 sio₂ 5.0 18.7 ± 0.9 0.42 no dnp-0.5 diamond 0.5 18.7 ± 0.7 0.38 no dnp-1.0 diamond 1.0 19.2 ± 0.8 0.19 no dnp-2.5 diamond 2.5 18.8 ± 0.9 0.32 no dnp-5.0 diamond 5.0 18.4 ± 1.0 0.75 no effect of nanoparncles on the mechanical propernes of polymethyl methacrylate denture base material vol 13, no 1 (2025) doi 10.5195/d3000.2025.1069 http://dentistry3000.pitt.edu 8 table 4. sem qualitative assessment of surface morphology and nanoparticle dispersion. group dispersion quality (1–5) surface morphology description agglomeration observed representative features control 5 smooth, continuous matrix no homogeneous surface, no inclusions znp-0.5 5 uniform, minor roughness no even particle-matrix interface znp-1.0 5 homogeneous, optimal integration no nanoparticles integrated with matrix znp-2.5 3 slightly rougher, some clustered areas mild limited zones of nanoparticle clusters znp-5.0 2 visible agglomeration, uneven surface yes agglomeration, microvoids noted snp-0.5 4 largely smooth, isolated minor roughness no good dispersion, minor inclusions snp-1.0 4 smooth, qine nanoparticle distribution no uniform distribution snp-2.5 3 noticeable clusters on topography mild clustering at certain regions snp-5.0 2 irregular, numerous agglomerates yes disrupted matrix with cluster artifacts dnp-0.5 4 smooth, slight undulation rare some isolated nano-clusters dnp-1.0 4 moderately smooth, qine particulate rare dispersed, but some cluster formation dnp-2.5 2 uneven, moderate numbers of clusters moderate more frequent cluster sites dnp-5.0 1 discontinuous, high agglomeration high large clusters, many voids, matrix gaps figure 2. dispersion quality assessment of nanoparticles in pmma specimens at varying concentrations. table 5. comparative analysis of surface properties and dispersion quality of pmma denture base material modiqied with zro₂, sio₂, and diamond nanoparticles. parameter highest value group lowest value group groups with statistically signiqicant difference (p < 0.05) vs control overall anova result surface roughness dnp-5.0 znp-1.0 znp-2.5, znp-5.0, snp-2.5, snp-5.0, dnp-2.5, dnp-5.0 p < 0.001 surface hardness znp-1.0 control/dnp-5.0 znp-1.0, znp-2.5, snp-1.0, snp-2.5 p < 0.001 dispersion quality control/znp-1.0 dnp-5.0 znp-2.5, znp-5.0, snp-5.0, dnp-2.5, dnp-5.0 (all signiqicantly lower) p < 0.001 effect of nanoparncles on the mechanical propernes of polymethyl methacrylate denture base material vol 13, no 1 (2025) doi 10.5195/d3000.2025.1069 http://dentistry3000.pitt.edu 9 table 6. summary of concentration-dependent effects of different nanoparticles on surface roughness (ra) and hardness values of pmma denture base material. nanoparticle 0.5% 1.0% (optimal) 2.5% 5.0% znps (zro₂) ~control values ↓ra, ↑↑ hardness ↑ra, ↑ hardness ↑↑ra, hardness↓ snps (sio₂) ~control values slight ↓ra, ↑hn ↑ra, ↑ hardness ↑↑ra, ~hn dnps (diamond) ~control values slight ↓ra, ~hn ↑ra, ~hn ↑↑ra, hn↓ figure 3. sem images of pmma specimens with (3a) zirconium dioxide 0.5, (3b) zirconium dioxide (2.5), (3c) silicon dioxide 0.5, (3d) pmma + silicon dioxide 5%, (3e) pmma + diamond 0.5%, (3f) pmma + diamond 2.5%, (3g) pure pmma, and 3h comparative reference group (for visual benchmarking). 1067 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1067 http://dentistry3000.pitt.edu 3d printed co-cr alloy surface treatments effect on shear strength of heat cured resins luay abdullah khalaf, luma mudhafar al-nema college of den*stry, university of mosul, mosul, iraq abstract objec7ve: to investigate the effect of different surface treatments—hydrochloric acid etching, sandblasting, metal primer application, and a combination of sandblasting with metal primer on the shear bond strength (sbs) of heat-cured polymethyl methacrylate (pmma) bonded to 3d-printed co-cr alloy, and to assess the failure modes. materials and methods: fifty disk-shaped 3d-printed co-cr specimens were divided into give groups (n=10 each): control (c), acid etch (a), sandblast (s), metal primer (p), and sandblast + primer (sp). specimens were bonded to heat-cured pmma resin and tested for shear bond strength using a universal testing machine. failure modes were examined microscopically. data were analyzed using one-way anova and duncan’s multiple range test. results: signigicant differences in sbs were observed among groups (p ≤ 0.05). the sp group showed the highest sbs (15.78 ± 1.19 mpa), followed by p (12.77 ± 2.61 mpa) and s (5.80 ± 1.01 mpa). the a (0.43 ± 0.08 mpa) and c (0.37 ± 0.07 mpa) groups exhibited the lowest sbs values. failure mode analysis revealed adhesive failures in c, a, and s groups, while p and sp showed predominantly mixed failures. sandblasting increased surface roughness, while primers enhanced chemical bonding without altering topography. conclusions: the combination of sandblasting and metal primer achieved the highest bond strength, congirming a synergistic effect between micromechanical and chemical bonding. acid etching alone was ineffective. combined treatments are recommended to improve the clinical performance of 3d-printed co-cr rpd frameworks. open access cita%on: khalaf la, et al. (2025) 3d printed co-cr alloy surface treatments effect on shear of heat cured resins. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1067 received: october 8, 2025 accepted: october 15, 2025 published: november 10, 2025 copyright: ©2025 khalaf la, et al. this is an open access ar%cle licensed under a crea%ve commons avribu%on work 4.0 united states license. email: luma2005@uomosul.edu.iq introduc)on a removable pareal denture (rpd) is an accepted device for treaeng paeents with pareal tooth loss. they help replace missing teeth and improve funceonality. rpd usually consists of a base that supports the areficial teeth, along with a metal framework that provides structural integrity and stability. this combinaeon ensures proper fit and funceon, enhancing the wearer's ability to chew and speak effecevely. cobalt-chromium (co-cr) alloy is owen chosen for the framework due to its favorable properees, which include cost-effeceveness, elevated mechanical strength, excellent resistance to corrosion, and suitability for manufacturing processes [1,2]. polymethyl methacrylate (pmma) is commonly uelized as a denture base material for rpd due to essue compaebility, aestheec qualiees, and favorable material characterisecs [3,4]. addieve manufacturing (am) technology, often referred to as three-dimensional (3d) prineng technology, has increasingly been uelized to produce various dental prostheses [5,6]. with addieve am, we can create the metal framework using materials that are surprisingly like those tradieonally used, like co-cr powder. this innovaeve approach opens up excieng possibiliees for enhancing design and funceonality [6,7]. any flaws or separaeons occurring between the acrylic resin and the metal framework, especially along the finishing line, may lead to cracks or crazing in the acrylic resin. as a result, a weak bond can have a direct effect on the junceon between the metal and the resin [4]. tradieonally, metal frameworks are secured to denture base resins using mechanical reteneon techniques, which can involve loops, meshes, beads, nail heads, and undercut finish lines. shear strength between the acrylic resin base and metal differs depending on the type of minor connector (labcework or mesh) [8]. the applicaeon of tradieonal mechanical reteneon methods for bonding metal to resin has 3d printed co-cr alloy surface treatments effect on shear strength of heat cured resins vol 13, no 1 (2025) doi 10.5195/d3000.2025.1067 http://dentistry3000.pitt.edu 2 been extensively invesegated, yet it fails to eliminate microleakage [9]. deficiencies in the chemical bond between metal and acrylic resin can lead to significant clinical issues, including microleakage of oral fluids. such complicaeons may be miegated through the establishment of a robust chemical bond [10]. the chemical bonding of the denture base resin to the metal is preferred over mechanical reteneon in construceng rpd. metal primers present a straigheorward approach. they are a more economical choice relaeve to tradieonal methods. special equipment is not necessary, and the applicaeon process is not parecularly sensieve to technique [10,11]. this study was designed to evaluate the effect of various surface treatments on shear bond strength of heat polymerized acrylic resin bonded to 3d printed co-cr alloy. the null hypothesis was that the shear bond strength between the co-cr alloy and 3d printed co-cr alloy would not differ, regardless of the types of various surface treatments. materials and methods study design in this study, 50 specimens were disk-shaped and used for the shear bond strength test. the specimens were divided into five groups based on the surface treatment performed on the specimens, each group had 10 specimens and as follows: group c: this is the control group in which no surface treatment was done on the bonding surface of the specimens. group a: the bonding surface of alloy samples in this group was treated with hydrochloric acid (10%). group s: the bonding surface of alloy samples in this group was sandblasted with110 μm aluminum oxide parecles. group p: the bonding surface of alloy samples in this group was treated with metal primer. group sp: the bonding surface of alloy samples was sandblasted with 110 μm aluminum oxide parecles, and then metal primer was applied. specimen design sketchup (trimble, usa) version 2024 was used to design the geometrical shape of the specimens. the specimens prepared for the assessment of shear bond strength were fabricated as disk-shaped specimens with a 10 mm diameter and 2 mm height [11-13]. all the designs were exported in an stl format. specimen prepara7on the specimens were fabricated by a seleceve laser meleng (slm) machine (d-150, riton, china), first, the stl files of the specimens' design were imported into the slm sowware system and muleplied to the required number of specimens. all specimens were distributed virtually in sowware within the boundaries of the stainless-steel plate to be printed. the samples were arranged on the plaeorm so that their 3-mm wide sides were aligned with the z-axis, and the slm was performed in a nitrogen environment with a laser energy of 10 kilowals and an energy density of 1.964 j/mm2. the laser power (p) determined the energy density of 165w. the building direceon was 90 degrees, the speed of the scan was 1050 (mm/s), and the scan line spacing was s. (mm). the parecle size of the co-cr powder alloy (riton, china) was between 12 and 65 microns, and the layer thickness was fixed to 25 microns [14]. the co-cr powder was applied to the stainlesssteel plate. the machine started meleng the powder layer by layer unel the samples were fully constructed according to the manufacturer's guidelines. when specimens’ fabricaeon is finished, removing the support is easily done through the band saw (dly-1bf1, riton, china). finishing of the specimens was carried out with diamond grinding stones, then all the specimens were finished by using the abrasive wheel to remove any irregulariees from the margin of the specimen [15,16]. for gebng a good polished specimens surfaces a grinder polisher device (mp-160e,goyojo, hong kong) was used, the abrasive silicon carbide paper number 600 was used under running water for 10 seconds and the grinder speed was set to 300 rpm to provide a flat and uniform surface [11,12,17-19]. applica7on of heat-cured acrylic resin to facilitate the fabricaeon of the acrylic wax palern, a special split mold was made from stainless steel by a cnc machine to produce a wax palern, a disk shape with 5 mm in diameter and 2 mm in thickness. the mold was fabricated to accommodate the co-cr disk with a dimension of 10mm in diameter and 2mm in thickness. above the disk specimen, the mold would be narrowed in diameter up to 5 mm and extended 2 mm. this created a space of 5mm in diameter and 2mm in thickness in the center of the disk specimens, as displayed in figure 1, where modeling wax (nk, china) was applied [11,12]. the final shape of specimen awer applicaeon of molten wax is shown in figure 2. these specimens (co/cr alloy disk modeling wax assemblies) were flasked in a standard flasking technique for acrylic dentures with dental stone (singletypo3,lascod, italy) and then the specimens were dewaxed and cleaned using boiled water [4]. before the packing procedure, the specimens were divided into five groups, each with ten specimens, and then labelled according to the surface treatment they received. for control group, the co-cr specimens without surface treatment. for acid etch group, apply the hydrochloric acid 10% (chemlab hydrochloric acid, analyechem, belgium) over the bonding surface of the co-cr specimens. awer 30 minutes, the specimens were rinsed with deionized water for 30 seconds [20]. for air abrasive group, the specimens were abraded with aluminum oxide 110µm (cobra abrasive, renfert, germany) using a sandblasting machine (sandblaster t1mestra, spain), at 4 bar pressure, and carried out for 14 seconds. the distance between the nozzle ep and the surface of the specimens was maintained at 2 cm, and held perpendicular to the ep [11]. for metal primer group, 23 drops of the metal primer (metal primer z, gc; japan) were dispensed into a clean dispensing dish, and then, using a brush, a thin layer was applied to the bonding surface and allowed to dry for 5 seconds. immediately awer that, the heatcure acrylic was applied to the treated surface [11]. for specimens , to be treated with both sandblaseng and metal primer, sandblaseng was followed by applicaeon of metal primer [11]. awer the surface treatment of samples, the heat cured acrylic resin(sr triplex hot, ivoclar, liechtenstein) mixed with powder: liquid raeo of 3:1 and packed by placing the flask in a hydraulic press (flask press, quayle dental, uk), with an applied pressure of 2000 psi, and then clamped for curing [21]. the clamped flasks were then placed in a water bath; the water was heated gradually from room temperature for about 45 minutes to a boiling temperature of 100сo and maintained at this temperature for 30 minutes according to the manufacturer's instruceons. the flask was allowed to bench cool slowly at room temperature [15]. the specimens were then deflasked and cleaned by using an ultrasonic cleaner(electronic scale) for 20 minutes, and then stored in diselled water(al-joud, iraq) for 24 hours according to ada specificaeon number 12 [22]. shear bond strength test the specimens inserted in the center of auto polymerizing acrylic resin base (dentway selfcure, dentway, turkey) by using a special silicon mold. to obtain the bond strength. the specimens were mounted in the universal testing machine (hsa-ut, dongguan hongjin test instruments (dhti), china) with the help of the specimen holder in a way that the treated specimen's surface was parallel to the loading piston, shown in figure 3. the loading piston had a chisel configuraeon, which was applied with a crosshead speed of 3d printed co-cr alloy surface treatments effect on shear strength of heat cured resins vol 13, no 1 (2025) doi 10.5195/d3000.2025.1067 http://dentistry3000.pitt.edu 3 0.5mm/min [11].the maximum load required at failure was recorded, and the shear bond strength was calculated by dividing the load by the surface area using the following equaeon: sbs (mpa) = f (n)/ s (mm2). sbs = shear bond strength (mpa), f = the load at failure (n), and s = surface area (mm2). the load is the value at which the specimen deboned, and the surface area is the bonding area of the circular specimen that was 5 mm in diameter. the surface area was calculated according to the equaeon (s)=πr2, where (r) represents the radius of the bonding area [23]. following the shear bond strength test, the types of failures were observed under an opecal microscope (hi-scope; hirox co. ltd.) at 10 x magnificaeon. the mode of failure was categorized as adhesive failure if it took place at the interface between the resin and the metal, or cohesive failure if the separaeon occurred within either the metal or the resin. in instances where both types of failure were noted, the failure mode was classified as mixed-type failures [21,24,25]. sta7s7cal analysis the shapiro-wilk normality test was first done. mean, standard deviaeon, analysis of variance and duncan's muleple rang test were carried out as staesecal analysis for data using a staesecal sowware program (spss version 25.0; ibm corp, usa). the level of significance was set to α=0.05. results all groups saesfied the normality assumpeon (all p > 0.05), and therefore one-way anova was performed. table 1 shows descripeve staesecs, including the mean values, standard deviaeon, standard error, and extreme values (minimum and maximum). the unit of measurement used is mpa. staesecally, the lowest mean values were found in the control group (0.369 ± 0.074 mpa) and then the acid group (0.433 ± 0.081 mpa). the sandblast group had a significantly higher mean value of (5.797 ± 1.011 mpa), while the primer group had a staesecally higher mean value of (12.769 ± 2.614 mpa). the combinaeon group of sandblast and metal primer had the highest mean value (15.783 ± 1.193 mpa) (table 1). one-way anova test was performed to determine if there was a significant difference between any of the studied groups. one-way anova had a significance value of 0.000, so there was a significant difference between at least two groups, as shown in table 2. duncan’s muleple range test was performed, which showed that there was no significant difference between only the control and the acid groups. however, there was a highly significant difference between all the other groups, as shown in figure 4. duncan’s muleple range test was performed, which showed that there was no significant difference between only the control and the acid groups. however, there was a highly significant difference between all the other groups, as shown in figure 4. failure mode evalua7on the control (c), acid (a), and sandblast (s) groups all exhibited 100% adhesive failures, with no instances of cohesive or mixed failure observed. the metal primer (p) group showed a clear shiw in failure behavior: 60% of the samples exhibited mixed failures, while only 40% were adhesive. in the combinaeon group sandblast and metal primer (sp), failure mode analysis revealed 70% mixed failures and only 30% adhesive failures, as showed in table 3 and figure 5. discussion the findings of this study reveal significant differences in mean shear bond strength between 3d printed co-cr alloy and heat-cured acrylic resin when subjected to various surface treatments, such sandblast (s), metal primer (p), and sandblast +metal primer (sp), compared to the control group. however, there was no significant difference observed with the acid etch treatment in relaeon to the control.so, the null hypothesis was pareally rejected. the control group (c) and acid-etched group (a) exhibited the lowest shear bond strength values (0.369 ± 0.074 mpa and 0.433 ± 0.081 mpa, respecevely), as showed in table 1, indicaeng poor adhesion at the interface. these results suggest that acid etch or no surface treatment is insufficient for achieving clinically acceptable bond strength [26]. the sandblast group (s) showed a substaneal increase in the shear bond strength (5.797 ± 1.011 mpa, as showed in table 1, refleceng the posieve effect of mechanical surface roughening. sandblaseng enhances micromechanical reteneon, which is well-documented as a mean to improve the bond between dental alloys and resin materials [25,27]. however, this group achieved significantly lower sbs values than the primer groups. the primer group (p) and the combinaeon sandblast +metal primer group (sp) achieved the highest shear bond strength values (12.769 ± 2.614 mpa and 15.783 ± 1.193 mpa, respecevely) as showed in table 1. the sandblast + metal primer (sp) group demonstrated staesecally superior performance compared to all other groups. the synergisec effect of mechanical and chemical surface treatments is supported by literature, which showed that combining surface roughening with a suitable primer maximizes both micromechanical and chemical adhesion, resuleng in a more durable and reliable bond [25,27]. the acid-etch surface group had lower shear bond strength values than the other surface treatments. this is maybe due to the presence of oxide film, which plays a vital role in the corrosion resistance of the co-cr material. surface oxide film forms spontaneously on alloy surfaces awer ambient oxygen exposure, aceng as a barrier to electron flow (resistor) between the electrolyte and the alloy surface, thus proteceng it against corrosion [28]. the oxide layer of the 3d-printed cr-co alloys has greater thickness and density than the cast alloys [29]. furthermore, it is known that a harder material is capable of maintaining a thicker oxide layer more firmly as compared with a sower material [30], and it was found that the slm-formed cocr alloy has higher hardness than the cast alloy [14]. the result of this study agrees with previous work [31,32] that concluded that the use of hcl did not improve etanium–ceramic bonding strength values. for aluminum oxide surface treatment group, the present study revealed that sandblaseng significantly enhanced the shear bond strength (sbs) between the co-cr alloy and acrylic resin compared to the untreated control and acidetched groups. this finding aligns with several previous studies that confirmed the positive correlation between surface roughness and bond strength in metal-resin systems [20,33]. these findings are consistent with previous reports [20,34,35], which concluded that the al2o3 abrasive significantly affects the quality of the co-cr alloy connection with the acrylic resin. for metal primer surface treatment group, the findings of the current study revealed that the applicaeon of metal primer had a staesecally significant posieve effect on the shear bond strength (sbs) between the co-cr alloy and acrylic resin. these results can be attributed to the chemical interaction facilitated by the functional monomers in the metal primer, most notably 10methacryloyloxydecyl dihydrogen phosphate (10-mdp). this monomer formed strong chemical bonds with the oxide layer of co-cr alloys, promoting adhesive interaction with the methacrylate groups in the resin matrix. this dual affinity enables the formation of a durable and stable hybrid layer at the alloy–resin interface, which significantly enhances sbs [36-40]. these findings are consistent with previous reports [13,20,41], which concluded that the metal primer significantly improved shear bond strength between acrylic resin and different metal alloys. 3d printed co-cr alloy surface treatments effect on shear strength of heat cured resins vol 13, no 1 (2025) doi 10.5195/d3000.2025.1067 http://dentistry3000.pitt.edu 4 combinaeon sandblast and metal primer surface treatment group (sp), the current study revealed that the combinaeon treatment, resulted in the highest shear bond strength (15.783 ± 1.193 mpa) among all the experimental groups, as showd in table 1. this remarkable increase in bond strength can be attributed to the synergisec effect of mechanical and chemical surface modificaeons [16]. these findings are consistent with previous reports [11,20,42], which concluded that the use of metal primers along with sandblaseng significantly improved the bonding of acrylic denture base resin with the co-cr alloy. however, this study disagrees with ghazwan and nabeel [43], who concluded that using metal primer combined with air abrasion improved the shear bond strength, but less than that obtained from specimens treated with metal primer only. this may be due to differences between the two studies, they used valpast (flexible) denture base material and a different metal primer than that used in this study. limitaeons of this study included the in vitro design and the dimensions of the test sample used did not represent the actual clinical condieons. the difference in the geometry may affect the stress distribueon and hence the shear bond strength and a single brand of metal primer was evaluated and there are addieonal surface treatments different from those used in this study that were not tested, like fiber laser and silica. conclusion within the limitaeons of this study, the following conclusions were drawn: 1. the combinaeon of sandblaseng and metal primer applicaeon demonstrated the highest shear bond strength, indicaeng a synergisec effect between micromechanical and chemical bonding. in contrast, untreated (control) and acid-etched groups showed low bond strength, emphasizing the limited impact of hcl alone. 2. all samples including: control, sandblasted, and acid-treated groups exhibited 100% adhesive failures, indicating weak bonding, while the primer and sp groups displayed (60%-70%) mixed failures, confirming superior interfacial strength when chemical bonding is involved. acknowledgements the faculty and staff of the university of mosul, college of denestry have been tremendously helpful throughout this process, and we are very grateful to them. ethical approval before stareng this study, documented approval was obtained from the research ethics commilee (rec) at the college of denestry, university of mosul, iraq, with a license numbered (uom.dent/25/1011). financial support and sponsorship none. conflict of interest none. data availbility data are available upon reasonable request to the authors. references 1. alexandrino, l.d., et al., mechanical and surface proper*es of co–cr alloy produced by addi*ve manufacturing for removable par*al denture frameworks. the journal of prosthe9c den9stry, 2023. 130(5): p. 780-785. 2. wataha, j.c., alloys for prosthodon*c restora*ons. the journal of prosthe9c den9stry, 2002. 87(4): p. 351-363. 3. alageel, o., et al., bonding metals to poly (methyl methacrylate) using aryldiazonium salts. dental materials, 2015. 31(2): p. 105-114. 4. al-harbi, f.a. and m.a. saber, bond strength of poly (methyl methacrylate) denture base to cast titanium and cobalt-chromium frameworks of different designs. life science journal-acta zhengzhou university overseas edi9on, 2012. 9(1): p. 610-6. 5. rosso, s., et al., an op*miza*on workflow in design for addi*ve manufacturing. applied sciences, 2021. 11(6): p. 2572. 6. yoon, s.-n., et al., tissue surface adapta*on of cad-cam maxillary and mandibular complete denture bases manufactured by digital light processing: a clinical study. the journal of prosthe9c den9stry, 2020. 124(6): p. 682-689. 7. chen, h., et al., adapta*on of removable par*al denture frameworks fabricated by selec*ve laser mel*ng. the journal of prosthe9c den9stry, 2019. 122(3): p. 316-324. 8. al-ali, a.a., shear strength of acrylic resin base to nickel chromium minor connector of removable par*al denture. al-rafidain dental journal, 2010. 10(2). 9. sanohkan, s., et al., shear bond strength between autopolymerizing acrylic resin and co-cr alloy using different primers. dental materials journal, 2012. 31(5): p. 765-771. 10. kawaguchi, t., et al., effect of surface prepara*on on the bond strength of heatpolymerized denture base resin to commercially pure *tanium and cobalt-chromium alloy. dental materials journal, 2011. 30(2): p. 143-150. 11. kalra, s., v. kharsan, and n.m. kalra, compara*ve evalua*on of effect of metal primer and sandblas*ng on the shear bond strength between heat cured acrylic denture base resin and cobalt-chromium alloy: an: in vitro: study. contemporary clinical den9stry, 2015. 6(3): p. 386391. 12. hasson, e.a., m.a.r. jaber, and a.e. abdulmaged, evalua*on the effect of diazonium salt and aluminum oxide on the shear bond strength of heat cure acrylic resin at co/cr and titanium alloys interface. tikrit journal for dental sciences, 2021. 9(1): p. 34-42. 13. dewan, h., h. chohan, and a.k. gujjari, the effect of metal primer on the shear bond strength of denture base resin to base metal alloy auer electroly*c etching and laser etching: an in vitro study. journal of interna9onal oral health, 2015. 7(12): p. 110. 14. ghadhban, a.h. and i.h. hasan, transverse strength and microstructure of cobaltchromium alloy produced by selec*ve laser mel*ng and cas*ng techniques (an in vitro study). j. res. med. dent. sci, 2023. 11: p. 176-182. 15. farhan, f.a. and s.s. al-ameer, effect of different metal surface treatments on microleakage of the two types of acrylic resin: heat cure and light cure at co/cr interface. journal of baghdad college of den9stry, 2009: p. 31. 16. yun, j.y., et al., effect of sandblas*ng and various metal primers on the shear bond strength of resin cement to y-tzp ceramic. dental materials, 2010. 26(7): p. 650-8. 17. ohkubo, c., et al., shear bond strengths of polymethyl methacrylate to cast *tanium and cobalt-chromium frameworks using five metal primers. the journal of prosthe9c den9stry, 2000. 83(1): p. 50-57. 18. denizoglu, s., c.s. hanyaloglu, and b. aksakal, tensile bond strength of composite lu*ng cements to metal alloys auer various surface treatments. indian journal of dental research, 2009. 20(2): p. 174-179. 19. kim, g., et al., an in vitro evalua*on of bond strength and failure behavior between 3dprinted cobalt-chromium alloy and different types of denture base resins. j dent, 2024. 147: p. 105119. 20. sadoon, m., effect of surface treatment on shear bond strength of acrylic resin to cobaltchromium alloy. al-rafidain dental journal, 2014. 14(2): p. 252-258. 21. bulbul, m. and b. kesim, the effect of primers on shear bond strength of acrylic resins to different types of metals. the journal of prosthe9c den9stry, 2010. 103(5): p. 303-308. 22. jassim, n.a. and m.a.-r. jaber, evalua*on of the effect of different surface treatments on bond strength of heat cured acrylic resin at co-cr alloy and peek polymer interface. interna9onal journal of medical research & health sciences, 2019. 8(1): p. 71-83. 23. duran, i̇., et al., effect of er-yag laser applica*on on shear bond strength of polymethyl methacrylate to cr-co alloy. selcuk dental journal, 2016(3): p. 87-91. 24. kim, j.-y., p. pfeiffer, and w. niedermeier, effect of laboratory procedures and thermocycling on the shear bond strength of resin-metal bonding systems. the journal of prosthe9c den9stry, 2003. 90(2): p. 184-189. 25. kim , m., et al., evalua*on of shear bond strengths of 3d printed materials for permanent restora*ons with different surface treatments. polymers, 2024. 16(13): p. 1838. 26. el mourad, a.m., assessment of bonding effec*veness of adhesive materials to tooth structure using bond strength test methods: a review of literature. the open den9stry journal, 2018. 12: p. 664. 27. tanaka, a., et al., shear bond strength of ultraviolet-polymerized resin to 3d-printed denture 3d printed co-cr alloy surface treatments effect on shear strength of heat cured resins vol 13, no 1 (2025) doi 10.5195/d3000.2025.1067 http://dentistry3000.pitt.edu 5 materials: effects of post-polymeriza*on, surface treatments, and thermocycling. journal of prosthodon9c research, 2024. 69(1): p. 21-29. 28. tuna, s.h., n. özçiçek pekmez, and i. kürkçüoğlu, corrosion resistance assessment of cocr alloy frameworks fabricated by cad/cam milling, laser sintering, and cas*ng methods. the journal of prosthe9c den9stry, 2015. 114(5): p. 725-34. 29. presolo, a.g.c., et al., feasibility of 3d printed co–cr alloy for dental prostheses applica*ons. journal of alloys and compounds, 2021. 862: p. 158171. 30. cvijović-alagić, i., et al., wear and corrosion behaviour of ti–13nb–13zr and ti–6al–4v alloys in simulated physiological solu*on. corrosion science, 2011. 53(2): p. 796-808. 31. troia jr, m.g., et al., the effect of surface modifica*ons on *tanium to enable *tanium– porcelain bonding. dental materials, 2008. 24(1): p. 28-33. 32. lee, b.-a., et al., effect of surface treatment on bond strength of ti-10ta-10nb to lowfusing porcelain. the journal of prosthe9c den9stry, 2013. 109(2): p. 95-105. 33. kurtz, k., et al., bond strength of prosthe*c composite to primed and silicoated cast *tanium. interna9onal chinese journal of den9stry, 2005. 5: p. 12-6. 34. mina, r.k., effect of surface treatment on resin bonding to 3d printed co-cr dental alloy. egyp9an dental journal, 2023. 69(4): p. 2881-2888. 35. wosińska, o., e. wołowiec-korecka, and l. klimek, influence of the al 2 o 3 abrasive to abrasive blas*ng on the quality of the bond between the co-cr-mo alloy and the lowtemperature polymerisable acrylic. archives of materials science & engineering, 2024. 129(2). 36. yanagida, h., et al., adhesive bonding of composite material to cast *tanium with varying surface prepara*ons. journal of oral rehabilita9on, 2002. 29(2): p. 121-126. 37. koizuka, m., et al., the effect of different surface treatments on the bond strength of a gingiva-colored indirect composite veneering material to three implant framework materials. clinical oral implants research, 2013. 24(9): p. 977984. 38. yilmaz, a., m.ş. akyil, and b. hologlu, the effect of metal primer applica*on and nd: yag laser irradia*on on the shear-bond strength between polymethyl methacrylate and cobalt-chromium alloy. photomedicine and laser surgery, 2011. 29(1): p. 39-45. 39. shimizu, h., et al., use of metal condi*oners to improve bond strengths of autopolymerizing denture base resin to cast ti–6al– 7nb and co–cr. journal of den9stry, 2006. 34(2): p. 117-122. 40. korkmaz, f.m. and s. aycan, effect of fiber laser irradia*on on the shear bond strength between acrylic resin and *tanium. scanning, 2019. 2019(1): p. 5452919. 41. nakhaei, m., et al., bonding the titanium afachment matrix housing to acrylic overdentures using different primers, adhesives, and resin materials: an in vitro study. interna9onal journal of den9stry, 2025. 2025(1): p. 9702318. 42. pereira, a.l.c., et al., which metal surface treatment improves the bond strength between metal alloys and acrylic resin in removable par*al dentures? a systema*c review. the journal of prosthe9c den9stry, 2025. 134(1): p. 114-121. 43. ghazwan, a. and a. nabeel, effect of different metal surface treatments and thermocycling on shear bond strength and microleakage of the flexible acrylic at co/cr interface. journal of baghdad college of den9stry, 2011. 23: p. 10-4. . table 1. descripeve staesecs of the shear bond strength test for the studied groups. groups n mean (mpa) standard deviaeon minimum maximum c 10 0.369 0.07415 0.27 0.46 a 10 0.433 0.08111 0.32 0.59 s 10 5.797 1.01101 4.43 7.43 p 10 12.769 2.61366 10.13 17.13 sp 10 15.783 1.19342 13.72 17.36 n: total number of measurements. c: control; a: acid; s: sandblast; p: metal primer; sp: sandblast + metal primer. table 2. one-way anova test for the shear bond strength test. sum of squares degrees of freedom mean square f p-value between groups 1989.607 4 497.402 267.718 0.000 within groups 83.607 45 1.858 total 2073.214 49 3d printed co-cr alloy surface treatments effect on shear strength of heat cured resins vol 13, no 1 (2025) doi 10.5195/d3000.2025.1067 http://dentistry3000.pitt.edu 6 table 3. distribueon of failure modes between metal and denture base resin bonding following the shear bond strength test. group adhesive cohesive mixed c 10 0 0 a 10 0 0 s 10 0 0 p 4 0 6 s p 3 0 7 c: control; a: acid; s: sandblast; p: metal primer; sp: sandblast + metal primer. figure 1. diagram of the metal mold for wax applicaeon [11]. 3d printed co-cr alloy surface treatments effect on shear strength of heat cured resins vol 13, no 1 (2025) doi 10.5195/d3000.2025.1067 http://dentistry3000.pitt.edu 7 figure 2. final shear bond strength specimen shape awer molten wax applicaeon. figure 3. specimens mounted in the universal teseng machine. 3d printed co-cr alloy surface treatments effect on shear strength of heat cured resins vol 13, no 1 (2025) doi 10.5195/d3000.2025.1067 http://dentistry3000.pitt.edu 8 figure 4. duncan’s muleple range test for the shear bond strength test for the studied groups. figure 5. types of failures: (a) adhesive failure. (b) mixed-type failure. 1001 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1001 http://dentistry3000.pitt.edu shear bond strength and sem/edx of two bioactive bulk fill resin base composite restorations rana fahad ahmed al azawee, nadia modhafer ahmed al shakir college of den*stry, university of mosul, iraq abstract objec;ve: to compare the shear bond strength of two bioackve bulk-fill dental restorakve materials to sound denkn aper aging in arkficial saliva and to evaluate the mode of failure and elemental analysis by sem/edx. material and methods: forty maxillary premolars extracted due to orthodonkc reasons from subjects with ages between 14 and 30 years were used in this study. the teeth were divided into two groups according to the type of composite, predicta bioackve or cenkon forte. each group had samples stored or not stored in arkficial saliva. occlusal denkn of all teeth was exposed by cu\ng 2mm below the deepest point on the occlusal surface of each tooth's crown. each restorakve material was applied according to the corresponding manufacture instruckons. the shear bond strength was measured between the composite and denkn and by using sem-edx to evaluate the mode of failure and element analysis. results: the study revealed that cenkon forte, aper storage, exhibited the highest shear bond strength (10.13 mpa), followed by its non-stored samples (8.38 mpa), while predicta bioackve showed the lowest values, especially in the non-stored samples (6.38 mpa). conclusion: cenkon forte had stronger bonding than predicta bioackve. open access cita%on: al azawee rfa, et al. (2025) shear bond strength and sem/edx of two bioac%ve bulk fill resin base composite restora%ons. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1001 received: july 27, 2025 accepted: august 4, 2025 published: september 9, 2025 copyright: ©2025 al azawee rfa, et al. this is an open access ar%cle licensed under a crea%ve commons avribu%on work 4.0 united states license. email: nadia.mudhafar@uomosul.edu.iq introduc)on composite restorations are widely used in direct esthetic dentistry due to their ability to mimic various shades and translucencies, providing high-quality aesthetic outcomes. additionally, the incremental placement of conventional composite materials can introduce voids and interfacial bonding failures, leading the remaining tooth structure and restoration interface to greater stress [1,2]. a main affecting factor for maintaining a restoration chemically bonded to the tooth structure is its ability to withstand shear stresses (that can be debined as the stresses created at the interface between restoration and tooth surface) which are caused by perpendicular or parallel forces acting on the tooth surface [3]. the oral environment is dynamic and complex, exposing restorative materials to variations in ph, temperature, microbial activity, and mechanical stress, dental tissues are in constant ionic exchange with bluoride, calcium, and phosphate, regulated by saliva, which plays a crucial role in maintaining a balanced mineral environment [4]. with this understanding, the focus of restorative dentistry has gradually shifted from biocompatibility to bioactivity, emphasizing materials that can interact with the oral environment to promote remineralization and inhibit bacterial growth [5]. bioactive resin composites are designed to discharging remineralizing ions, elevating ph levels, and generating hydroxyapatite, thereby enhancing the longevity and effectiveness of restorations [6]. several bioactive composites have been developed with properties aimed to combating caries while maintaining their stability under occlusal forces, temperature changes, and enzymatic degradation [7,8]. among these materials and for direct restorations, predicta bioactive which is a bulk-bill resin composite was designed. according to the manufacturer, it demonstrates high compressive, tensile, and blexural strengths while also releasing bluoride, calcium, and phosphate ions to facilitate the formation and mineral apatite remineralization at the tooth-restoration interface [9]. similarly, cention forte (ivoclar vivadent) belongs to the alkasite material family and is characterized by its high blexural strength and esthetic properties. it contains an alkaline biller that releases hydroxide ions to shear bond strength and sem/edx of two bioackve bulk fill resin base composite restorakons vol 13, no 1 (2025) doi 10.5195/d3000.2025.1001 http://dentistry3000.pitt.edu 2 regulate ph during acid attacks, preventing demineralization and promoting remineralization through the release of calcium and bluoride ions. additionally, it exhibits moderate viscosity and strong mechanical properties [10]. the aim of this study was to evaluate and compare shear bond strength of two bioactive bulk-bill dental restorative materials to sound dentin after aging in artibicial saliva, and to evaluate their mode of failure and element analysis by sem/edx. material and methods an ethical approval was received from the “research ethics committee” of the college of dentistry, university of mosul (no. uom. dent.25/1013) because the research study used extracted human teeth. forty maxillary premolar teeth extracted for orthodontic purposes from patients aged 14 to 30 years were collected for this study from different health center in mosul, each tooth was examined using a stereomicroscope at10x magnibication. only teeth that were free of caries, cracks, attrition, abrasion, or restorations were included. the teeth cleaned to remove debris using an ultrasonic scaler, then were immersed in a 0.1% thymol solution (dbh, england) for 48 hours to ensure disinfection and subsequently stored in deionized water (almansur factory, iraq) until the time of the study, for the next mounting step the teeth samples were prepared [11-13]. for mounting the teeth a retentive tube polyvinyl chloride (pvc), 2cm in diameter and 2cm in length, was used as a mold, each tooth was positioned with the aid of a dental surveyor and a sticky wax (hoppegarten, germany) was used to attach the tooth to the rod of the surveyor, the selected teeth with their roots were embedded at the tube center and parallel to its long axis to a level of 2 mm below the cement-enamel junction simulate the position of the tooth in the alveolar bone [14,15]. forty teeth were divided randomly into two groups according to the type of composite restorative materials (20 teeth for each group). then, each group was subdivided into two subgroups. the birst 10 teeth were not stored in artibicial saliva, while the second 10 teeth were stored for 30 days in artibicial saliva. all teeth were tested for shear bond strength using a universal testing machine (hongjin has-ut-5pc, china) and for mode of failure and element analysis using sem/edx (axia chemisem, holland). the occlusal dentin of each tooth was exposed by cutting 2 mm below the deepest point on the occlusal surface of each tooth's crown. this cut was made using a diamond coated separating disc attached to a slowspeed hand piece with water coolant. perpendicular to the long axis of the tooth the cutting was made. occlusal dentin surface of all specimens was polished using 600-grit wet silicon carbide abrasive papers for ten time in a circular motion under running tap water to obtain blat dentin bonding surface [12,16]. for predicta bioactive (parkell, usa) bulk bill resin composite, an etch and rinse protocol was used following the manufacturer’s recommendations, where the dentin surface was exposed, 37% phosphoric acid gel (spident, korea) was used for 15 seconds and then rinsed with water for 15 seconds. the teeth were dried with oil-free air for 5 seconds [17]. according to the manufacturer’s instructions, one layer of g-premio bond (universal bond quick, gc, japan) was rubbed onto the dentin surface for 20 seconds with a microbrush, the bond remained on the dentin surface for 10 seconds and was dried at the maximum airblow rate for 5 seconds. finally, the adhesive layer was light cured at a light intensity of 1000 mw/cm2 for 10 seconds using light emitting diode light curing unit (rogin dental, china) at a 1mm distance [18]. for a standardized distance we used celluloid crown which cut 1mm from cervical margin so the distance between occlusal surface of celluloid crown and occlusal surface of coronal dentin is 1mm. a custom-made teblon mold was designed to standardize bulk bill composite application on bonded dentin to produce a 4mm diameter and 2mm height [16]. each restorative material was applied according to the corresponding manufacture instructions utilizing the bulk placement technique. for the cention forte (ivoclar vivadent, schaan, liechtenstein), the primer was dispensed as one drop in a dish and mixed with the applicator for 5 seconds, applying on dentin for 10 s, blowing by air pressure for 5 seconds (selfcure) [19]. cention forte capsules were activated by pressing the plunger on a blat surface to allow mixing of the powder and liquid. immediately after activation, the capsule was inserted in the amalgamator (capsule mixer) (softly8, italy) and mixed for 17 seconds at room temperature (21°c) (low temperature led to delay setting of material). after mixing, immediately the capsule was placed into the applicator, clicked 3 clicks and dispensed to bill the mold, keeping the tip immersed in the material to prevent the formation of air bubbles [20]. the predicta bulk bioactive (parkell, usa) composite was delivered on bonded dentin by a spiral nozzle to bill the mold and slowly express the composite as we withdraw the tip, keeping the tip immersed in the material to eliminate air entrapment. a celluloid strip was placed over the restoration to achieve a smooth, blat surface to prevent oxygen inhibition surface layer, then the material was light-cured using a lightemitting diode (led) curing unit with an intensity of 1000 mw/cm² (rogin dental, china) in a one step for 20 seconds. the tip of light-curing was positioned perpendicularly to the occlusal surface of the restorative material, maintaining a standardized distance of 1 mm (equal to the thickness of a metal plate from a custom-made teblon mold). additional curing was performed for 20 seconds on each side of the restorative material to optimize polymerization [21,22]. each group were subdivided into two subgroups. the birst subgroup was not stored in artibicial saliva. all samples were stored in plastic container containing 30 ml of deionized water in an incubator (binder, germany) at 37± 2°c in 95% humidity for 48 hours until subjected to shear bond strength test [13,23]. the second subgroup was stored in artibicial saliva. samples were placed upright inside a plastic container. each container was then billed with 30 ml of artibicial saliva (ph 7) and securely covered. the containers were incubated at 37 ± 2 °c and 95 % humidity for 30 days, and they were replenished every week until the endpoint time (30 days) [24,25]. an universal testing machine (hongjin hasut-5pc, china) was used to measure the shear bond strength between the composite and dentin. all samples were loaded at a speed of 0.5 mm per minute until de-bonding occurred with a maximum failure load that was recorded automatically in newton (n) by a computer connected to the testing machine [16]. to calculate shear bond strength in mpa, the maximum force was divided by the surface area of the sample. shear bond strength = (𝐅) fracture load (n)/ (𝐀) surface area (mm2) the surface area (a) was calculated from the following equation: a= π r 2 where π = 3.14 r = radius of each specimen (2mm), a=3.14*2 2 =12.56mm2. to identify the mode of failure, all samples (de-bonded dentin surfaces) following shear bond strength test were examined using a stereromicroscope at 40x magnibication [27]. also, scanning electron microscopy (sem) of three de-bonded surface samples were done to determine the micromorphological topography at 5000x and the mode of failure at 1300x [26]. the mode of failure was categorized as adhesive (failure at the adhesive-substrate interface), cohesive (within the material mass), or shear bond strength and sem/edx of two bioackve bulk fill resin base composite restorakons vol 13, no 1 (2025) doi 10.5195/d3000.2025.1001 http://dentistry3000.pitt.edu 3 mixed (a combination of adhesive and cohesive) [23]. all corresponding samples (de-bonded surfaces and fractured composite stubs) were collected after shear bond strength testing to prepared for sem/edx (axia chemisem, holland), the teeth samples from each subgroup (non-storage and storage in artibicial saliva for 30 days) were subjected to longitudinal sectioning (sectioning at the middle of mesio-distal direction) to the surface of acrylic base and then by using slow speed hand piece with cutting diamond disc each half sectioned horizontally below cementenamel junction near the acrylic base, then one of the obtained sections from each tooth sample was randomly selected and cleaned in an ultrasonic water bath (granbo, china) for 3 min, in order to remove the debris and left to dry for 24 hours [28]. the corresponding samples were secured to the aluminum stubs using carbon double-sided tape. after that, a thin gold coating (15nm) was spattered on the surface for 20 seconds to determine the mode of failure and micromorphological topography. the sem system was adjusted to a 30 kv accelerating voltage for this purpose [29]. the weight percentages of chemical elements in the de-bonded dentin and restorative material core for each tooth have been identibied by analyzing the chemical composition using edx data. in addition, the calcium-to-phosphorus (ca/p) ratio was determined for both the storage and nonstorage groups [30]. the statistical analysis included the following tests: “two-way analysis of variance” (anova) was analyzed the results for shear bond strength and “duncan's multiple range” to identify the signibicance among groups at p≤0.05; independent sample t-test was used to compare different groups to assess the effect of storage in artibicial saliva on the shear bond strength. statistical signibicance was set at p<0.05; and chi-square test to compare different groups to assess the effect of storage in artibicial saliva on the mode of failure. results shapiro wilks test showed that the data were normally distributed (p<0.05). in most of the groups, the results of the descriptive statistics that included the minimum, maximum, mean, and standard deviation value of shear bond strength of all groups of the study as shown in table 1. the highest shear bond strength was observed for cention forte, whereas the lowest shear bond strength was observed for predicta bioactive. mode of failure de-bonded tooth samples were inspected using a steromicroscope at 40x magnibication as shown in figure 1. the most common failure pattern was mixed except for cention forte stored in artibicial saliva, which had a cohesive failure. sem\edx analysis sem imaging was performed at 5000x magnibication to examine the de-bonded areas of all restorative material core and the coronal dentin samples stored or not in artibicial saliva. the de-bonded samples were analyzed to assess morphological changes at these critical regions (figures 2 and 3). sem for predicta bioactive showed a slightly precipitation of hydroxyapatite (ha) at the de-bonded area of composite for both groups (figure 4). sem for cention forte showed precipitation of hydroxyapatite (ha) at the de-bonded area of composite for both the samples not stored in artibicial saliva (b1) and stored (b2) (figure 5). sem images showed the presence of "crystallike" deposits at de-bonded coronal dentin areas in the predicta bioactive samples in both stored and not stored in artibicial saliva (figure 6). sem images of cention forte samples, both not stored and stored in artibicial saliva showed that de-bonded coronal dentin areas had “crystal-like” depositions (figure 7). edx analysis edx analysis was conducted to evaluate the elemental composition of the de-bonded areas of all restorative materials core and the coronal dentin samples. this analysis aimed to detect compositional changes associated with aging in artibicial saliva. the weight percentage of calcium and phosphorus elements were used to calculate the calcium/phosphorus ratios as illustrated in the following equation: the edx spectra showed low concentration of phosphorous (p) for samples not stored in artibicial saliva (figure 8). the edx spectra showed low concentration of phosphorous (p) centrion forte samples (figure 9). the edx spectra of predicta bioactive for both not stored and stored in artibicial saliva showed a change in the spectra for both phosphorous (p) and calcium (ca). predicta bioactive samples showed increase in the spectra of calcium (ca) with slightly decrease in the spectra of phosphorous (p) (figure 10). the edx spectra for cention forte both samples showed a change in the spectra for both phosphorous (p) and calcium (ca). cention forte samples stored in artibicial saliva showed an increase in the spectra of calcium (ca) that was higher than predicta bioactive with slightly decrease in the spectra of phosphorous (p) (figure 11). the mean ca/p ratios was (1.84±0.71) in predicta bioactive samples stored in artibicial saliva, which is higher than the ratio of natural ha (1.67). this indicated apatite deposition, which conbirms the sem analysis that showed apatite deposition at the restoration surface. the mean ca/p ratio for cention forte samples stored in artibicial saliva was (2.63±0.62), which was higher than the ratio for natural ha (1.67). this indicated apatite deposition, which would support the sem analysis that showed apatite deposition at the restoration surface. for predicta bioactive samples not stored in artibicial saliva exhibited a mean ca/p ratio of (1.81±0.45), signibicantly above the natural hydroxyapatite (ha) ratio of dentin (1.67), suggesting a composition like native minerals. in samples stored in artibicial saliva, the ca/p ratio at the de-bonded area of the coronal dentin samples increased to (2.71 ± 0.38). the rising ratio, signibicantly above that of natural dentin hydroxyapatite, indicates the development and deposition of a hydroxyapatite-like crystals. the edx elements for samples not stored in artibicial saliva of cention forte detected the mean ca/p ratio (1.97±0.38) slightly higher than natural ha ratio (1.67) for dentin. while after storage in artibicial saliva, the coronal dentin area of cention forte showed ca/p ratio about (4.01±1.98), which is higher than that for natural ha ratio for dentin. this indicates ha precipitation. discussion alkasite-based tooth-colored restorative material is a hybrid that releases calcium, bluoride, and hydroxyl ions, which exhibit effective anti-cariogenic properties. this novel material combines the advantages of glass ionomer cements (gics) and resin-based composites. dual-cure capability allows bulk placement with or without adhesive [31]. shear bond strength test was selected to evaluate bonding strength of the two different restorative materials when applied to blat (mid-coronal) dentin. the clinical importance of this test because of it closely simulates the shearing forces found at the tooth– restoration interface [32]. the aging procedure is essential in determining the bond durability, one of the factors which affect the properties of restorative material is saliva which is a slightly acidic body bluid having a ph scale of 6–7 and whose main ingredient is water (99%). shear bond strength and sem/edx of two bioackve bulk fill resin base composite restorakons vol 13, no 1 (2025) doi 10.5195/d3000.2025.1001 http://dentistry3000.pitt.edu 4 artibicial saliva was used to simulate the wet oral environment. the teeth were stored in artibicial for a period as the restoration remains in continuous contact with saliva in the oral cavity [33]. in the current study, teeth were stored in artibicial saliva in an incubator at (37 ℃ ± 1) for 30days at ph 7. this was performed to mimic the oral cavity’s environment for assessing the behavior of restorative materials [34]. in this vitro study, bonding effectiveness and bond durability after artibicial aging (30 days in artibicial saliva) were investigated through evaluate 1) shear bond strength of these tested material according to storage condition 2) micromorphological analysis of these materials at debonded dentin area by sem identify the main failure modes 3)elementary analysis of both core of materials and debonded dentin area by sem/edx analysis. the null hypothesis was rejected since there were signibicant differences of two bioactive bulk-bill dental restorative materials in values of bonding to sound dentin and failure patterns after aging in artibicial saliva in the current study, cention forte showed the greatest value of shear bond strength. the highest mean in the cention forte with storage group (10.13 mpa), followed by cention forte without storage (8.38 mpa). these values were signibicantly higher compared to predicta bioactive, indicating that cention forte exhibited superior bonding performance, particularly after being stored in artibicial saliva. this could be due to the strong mechanical properties of cention forte which is due to its chemical composition as its monomer matrix consists of a mixture of urethane dimethacrylates (udma), tricyclodecan-dimethanol dimethacrylate (dcp), tetramethyl-xylylendiurethane dimethacrylate (aromatic aliphatic-udma) and polyethylene glycol 400 dimethacrylate (peg-400 dma), which interconnects (cross-links) during the process of polymerization leading to stronger mechanical properties. this agrees with bassiouny et.al. (2024) [23], who valuate the shear bond strength (sbs) of cention forte and tetric n-ceram bulk fill composite methacrylate-modibied polyacrylic acid present in the primer offering dual adhesion mechanisms (mechanical and chemical) by forming micro-mechanical interlocking provided by the surface roughness, most likely combined with chemical interaction through its acrylic / itaconic acid copolymers. polyacrylic acid can remove the smear layer and leave the smear plug, producing a partial demineralization of the dentin, leaving hydroxyapatite around the collagen bibers, allowing the chemical interaction of the carboxylic groups with dentin hydroxyapatite [35]. these results agree with previous work [36], who determine and compare blexural strength and microhardness of cention n with other materials at a distinctive period in artibicial saliva. the current study also agrees with previous work [37,38] that compared the bonding efbicacy of three bioactive self-adhesive restorative systems to dentin after storage in artibicial saliva revealed that using of primers prior to application of alkasite-based restorative material is highly recommended and show high microshear bond strength values for both immediate and storage groups of cention forte with primer among other tested groups. the contrary, predict bioactive showed a lower sbs (with p value less, than or equal 0.05) which could be attributed to its unique monomer composition , a novel monomer (poly-2-hema), which has been advocated to reduce the risk of potentially toxic effects of bisgma-based compounds that cause increased solubility[39] by promotes the formation of an unstable aqueous gel that is susceptible to hydrolytic degradation ,hema has a negative interaction with 10-mdp in the adhesive agent (g-premio bond) used in this study, which signibicantly reduces the demineralization of hydroxyapatite. this decreases the formation of mdp ca salts and partially inhibits the deposition of the nanolayers, which are necessary to obtain an adequate chemical interaction with the dentin substrate [40]. this may be explained the low viscosity of predicta bulk bioactive composite (as the predicta bioactive type used in the current study is low viscosity type as claimed by manufacture). the morphology of the biller particles and the amount of biller loading that improved the mechanical and physical properties of the resin-based composites. reduced biller content of bulk-bill composites results in an increase in the polymerization shrinkage and shrinkage stresses to potentially debond the material from dentin during polymerization [41,43].this come in agreement with the study conducted by hegde et al., 2023) [44] , who compare microtensile bond strength of high-viscosity bulk-bill composites and low-viscosity bulkbill composites revealed that the high-viscosity bulk-bill composites exhibited the highest microtensile bond strength in comparison to other resin-based composites, nanocomposites, and bulk-bill blowable composites. although aging in artibicial saliva did not yield a statistically signibicant overall effect. cention forte demonstrated the highest sbs after storage, predicta bioactive also exhibited improved sbs following storage. the use of ion-releasing materials in restorative dentistry may contribute to the reduced activity of proteases such as metalloproteinases (mmps) and cathepsins involved in collagen degradation. such enzymes are considered one of the main causes for reduction of bonding longevity when simplibied bonding systems are applied in dentine with self-etching or etch-and-rinse protocols [45], thus reducing the enzymatic degradation at the bonding interface. it may be also possible that in the case of diffusion of calcium and phosphate ions through permeable hybrid layers, these may precipitate and crystallize in complex calcium-phosphates and inhibit mmps through the formation of a ca-po/mmp complex [46]. cention forte storage group had the mean value (10.13±2.11) in which the increase was statistically signibicant in comparison to the non-storage group of the same material (8.38 ± 1.12) after storage, predicta bioactive showed some improvement, but not nearly as much as cention forte, according to the results. for predicta bioactive, its capacity to release ions that promote bond stability implies it can aid in remineralisation and improve the connection. this material demonstrates great potential as a bioactive bulk-bill composite, which can release f, ca, and p ions, for long-term restoration treatments. this bulkbill resin-based composite is simple to apply, cures in two stages, and has optical properties [47]. presence of "hema" in predicta bioactive, a hydrophilic monomer with enhanced solubility which may explain its ability to release more ions and enhance its bioactivity[48] also downsizing bioactive glass particles to nano-size improves the alkalizing and hydroxyapatite-forming based on the information provided by the manufacturer and validated by the edx study, one of the unique compositions of predicta bioactive as manufacture claimed is titanium dioxide (tio2 ) tio2 nanobller incorporation has no effect on the shear bond strength of the blowable composites[49] however ,may enhance the bioactivity and hydroxyapatite (ha) formation of this material. this in agreement with (witkowska et al., 2024) [50], who indicates the good bioactivity of titanium oxide layers. under simulated biological conditions at ph 7.4 found that tio2 surface shows predominantly negative properties, creating an electrostatic environment that attracts oppositely charged ions, such as calcium. subsequently, the positively charged ca2+ undergoes a reaction with negatively charged po43− and co32−, leading to the formation of a surface layer containing ca–p. over time, this layer may crystallize into hydroxyapatite [51,52]. the current study found that the storage group of cention forte exhibited the highest shear bond strength and sem/edx of two bioackve bulk fill resin base composite restorakons vol 13, no 1 (2025) doi 10.5195/d3000.2025.1001 http://dentistry3000.pitt.edu 5 shear bond strength, having a low incidence of mixed failure and a higher incidence of cohesive failure. on the other hand, the nonstorage group of predicta bioactive revealed the lowest shear bond strength, associated with a greater rate of adhesive failure. these bindings align with sabatini’s work, [53] which demonstrated a link between the highest bond strength and mixed failure, while the lowest strength corresponded to adhesive failure. evidence from several studies supports the hypothesis that cohesive failure in dentin is related to high bond strength [54] perhaps because of the strong bonding to dentin developed by universal adhesives [55]. scanning electron microscopy (sem) is often used for the morphological analysis of adhesive-dentin interfaces. many studies have investigated the relationship between bonding performance and interfacial properties [56], (sem) analysis for cention forte showed the interface was completely sealed as an acid-resistant, resin-dentin interdiffusion zone with resin tags extending into the dentinal tubules. close the dentinal tubules in storage group while partially cover dentinal tubules in non-storage group, indicating that the primer effectively demineralized the dentin surface and facilitated the diffusion of resin into exposed collagen bibrils. both cention forte and predicta bioactive demonstrated the ion releasing property, which supported the formation of a hybrid layer and improved bond strength after storage in artibicial saliva. this binding supported by (sem) analysis that show mixed failure in storage group of predicta bioactive. the combined scanning electron microscopy (sem) and energy-dispersive x-ray (edx) analytical technique was utilized to assess both the morphology and mineral content of dentin surfaces. this technique allowed for qualitative evaluation of surface morphology and quantitative assessment of mineral changes resulting from experimental interventions. the preservation of the crystalline structure and the measured ca/p ratio were used to determine the extent of remineralization, reblecting the effectiveness of the applied treatments. sem, with magnibication ranging from 50x to over 10,000x, is a valuable tool in dental research, especially when paired with edx, which detects elemental composition and can indicate the presence of hydroxyapatite through calcium phosphate analysis [57,58]. the debinition of "bioactive" will vary based on the application. in restorative dentistry, the capacity of materials to release ions like as calcium, phosphorus, and bluoride, hence facilitating remineralisation and the formation of hydroxyapatite crystals upon contact with physiological bluids, is referred to as bioactivity [59,60]. an increase in the ca/p ratio is a critical indicator of remineralisation, which allows for the assessment of a material's suitability for use on demineralised dental tissue. the typical ca/p ratio in natural dentition is 1.67, when the ca/p ratio is less than 1.67, it can be classibied as a nonstoichiometric crystal that is debicient in calcium [61,62]. the study found that under controlled storage conditions, sem/edx revealed differences in both the restorative materials surface and the area of the coronal dentin-restoration interface on tooth structure in cention forte and predicta bioactive indicated their bioactivity by increase the ca/p ratio of restorative materials surface for predicta bioactive and cention forte (1.21±0.20 to 1.84±0.71, 1.31±0.12to2.63±0.63, respectively) and also for coronal dentin-restoration interface area (1.75±0.35 to 2.71±0.34, 1.97±0.38 to 4.01±1.98, respectively) after storage in artibicial saliva at ph =7 in 37 c temperature for 30 days. this binding agreement with (di lauro et al., 2023) [63], in which this study evaluated the effect of ph and temperature on the ion release of a resin-based material containing alkaline billers and a self-setting high-viscous glass ionomer cement the highest amount detected at ph = 6.8 was at 37 °c after 28 days which is comparable to the condition of our study. in neutral and alkaline solutions, ca and po4 ions can be precipitated as apatite [64,65]. in this study sem images supported these bindings which showed apatite-like crystal in both the restorative materials surface and the area of the coronal dentin-restoration interface on tooth structure in cention forte and predicta bioactive. the results of the present study showed no signibicant different in ca/p ratio in both the restorative materials surface and the area of the coronal dentin-restoration interface on tooth structure in cention forte and predicta bioactive in non-storage and storage groups. in non-storage groups of this study cention forte and predicta bioactive showed no signibicant different in ca/p ratio and less than storage groups .for cention forte this can be due to the billers in which three inorganic glasses: barium alumino-silicate glass, calcium barium alumino-bluoro-silicate and a basic calcium bluoro-silicate glass referred to as an “alkasite”bille [66] that lead to the formation of a superbicial layer of calcium bluoride and calcium phosphate with 0.5 mm thickness on the surface of cention forte at the initiation of setting reactions, which resists dissolution with deionized water for some time[67] . previous work [68] showed that the phosphate ion release from cention n bioactive materials signibicantly increased as the storage time increased 24 h to 4 h, and 6 months in distilled water. cention forte primer and predicta bioactive contain hema, which is hydrophilic monomer, high hydrophilicity promotes increased water acceptance that results in the hydrolytic degradation [69], with increased their solubility, that explain their ability to release more ions and enhance their bioactivity. in this study, the storage media was artibicial saliva with ph=7 at 37°c temperature for 30 days that is preferable environment for cention®forte to release ca, f, and p ions, which results in the formation of apatite on its surface, like previous results [7,70]. in the current study, even there is no signibicant different in ca/p ratio in both cention forte and predicta bioactive in non-storage and storage groups. however, in storage groups, the ca/p ratio of cention forte is higher in both the restorative materials surface and the area of the coronal dentin-restoration interface on tooth structure (2.63±0.63), (4.01±1.98) respectively than predicta bioactive (1.84±0.71, 2.71±0.34, respectively). this can be attributed to the fact that it contains alkaline billers with a strong afbinity for water [71] .when alkaline billers come in contact with the saliva three salts are connected (na2o, cao, caf2) in sio2 are dissolved and released ca, f and oh ions depend on the ph, forming apatite in vitro on dentine at ph 7 if phosphate available combine with a specibic primer [72]. clinical trials are essential to assess the durability of these bioactive restorative materials, their performance in patients, and the mechanisms by which these hybrid materials release ions and interact with decayed dentin. cention forte appears suitable for durable restorations, however predicta bioactive may still be advantageous in cases where bioactivity and remineralisation properties are required. conclusion this study showed that employing ion-releasing restorative material, specibically alkasite-based primers before applying it, is highly suggested because this method seems to be the best way to get a stronger binding with dentin. as a result, this study's binding that applying primer can improve bonding to dentin goes against the manufacturer's initial advice and classibication of this type of restoration as self-adhesive. compared to predicta bioactive, cention forte had a stronger connection and was better at generating apatite. higher ca/p ratios and sem images suggest that storing in artibicial saliva made mineral deposition at the contact better. these results support the idea that cention forte can interact with living things, especially when it is moist and old. shear bond strength and sem/edx of two bioackve bulk fill resin base composite restorakons vol 13, no 1 (2025) doi 10.5195/d3000.2025.1001 http://dentistry3000.pitt.edu 6 references 1. junior, a. s., penha, j. k., luia, c. e., and lima, m. d. (2017). is there correlation between polymerization shrinkage, gap formation and void in bulk-sill composite? a μct study. braz oral res. 31(100):1-10. 2. hussein, a. a. a., & al-shamma, a. m. (2019). effect of chlorhexidine and/or ethanol pre-bonding treatment on the shear bond strength of resin composite to dentin. int. j. med. res. heal. sci, 8, 150-159. 3. shackori, m. w., hasan, n. h., qasim, a. a., & abdulmajeed, a. a. (2024). evaluate the shear bond strength for alkasite in comparison with other esthetic restorative materials. al-rasidain dental journal, 24(1), 109-119. 4. ladino, l. g., bernal, a., calderón, d., & cortés, d. (2021). bioactive materials in restorative dentistry: a literature review. svoa dent, 2, 74-81. 5. marovic, danijela, matej par, matea macan, nikolina klarić, iva plazonić, and zrinka tarle. 2022. "aging-dependent changes in mechanical properties of the new generation of bulkfill composites" mater. 15, no. 3: 902a. 6. angadala, p., mandava, j., ravi, r., hanumanthu, k.r., penmatsa, p., and pulidindi, h. (2022). an in vitro micro-ct assessment of bioactive restorative materials interfacial adaptation to dentin. dent res j. 19(1): 56-64. 7. marovic, d.; haugen, h.j.; negovetic mandic, v.; par, m.; zheng, k.; tarle, z.; boccaccini, a.r. incorporation of copper-doped mesoporous bioactive glass nanospheres in experimental dental composites: chemical and mechanical characterization. mater. 2021, 14, 2611b. 8. abuljadayel, r., aljadani, n., almutairi, h., & turkistani, a. (2023). effect of antibacterial agents on dentin bond strength of bioactive restorative materials. polymers, 15(12), 2612. 9. jumaah, s. s., al-shamma, a. m. (2021). immediate and long term gingival marginal leakage of two bioactive bulk fill restorative materials (a comparative in vitro study). j res med dent sci. 9(7): 120-126. 10. abdelkafy, h., salem, n. a., marzouk, r. m., & eldehna, a. m. (2024). interfacial assessment of cention forte vs. equia forte and two forms of calcium silicate cements at two time intervals. bdj open, 10(1), 68. 11. khalil, r. j., & al-shamma, a. m. (2015). early and delayed effect of 2% chlorhexidine on the shear bond strength of composite restorative material to dentin using a total etch adhesive.jbcd. 325(2219), 1-17 12. heba, e., hussein, y., & wedad, e. (2020). shear bond strength of bioactive dental restorative materials to dentin. iosr j dent med sci, 19(11), 15-25. 13. rifai, h., qasim, s., mahdi, s., lambert, m. j., zarazir, r., amenta, f., ... & mehanna, c. (2022). in-vitro evaluation of the shear bond strength and sluoride release of a new bioactive dental composite material. journal of clinical and experimental dentistry, 14(1), e55. 14. assiri, a., alomairy, a., & nashaat, m. (2018). marginal adaptation of bulk-fill versus layered resin composite restorations. inter j health sci res, 8(6), 65-74. 15. baban, l.m. s. (2019). the insluence of slowable composite liner on microleakage of class ii packable composite resin restoration with different application techniques (comparative study). j bagh coll dent. 24(4): 19-24. 16. mohammad, m. h., & thyab, s. a. (2024). assessment of bond strength of different bonding systems to eroded dentin using sonic activation: in vitro study. eur j gen den . 17. martıńez-sabio, l., peñate, l., arregui, m., veloso duran, a., blanco, j. r., & guinot, f. (2023). comparison of shear bond strength and microleakage between activa™ bioactive restorative™ and bulk-sill composites—an in vitro study. polymers, 15(13), 2840. 18. fathpour, k., bazazzade, a., & mirmohammadi, h. (2021). a comparative study of cervical composite restorations microleakage using dental universal bonding and two-step self-etch adhesive. j contemp dent pract, 22(9), 1035-40. 19. sahli, agnès, laurent daeniker, isaline rossier, luciana caseiro, enrico di bella, ivo krejci, and tissiana bortolotto. (2024)."comparison of class ii bulk-fill, self-adhesive composites, alkasite, and high-viscosity glass ionomer restorations in terms of marginal and internal adaptation" mater. 17, no. 17: 4373. 20. obaees, r. k., alkhalidi, e. f., & hamdoon, s. m. (2024). fluoride release and antibiosilm of alkasite restorative materials. edelweiss applied science and technology, 8(4), 447459. 21. al-ashou, w. m., taher, r., & ali, a. h. (2022). shear-bond strength of different self-etching adhesive systems to dentin with or without laser irradiation before photopolymerization (a comparative study). the saudi dental journal, 34(8), 779-787. 22. kiliç, e., sirin karaarslan, e., hatirli, h. ut . s. e. y. i. n., & yilmaz, f. (2021). effects of polishing systems and storage media on color stability and surface roughness of bulk-sill restorative composites. am j dent, 34(5), 250-256. 23. bassiouny, a. a., bakry, n., aboulgheit, s., & zeitoun, s. (2024). shear bond strength of an alkasite based restorative material to silver diamine sluoride pretreated dentin (in vitro study). alexandria dental journal, 49(3), 165-170. 24. kapshe, n. (2020). comparative evaluation of shear bond strength of bulk fill and nanohybrid composites to resin modisied glass ionomer cement in presence of saliva at different storage period-an in vitro study (master's thesis, rajiv gandhi university of health sciences (india)). 25. deeb, a., & rmdan, g. a. (2022). effect of different storage conditions on the mechanical properties of silorane based composite versus microhybride and nanohybrid methacrylate based composite. sch j dent sci, 7, 118-146. 26. nada h, e., ahmed s, e., & fayza h, a. (2016). shear bond strength of ceramic laminate veneers to enamel and enamel–dentine complex bonded with different adhesive luting systems. alexandria dental journal, 41(2), 131-137. 27. raji, z., hosseini, m., & kazemian, m. (2022). micro-shear bond strength of composite to deep dentin by using mild and ultra-mild universal adhesives. dental research journal, 19(1), 44. 28. kaisarly, d., meierhofer, d., el gezawi, m., rösch, p., and kunzelmann, k. h. (2021). effects of slowable liners on the shrinkage vectors of bulksill composites. clin oral invest. 25(8): 4927-4940. 29. sh ibrahim, h., & a al-askary, r. (2023). marginal leakage evaluation of bioactive bulksill restorative materials in class ii cavities: an in vitro comparative study. 30. abdallah, a. (2022). elemental and micromorphological analysis of new alkasite based restorative material/tooth interface. egypt dent j. 68(1): 1065-1072. 31. mehesen, r., jazar, h., sheta, n., & montaser, m. (2022). scan electron microscope evaluation of marginal adaptation of alkasite, bulk-sil resin composite, resin modisied glass ionomer, and high viscosity glass ionomer restorative materials. egyptian dental journal, 68(2), 1859-1866. 32. nanavati, k., katge, f., chimata, v. k., pradhan, d., kamble, a., & patil, d. (2021). comparative evaluation of shear bond strength of bioactive restorative material, zirconia reinforced glass ionomer cement and conventional glass ionomer cement to the dentinal surface of primary molars: an in vitro study. journal of dentistry, 22(4), 260. 33. badole, g. p., shenoi, p. r., kubde, r., & bengal, s. (2023). comparative evaluation of surface roughness and microhardness of bulk-sill composite placed in artisicial saliva at three different ph. world j dent, 14(4), 331-335. 34. leung, v. h., & darvell, b. w. (1997). artisicial salivas for in vitro studies of dental materials. journal of dentistry, 25(6), 475-484. 35. rivera, z. j. t., tarazona, j. a. f., & de souza, a. s. (2020). insluence of dentin conditioning with polyacrylic acid on the shear bond strength of a nano-silled resin-modisied glass ionomer cement. journal of oral research, 9(4), 319325. 36. adsul, p. s., dhawan, p., tuli, a., khanduri, n., & singh, a. (2022). evaluation and comparison of physical properties of cention n with other restorative materials in artisicial saliva: an in vitro study. international journal of clinical pediatric dentistry, 15(3), 350. 37. samy, f. m., el-kholany, n. r., & hamama, h. h. (2025). evaluation of bond durability of different self-adhesive bioactive restorative systems to dentin. scientisic reports, 15(1), 3667 38. sadeghyar, a., lettner, s., watts, d. c., & schedle, a. (2022). alternatives to amalgam: is pretreatment necessary for effective bonding to dentin?. dental materials, 38(11), 1703-1709. 39. kunert, m., rozpedek-kaminska, w., galita, g., sauro, s., bourgi, r., hardan, l., ... & lukomska-szymanska, m. (2022). the cytotoxicity and genotoxicity of bioactive dental materials. cells, 11(20), 3238. 40. wendlinger, m., nuñez, a., moreira, p. h. a., carneiro, t. s., cochinski, g. d., siqueira, f. s. f., ... & loguercio, a. d. (2023). effect of the absence of hema on the bonding properties of universal adhesive systems containing 10-mdp: an in vitro study. operative dentistry, 48(5), 500-512. 41. kim, r. j. y., kim, y. j., choi, n. s., & lee, i. b. (2015). polymerization shrinkage, modulus, and shrinkage stress related to tooth-restoration interfacial debonding in bulk-sill composites. journal of dentistry, 43(4), 430-439.. 42. yu, p., yap, a. u. j., & wang, x. y. (2017). degree of conversion and polymerization shrinkage of bulk-sill resin-based composites. operative dentistry, 42(1), 82-89. 43. satterthwaite, j. d., maisuria, a., vogel, k., & watts, d. c. (2012). effect of resin-composite siller particle size and shape on shrinkage-stress. dental materials, 28(6), 609-614. shear bond strength and sem/edx of two bioackve bulk fill resin base composite restorakons vol 13, no 1 (2025) doi 10.5195/d3000.2025.1001 http://dentistry3000.pitt.edu 7 44. hegde, v., vaidya, m. j., & jadhav, k. s. (2023). microtensile bond strength of packable and slowable bulk-sill composite resin-based restorative materials to dentin. journal of operative dentistry & endodontics, 7(1), 6-10. 45. sauro, s., watson, t., moscardó, a. p., luzi, a., feitosa, v. p., & banerjee, a. (2018). the effect of dentine pre-treatment using bioglass and/or polyacrylic acid on the interfacial characteristics of resin-modisied glass ionomer cements. journal of dentistry, 73, 32-39. 46. makowski, g. s., & ramsby, m. l. (2004). differential effect of calcium phosphate and calcium pyrophosphate on binding of matrix metalloproteinases to sibrin: comparison to a sibrin-binding protease from inslammatory joint sluids. clinical & experimental immunology, 136(1), 176-187. 47. phyo, w. m., saket, d., da fonseca, m. a., auychai, p., & sriarj, w. (2024). in vitro remineralization of adjacent interproximal enamel carious lesions in primary molars using a bioactive bulksill composite. bmc oral health, 24(1), 37. 48. tiskaya, m., al-eesa, n. a., wong, f. s. l., & hill, r. g. (2019). characterization of the bioactivity of two commercial composites. dental materials, 35(12), 1757-1768. 49. nayak, p. p., kini, s., ginjupalli, k., & pai, d. (2023). effect of shape of titanium dioxide nanosillers on the properties of dental composites. odontology, 111(3), 697-707. 50. witkowska, justyna, tomasz borowski, krzysztof kulikowski, karol wunsch, jerzy morgiel, jerzy sobiecki, and tadeusz wierzchoń. (2024). "structure and properties of bioactive titanium dioxide surface layers produced on niti shape memory alloy in low-temperature plasma" micromachines 15, no. 7: 886. 51. ponomarev, v. a., popova, a. d., sheveyko, a. n., permyakova, e. s., kuptsov, k. a., ilnitskaya, a. s., ... & shtansky, d. v. (2022). microstructure and biological properties of titanium dioxide coatings doped with bioactive and bactericidal elements. applied surface science, 575, 151755. 52. wang, x. x., hayakawa, s., tsuru, k., & osaka, a. (2002). bioactive titania gel layers formed by chemical treatment of ti substrate with a h2o2/hcl solution. biomaterials, 23(5), 13531357. 53. sabatini, c. (2013). effect of phosphoric acid etching on the shear bond strength of two self-etch adhesives. journal of applied oral science, 21, 56-62. 54. takamizawa t, barkmeier ww, tsujimoto a, berry tp, watanabe h, erickson rl et al. (2016) insluence of different etching modes on bond strength and fatigue strength to dentin using universal adhesive systems. dent mater 32, e9e21, 55. kawazu m, takamizawa t, hirokane e, tsujimoto a, tamura t, barkmeier ww et al. (2020) comparison of dentin bond durability of a universal adhesive and two etch-and rinse adhesive systems. clin oral investig 24, 2889-2897; 56. abdallah, a. (2022). elemental and micromorphological analysis of new alkasite based restorative material/tooth interface. egyptian dental journal, 68(1), 1065-1072. 57. molaasadolah, f., hosseinipour, z. s., afzali, f., parhizkar, a., & poorzandpoush, k. (2022). the effect of two calcium phosphate-containing agents on the enamel resistance of permanent molars to demineralization: an experimental study. clinical and experimental dental research, 8(6), 1533–1539. 58. thimmaiah, c., shetty, p., shetty, s. b., natarajan, s., & thomas, n. a. (2019). comparative analysis of the remineralization potential of cppacp with fluoride, tri-calcium phosphate and nano hydroxyapatite using sem/edx an in vitro study. journal of clinical and experimental dentistry, 11(12), e1120–e1126. 59. par, m., gubler, a., attin, t., tarle, z., tarle, a. and taubock, t. t. (2021). experimental bioactive glass-containing composites and commercial restorative materials: anti-demineralizing protection of dentin. biomedicines. 9(11): 1616. 60. spagnuolo, g. (2022). bioactive dental materials: the current status. materials. 15(6): 2016 61. khan, a. s. and syed, m. r. (2018). a review of bioceramics-based dental restorative materials. dent mater j. 38(2): 163-176. 62. ghilotti, j., fernández, i., sanz, j. l., melo, m. and llena, c. (2023). remineralization potential of three restorative glass ionomer cements: an in vitro study. j clin med. 12(6): 2434. 63. di lauro, a., di duca, f., montuori, p., dal piva, a. m. d. o., tribst, j. p. m., borges, a. l. s., & ausiello, p. (2023). fluoride and calcium release from alkasite and glass ionomer restorative dental materials: in vitro study. journal of functional biomaterials, 14(2), 109. 64. al-eesa, n. a., karpukhina, n., hill, r. g., johal, a., & wong, f. s. l. (2019). bioactive glass composite for orthodontic adhesives—formation and characterisation of apatites using mas-nmr and sem. dental materials, 35(4), 597-605. 65. al-eesa, n. a., karpukhina, n., hill, r. g., johal, a., & wong, f. s. l. (2019). bioactive glass composite for orthodontic adhesives—formation and characterisation of apatites using mas-nmr and sem. dental materials, 35(4), 597-605. 66. abdallah, a. (2021). gingival microleakage of dental amalgam, bulk fill composite resin and alkasite based composite restorations in class ii cavities. egyptian dental journal, 67(4), 3759-3765. 67. todd, j. c. (2016). scientisic documentation: cention n. ivoclar-vivadent press: schaan, liechtenstein, 1-58. 68. kasraei, s., haghi, s., valizadeh, s., panahandeh, n., & nejadkarimi, s. (2021). phosphate ion release and alkalizing potential of three bioactive dental materials in comparison with composite resin. international journal of dentistry, 2021(1), 5572569. 69. wendlinger, m., nuñez, a., moreira, p. h. a., carneiro, t. s., cochinski, g. d., siqueira, f. s. f., ... & loguercio, a. d. (2023). effect of the absence of hema on the bonding properties of universal adhesive systems containing 10-mdp: an in vitro study. operative dentistry, 48(5), 500-512. 70. shaymaa, a. d., al-naimi, a., & suliman, a. (2024). a descriptive study of alkasite, bulk fill, and fiber-reinforced restoration bioactivities. alrasidain dental journal, 24(1), 120-129. 71. mederos, m., león, e. d., garcıá, a., cuevas-suárez, c. e., hernández-cabanillas, j. c., rivera-gonzaga, j. a., & grazioli, g. (2024). in vitro characterization of a novel resin-based restorative material containing alkaline sillers. journal of applied oral science, 32, e20230219. 72. slimani, a., sauro, s., gatón hernández, p., gurgan, s., turkun, l. s., miletic, i., banerjee, a., & tassery, h. (2021). commercially available ionreleasing dental materials and cavitated carious lesions: clinical treatment options. materials (basel, switzerland), 14(21), 6272. table 1. shear bond strength values in all groups of the study in mpa. restorative materials saliva artibicial n minimum maximum mean std. deviation predicta bioactive non-stored 10 5.10 8.52 6.38 1.15 stored 10 4.57 8.99 7.18 1.31 cention forte non-stored 10 7.05 10.31 8.38 1.12 stored 10 7.22 13.42 10.13 2.11 shear bond strength and sem/edx of two bioackve bulk fill resin base composite restorakons vol 13, no 1 (2025) doi 10.5195/d3000.2025.1001 http://dentistry3000.pitt.edu 8 figure 1. the mode of failure: (a) adhesive failure, (b) mixed failure, and (c) cohesive failure. then, scanning electron microscopy (sem) at 1300x was used in three samples from each subgroup to determine the mode of failure of each de–bonded coronal dentin samples (figures 2 and 3). figure 2. sem images at 1300x magnibication for predicta bioactive representing the mode of failure of de-bonded coronal dentin samples; adhesive failure of a sample not stored in artibicial saliva (a); mixed failure of a sample stored in artibicial saliva (b). notes: od = open dentinal tubules, c = composite material, d = dentin, da = de-bonded area. figure 3. sem images at 1300x magnibication for cention forte representing the mode of failure of de-bonded coronal dentin sample; mixed failure of a sample not stored in artibicial saliva (a); cohesive failure of a sample stored in artibicial saliva (b). notes: cd = closed dentinal tubules, od = open dentinal tubule, d = dentin, da = de-bonded area. do c da da c a b ha ha ha ha ha ha c shear bond strength and sem/edx of two bioackve bulk fill resin base composite restorakons vol 13, no 1 (2025) doi 10.5195/d3000.2025.1001 http://dentistry3000.pitt.edu 9 figure 4. the sem image at 5000x magnibication for:(a); predicta bioactive composite not stored in artibicial saliva (a1) showed slightly precipitation of hydroxyapatite (b); after storage in artibicial saliva (a2) showed hydroxyapatite deposition (ha) at the de bonded area of composite (c). figure 5. the sem image at 5000x magnibication for (a) cention forte composite not stored in artibicial saliva and (b) after storing in artibicial saliva showed hydroxyapatite (ha) deposition at the de-bonded area of the composite (c). figure 6. sem image at 5000x magnibication for de-bonded areas of coronal dentin samples: (a) predicta bioactive not stored in artibicial saliva and (b) after storage in artibicial saliva. both images showed crystal-like deposition of hydroxyapatite (ha) at the de-bonded coronal dentin area. d = dentin, da = de-bonded area. ha ha ha ha ha ha ha ha ha ha ha ha a b a b ha ha ha ha ha ha ha ha da d da d shear bond strength and sem/edx of two bioackve bulk fill resin base composite restorakons vol 13, no 1 (2025) doi 10.5195/d3000.2025.1001 http://dentistry3000.pitt.edu 10 figure 7. sem images at 5000x magnibication for de-bonded areas of coronal dentin samples: (a) cention forte not stored in artibicial saliva and (b) after storage in artibicial saliva. both images showed crystal-like hydroxyapatite (ha) depositions at the de-bonded coronal dentin areas. d = dentin, da = de-bonded area. figure 8. the edx spectra for one core sample of predicta bioactive not stored in artibicial saliva (a1) and after storage for 30 days in artibicial saliva (a2) at ph=7 showing the identibication of the phosphorus peak after storage. figure 9. the edx spectra for one core sample of cention forte not stored in artibicial saliva (b1) and after storage for 30 days in artibicial saliva at ph=7 (b2) showing the phosphorus peak identibication after storage. a b ha ha d ha ha ha ha ha ha da da d shear bond strength and sem/edx of two bioackve bulk fill resin base composite restorakons vol 13, no 1 (2025) doi 10.5195/d3000.2025.1001 http://dentistry3000.pitt.edu 11 figure 10. the edx spectra of one tooth sample (de-bonded coronal dentin) for predicta bioactive not stored in artibicial saliva (a1) and after storage for 30 days in artibicial saliva (a2) showing a change in the spectra for both phosphorous (p) and calcium (ca). figure 11. the edx spectra of one tooth sample (de-bonded coronal dentin area) for cention forte not stored in artibicial saliva (b1) and after storage for 30 days in artibicial saliva (b2) showing a change in the spectra for both phosphorous (p) and calcium (ca). 1014 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1014 http://dentistry3000.pitt.edu effect of strontium titanate addition on antifungal, physical and mechanical properties of soft-liners zakaria alabed, aseel mohammed al-khafaji college of den*stry, university of baghdad, baghdad, iraq abstract objec8ve: so? denture liners are esseneal in prosthodonecs due to their ability to cushion masecatory forces and provide comfort to paeents with compromised oral essues. despite their clinical benefits, conveneonal so? liners suffer from significant limitaeons, including poor bond strength with the denture base, surface degradaeon, microbial colonizaeon, and limited mechanical resilience. the goal was to assess improvements in shear bond strength and other related surface properees such as surface hardness, and a reduceon in c. albicans adherence. material and methods: stroneum titanate (srtio₃) nanoparecles were obtained from, and a self-cured so? denture liner was sourced from. all procedures were performed under condieons simulaeng intraoral temperature, following the manufacturer’s instruceons. a dry-heat oven was used for sample condieoning. a total of 45 specimens were fabricated and equally allocated into three experimental groups containing 0%, 1%, and 1.5% concentraeons of stroneum etanate (srtio₃), respecevely. results: the results indicated that srtio₃ can play a valuable role in the development of advanced so? lining materials with enhanced durability, biocompaebility, and clinical effeceveness. conclusion: the incorporaeon of stroneum titanate (srtio₃) nanoparecles at concentraeons of 1% and 1.5% significantly improves the mechanical properees, surface roughness, and reduceon in anefungal behavior of acrylic-based so? denture liners. open access cita%on: alabed z, et al. (2025) effect of stron%um titanate addi%on on an%fungal, physical and mechanical propor%es of soh-liners. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1014 received: august 6, 2025 accepted: august 20, 2025 published: september 10, 2025 copyright: ©2025 alabed z, et al. this is an open access ar%cle licensed under a crea%ve commons atribu%on work 4.0 united states license. email: zakariya.azam2401m@codental.uobaghdad.edu.iq introduc)on complete and partial removable dentures have used soft denture liner materials for over a century to disperse functional loads on the supporting tissues of the denture. this has allowed for material manipulation, improving the materials' physical, chemical, biological, and electrical qualities in comparison to their larger-scale counterparts [1]. function, comfort, and appearance all affect how effective full or partial dentures are. a precise denture foundation that appropriately adapts to the underlying tissue is crucial. when building complete dentures, this is a crucial goal [2]. a complete denture is used when a person has lost all of their teeth, while a partial denture is used if they still have some teeth [3]. the fundamental reason removable dentures may retain their support, stability, and retention is because their bases are fine-suited to the oral mucosa [4]. strontium titanate (srtio₃) is a ceramic oxide with remarkable mechanical and thermal properties, making it valuable in various dental applications. its incorporation enhances strength, hardness, and durability of dental materials without affecting their essential flexibility. this contributes to improved performance and longevity of restorations in the oral environment [5]. progressive bone resorption compromises denture fit, resulting in discomfort and even significant pain, ultimately affecting the patient’s comfort and quality of life [6]. material and methods a total of 45 specimens of a self-cured, roomtemperature polymerizing denture liner were prepared for evaluating candida albicans adherence, surface roughness, and shear bond strength. the specimens were divided equally into three groups containing 0%, 1%, and 1.5% concentrations of strontium titanate (srtio₃), as shown in the table 1. surface roughness was assessed using a profilometer, while c. albicans adherence was quantified via optical density measurements using a spectrophotometer. shear bond strength was evaluated using a testing machine (wdw-50). effect of stroneum titanate addieon on anefungal, physical and mechanical properees of so?-liners vol 13, no 1 (2025) doi 10.5195/d3000.2025.1014 http://dentistry3000.pitt.edu 2 statistical analysis statistical comparisons between groups were performed using one-way anova in the spss software, with a significance level set at α = 0.05. the analysis revealed statistically significant differences across all tested parameters, indicating that the incorporation of srtio₃ had a substantial effect on the mechanical and biological properties of liner material as shown in table 1. shear bond strength test sample design using acrylic block dimensions, the shear bonding strength between the soft lining material and the acrylic denture base was assessed. two heat-cured acrylic blocks measuring 75*25*5 mm in diameter, length, width, and thickness, as well as a stopper that was roughly 3 mm deep, made up the shear bond sample, as shown in figure 1 [7]. to apply reline material, the two acrylic blocks are positioned one on top of the other, leaving a 25 mm*25 mm* 3 mm space between them. the handle needs to be precisely 13 mm thick to guarantee that the applied forces are parallel and that the testing machine is gripping the sample well [8]. preparation of the heat-cure acrylic samples mold preparation it is possible to create acrylic blocks using an aluminum template. it was manufactured by employing substances mold and a machine that cuts by laser, in addition to other measuring tools that it is essential to the inferior portion includes both the pattern and the mold, it belongs to the dental flask of course. however, it includes a dental stone of the third type which originally had been designed to comply with the instructions of the producing party (p/l) ratio of 25ml/100q). as soon as the stone is set, both the stone and the aluminum model would be covered with an isolating material and thus be liable to dry. the higher section of the flask would later be placed over the lower part. next, a stone that was recently mixed would be added up. the aluminum sculpture was extracted from the mold. consequently, we can open the flask when we had solidified the stone. proportioning and mixing of heat cure acrylic maarc dental rapid heat-cure acrylic was used to create the acrylic sample. the mixture was combined in a dry, clean glass container with a powder-to-liquid monomer ratio of 10 cc of powder per 5 ml, as directed by the manufacturer. after being covered, the mixture was let to solidify into dough. packing we covered the two parts of a flask with an isolating material that will be dried up later. next, we erased the sculpture and locate the dough like acrylic resin into a hollow room. later, we cover the acrylic with a polythene layer. we close thus the flask. a hydraulic pressure is employed to scatter the substance equally within the mold. so, it can get pressure step by step. curing we used a digital water bath, as shown by the producer, in order to cure the material of the acrylic resin. the resin is then placed in a water bath at 74°c for approximately 120 minutes. subsequently, the temperature is increased to 100 °c to complete the curing process. since cooling is achieved at normal heat of room. for 30 minutes, we soak the metal flask in a faucet water, then we can get it from the flask. preparation of final samples for shear bond strength test mold preparation one sample was created by using two acrylic blocks to test the shear bonding strength. when the two blocks are brought together, they make a space of 25mm × 25mm × 3mm (length, width, and depth accordingly), as shown in figure 1. this space is then filled with material. then, the samples were entirely covered with laboratory silicone and allowed to set properly the acrylic blocks and silicone mold are placed together after the silicon has been set in a custom-made flask filled with freshly mixed type iii dental stone the custom flask was made from and consisted of upper and lower parts with a dimension of (33cm length, 23cm width, and 4cm height) for each part. the parts came together and tightly screwed until edge-toedge contact was achieved. incorporation of srtio3 in a dry, clean glass container, the strontium titanate nanoparticles were weighed with an electronic balance. after being measured using a medical syringe, the soft-liner monomer was applied to the nanoparticles. after that, the mixture was put through a probe sonication device. for 3 minutes at 120w and 60 khz to break up the nanoparticles into separate particles [9]. to avoid particle aggregation, the resultant srtio3-soft liner monomer dispersion was combined right away with soft liner powder. by deducting their weight from the soft-liner polymer powder, it was thought that adding srtio3s to the monomer would result in the proper p/l ratio. soft liner application we weigh the (srtio₃) within a spotless, dry glass container using an electronic balance. next, we measure employing a medical syringe, thus later we expose the soft-liner monomer dispersion suddenly with soft liner powder. it was believed, through reducing the weight from the soft-liner polymer powder, that if we add srtio3 soft liner monomer would cause the cause the suitable p/l percentage, as shown in table. curing according to the manufacturer's instructions, the material should be placed at a temperature like that of the mouth. the samples were placed in the designated places in the mold and transferred to the oven for 4 to 5 minutes at a temperature of 37 degrees celsius. the samples were then removed from the oven and removed from the mold. testing procedure the models underwent testing at, utilizing an instron testing machine (wdw-50 from laryee technology co., a computer-controlled electronic universal testing machine) with a load cell possessing a capacity of 50 kn and the crosshead speed set to 0.5 millimeters per minute, as shown in figure 1. according to astm specification d-638m, the shear bond values were determined by dividing the ultimate load needed to cause sample failure by the samples' cross-sectional area [10]. surface hardness test sample design and preparation the disc like plastic pattern was manufactured to comply with iso 10139, 2, 2016, it had a diameter of 35 millimeter and 6 thickness. we employ the plastic models to create laboratory soft denture liner manipulation samples as templates. those soft lining substances of samples were created, mixed, stuffed in and dried in compliance with the manual [11]. testing surface hardness testing was carried out in accordance with iso 10139-2:2016, which is the international organisation for standardisation (iso) guideline for permanent soft denture liners. before being measured, all samples were incubated for five minutes in a perfect oven setting that about matched the manufacturer's recommended oral temperature [12]. the hardness was measured using the shore a durometer (produced by zwick, germany); it was calibrated in compliance with astm d2240. we reported the data using shore units. the device is comprised of a 1.6 mm cylinder that gradually shrinks to a blunted indenter with a diameter of 0.8 mm. an indentation point-holding lever is connected to a scale with gradations from 0 to 100 units. zero signifies that the indenter has completely pierced the sample, whereas 100 denotes no penetration at all. to test the discs, five locations were selected. the sample was loaded evenly across its surface, away from its edges, to determine the average shore hardness [13]. fabrication of soft-liner specimens to evaluate c. albicans adherence ability specimen preparation effect of stroneum titanate addieon on anefungal, physical and mechanical properees of so?-liners vol 13, no 1 (2025) doi 10.5195/d3000.2025.1014 http://dentistry3000.pitt.edu 3 disk-shaped plastic patterns, each measuring 10 mm in diameter and 2 mm in thickness, were used to prepare the soft-liner specimens [14]. these plastic disks were positioned in the lower half of a dental flask already filled with dental stone. once the stone had fully set, the surface—including the plastic patterns—was coated with a thin layer of separating medium and allowed to dry. subsequently, the upper half of the flask was assembled and filled with dental stone using vibration to eliminate any air bubbles, then sealed with its cover. after the second layer of stone had completely set, the flask was opened, and the plastic disks were carefully removed [15]. proportioning and mixing of room temperature acrylic based soft liner control negative specimens’ preparation according to the manufacturer's instructions (powder-to-liquid ratio of 2.2 g:1.8 ml), the required amounts of soft-liner powder and liquid, as shown in figure 3 were measured and mixed in a clean, dry glass jar, then covered with a lid. both the upper and lower halves of the dental flask were coated with a separating medium to prevent the soft liner from adhering to the stone. once the mixture reached the dough stage, it was handkneaded and adapted onto the lower half of the flask in preparation for packing. positive control specimen preparation to comply with the producer’s recommendation, powder to liquid percentage of 8.6, 8.1ml.wt% of nystatin powder was precisely calculated and taken out of the total weight of the sift liner powder. we add the left soft liner powder with nystatin and mix all with the soft liquid by a tiny electric manual mixer for 60 seconds. by doing so, the targeted quantity of monomer will be added up to the mixture [16]. incorporation of srtio3 nanoparticles we weigh the srtio3 nano particles within a spotless dry glass container then add up the soft-liner monomer. the total mixture will be exposed to probe sonification for almost 3 minutes at 120 and 60 khz to scatter. those nano particles by dividing up any agglomerates and attaining uniform distribution [10]. to prevent particle aggregation, the resulting srtio₃–soft liner monomer suspension was immediately mixed with the soft-liner powder. to maintain the manufacturer's specified powder-to-liquid (p/l) ratio, the weight of the srtio₃ nanoparticles was subtracted from the total weight of the soft-liner powder. a medical syringe was used to measure the volume of the monomer, while an electronic balance was employed to accurately weigh the srtio₃ nanoparticles and soft-liner powder. packing since the soft liner gets into the dough period. we knead it by hand and used this for the ready mold. we put a polyethylene sheet over the substance. the lid will be placed on the top part of the flask. to guarantee the uniform scattering of the soft lining material in the mold to get rid if any extra value we use hydraulic press to maintain permanent pressure of 100kg/cm2. fir about 5 minutes. the mixture will be positioned inside an oven with a temperature that’s close to the oral environment. this will be achieved to comply with the manual recommendations. when cutting is over, we opened the flask after i was totally cold, and we took the specimen out of the mold [10]. after curing completion, the flask was opened when became completely cold and the specimens were removed from the mold. finishing and sterilization after trimming the excess material from the specimens using a sharp blade, they were finished using a 240-grit silicon polishing bur followed by fine-grit sandpaper. the specimens were then rinsed with distilled water, properly positioned, and sterilized under uv light for 20 minutes on each side at a wavelength of 254 nm. finally, the specimens were stored in test tubes containing 250 ml of distilled water for 24 hours [10]. isolation of c. albicans candida albicans was collected from the oral cavities of patients showing clinical signs of denture stomatitis who visited the prosthodontics clinic at the college of dentistry, kufa university, for treatment. sterile cotton swabs were used to gently collect samples from oral lesions, which were then cultured on sabouraud dextrose agar (sda), a standard medium for fungal growth. the culture plates were incubated at 37°c under aerobic conditions for 48 hours. following incubation, the fungal isolates were preserved at 4°c for future testing [17,18]. preparation of sabouraud dextrose agar according to the manufacturer’s instructions, 62 g of sabouraud dextrose agar (sda) was weighed and fully dissolved in 1000 ml of distilled water. the mixture was then sterilized using an autoclave at 121°c and 15 psi for 15 minutes. after sterilization, the medium was allowed to cool to approximately 47 °c to prevent damage to the petri dishes. to inhibit bacterial growth, 0.05 g of the broad-spectrum antibiotic chloramphenicol was added per 1000 ml of prepared medium. the sda was then poured into petri dishes, allowed to cool and solidify, and subsequently stored at 4 °c until use [19]. identification of c. albicans morphological examination a smooth, creamy, and pasty candida colonies appear in sabouraud dextrose agar medium. microscopical examination a small amount from a single isolated candida colony was picked and mixed with a drop of normal saline on a clean glass slide to create a suspension. this suspension was evenly spread across the slide, left to air dry at room temperature, and then heat-fixed by passing the slide several times through the flame of a bunsen burner. gram staining was performed according to the method outlined by marler (2001) [23], as follows: 1. the slide was stained with crystal violet for one minute, then rinsed with distilled water. 2. gram’s iodine was applied for one minute and rinsed off. 3. decolorization was carried out using acetone-alcohol until the dark violet color lightened, followed by a rinse with distilled water. 4. the slide was counterstained with safranin for one minute, rinsed again, and left to dry. microscopic examination under a light microscope revealed candida as round or oval cells. the prepared slide was examined under a light microscope, where candida appeared as round or oval-shaped cells [26]. germ tube formation a loopful of yeast cells was taken from a single colony and suspended in tubes containing 0.5 ml of serum. the tubes were then incubated at 37 °c for 3 hours. after incubation, a drop of the suspension was placed on a clean glass slide and examined under a light microscope to observe the presence of germ tubes [22]. biochemical identijication the vitek 2 system is a fully automated device that utilizes sensitive fluorescencebased technology for the identification of microorganisms. before testing, a yeast suspension was prepared in sterile saline, adjusted to a turbidity equivalent to a 2.0 mcfarland standard, as verified using the densichek instrument. the vitek id-yst card was then automatically filled with the suspension, sealed, and incubated at 35.5°c for 18 hours within the vitek 2 system. optical density readings were taken automatically every 15 minutes throughout incubation. after completion, the results were compared with the device’s database, enabling identification of the unknown microorganism. final identifications labeled in the laboratory report as “very good “were considered accurate [23]. preparation of sabouraud dextrose broth (sdb) according to the manufacturer’s instructions, 30 g of broth powder was dissolved in 1000 ml of distilled water and sterilized by autoclaving at 121°c and 15 psi for 15minutes. after sterilization, the broth was cooled to 47°c, and to prevent bacterial effect of stroneum titanate addieon on anefungal, physical and mechanical properees of so?-liners vol 13, no 1 (2025) doi 10.5195/d3000.2025.1014 http://dentistry3000.pitt.edu 4 growth, 0.05 g of chloramphenicol antibiotic was added [24]. evaluating the effect of chitosan / soft denture liner specimens on adherence of c. albicans adherence sterile tubes were filled with freshly prepared sabouraud dextrose broth (sdb), into which a small amount of the isolated yeast culture was introduced. the yeast suspension was then adjusted to a 0.5 mcfarland standard using a mcfarland densitometer. pre-sterilized soft lining material specimens were immersed in the inoculated broth and incubated at room temperature for one hour. after incubation, the specimens were carefully removed and gently rinsed with phosphate-buffered saline (pbs) for one minute with mild rocking to remove loosely attached yeast cells, then dried using filter paper. the adherent candida cells were fixed on the lining surface using methanol, stained with crystal violet for 60 seconds, rinsed again with pbs for 30 seconds, and finally dried with filter paper. microscopic examination was carried out using an inverted light microscope [25]. for each specimen, the attached candida cells were counted in two standardized microscopic fields, and the mean value of the two counts was calculated and recorded. results and discussion scanning electron microscope (sem) (figure 1) results demonstrated that the incorporation of srtio₃ at concentrations of 1% and 1.5% contributed to improved surface morphology and reduced porosity. the nanoparticles exhibited good dispersion at the lower concentration, while slight agglomeration was observed at the higher concentration. these surface modifications may enhance the bond strength between the soft liner and the denture base, as well as improve mechanical properties such as hardness and resistance to fungal adhesion. figure 1. sem images of soft liner (control,1%1.5%). candida albicans adherence the results of the multiple comparison table using the games-howell test designated significant differences between group means (p < 0.05), confirming the heterogeneous variances. this demonstrated a clear effect of strontium titanate addition on the soft liner in comparison with the control group. among the tested groups, nystatin exhibited the greatest antifungal efficacy, followed by 1.5% and then 1% strontium titanate, all of which significantly outperformed the control in reducing candida growth (figure 2). figure 2. candida albicans adherence. shore a hardness the source of the difference was more investigated by the analysis of the data, tukey (figure 3). through the mean differences, we noted the superiority of the group followed by 1.5 and then 1% over the control group in increasing hardness. figure 3. differences in hardness. shear bond strength statistically significant changes in shear strength of bonding between the control group and the groups treated with either 1% or 1.5% strontium titanate was observed, according to tukey's post hoc analysis. the findings indicate that both concentrations improved shear bond strength compared to the control, with the 1% concentration demonstrating the highest mean value, outperforming even the 1.5% group (figure 4). figure 4. differences in shear bond strength. conclusion within the parameters of this study, it was shown that addition silicone impression material can be safely disinfected by immersion in tto for ten minutes without affecting the details of the reproduction and dimensional accuracy of the impressions. references 1. elmwafy da, abdelghany am. strontium titanate nanoparticles modieied denture base materials. egypt dent j. 2024 jan;70(1):403–11. doi:10.21608/edj.2023.238541.2725 2. mohammed ali b. preparation and evaluation of some properties of heat-cured, acrylicbased soft denture liner. [phd thesis]. baghdad: university of baghdad; 2006. 3. shankargouda s. to evaluate different soft tissue liners, as functional impression materials, by determining its effect on surface roughness of dental stone over a time period: an in-vitro study. mod appr dent oral health care. 2018;3(2):247–55. 4. hussein bm, salem sa, aliwi sm. preparation and evaluation of some properties of heat cured acrylic based denture soft liner. j bag coll dent. 2015;27(4):32–6. control ol 1 % 1.5 % 0.1252 0.0321 0.0665 0.0642 0 0.05 0.1 0.15 0.2 control nystatin 1.00% 1.50% candida albicans adherence test groups 15.28 21.21 22.42 0 10 20 30 control 1% 1.50% shore a hardness test groups 0.7959 1.1418 1.0195 0 0.5 1 1.5 control 1% 1.50% shear bond strength test groups effect of stroneum titanate addieon on anefungal, physical and mechanical properees of so?-liners vol 13, no 1 (2025) doi 10.5195/d3000.2025.1014 http://dentistry3000.pitt.edu 5 5. kreve s, dos reis ac. denture liners: a systematic review relative to adhesion and mechanical properties. sci world j. 2019;2019:1–9. 6. ibraheem em, elgabry hs. effect of mandibular complete dentures relining on occlusal force distribution using t-scan system. bull natl res cent. 2021;45(1):93. 7. qanber lm, hamad ti. effect of plasma treatment on the bond of soft denture liner to conventional and high impact acrylic denture materials. j bag coll dent. 2021;33:9-17. 8. santos j, et al. strontium titanate for dental remineralization: a review of its role in enamel repair. j dent res. 2019;98(5):489-97 9. al-azzawi r. evaluation of some properties of three types of denture reline materials with miconazole (antifungal agent) preparation [master's thesis]. baghdad (iq): university of baghdad, college of dentistry, department of prosthodontics; 2007. 10. yasser ad, fatah na. the effect of addition of zirconium nanoparticles on antifungal activity and some properties of soft denture lining material. j bag coll dent. 2017;29(4):27-33. 11. astm d-638. standard test method for tensile properties of plastics. west conshohocken: american society for testing and materials; 2014. 12. iso dentistry-soft lining materials for removable dentures-part 2: materials for longterm use. iso; 2016. 13. maarc dental materials. soft-liner acrylic temporary relining kit: instructions for use. maarc dental materials; 2020 14. mohammed mj, abass sm. ineluence of hypochlorous acid on surface roughness and wettability of addition silicon impression material. egypt j hosp med. 2023;90(2):36-41. 15. karakis d, akay c, oncul b, rad ay, dogan a. effectiveness of disinfectants on the adherence of candida albicans to denture base resins with different surface textures. j oral sci. 2016;58(3):431-7. 16. mohad a, fatalla aa. the effectiveness of aluminum potassium sulfate micro-particles addition into soft denture lining material on candida albicans adherence. indian j public health res dev. 2019;10(9):935–941. 17. jorge jh, silva crc, pavarina ac, amaya mi, masetti p, pellissari cv. metabolism of l929 cells after contact with acrylic resins. part 2: soft relines. int j dent oral sci. 2015;1(6):6-11. 18. binns r, li w, wu cd, campbell s, knoernschild k, yang b. effect of ultraviolet radiation on candida albicans bioeilm on poly(methyl methacrylate) resin. j prosthodont. 2020;29(8):686-92. 19. das g, khokhar m, naeem s, reehana. comparison of solubility and water sorption of two different soft lining materials. j ayub med coll abbottabad. 2018;30(2):175-9. 20. marsh pd, martin m. oral microbiology. 5th ed. edinburgh: churchill livingstone; 2009 21. baveja c. textbook of microbiology for dental students. 3rd ed. delhi: arya publications; 2010. 22. forbes ba, sahm df, weissfeld as, bailey ss. diagnostic microbiology. 12th ed. st. louis: mosby elsevier; 2007 23. marler lm, siders ja, allen sd. direct smear atlas: a monograph of gram-stained preparations of clinical specimens. philadelphia: lippincott williams & wilkins; 2001 24. forbes ba, sahm df, weissfeld as, bailey ss. diagnostic microbiology. 12th ed. st. louis: mosby elsevier; 2007. 25. chander j. textbook of medical mycology. london: jp medical ltd; 2017. 26. byadarahally rs, rajappa s. isolation and identieication of candida from the oral cavity. int scholarly res netw dent. 2011;2011:1-6. table 1. the mixing ratios of srtio3 nanoparticles, soft liner(pmma)(mma), nystatin. test selected concentration % amount of srti o3 powder (g) amount of polymer powder (g) amount of monomer liquid (ml) shear control 0 10 8.1 1 0.1 9.9 8.1 1.5 0.15 9.85 8.1 hardness control 0 10 8.1 1 0.1 9.9 8.1 1.5 0.15 9.85 8.1 candida albicans control 0 10 8.1 control + 1.wt.% nystatin 1.4 8.6 8.1 1% 0.1 9.9 8.1 1.5% 0.15 9.85 8.1 918 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.918 http://dentistry3000.pitt.edu a cross-sectional study of environment stress among undergraduate dental students huda a. yaseen, dhaffar alwan majbil, wathba mohammed jabber, reyadh r. al-rashidi, umelbaneen j. alalawy, sarab m. zeki radhi college of den*stry, university of kut, wasit, iraq abstract objeceve: stress is a prevalent issue in the field of dental educaeon, with numerous studies highligheng its impact on students. creaeng a supporeve and stress-free environment is vital in dental educaeon, not only for the well-being of students but also to minimize hazards which are frequently linked to stress. the main objeceve of the study was to invesegate the top stressors among the clinical years’ dental students. material and methods: a modified "dental environment stress" queseonnaire-based convenient sample from fourthand fish-year dental students in the university of wasit and kut university college was collected from november 2023 to december 2023. prior to data colleceon, an ethical approval was taken from the kut university college research ethics commizee (krec) and each parecipant provided a wrizen informed consent. the data were analysed using the staesecal package for the social sciences staesecal sosware (spss version 25.0). results: 392 students parecipated with a female to male raeo (2.4: 1). the "self-efficacy belief" domain emerged as the most impaccul stressor. fish-year students experience higher stress related to eme management and clinical requirements compared to fourthyear students. stress levels are similar between students from private and public universiees. conclusion: the study highlights that dental educaeon induces significant stress among students. efforts to improve paeent organizaeon and engagement are recommended to alleviate stress among students. open access cita%on: yaseen ha, et al. (2025) a cross-sec%onal study of environment stress among undergraduate dental students. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.918 received: april 26, 2025 accepted: may 22, 2025 published: june 26, 2025 copyright: ©2025 hadi am, et al. this is an open access ar%cle licensed under a crea%ve commons atribu%on work 4.0 united states license. email: huda.yaseen@alkutcollege.edu.iq introduc)on the term "stress" according to who, refers a state of worry or mental tension caused by a dif;icult situation. stress is a natural human response that prompts us [1]. the brain plays a crucial role in perceiving and responding to stress with exceptional plasticity. " eustress" is a positive form of stress that can motivate and energize individuals, helping them to perform better and achieve their goals. on the other hand, when stressors overwhelm an individual's ability to cope, stress can transform into a negative form " distress" that can lead to anxiety, depression, and other health issues [2,3]. the crisis behind being stressed is the endless physical and mental consequences and ;inally, burnout [4]. physical consequences can be fatigue/tiredness, mood alteration, effect on performance, headaches and sleep disturbance. psychological distress manifests in terms of showing higher levels of depression, obsessive-compulsive disorders, and interpersonal sensitivity than agematched norms [5,6]. stress is not exclusive to medical and dental students but is more pronounced among them. what worsens the a cross-sec+onal study of environment stress among undergraduate dental students vol 13, no 1 (2025) doi 10.5195/d3000/2025.918 http://dentistry3000.pitt.edu 2 condition is the high expectation that they are capable of handling stress and not be greatly affected by it [5]. the current dental school curricula strongly emphasize developing a wide range of skills among students, such as theoretical knowledge, clinical expertise, and the ability to effectively interact with anxious patients and supervisors. [7]. in iraq, the cost of studying dentistry is the highest among all undergraduate programs, even at governmental universities, which adds another burden on dental students. many studies reported that dental students, compared with their counterparts in the medical ;ield, experience more stress particularly during their clinical study years [810]. stress among dental students has been assessed using various stress scales, including "dental environment stress scale" (des), the "maslach burnout inventory" (mbi), and the "psychosocial stress inventory" (pssi). among these, des is commonly used [11]. fourth-year dental students are anxious about their career prospects, while newly arrived students are greatly concerned about having little time for rest [12]. when investigating the main stressors in a multi-country study, divaris and polychronopoulou concluded that performance pressure, workload, and self-ef;icacy beliefs constituted the students' main concerns [13]. another study reported that the major stressors for ;ifthyear students were: examinations, patients being late to appointments and the amount of assigned work [14]. creating a healthy and relaxed environment without hazards is a priority in the dental setting. stress has been shown to be a common cause (50%) of needle-stick injuries [9], which can lead to disease transmission and harm to practitioners. the prevalence, frequency and the main contributors of stress among dental students in wasit governorate have not been previously investigated. wasit currently has two universities. therefore, an attempt was made to study the mental health issues among this population particularly the clinical years students. materials and methods a cross-sectional study conducted in the 1st semester of the academic year 2023/2024 during the period from november 2023 to december 2023. a convenience sampling of participants was recruited from fourthand ;ifth year dental students in the university of wasit and kut university college, wasit province, iraq. the questionnaire used in the study was the modi;ied "dental environment stress" (des) questionnaire by al-sowygh [2], which was selected due to the religious and cultural similarities between the two societies. prior to data collection, the questionnaire underwent translation into arabic by a professional language translator. the arabic version was then tested on a small sample of students (10%) to ensure that the questions were clear, understandable, and culturally relevant. the translated questionnaire in arabic showed a satisfactory cronbach’s alpha of 0.84. the questionnaire consisted of demographic data (the student’s gender, stage, and university) and 32 stress-provoking items. each item had ;ive potential responses: not stressful, slightly stressful, moderately stressful, severely stressful, and very severely stressful. these items were grouped into 6 major domains: academic challenge, workload, performance pressure, self–ef;icacy beliefs, clinical training and patient treatment. however, the distributed questionnaire among the participants did not include the grouped domains. the researchers received approval to conduct the study from the kut university college research ethics committee (krec). also, each participant provided a written informed consent. the questionnaires were distributed anonymously, and participants were assured that all information would be kept strictly con;idential and used solely for research purposes. the data were analysed using the statistical package for the social sciences statistical software (spss version 25.0). descriptive statistics (proportion, mean and standard deviation) were used to describe the study and outcome variables. independent sample t-test for difference of means between two groups for the categorial variables. all tests were conducted with a con;idence level of 95% and a signi;icant level of 0.05. results the research examined the stress levels among 392 dental students, with most participants being female (70.20%) compared to male (29.80%), as shown in table 1. regarding academic stage, more students were from the 5th grade (56.4%) compared to the 4th grade (43.6%). age of participants ranged from 21 to 24 years old, the mean age was 22.30 years, with a standard deviation of 0.763 years. most of participants (57.4%) pursued education in private institution. as demonstrated in table 2, the "self-ef;icacy belief" domain emerges as the most signi;icant stressor among the domains, recording a mean of 3.25±0.893. following closely is the domain of " academic challenge" with a mean score of 2.50±0.615. conversely, the domain of "patient management" registered the lowest stress levels with a mean score of 1.59±0.509, indicating relatively lesser concerns among participants in this aspect of their education. in table 3, signi;icant stress level differences were observed across domains based on gender, grade, and type of education. specifically, males exhibited lower stress in the "academic challenge" domain (mean: 2.39, p = 0.015) compared to females (mean: 2.55). while no signi;icant gender differences were found in "time management" (p = 0.393), males reported lower stress in "self-ef;icacy belief" (mean: 3.29, p = 0.58) than females (mean: 3.24). signi;icant grade disparities were noted in "time management" (p = 0.003), with 4th-grade participants reporting lower stress (mean: 2.03) compared to 5th-grade participants (mean: 2.21). no signi;icant grade differences were found in "self-ef;icacy belief" or "academic performance." regarding the comparison between stressors among the type of education, it is obvious that the "performance pressure " is higher in the pubic than the private colleges (mean: 2.50 vs. 2.34, p=0.003). stressor responses varied between genders. notably, males reported lower stress in "lack of con;idence in being a successful dental student" (mean: 3.64, p = 0.626) compared to females (mean: 3.71), as found in table 4, while no signi;icant gender differences were found in "thinking about the possibility of not succeeding in the profession in the future" (p = 0.212) or "lack of cooperation among colleagues" (p = 0.436), males exhibited lower stress levels in "facing criticism in how you do your work" (mean: 2.21, p = 0.006) compared to females (mean: 2.56). no signi;icant differences were noted in other stressors between genders. stressor responses also differed between grades. notably, signi;icant grade disparities a cross-sec+onal study of environment stress among undergraduate dental students vol 13, no 1 (2025) doi 10.5195/d3000/2025.918 http://dentistry3000.pitt.edu 3 were observed in "lack of cooperation among colleagues" (p = 0.000), with 4thgrade participants reporting lower stress (mean: 3.16) compared to 5th-grade participants (mean: 2.55), as shown in table 5. similarly, signi;icant grade differences were noted in "some teachers treated students inappropriately" (p = 0.002), with 4th-grade participants exhibiting lower stress levels (mean: 2.56) compared to 5th-grade participants (mean: 2.90). no signi;icant grade differences were found in other stressors. table 6 demonstrates varying stress levels between types of education. participants from private universities reported higher stress in domains such as "lack of con;idence in being a successful dental student" (mean: 3.84 vs. 3.48, p = 0.003), "thinking about the possibility of not succeeding in the profession in the future" (mean: 3.48 vs. 3.20, p = 0.019), and "language barrier and dif;iculty learning medical terminology" (mean: 2.92 vs. 3.24, p = 0.011) compared to those from public universities. conversely, public university students experienced higher stress levels in "fear of failure in the semester or academic year" (mean: 2.87 vs. 2.60, p = 0.022). however, no signi;icant differences were observed in domains such as "facing criticism in how you do your work," "some teachers treated students inappropriately," and "responsibility for obtaining suitable patients." discussion previous studies have found that dental education induces stress among students, regardless of the country or educational system. this highlights the challenging nature of dental education, which requires students to acquire extensive knowledge and professional hand skills while maintaining a positive attitude towards patient care within a limited timeframe. the primary goal of this study was to determine the perceived factors causing stress among dental students in their clinical study years, wasit governorate, iraq. out of the total 392 dental students, 70% were females. low participation rate of males was also observed in several studies [8,9,15]. female students scored higher level of stress than males in the overall mean score with signi;icance observed in both "academic challenge" and "clinical training" domains. this is consistent with most of the studies regardless of the main stressor and coinciding with the fact that stress level is higher in females in the general population due to the hormonal, physiological, and stressor variations between the genders. [2,8,16]. in contrast, ;indings reported by tangade et al. indicated that males had stress more than females and attributed that to the socioeconomic burden on men in indian society [7]. the mean scores of 4th and 5th-year students did not show a signi;icant difference. however, 5th-year students reported higher stress levels in "time management" and "clinical training" compared to 4th-year students. this could be due to the increased number and complexity of case requirements for 5th-year students. a study conducted in jordan yielded similar results. [9]. when considering the top ten stressors, "being treated inappropriately by teachers" was a signi;icant source of stress for 5th-year students. these ;indings agree with an indian study by tangade et al. but contradict the ;indings of al-sowygh et al. study conducted in saudi arabia, where the mentioned stressor was more signi;icant among 4thyear students [2,7]. regarding the type of education, the average scores were almost the same for students in private and public universities, which was concordant with a study from pakistan in 2024 [17]. this proximity may be due to the comparable social and ;inancial support, as well as similar challenges experienced by both groups. however, it was observed that in terms of domains, "performance pressure" and particularly "lack of cooperation among colleagues. ", "language barriers" and "fear of being unable to catch up with colleagues" were signi;icantly higher among public university students. furthermore, "lack of technical laboratories", one of the perceived top 10 stressors, was surprisingly signi;icantly more prominent among private university students. this could be because the private university has a larger student population than the public university. "fear of failure in the course’’ ranked among the top 10 stressors and was found to be signi;icantly higher among public university students. a study conducted in russia showed that it was among the top stressors but had no signi;icant difference regarding to the type of education [15]. the "self-ef;icacy belief" domain has emerged as the most impactful stressor among all the domains, which was inconsistent with most of the perceived local and outside studies where "workload " domain was the most signi;icant stressor [2,8,11,15]. this may be attributed to the social and academic stigma associated with private universities in iraq, as the majority of the present study participants were from a private university. in fact, the top two stressors "lack of con;idence in being a successful dental student" and "thinking about the possibility of not succeeding in the profession in the future" that fall under the umbrella of "self-ef;icacy belief" domain were signi;icantly higher among private compared to public university students. continuing in the list of the top stressors in the current study, females experienced higher levels of stress from being criticized by teachers and treated inappropriately compared to males, and ;ifth-year students reported feeling even more stressed by this criticism. however, a study by al-sowygh et al. found that males and fourth-year students experienced this fear more [2]. the difference could be due to variations in staff, rules, and other factors among the universities. patient-related factors and dif;iculty ;inding a suitable patient were listed the last of the top stressors among both 4th and slightly less in 5th-year students, regardless of gender, coinciding with the ;indings of a jordanian study in 2024 [9]. despite being insigni;icant, private university students were more stressed about this issue. this discrepancy might be due to being away from the city centre. therefore, kut university could improve efforts to organize the process of obtaining patients and consider offering incentives to promote patients’ engagement. finally, each university may have unique stressors depending on various factors related to students, the university and society environment. identifying these factors might help alleviate students' anxiety and, hence, improve their performance. conclusion the study highlights that dental education induces signi;icant stress among students, particularly female students, due to academic challenges and clinical training. fifthyear students experience higher stress related to time management and clinical requirements compared to fourth-year students. stress levels are similar between a cross-sec+onal study of environment stress among undergraduate dental students vol 13, no 1 (2025) doi 10.5195/d3000/2025.918 http://dentistry3000.pitt.edu 4 students from private and public universities, though speci;ic stressors like performance pressure and lack of technical laboratories vary. the "self-ef;icacy belief" domain emerged as the most impactful stressor. efforts to improve patient organization and engagement are recommended to alleviate stress among students. acknowledgement the authors would like to express their sincerest gratitude to all dental students, as this study would not have been possible without their cooperation and participation. moreover, the authors thank dr. hawra’s exceptional support in writing and reviewing this manuscript. references [1] who what is stress [https://www.who.int/news-room/questionsand-answers/item/stress] [2] al-sowygh zh, alfadley aa, al-saif mi, alwadei sh. perceived causes of stress among saudi dental students. king saud univ j dent sci. 2013; 4:7-15. [3] le fevre, m., matheny, j. and kolt, g.s. (2003), "eustress, distress, and interpretation in occupational stress", journal of managerial psychology, vol. 18 no. 7, pp. 726-744. https://doi.org/10.1108/026839403105024 12 [4] pohlmann k, jonas i, ruf s, harzer w. stress, burnout and health in the clinical period of dental education. eur j dent educ, 9, 2005, 7884 [5] jain a, bansal r. stress among medical and dental students: a global issue. iosr j dent med sci. 2012;1(5):5-7. [6] basudan s, binanzan n, alhassan a. depression, anxiety and stress in dental students. int j med educ. 2017 may 24;8:179-186. doi: 10.5116/ijme.5910.b961. [7] tangade ps, mathur a, gupta r, chaudhary s. assessment of stress level among dental school students: an indian outlook. dent res j (isfahan). 2011;8(2): spring. [8] murad a, alhajj m. evaluation of stress among undergraduate dental students in al-qadisiyah university-iraq. journal of stomatology. 2017;70(2):171-182. doi:10.5604/01.3001.0010.1843. [9] rasha a alamoush, sereen al-sawaeir, dima abu baker, sanaa a aljamani, salah a alomoush, mahmoud k al-omiri, stress experienced by dental students performing clinical training in different dental disciplines: a cross-sectional study, journal of occupational health, volume 66, issue 1, january-december 2024, uiae006, https://doi.org/10.1093/joccuh/uia e006 [10] abbasi sz, mubeen n, ayub t, khan ma, abbasi z, baig n. comparison of stress levels among medical and dental students in the clinical years of training and their coping strategies. j pak med assoc. 2020 jun;70(6):10061008. doi: 10.5455/jpma.294959. pmid: 32810097. [11] saleem t, yasmin r, aziz a, mahboob u, sethi a. perception of academic stressors among dental undergraduate students. jpda. 2021;30(4): oct-dec. [12] polychronopoulou a, divaris k. perceived sources of stress among greek dental students. j dent edu. 2005;69(6):687–692. [13] polychronopoulou a, divaris k. dental students' perceived sources of stress: a multicountry study. j dent educ. 2009 may;73(5):631-9. pmid: 19433538. [14] suha b. abu-ghazaleh, hawazen n. sonbol, lamis d. rajab. a longitudinal study of psychological stress among undergraduate dental students at the university of jordan. bmc medical education. 2016. [15] myrvold b. stress among dental students: a survey from arkhangelsk, russia [master’s thesis]. tromsø, norway: uit the arctic university of norway; 2017. available from: https://munin.uit.no/bitstream/handle/10037/12910/thesis.pdf?sequence=1 [16] al okaili ag, khalaf ah, hassan ts. a crosssectional study of stress and anxiety among dental students at ajman university. tikrit j dent sci. 2017;5(2):87-97. [17] ayub a, ali s, issrani r, sethi a, khattak o, iqbal a. burnout among dental students of private and public dental colleges in pakistan a cross-sectional study. pesqui bras odontopediatria clıń integr. 2024; 24:220176. doi:10.1590/pboci.2024.016. table 1. characteristics of all participants. frequency percent gender male 117 29.80% female 275 70.20% grade 4th 171 43.60% 5th 221 56.40% type of education public 167 42.6% private 225 57.4% age mean ± sd. (years) 22.30 ± 0.763 table 2. dental environment stress domains scores (n=392). domains mean ± sd academic challenge 2.50 ± 0.615 time management 2.12 ± 0.608 self-ef;icacy belief 3.25 ± 0.893 clinical training 2.09 ± 0.627 patient management 1.59 ± 0.509 performance pressure 2.41± 0.530 a cross-sec+onal study of environment stress among undergraduate dental students vol 13, no 1 (2025) doi 10.5195/d3000/2025.918 http://dentistry3000.pitt.edu 5 table 3. comparison between responses by domain according to gender, grade, and education. domain male (mean ± sd) female (mean ± sd) gender (p-value) 4th grade (mean ± sd) 5th grade (mean ± sd) grade (pvalue) public (mean ± sd) private (mean ± sd) education (pvalue) academic challenge 2.39 ± 0.63 2.55 ± 0.60 0.015 2.46 ± 0.60 2.54 ± 0.62 0.18 2.51 ± 0.63 2.49 ± 0.64 0.794 time management 2.17 ± 0.62 2.11 ± 0.60 0.393 2.03 ± 0.59 2.21 ± 0.60 0.003 2.14 ± 0.64 2.11 ± 0.56 0.556 self-ef;icacy belief 3.29 ± 0.84 3.24 ± 0.92 0.58 3.27 ± 0.94 3.24 ± 0.86 0.758 3.18 ± 0.97 3.30 ± 0.82 0.185 clinical training 1.96 ± 0.56 2.15 ± 0.65 0.008 1.99 ± 0.62 2.17 ± 0.62 0.007 2.02 ± 0.63 2.14 ± 0.61 0.075 patient management 1.59 ± 0.51 1.59 ± 0.51 0.926 1.62 ± 0.46 1.57 ± 0.54 0.344 1.62 ± 0.50 1.56 ± 0.51 0.307 performance pressure 2.38 ± 0.51 2.44 ± 0.54 0.312 2.47 ± 0.53 2.38 ± 0.53 0.088 2.50 ± 0.53 2.34 ± 0.51 0.003 table 4. comparison between responses by top ten stressors according to gender. top ten stressors mean ± sd (male) mean ± sd (female) p-value lack of con;idence in being a successful dental student 3.64 ± 1.200 3.71 ± 1.198 0.626 thinking about the possibility of failing in the profession in the future 3.48 ± 1.103 3.32 ± 1.204 0.212 lack of cooperation among colleagues 2.74 ± 1.212 2.85 ± 1.263 0.436 language barrier and dif;iculty learning medical terminology 2.97 ± 1.249 3.09 ± 1.192 0.369 fear of not being able to catch up with colleagues if you fall behind them 2.75 ± 1.299 2.91 ± 1.237 0.269 facing criticism in how you do your work 2.21 ± 1.097 2.56 ± 1.146 0.006 fear of failure in the semester or academic year 2.76 ± 1.215 2.69 ± 1.187 0.617 some teachers treated students inappropriately 2.64 ± 1.178 2.80 ± 1.052 0.178 lack of quali;ied technical laboratories 2.26 ± 1.054 2.62 ± 1.125 0.004 responsibility for obtaining suitable patients 1.71 ± .881 1.72 ± .939 0.915 a cross-sec+onal study of environment stress among undergraduate dental students vol 13, no 1 (2025) doi 10.5195/d3000/2025.918 http://dentistry3000.pitt.edu 6 table 5. comparison between responses by top ten stressors according to grade. top ten stressors mean ± sd (4th grade) mean ± sd (5th grade) p-value lack of con;idence in being a successful dental student 3.71 ± 1.192 3.67 ± 1.204 0.756 thinking about the possibility of failing in the profession in the future 3.44 ± 1.218 3.31 ± 1.142 0.275 lack of cooperation among colleagues 3.16 ± 1.287 2.55 ± 1.149 0.000 language barrier and dif;iculty learning medical terminology 3.08 ± 1.248 3.04 ± 1.181 0.739 fear of not being able to catch up with colleagues if you fall behind them 2.96 ± 1.285 2.78 ± 1.231 0.169 facing criticism in how you do your work 2.43 ± 1.203 2.47 ± 1.093 0.745 fear of failure in the semester or academic year 2.67 ± 1.178 2.75 ± 1.209 0.488 some teachers treated students inappropriately 2.56 ± 1.117 2.90 ± 1.051 0.002 lack of quali;ied technical laboratories 2.54 ± 1.189 2.50 ± 1.056 0.723 responsibility for obtaining suitable patients 1.75 ± .957 1.69 ± .893 0.478 table 6. comparison between responses by top ten stressors according to education. top ten stressors mean ± sd (public university) mean ± sd (private university) p-value lack of con;idence in being a successful dental student 3.48 ± 1.246 3.84 ± 1.138 0.003 thinking about the possibility of failing in the profession in the future 3.20 ± 1.205 3.48 ± 1.142 0.019 lack of cooperation among colleagues 2.99 ± 1.289 2.69 ± 1.202 0.021 language barrier and dif;iculty learning medical terminology 3.24 ± 1.142 2.92 ± 1.242 0.011 fear of not being able to catch up with colleagues if you fall behind them 3.01 ± 1.331 2.75 ± 1.189 0.039 facing criticism in how you do your work 2.43 ± 1.159 2.47 ± 1.130 0.732 fear of failure in the semester or academic year 2.87 ± 1.257 2.60 ± 1.134 0.022 some teachers treated students inappropriately 2.69 ± 1.081 2.80 ± 1.101 0.300 lack of quali;ied technical laboratories 2.28 ± 1.124 2.69 ± 1.078 0.000 responsibility for obtaining suitable patients 1.69 ± .883 1.73 ± .950 0.681 1088 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.1088 http://dentistry3000.pitt.edu effect of different surface treatment on hardness of dental alloys evan hussain alwan1, sara abdulbast turki2, zaid ezzat abdul majeed2 1medical technical ins.tute and 2college of health and medical technology, middle technical university, iraq abstract objec<ves: inves<gate different surface treatments on hardness of specimens produced by cad-cam milling techniques and selec<ve laser mel<ng technique. materials and methods: thirty disc-shaped specimens were put into three main groups. each group has ten examples of co-cr metal that were made of different materials. there were three kinds of milling: hard, selec<ve laser mel<ng (3d printer), and sop. everything was cut up into 15 mm wide pieces that were 3 mm thick so that they could be used for more tests. the last step was to finish them off and make their surfaces smooth with a diamond grinder. a scanning electron microscope (sem) was used to look at one example from each group and figure out how the surface of the metal frame was made. we tested how hard specimens were. we used the independent sample one-way anova to determine differences. results: hardness tests for all test groups in the study showed that 3d prin<ng usually gave the best hardness in the control and sandblast groups. hard cuxng gave the most hardness in the laser group. we used oneand two-way anova and levene's test to look at the data sta<s<cally. all the tests had p-values of 0.001, which means that the hardness values for each milling type are very different from one another. conclusion: 3d prin<ng generally resulted in the highest hardness values, and the control and sandblast groups hard milling showed superior hardness in the laser group. open access cita%on: alwan eh et al. (2025) effect of different surface treatment on hardness of dental alloys. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1088 received: october 21, 2025 accepted: november 12, 2025 published: december 12, 2025 copyright: ©2025 alwan eh et al. this is an open access ar%cle licensed under a crea%ve commons asribu%on work 4.0 united states license. email: evan.hussain.alwan@gmail.com introduc)on pre-treatment of different cad/cam materials, such as ceramics, zirconia, resin-infiltrated ceramic, and resin-based composites, is required and will show differences in roughness, surface energy, and imperfections [1]. there is a need to establish best practices for obtaining a more favorable surface roughness and wear. it is known that depending on the material and the surface treatment, surface roughness, microhardness, volumetric loss, and height loss of enamel, are affected [2]. the chemical stability of cad/cam materials is also affected by surface treatment (glazed or polished) and unique signatures of ion elution can be detected [3]. the goal of our work was to determine what is the best approach to pretreat surface of cad/cam materials. material and methods thirty metal specimens of 15 mm in dimensions and 3 mm thickness were used in this study. metal specimens were fabricated by using cad/cam system and selective laser melting (3dprinting) (figure 1). figure 1. specimens used in the study. the specimens were put into three groups based on the three different ways: effect of different surface treatment on hardness of dental alloys vol 13, no 1 (2025) doi 10.5195/d3000/2025.1088 http://dentistry3000.pitt.edu 2 hard milling: 10 specimens made from metal kera-disk hard milling (eisenbacher dentalwarened,gmbh). soft milling: 10 specimens metal made from kera-disk soft milling (eisenbacher dentalwarened,gmbh). selective laser melting: 10 specimens made of metal (riton d150dental 3d printer). after printing, a carbide cutting bur was used to vinish and a silicon diamond polisher bur was used to clean. last, extra care was taken to clean each specimen with steam to get rid of any metal oxide dust that was still on them. scanning electron microscope (sem) was used to study the metal surface topology and details of all groups (figures 2 to 10). hardness test was done with an hv-1000z digital micro vickers hardness tester from hst in china. before and after the surface treatment, a 9.8 n force for 20 seconds was used [4]. surface was treated with a 1,064 nm nd:yag laser. when it was used to treat the surface, the laser was set to these settings: each spot has a 1.30 kv potential, 84.4 mj of energy, 10 waves, and a 1hz tone. the top of the sample was facing the laser beam in the opposite direction. this laser beam was bent by something with a focal length of 10 cm. the sample was put 10 cm from the lens with the help of a wheel with markings on it. a laser spot was put on the scale every vive degrees to make a straight line around the edge of the item [5]. we used an aluminum oxide (al2o3) sandblaster that was 110 mm long and 0.25 mpa for 30 seconds to make the surface of each specimen rough. a 10-minute ultrasonic bath with water was then used to clean the surface [2]. we used anova, one-way, and two-way tests to compare hardness values for the groups. results/discussion the control group showed a mean hardness of 422.4 ± 1.32 hv. the laser-treated group showed an increased mean hardness of 548.7 ± 1.59 hv. the sandblast group exhibited the highest mean hardness of 554.2 ± 1.51 hv (p=0.93). by anova, we found that the mean hardness (hv) was significantly different between the groups (f=25,352.6, p=0.001). post hoc analysis using tukey's hsd test demonstrated that both the laser and sandblast groups had significantly higher mean hardness values compared to the control group with p-value of 0.001. specifically, the laser group showed a mean increase of 126.37 hv over the control group, while the sandblast group exhibited an even greater mean increase of 131.84 hv over the control group. furthermore, the sandblast group displayed a marginally higher hardness than the laser group, with a mean difference of 5.47 hv, which was also statistically significant (p=0.001) as shown in table 1. for soft milling, the control group had a mean hardness of 447.3 ± 0.35 hv. the laser group demonstrated a higher mean hardness of 452.1 ± 0.45 hv. in contrast, lower mean hardness value was observed in the sandblast group with 443.6 ± 0.41 hv (p=0.7). the analysis of variance (anova) for soft milling demonstrated a statistically significant difference in mean hardness (hv) among the three groups (p=0.001). post hoc analysis using tukey's hsd test revealed that the laser group had a significantly higher mean hardness compared to the control group, with a mean difference of -4.76 hv (p = 0.001). in contrast, the sandblast group exhibited a significantly lower mean hardness than the control group, with a mean difference of 3.75 hv (p=0.001). additionally, the laser group showed the highest mean hardness among all groups, significantly outperforming the sandblast group with a mean difference of 8.51 hv (p=0.001) (table 2). the control group had a mean hardness of 596.3 ± 0.16 hv. the laser group showed a lower mean hardness of 386.9 ± 0.39 hv. conversely, the sandblast group exhibited the highest mean hardness of 706.2 ± 0.42 hv (p=0.7). the analysis of variance (anova) for 3d printing demonstrated a statistically significant difference in mean hardness (hv) among the three groups (p=0.001). post hoc analysis using tukey's hsd test revealed that the control group had a significantly higher mean hardness compared to the laser group, with a mean difference of 209.43 hv (p=0.001). however, the control group exhibited significantly lower mean hardness than the sandblast group, with a mean difference of -109.85 hv (p=0.001). among the groups, the laser group demonstrated the lowest mean hardness, in comparison to the sandblast group, with a mean difference of 319.29 hv (p=0.001) (table 3). for the control (hv) group, the mean hardness values for hard milling, soft milling, and 3d printing were 422.41 ± 1.32, 447.38 ± 0.35, and 596.35 ± 0.17, respectively. among these, the 3d printing group exhibited the highest hardness value. in the laser (hv) group, the mean hardness values for hard milling, soft milling, and 3d printing were 548.79 ± 1.60, 452.15 ± 0.45, and 386.91 ± 0.39, respectively. unlike the control group, hard milling had the highest hardness values, while the 3d printing group demonstrated the lowest values in this category. for the sandblast (hv) group, the mean hardness values for hard milling, soft milling, and 3d printing were 554.26 ± 1.51, 443.64 ± 0.42, and 706.21 ± 0.42, respectively. like the control group, 3d printing demonstrated the highest hardness values, while soft milling showed the lowest. the analysis indicates a statistically signivicant differences in hardness values across the milling types with p-value is 0.001, suggesting a statistically signivicant difference in hardness among hard milling, soft milling, and 3d printing. when you use the bonferroni post hoc test, these gaps get even bigger. a big difference (p=0.001) was found between hard milling and soft milling. there was also a difference between hard milling and 3d printing and soft milling and 3d printing. there's no doubt that these two steps are different. the p-value analysis for specivic group differences convirms statistically signivicant differences in hardness across milling types within each group. in the control (hv) group, all comparisons yielded p-values of 0.001, indicating signivicant differences in hardness values. the difference between hard milling and soft milling was -24.97 (p=0.001), suggesting that soft milling resulted in slightly higher hardness. the difference between hard milling and 3dprinting was -173.93 (p=0.001), showing that 3d printing signivicantly increased hardness. similarly, soft milling vs. 3d printing exhibited a difference of -148.95 (p=0.001), convirming that 3d printing had the highest hardness values. in the laser (hv) group, the hard milling vs. soft milling comparison showed a difference of 96.63 (p=0.001), indicating that hard milling resulted in signivicantly higher hardness than soft milling. the difference between hard milling and 3d printing was 161.87 (p=0.001), convirming that 3d printing had the lowest hardness values. similarly, soft milling vs. 3d printing had a difference of 65.23 (p=0.001), further supporting the trend that 3d printing resulted in the lowest hardness in this group. in the sandblast (hv) group, signivicant differences were also observed, with hard milling vs. soft milling showing a difference of 110.61 (p = 0.001), convirming that hard milling resulted in signivicantly higher hardness. the difference between hard milling and 3d printing was -151.95 (p = 0.001), indicating that 3d printing exhibited signivicantly higher hardness than hard milling. the largest difference was seen between soft milling and 3d printing, at -262.56 (p = effect of different surface treatment on hardness of dental alloys vol 13, no 1 (2025) doi 10.5195/d3000/2025.1088 http://dentistry3000.pitt.edu 3 0.001), highlighting a substantial increase in hardness with 3d printing. overall, the p-values of 0.001 across all comparisons convirm highly signivicant differences in hardness values between milling types within each group. the vindings reinforce that 3d printing generally resulted in the highest hardness in the control and sandblast groups, whereas hard milling showed superior hardness in the laser group (figure 11). when orthodontic brackets had their surface treated with laser [6], they showed the highest shear bond strength when compared to sandblasting. on the other hand, bond strength of posts to dentin used in endodontic treatment was affected by the type of material but not by the surface treatment [7]. laser surface treatment of composites used to repair bond strength of cad/cam resin nanoceramic showed improved micro-shear bond strength values, particularly shortpulse lasers [8]. like our results, previous work has shown that surface treatment by sandblasting followed by acid etching was more effective that their individual applications and laser irradiation in terms of resin cad/cam ceramics retention [9]. however, combining laser and sandblasting for surface treatment of zirconia and metal resulted rougher surfaces than sandblasting alone, especially for metal [10]. 3d printing can produce very strong dental alloys with high durability. alloys like titanium and cobalt-chrome are commonly used for 3d printing and offer excellent strength for dental prosthetics. certain 3d printed metals can be even stronger than their conventionally manufactured counterparts. the final hardness of the printed alloy is influenced by the base metal, the energy input of the printing process, and any subsequent heat treatments [11-13]. conclusions hardness values varied. 3d printing generally resulted in the highest hardness values in the control and sandblast groups, whereas hard milling showed superior hardness in the laser group. references 1. roughness, surface energy, and super;icial damages of cad/cam materials after surface treatment. strasser t, preis v, behr m, rosentritt m. clin oral investig. 2018 nov;22(8):2787-2797. doi: 10.1007/s00784-018-2365-6. 2. effect of surface treatments on wear and surface properties of differentcad-cam materials and their enamel antagonists. çakmak g, subaşı mg, sert m, yilmaz b.j. prosthet dent. 2023 mar;129(3):495-506. doi: 10.1016/j.prosdent.2021.06.023. 3. effect of different surface treatments and thermomechanical aging on the ion elution of cadcam materials. sert m, çakmak g, subaşı mg, donmez mb, yilmaz b. j prosthet dent. 2022 jun;127(6):926.e1-926.e10. doi: 10.1016/j.prosdent.2022.03.021. 4. al jabbari ys, barmpagadaki x, psarris i, et al. microstructural, mechanical, ionic release and tarnish resistance characterization of porcelain fused to metal co–cr alloys manufactured via casting and three different cad/cam techniques. j prosthodont res 2019; 63:150-156. 5. yilmaz a, akyil mad , hologlu b. the effect of metal primer application and nd: yag laser irradiation on the shear-bond strength between polymethyl methacrylate and cobalt-chromium alloy. photomedicine and laser surgery. 2011;29:39-45. 6. shear bond strength of orthodontic brackets to specimens fabricated from temporary restorative materials by 3d-printing, cad/cam technology, and the conventional technique after surface treatment by sandblasting and laser. alijani s, fotovat f, rezaei sou;i l, alafchi b, mohammadkhani mh. int orthod. 2023 dec;21(4):100790. doi: 10.1016/j.ortho.2023.100790. 7. surface treatment effects on bond strength of cad/cam fabricated posts to root canal dentin. oguz ahmet bs, egilmez f, ergun g, cekic nagas i. am j dent. 2019 jun;32(3):113-117. 8. effects of er,cr:ysgg laser surface treatments and composites with different viscosities on the repair bond strength of cad/cam resin nanoceramic. degirmenci a, unalan degirmenci b. polymers (basel). 2024 aug 2;16(15):2212. doi: 10.3390/polym16152212. 9. the impact of various surface treatments and er-yag laser irradiation on the bond strength of resin matrix cad/cam ceramics to resin cement. usta kutlu ik, yerliyurt k. lasers med sci. 2025 apr 21;40(1):201. doi: 10.1007/s10103-025-04460-5. 10. effect of combined surface treatments on surface roughness and resin bond strength to y-tzp ceramic and nickel-chromium metal alloy. sayin ozel g, okutan y, oguz ahmet bs, ozdere e. photobiomodul photomed laser surg. 2019 jul;37(7):442-450. doi: 10.1089/photob.2018.4590. 11. 3d printed zirconia used as dental materials: a critical review. su g, zhang y, jin c, zhang q, lu j, liu z, wang q, zhang x, ma j.j biol eng. 2023 dec 21;17(1):78. doi: 10.1186/s13036-023-00396-y. 12. three-dimensional printing and cad/cam milling in prosthodontics: a scoping review of key metrics towards future perspectives. cioloca holban c, tatarciuc m, vitalariu am, vasluianu ri, antohe m, diaconu da, stamatin o, dima am.j clin med. 2025 jul 8;14(14):4837. doi: 10.3390/jcm14144837. 13. effect of surface treatments on the bond strength of computer-aided design and computer-aided manufacturing lithium disilicate to restorative materials: a systematic review. shoorgashti r, ehsani ss, ducret m, rokhshad r.eur j prosthodont restor dent. 2024 nov 29;32(4):423-433. doi: 10.1922/ejprd_2777shoorgashi11. effect of different surface treatment on hardness of dental alloys vol 13, no 1 (2025) doi 10.5195/d3000/2025.1088 http://dentistry3000.pitt.edu 4 table 1. summary of comparisons for hard milling specimens. mean sd std. error "95% confidence interval for mean" f-test p value lower bound upper bound hard milling control (hv) 422.4 1.32 0.41 421.46 423.36 25352 0.001 laser (hv) 548.7 1.59 0.50 547.64 549.92 sand blast (hv) 554.2 1.51 0.47 553.17 555.33 total 508.4 61.9 11.31 485.34 531.62 post hoc analysis (tukey hsd) mean difference p-value control (hv) laser (hv) -126.37364 0.001 sand blast (hv) -131.84160 0.001 laser (hv) sand blast (hv) -5.46797 0.001 table 2. summary of comparisons for soft milling specimens. mean sd std. error "95% confidence interval for mean" f-test p value lower bound upper bound soft milling control (hv) 447.3 0.35 0.11 447.1 447.6 1086 0.001 laser (hv) 452.1 0.45 0.14 451.8 452.4 sand blast (hv) 443.6 0.41 0.13 443.3 443.9 total 447.7 3.56 0.65 446.3 449.0 post hoc analysis (tukey hsd) mean difference p-value control (hv) laser (hv) -4.76252 0.001 sand blast (hv) 3.74538 0.001 laser (hv) sand blast (hv) 8.50789 0.001 table 3. summary of comparisons for laser treatment. mean sd std. error "95% confidence interval for mean" f-test p value lower bound upper bound 3d printer control (hv) 596.3 0.16 0.05 596.2 596.4 21958 0.001 laser (hv) 386.9 0.39 0.12 386.6 387.1 sand blast (hv) 706.2 0.42 0.13 705.9 706.5 total 563.1 134.7 24.5 512.8 613.4 post hoc analysis (tukey hsd) mean difference p-value control (hv) laser (hv) 209.43 0.001 sand blast (hv) -109.85 0.001 laser (hv) sand blast (hv) -319.29 0.001 effect of different surface treatment on hardness of dental alloys vol 13, no 1 (2025) doi 10.5195/d3000/2025.1088 http://dentistry3000.pitt.edu 5 figure 2. the scanning electron microscope (sem) of 3d printer before surface treatment. figure 3. the scanning electron microscope (sem) of soft metal before surface treatment. figure 4. the scanning electron microscope (sem) of hard metal before surface treatment. effect of different surface treatment on hardness of dental alloys vol 13, no 1 (2025) doi 10.5195/d3000/2025.1088 http://dentistry3000.pitt.edu 6 figure 5. the scanning electron microscope (sem) of soft metal after surface treatment sandblast. figure 6. the scanning electron microscope (sem) of soft metal after surface treatment laser. figure 7. the scanning electron microscope (sem) of (3dprinter metal) after surface treatment sandblast. effect of different surface treatment on hardness of dental alloys vol 13, no 1 (2025) doi 10.5195/d3000/2025.1088 http://dentistry3000.pitt.edu 7 figure 8. the scanning electron microscope (sem) of (3dprinter metal) after laser surface treatment. figure 9. the scanning electron microscope (sem) of (hard metal) after sandblast surface treatment. effect of different surface treatment on hardness of dental alloys vol 13, no 1 (2025) doi 10.5195/d3000/2025.1088 http://dentistry3000.pitt.edu 8 figure 10. the scanning electron microscope (sem) of (hard metal) after laser surface treatment. figure 11. hardness comparisons for the study. 868 final the impact of aloe vera herbal extract of the an6fungal and mechanical proper6es of maxillofacial prosthe6c silicones vol 13 no 1 (2025) doi 10.5195/d3000.2025.868 h#p://den*stry3000.pi#.edu the impact of aloe vera herbal extract on the an6fungal and mechanical proper6es of maxillofacial prosthe6c silicones nusaiba mustafa muhammed, safwan abid-alhameed suliman, luma nasrat arab college of dentistry, tikrit university, tikrit, iraq abstract objec&ves: the study aimed to evaluate the an;microbial efficacy and some mechanical proper;es of maxillofacial silicone aaer incorpora;ng aloe vera oil. materials and methods: two concentra;ons of aloe vera oil were added to the room temperature vulcanized maxillofacial silicone. three tests were conducted (disk diffusion test, tear strength, and shore a hardness) using standardized condi;ons. sta;s;cal analysis involved the use of one-way anova and tukey’s post-hoc tests. results: it was revealed that aloe vera oil has strong an;fungal proper;es for 1% and 2% incorporated specimens as there was large inhibi;on zone (5.94 mm and 8.45 mm respec;vely) around the incorporated specimens compared to control specimens. tear strength was decreased aaer the addi;on, while hardness was increased especially for 2% incorporated specimens but s;ll within acceptable limits. conclusions: the study contributes valuable insights into the development of an;microbial silicone material reinforced with aloe vera oil, advancing maxillofacial materials towards improved pa;ent outcomes and enhanced health. keywords: aloe vera; candida albicans; disk diffusion; shore a hardness; tear strength. cita:on: muhamed nm, et al. (2025) the impact of aloe vera herbal extract on the an:fungal and mechanical proper:es of maxillofacial prosthe:c silicones. den:stry 3000. 1:a001 doi:10.5195/d3000.2025.868 received: march 2, 2025 accepted: march 14, 2025 published: april 21, 2025 copyright: ©2025 muhammed nm, et al. this is an open access ar:cle licensed under a crea:ve commons avribu:on work 4.0 united states license. email: nooosamustafamustafa@gmail.com introduction for those with facial damage, congenital malformations, or postsurgical interventions, maxillofacial silicone prostheses are essential in restoring facial aesthetics and function [1,2]. wearers must receive the necessary care to ensure their longevity and wellbeing, and disinfection is an essential component in preventing microbial colonization and maintaining hygiene [3,4]. one of the most significant issues with using silicone prostheses is the microbial colonization of their surface by bacteria, fungus, and plaque, which can lead to mucosal infection and/or inflammation [5,6]. a medicinal cost plant might hold the key to finding a novel antimicrobial agent considering the global consumption of antimicrobial medications, which has resulted in the evolution of resistant bacteria species [7]. many plant extracts can serve as natural alternatives to manufactured medications since medicinal plants are affordable, safe, and efficacious [8,9]. aloe vera is regarded as the most powerful, significant to the economy, and well-liked plant in the scientific community. about 200 active chemicals, including amino acids, carbohydrates, enzymes, vitamins, minerals, saponins, anthraquinones, lignin, and salicylic acid, are present in various plant sections, along with about 75 nutrients [10]. the rind the impact of aloe vera herbal extract of the an6fungal and mechanical proper6es of maxillofacial prosthe6c silicones vol 13 no 1 (2025) doi 10.5195/d3000.2025.868 h#p://den*stry3000.pi#.edu contains carbohydrates, lignin, pectin, hemicellulose, and cellulose, while the blossoms include volatile substances and ascorbic acid. analogously, different organic acids, enzymes, phenolic compounds, minerals, and vitamins can be obtained from the leaves [11,12]. aloe vera is widely recognized for its anti-inflammatory, antioxidant, antidiabetic, immuneboosting, anti-aging, and anticancer qualities. it also relieves sunburn. owing to its distinct composition, aloe vera has been used in a variety of industrial applications [13]. this study provides an overview of the medicinal properties of aloe vera as well as its uses in culinary and cosmetic products. furthermore, spoken about are risks connected to using aloe vera and related safety measures. materials and methods materials used in this study, aloe vera oil (nature in bottle, india) and vst-50 room temperature vulcanized silicone (factor ii inc., usa) were used. specimen grouping in this in-vitro prospective investigation, 90 silicone specimens (30 for antifungal testing, 30 for hardness testing, and 30 for tear testing) were used. the specimens were separated into three groups (10 each) based on the amount of oil combined with silicone (0% control, 1%, and 2%). acrylic molds preparation autocad 2015, developed by autodesk inc. in san rafael, ca, usa, was used to create the dimensions, which were then cut using a laser cutting device (jl1612, jinan link manufacture and trading co., ltd., china) according to each test. the mold consists of three parts: the base, the mold space into which silicone was poured, and the lid. g-clamps, nuts, and screws are used to attach the mold pieces together. mixing as advised by the manufacturer, the mixing ratio was 10 parts a (base) to 1 parts b (catalyst) by weight. the manufacturer also suggests utilizing a vacuum mixer or vacuum chamber (figure 3) to prevent air entrapment, which affects mechanical qualities. to achieve the greatest results from mixing and curing, the operation should be carried out at 23°c and 50% relative humidity [19]. the samples with additional oil were created by adding the oil to silicone material at two different concentrations (1%, and 2% by weight). to establish an appropriate base to catalyst ratio, the additional oil’s weight was deducted from the weight of the base (part a) and then added to the catalyst (part b). preparation and storage of specimens specimens were made by covering the mold cover with two layers of alginate solution as a separating medium, then gently pouring silicone mixture into all sample areas, slightly overfilling to assure completeness. the top piece of the mold was then joined over the matrix part with mild hand pressure, screws, nuts, and gclamps to remove air bubbles and excess silicone. polymerization took 24 hours at room temperature (23±2°c), following which samples were carefully removed from the mold. cleaning included washing with tap water, drying with paper towels, and finetuning with a scalpel to remove excess material [14]. disk diffusion test to conduct the disc diffusion test, sabouraud dextrose agar (oxoid, england) was prepared and put into sterile petri plates per manufacturer directions. the disc the impact of aloe vera herbal extract of the an6fungal and mechanical proper6es of maxillofacial prosthe6c silicones vol 13 no 1 (2025) doi 10.5195/d3000.2025.868 h#p://den*stry3000.pi#.edu diffusion technique, as recommended by who, was used to evaluate the antifungal properties of aloe vera oil put into silicone material specimens. to generate a fungal suspension, 4-8 isolated candida albicans colonies were selected from the incubated culture and placed in a test tube containing 4 ml of normal saline. a sterile cotton swab was then used to inoculate a tiny quantity of the fungal solution and streak it equally across the surface of the sabouraud dextrose agar medium. the infected plates were then allowed to dry for ten minutes. disk shaped specimens (10 mm diameter and 2 mm thickness) were used. the specimen discs were carefully placed on the agar with sterile forceps and gently pushed to establish appropriate contact with the agar surface. plates were then incubated at 30 ºc for 18-24 hours. the inhibition zones were then measured using a millimeter scale. tear strength specimens with an apex and two ends were designed and tested in accordance with the iso 34-1:2015 standard. the thickness measures 2±0.2mm. a digital caliper was used to determine the thickness of the angled section where tearing may occur. the testing was carried out with a universal testing equipment. the testing speed was 500mm/minute. to uniformly distribute the force within the specimen, it must be held accurately between the grips of the machine. the specimens were held 30±0.5 mm apart. the lower grip was fixed while the upper grips were movable. the highest force at break (n/mm) was measured to determine tear strength (figure 1). figure 1. tear strength test using a universal testing machine. shore a hardness the test samples were made in line with iso 7619-1 (2010) criteria, with each measuring 40mm long, 40mm wide, and 6mm thick. these dimensions were chosen to guarantee that there was enough exterior surface area to conduct five measurements with a shore a durometer (ezitown, china) at 6mm between each spot. furthermore, a 12mm distance from the sample margin was maintained to ensure accurate hardness testing in compliance with the standards. the average of the five hardness values was then determined for each sample. statistical analysis the statistical analysis used spss version 26. to establish the difference between groups, independent t test, a one-way anova and a tukey’s post hoc test were used with a significance threshold of p=0.05. p < 0.05 was judged significant, while p < 0.01 was regarded highly significant. results disk diffusion test the results revealed an increase in the inhibition zone around the specimens that were incorporated with 2% aloe vera oil compared to those incorporated with 1% aloe vera oil (table 1 and figure 2), while there was no inhibition zone around control specimens (figure 3). control group was not included in the statistical analysis as there was no inhibition zone around control specimens. groups n mean standard deviation standard error +mean 1% 10 5.949 0.37 0.117 2% 10 8.4521 0.74329 0.23505 table 1. descriptive statistics and one-way anova test for disk diffusion test. the impact of aloe vera herbal extract of the an6fungal and mechanical proper6es of maxillofacial prosthe6c silicones vol 13 no 1 (2025) doi 10.5195/d3000.2025.868 h#p://den*stry3000.pi#.edu figure 2. bar chart representing the means and standard deviation for 2% and 1% incorporated specimens. figure 3. disk diffusion test result for each group. independent t test revealed highly significant difference between 2% specimens and 1% specimens as indicated in (table 2). shore a hardness the experimental groups (1% and 2%) revealed higher hardness levels than control group (figure 4). also, there was highly significant difference between the control group and group 1% and 2% respectively, while there was nonsignificant difference between 1% group and 2% group (table 3). figure 4. bar chart representing the means and standard deviation of shore a hardness for the groups. tear strength tear strength was decreased after the addition of aloe vera oil especially for 2% incorporated levene's test for equality of variances t-test for equality of means f p-value t degrees of freedom p-value (2tailed) equal variances not assumed 6.396 0.021 -9.533 13.202 .000 anova tukey hsd group minimum maximum mean ±sd f pvalue groups pvalue b 44.14 44.83 44.527 0.24676 30.995 0.000 a b 0.000 c 44.88 45.21 45.086 0.10824 a c 0.000 d 44.88 45.63 45.207 0.234 b c 0.395 levene statistics=3.826, p-value=0.34 table 2. independent t test for disk diffusion test. table 3. descriptive statistics, one-way anova, and post hoc test for shore a hardness test. table 4. descriptive statistics, one way anova, and post hoc test for tear strength test. the impact of aloe vera herbal extract of the an6fungal and mechanical proper6es of maxillofacial prosthe6c silicones vol 13 no 1 (2025) doi 10.5195/d3000.2025.868 h#p://den*stry3000.pi#.edu specimens (figure 5) as there was a highly significant difference between 2% group and control group, also a highly significant difference between 2% and 1% groups, while there was a nonsignificant difference between control group and 1% group (table 4). figure 5. bar chart representing the means and standard deviation of tear strength for the groups. table 4. descriptive statistics, one way anova, and post hoc test for tear strength test. discussion the rich composition of bioactive substances in aloe vera, such as acemannan, emodin, and aloin, which are known for their antimicrobial qualities, was the key behind its incorporation into rtv silicone. through a variety of interactions, these components compromise fundamental cellular activities necessary for fungal growth and proliferation and damage the integrity of fungal cell membranes. for example, anthraquinone derivatives such as aloin and emodin can pass through the cell walls of fungi to change the permeability of the membrane and allow internal elements like proteins and ions to seep out. the result of this disturbance is cellular malfunction and apoptosis, which effectively stops fungal growth [15]. the present investigation thoroughly assessed the efficacy of aloe vera oil at 1% and 2% concentrations in conferring antifungal characteristics on rtv silicone. clear inhibition zones surrounding the silicone samples containing aloe vera oil were shown by agar diffusion tests, indicating that the oil had the capacity to stop fungal growth. the reason for this is that bioactive molecules from the oil diffuse into the surrounding agar media. there, they interact with the fungal cells, causing damage to their membrane integrity and preventing them from proliferating [16]. additionally, aloe vera contains acemannan, a polysaccharide that is essential for boosting the immune system's defenses against fungal infections at the molecular level. acemannan stimulates the generation of antimicrobial peptides and increases phagocytic anova tukey hsd group minimum maximum mean ±sd f pvalue groups pvalue b 24.53 27.53 26.0300 .97183 13.728 .000 a b 0.39 c 22.51 28.51 25.2100 1.82878 a c 0.000 d 21.42 25.42 22.9200 1.17851 b c 0.003 levene statistics=2.017, p-value=0.153 the impact of aloe vera herbal extract of the an6fungal and mechanical proper6es of maxillofacial prosthe6c silicones vol 13 no 1 (2025) doi 10.5195/d3000.2025.868 h#p://den*stry3000.pi#.edu activity through interactions with immune cells and cytokines. acemannan may improve rtv silicone's capacity to control immune responses at the application site, which would increase the material's antifungal activity in concert [17,18]. aloe vera oil and rtv silicone interact chemically when bioactive substances are incorporated into the silicone matrix during the curing process. aloe vera oil molecules are incorporated into silicone polymer chains by crosslinking, which enables an antibacterial agent to release continuously throughout time. aloin, emodin, acemannan, and other active ingredients are continuously exposed to fungal infections thanks to this sustained release mechanism, which prolongs the antifungal action [19]. physically, aloe vera oil may change the hydrophobicity and surface characteristics of rtv silicone, affecting how well it interacts with fungi and their biofilms. fungal colonization and persistence can be further inhibited by including hydrophilic and amphiphilic components from aloe vera oil, which can interfere with biofilm development and adhesion to silicone surfaces [20]. notwithstanding the advantageous antifungal characteristics noted, there was a discernible compromise in the silicone's mechanical attributes, specifically regarding tear strength. in applications like maxillofacial prosthesis, where durability and longevity are critical for long-term performance, the tearing of silicone materials is important. there are several reasons why adding aloe vera oil, particularly at higher doses (2%), resulted in a reduction in tear strength. disruption in the silicon network since aloe vera oil is a non-reactive filler, it might damage the silicone matrix's crosslinked structure. oil can prevent silicone chains from forming strong bonds during the vulcanization process, which is when silicone chains crosslink to form a sturdy network. as a result, the material develops isolated weak points that make it more [21]. aloe vera oil serves as a plasticizer, improving the flexibility and decreasing the overall stiffness of silicone. while this can improve some qualities, too much plasticization can reduce tear resistance by weakening cohesiveness between silicone strands. the inclusion of aloe vera oil may cause gaps or abnormalities in the silicone matrix. these microstructural alterations can operate as stress concentration areas, allowing tears to form and spread more easily under mechanical loads [22,23]. interestingly, despite the decreased tear power, the addition of aloe vera oil resulted in a modest rise in shore a hardness. shore hardness is a measure of a material's resistance to indentation, with higher values suggesting stiffer material. this rise can be ascribed to oil filling up micro voids or holes in the silicone matrix, resulting in a denser material structure [24]. however, it is worth noting that the increase in hardness was minor in comparison to the loss in tear strength, indicating a complicated interplay between material attributes. conclusions in conclusion, while aloe vera oil shows promise as a natural antimicrobial additive for rtv maxillofacial silicone, its incorporation necessitates a careful balance between enhancing antifungal properties and maintaining mechanical integrity. further research is essential to refine formulations and optimize performance for practical medical applications. funding self-funded. the impact of aloe vera herbal extract of the an6fungal and mechanical proper6es of maxillofacial prosthe6c silicones vol 13 no 1 (2025) doi 10.5195/d3000.2025.868 h#p://den*stry3000.pi#.edu references 1. slijepcevic aa, afshari a, vitale ae, couch sm, jeanpierre lm, chi jj. a contemporary review of the role of facial prostheses in complex facial reconstruction. plastic and reconstructive surgery. 2023 feb 1;151(2):288e-98e. 2. plana nm, barbosa jm, diaz-siso jr, brecht le, rodriguez ed. dental considerations and the role of prosthodontics and maxillofacial prosthetics in facial transplantation. the journal of the american dental association. 2018 feb 1;149(2):90-9. 3. hung st, cheng yc, wu cc, su ch. examining physical wellness as the fundamental element for achieving holistic well-being in older persons: review of literature and practical application in daily life. journal of multidisciplinary healthcare. 2023 dec 31:1889-904. 4. marquez dx, aguiñaga s, vásquez pm, conroy de, erickson ki, hillman c, stillman cm, ballard rm, sheppard bb, petruzzello sj, king ac. a systematic review of physical activity and quality of life and well-being. translational behavioral medicine. 2020 oct;10(5):1098-109. 5. van den berg d, asker d, awad ts, lavielle n, hatton bd. mechanical deformation of elastomer medical devices can enable microbial surface colonization. scientific reports. 2023 may 11;13(1):7691. 6. spałek j, deptuła p, cieśluk m, strzelecka a, łysik d, mystkowska j, daniluk t, król g, góźdź s, bucki r, durnaś b. biofilm growth causes damage to silicone voice prostheses in patients after surgical treatment of locally advanced laryngeal cancer. pathogens. 2020 sep 26;9(10):793. 7. vaou n, stavropoulou e, voidarou c, tsigalou c, bezirtzoglou e. towards advances in medicinal plant antimicrobial activity: a review study on challenges and future perspectives. microorganisms. 2021 sep 27;9(10):2041. 8. nasim n, sandeep is, mohanty s. plant-derived natural products for drug discovery: current approaches and prospects. the nucleus. 2022 dec;65(3):399-411. 9. atanasov ag, zotchev sb, dirsch vm, supuran ct. natural products in drug discovery: advances and opportunities. nature reviews drug discovery. 2021 mar;20(3):200-16. 10. sánchez gómez-serranillos m, gonzález burgos e, iglesias peinado i, gómez-serranillos cuadrado mp. pharmacological update properties of aloe vera and its major active constituents. 11. rao j, lv z, chen g, peng f. hemicellulose: structure, chemical modification, and application. progress in polymer science. 2023 may 1;140:101675. 12. abdo khairallah mk, alkabani y, refai w, mohamed km. in vitro comparison of the mechanical properties of single and triple layered clear aligner materials. ain shams dental journal. 2024 sep 1;35(3):53-72.. 13. naini ma, zargari-samadnejad a, mehrvarz s, tanideh r, ghorbani m, dehghanian a, hasanzarrini m, banaee f, koohi-hosseinabadi o, tanideh n, iraji a. antiinflammatory, antioxidant, and healing-promoting effects of aloe vera extract in the experimental colitis in rats. evidence-based complementary and alternative medicine. 2021;2021(1):9945244. 14. mishra p, paul m, vinod p, sarathi r, kornhuber s. performance evaluation of room temperature vulcanized silicone rubber nanocomposites aged in strong aqueous solutions. polymer engineering & science. 2022 may;62(5):1619-30. 15. stompor-gorący m. the health benefits of emodin, a natural anthraquinone derived from the impact of aloe vera herbal extract of the an6fungal and mechanical proper6es of maxillofacial prosthe6c silicones vol 13 no 1 (2025) doi 10.5195/d3000.2025.868 h#p://den*stry3000.pi#.edu rhubarb—a summary update. international journal of molecular sciences. 2021 sep 1;22(17):9522. 16. yeshi k, wangchuk p. essential oils and their bioactive molecules in healthcare. inherbal biomolecules in healthcare applications 2022 jan 1 (pp. 215237). academic press. 17. bai y, niu y, qin s, ma g. a new biomaterial derived from aloe vera—acemannan from basic studies to clinical application. pharmaceutics. 2023 jul 9;15(7):1913. 18. sadgrove nj, simmonds ms. pharmacodynamics of aloe vera and acemannan in therapeutic applications for skin, digestion, and immunomodulation. phytotherapy research. 2021 dec;35(12):657284. 19. sánchez gómez-serranillos m, gonzález burgos e, iglesias peinado i, gómez-serranillos cuadrado mp. pharmacological update properties of aloe vera and its major active constituents. 20. nizam nh, rawi nf, ramle sf, abd aziz a, abdullah ck, rashedi a, kassim mh. physical, thermal, mechanical, antimicrobial and physicochemical properties of starch based film containing aloe vera: a review. journal of materials research and technology. 2021 nov 1;15:1572-89. 21. barney cw, helgeson me, valentine mt. network structure influences bulk modulus of nearly incompressible filled silicone elastomers. extreme mechanics letters. 2022 apr 1;52:101616. 22. shawky ym, maged ar. evaluation of maximum biting force and chewing efficiency in patients with digital complete dentures constructed using two different workflows: a cross-over randomized controlled trial. ain shams dental journal. 2024 1;35(3):1-1. 23. eslami z, elkoun s, robert m, adjallé k. a review of the effect of plasticizers on the physical and mechanical properties of alginatebased films. molecules. 2023 sep 15;28(18):6637. 24. kashi s, de souza m, al-assafi s, varley r. understanding the effects of in-service temperature and functional fluid on the ageing of silicone rubber. polymers. 2019 feb 26;11(3):388. 1004 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1004 http://dentistry3000.pitt.edu impact of sinalized silicon carbide nanoparticles on the physical characteristics of a heat-cured acrylic soft liner zeena majid abdul amir, ghasak husham jani college of den*stry, university of baghdad, iraq abstract objec9ve: the purpose of this research was to examine how adding salinized silicon carbide np to the soo denture lining material affected its thermal conducqvity, water sorpqon, and solubility. material and methods: silicon carbide nps were added to the heat-cured soo lining material at quanqqes of 0.4%, and 0.6% by weight. the sixty specimens have been generated in compliance with the prescribed tests. results: when compared to control specimens, soo lining material with 0.6% and 0.4% wt. sic np had a highly significant decrease in solubility, a non-significant increase in thermal conducqvity, and a non-significant decrease in water absorpqon. conclusion: the addiqon of silicon carbide nanoparqcles into heat cured acrylic denture soo liner material improved thermal conducqvity and decrease water sorpqon and solubility. open access cita%on: amir maa, et al. (2025) impact of sinalized silicon carbide nanopar%cles on the physical characteris%cs of a heat-cured acrylic sog liner. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1004 received: july 28, 2025 accepted: july 31, 2025 published: september 9, 2025 copyright: ©2025 amir maa, et al. this is an open access ar%cle licensed under a crea%ve commons avribu%on work 4.0 united states license. email: zeenaalkabi555@gmail.com introduc)on dentists have been using soft denture liners for almost a century, with natural rubber being the 9irst [1]. resilient denture liners can 9ix the problem of dentures becoming ill-9itting from residual ridge resorption, which causes discomfort and agony to the patient, while retention is necessary for effective dentures [2]. for complete dentures to be comfortable, 9lexible lining materials must be used. for example, if a patient has thin, non-pliable mucosal tissue covering their irregular alveolar margin, chewing forces applied through a solid acrylic base could be painful. by using soft lining materials, this pain could be alleviated, and the patient would be more likely to accept their dentures [3,4]. the soft lining materials consist of a variety of synthetic and natural substances, including hydrophilic polymers, silicone rubber, polyphosphazine 9luoropolymers, 9luoroethylene, and silicones added to polyvinyl siloxane. silicone rubber and plasticized acrylics are the two most popular types of soft liners, and they are activated either by heat or chemicals [5]. to be ideal, soft denture liners should exhibit certain properties to ensure a maximum bene9it for denture wearers; among these properties are the biocompatibility, dimensional stability, good resiliency, softness, proper wettability, color stability, low water solubility, suf9icient bond strength with the underlying denture base and the ability to inhibit or reduce the microbial growth [6]. the patient's level of satisfaction with their dentures is affected by the soft denture liners' inability to adequately transfer heat from the denture base to the oral mucosa, furthermore, the parotid gland's secretion and the health of the underlying supporting tissues are signi9icantly impacted by the denture base's poor heat conductivity [7,8]. soft lining materials have a lot of issues with water solubility and sorption. with time, the plasticizers and other important ingredients in the material will leach out, creating space for other particles and water to enter. this changes the material's chemical structure and physical properties [9]. due to the gradual loss of plasticizer, the modulus of elasticity of soft-liner material would rise; consequently, its resilience, an essential property, will be compromised. in addition, the epithelial tissue may have a negative response to the release of some plasticizers, such phthalate ester [10]. silicon carbide is a great material for structural and functional materials due to its high mechanical strength, minimal friction, chemical stability, and outstanding thermal conductivity [11]. impact of sinalized silicon carbide nanoparqcles on the physical characterisqcs of a heat-cured acrylic soo liner vol 13, no 1 (2025) doi 10.5195/d3000.2025.1004 http://dentistry3000.pitt.edu 2 nanoparticles' exceptional physical features, including high-9lexibility, enhanced strength, and ease of form replication, have garnered a lot of interest for their incorporation into polymer-matrices [12]. without using dispersing agent, inorganic particles are not easily distributed in polar organic matrix. for this reason, to produce stable chemical bonds with both inorganic and organic materials silane coupling agents are often used. so, the silane function as adhesion promoter that is to improve the binding of nanoparticles to the matrix of polymer [13]. material and methods silicon carbide surface modi1ication the nanoparticles of silicon carbide (sic, beta, 99.9% purity, less than 80 nm, cubic, nanoshel, usa) were treated with a silane coupling agent (trimethoxysilylprpoyl methacrylate tmspm cheng du micxy chemical co. ltd. 2530-86-0. china) to enhance their binding to the polymer matrix and to introduce reactive groups into the nanoparticles. typical process as followed, 30g of nanoparticles and 200 milliliters pure toluene were placed into a 9lask then sonicated at ambient temperature for 20 minutes. the standard procedure involved adding 30 grams of nanoparticles to 200 milliliters of pure toluene in a 9lask and sonicating the mixture at room temperature for 20 minutes. twenty hours were spent drying the modi9ied nano 9iller in a vacuum oven set at 60°c. the nano9iller is thereafter left at room temperature until it is needed [14,15]. by examining the distinctive vibrations of functional groups, the ftir spectrophotometer can identify if mps functional groups are bonded to nanoparticles [16]. preparation of specimens the soft denture liner material was made of heat-cured acrylic and was sourced from moonstar in turkey. nanoparticles of silicon carbide were included into the soft liner's liquid component. results from the pilot investigation indicated that concentrations of silicon carbide nps between 0.4% and 0.6% produced the best shear bond strength and hardness increases. the monomer was combined with the measured amount of nano sic to form the dough with the correct quantity of nano9iller. the nanoparticles were then uniformly dispersed throughout the monomer by use of a probe sonication equipment operating at 120 w and 60 khz for a duration of three minutes [17]. the manufacturer's instructions required the creation of acrylic specimens; thus, a new plastic design was made. the mold was then constructed following the standard procedure for making full dentures. experimental groups control group without addition of nanoparticles (n=10, for each test). experimental group with 0.4 % wt. sci nanoparticles (n=10, for each test). experimental group with 0.6 % wt. sci nanoparticles (n=10, for each test). evaluation of thermal conductivity disks with 40 mm diameters and 5 mm thicknesses have been manufactured for thermal property testing in accordance with instrument standards iso 22007-2. the specimens were subjected to thermal conductivity testing using the hot disk thermal analyzer (tps 500, kiteley, sweden), which uses a double spiral design that extends from a thick sheet of nickel foil. once the polymer parameter is selected, the system may be tested with a thermal conductivity value 15 minutes after the hot disk is turned on, which should be done around 1 hour before to the testing. evaluation of water sorption and solubility the dissecator was used to dry the specimens using recently dried silica gel. after being incubated at 37°c ±2 °c for 24 hours, the specimens were allowed to cool to ambient temperature for an additional hour. afterwards, they were precisely weighed using a digital scale, which had an accuracy of (0.000lg). after 9ive days of this, we obtained a constant mass "conditioned mass" (m1), which indicates that the weight loss from each disc was less than 0.2 mg in 24 hours (ada speci9ication no.12, 2000). following a 7-day immersion in distilled water at 370c ± 2.0c, the specimens were taken out of the water using tweezers and dried with a clean, dry hand towel for 30 seconds. after that, they were permitted to air for 15 seconds before being weighed; the resulting value represents m2. as was done before for the sorption test, the discs were reconditioned to a constant mass in the desiccators at 37°c ±2 °c to get the solubility value; this time, the reconditioned mass was recorded as (m3). within 9ive days, the entire group arrived at m3. the following equations were used to calculate the solubility and water sorption: water sorption = (m2 – m1) / s water solubility= (m1m3) / s m1=initial weight of specimen m2=weight after specimen immersion in water m3=weigh after specimen placed in desiccators s=surface area atomic force microscopy (afm) analysis the surface topography and morphology of specimens with 0%, 0.4%, and 0.6% concentrations were examined using atomic force microscopy. the prepared specimens were identical to the hardness test specimens in terms of dimensions (35 mm wide and 6 mm deep). results investigating the properties of nano1illers made of silanized silicon carbide (sic) as shown in figure 1, it is demonstrated that the ftir result of sic after silanization displays the same absorption peaks as sic before salinization. this indicates that the sic nanoparticles and the silane coupling agent do not form a chemical connection. figure 2 displays the twoand three-dimensional pictures captured by the afm investigation. the twoand three-dimensional images of all have shown unevenly distributed granular 9ilms with large diameters of protuberances. grain number and average roughness have increased after nanoparticles addition when compared to untreated specimens. results for both the untreated and treated samples were compared in terms of thermal conductivity, water sorption, and water solubility. to establish the importance of adding sci nps to acrylic soft-liner specimens, descriptive statistics and one-way anova were used to conduct comparative analysis for each group. table 1 displays the results of the thermal conductivity test, which showed a high mean value in the 0.6% wt. sci np group and a statistically non-signi9icant rise in the one-way anova across all groups (p>0.05). water sorption test results reveal a non-signi9icant decrease in water sorption when comparing all groups in a one-way anova with a pvalue of more than 0.05. the water solubility showed a statistically signi9icant reduction in water solubility when comparing all groups in a one-way anova with a signi9icance level of p<0.05. discussion the use of denture lining materials has grown in signi9icance in the 9ield of dental prosthetics due to the following reasons: increased patient comfort, more uniform force distribution, less localized pressure, and better denture retention for patients whose numbers are projected to rise over the next 20 years [18]. results from integrating sic np into pema were assessed in this study. a study was impact of sinalized silicon carbide nanoparqcles on the physical characterisqcs of a heat-cured acrylic soo liner vol 13, no 1 (2025) doi 10.5195/d3000.2025.1004 http://dentistry3000.pitt.edu 3 conducted to examine the physical features of the soft liner, including its heat conductivity, water sorption, and solubility. a material's thermal conductivity may be de9ined as its capacity to evaluate the rate of heat transfer over a certain period in a speci9ic cross-sectional area of the material samples [19]. poor thermal conductivity is a major concern with poly ethyl methacrylate (pema) resin, which impacts both the patient's acceptance of the prosthesis and the health of the tissues supporting the denture [19]. the thermal conductivity reveals a non-signi9icant increase of 0.4% and 0.6% with the introduction of sic nps into pema. this may happen because of particles coming into touch with one another over time, creating a network like a structure known as thermal pathways. these routes facilitate the transfer of heat from one area of the sample to another, effectively bridging the insulating effect of the polymer. solubility and water sorption were assessed concurrently by measuring the amount of water gained or lost by soluble components [20]. the bonding strength of the liner-denture base contact weakens because the water sorption by the lining material causes changes in dimension and stress concentration. how much water a polymer absorbs depends on the 9iller and how it bonds to the polymer. incorporating silicon carbide nps into soft denture liner at the concentrations of 0.4% and 0.6% utilized in this research reduced the water sorption mean value, while the effect was not statistically signi9icant. adding sci nps may have reduced the number of pema molecules on the specimen's surface, which in turn reduced water diffusion. the hydrophobic property of sci aids in minimizing the in9iltration of water into the polymer. another possibility is that the microporosity that forms during polymerization makes it easier for the polymer to absorb 9luids. because of their 9iller properties, the inclusion of sci nps reduced the water sorption by decreasing the size of these spaces. this study found that adding 0.4%, 0.6% sic nps signi9icantly reduced water solubility compared to the control group. this might be because the soft lining material's water sorption characteristics decreased as the amount of sci nps increased. because of this restriction in the dispersed water, molecular 9lexibility and the extraction of soluble components from the polymer mass are less likely to occur. another possible explanation is that the solubility in this study was measured by the loss of specimen weight. since sci nps are insoluble in water, they added mass to the specimens and acted as impurities, reducing their solubility mean value. this could explain why there was a decrease in solubility mean value as the amount of sic nps increased. conclusion according to the study's criteria, adding sci nps to soft liner material can improve its heat conductivity, minimize water sorption, and make it less soluble. funding no speci9ic grant was given to this research by funding organizations in the public, private, or not-for-pro9it sectors. author’s contribu)ons each author agreed to be accountable for every part of this work and contributed to data analysis, writing, and revision of the article. conflict of interest according to the author, there is no con9lict of interest. references 1. garcia lt, jones jd. soft liners. dent clin north am 2004;48(3):709-720. 2. qanber lm, hamad ti. effect of plasma treatment on the bond of soft denture liner to conventional and high impact acrylic denture materials. j bagh coll dent . 2021 sep. 15 [cited 2024 sep. 29];33(3):9-17. 3. y. k. o[ zkan, "long-term 6 dentures," complete denture prosthodontics: treatment and problem solving, p. 197, 2019. 4. m. s. alaa’a, "effect of aging on bond strength of two soft lining materials to a denture base polymer," the journal of indian prosthodontic society, vol. 14, no. 1, pp. 155-160, 2014. 5. zarb ga, bolender cl, eckert s, jacob r, fenton a, mericske stern r. prosthodontic treatment for edentulous patients. complete dentures and implant-supported prostheses. 12th. ed. st. louis: mosby 2004. 6. bushra m alamen, ghassan abdul-hamid naji, mohammed a alsmael, the effect of virgin coconut oil addition on the hardness and wettability of acrylic based denture soft lining material, j res med dent sci, 2020, 8(1):96-106. 7. kapur kk, fischer ee. effect of denture base thermal conductivity on gustatory response. j prosthet dent 1981; 46:603-609. 8. samir kamil abdulmajeed, hikmat jameel abdulbaqi, the impact of calcium carbonate nanoparticles incorporation into heat cured soft denture lining material on thermal conductivity and some other properties, j res med dent sci, 2023, 11 (01): 208-214. 9. v. m. urban et al., "effect of the addition of antimicrobial agents on shore a hardness and roughness of soft lining materials," journal of prosthodontics, vol. 24, no. 3, pp. 207-214, 2015. 10. b. m. hussein, i. j. ismail, and h. a. khalaf, "effect of some disinfectant solutions on the hardness property of selected soft denture liners after certain immersion periods," journal of the faculty of medicine baghdad, vol. 51, no. 3, pp. 259-264, 2009. 11. chen y, cao m, xu q, et al. electroless nickel plating on silicon carbide nanoparticles. surf coat technol 2003; 172:90-4. 12. guo z, kim ty, lei k, et al. strengthening and thermal stabilization nanocomposites nanoparticles with by of polyurethane silicon a carbide surface-initiated polymerization approach. compos sci technol 2008; 68:164-70. 13. pavan s, arioli filho jn, dos santos ph, et al. effect of disinfection treatments on the hardness of soft denture liner materials. j prosthodont 2007; 16:101-116. 14. mancuso dn, goiato mc, zuccolotti bcr, et al. effect of thermocycling on hardness, absorption, solubility and colour change of soft liners. gerodontology 2012; 29:215-219. 15. avella m, errico me, martuscelli e. novel pmma/ caco3 nano composites abrasion resistant prepared by an in situ polymerization process. nano lett 2001; 1:213–217. 16. abdullayev e, lvov y. halloysite clay nanotubes as a ceramic skeleton for functional biopolymer composites with sustained drug release. j mater chem b 2013; 1:2894-2900. 17. taurozzi js, hackley va, wiesner m. preparation of nanoparticle dispersions from powdered material using ultrasonic disruption. nist special publication 2012; 1200:1200-1202. 18. akin, h., et al. (2011). "effect of different surface treatments on tensile bond strength of silicone-based soft denture liner." lasers in medical science 26: 783-788. 19. sakaguchi rl, powers jm. craig's restorative dental materials e-book. elsevier health sciences, 13th edition, united state, 2012. 20. issa mi, abdul-fattah n. evaluating the effect of silver nanoparticles incorporation on antifungal activity and some properties of soft denture lining material. j bagh coll dent [internet]. 2015 jun. 15 [cited 2024 sep. 30];27(2):17-23. impact of sinalized silicon carbide nanoparqcles on the physical characterisqcs of a heat-cured acrylic soo liner vol 13, no 1 (2025) doi 10.5195/d3000.2025.1004 http://dentistry3000.pitt.edu 4 table 1. thermal conductivity, water sorption and solubility. thermal conductivity test groups n mean std. deviation p-value 0% 10 0.1909 0.01296 0.4% 10 0.1915 0.04040 0.6% 10 0.1987 0.03562 total 30 0.5811 0.08898 0.833 water sorption 0% 10 2.09 11.72 0.4% 10 1.63 4.12 0.6% 10 0.31 5.40 total 30 4.03 21.24 0.082 water solubility 0% 10 16.35 24.40 0.4% 10 10.39 35.21 0.6% 10 6.22 22.82 total 30 32.96 82.43 0.021 figure 1. ftir spectrum of silicon carbide nanoparticles (a) before surface modi9ication and (b) after surface modi9ication. a b impact of sinalized silicon carbide nanoparqcles on the physical characterisqcs of a heat-cured acrylic soo liner vol 13, no 1 (2025) doi 10.5195/d3000.2025.1004 http://dentistry3000.pitt.edu 5 e a b c d impact of sinalized silicon carbide nanoparqcles on the physical characterisqcs of a heat-cured acrylic soo liner vol 13, no 1 (2025) doi 10.5195/d3000.2025.1004 http://dentistry3000.pitt.edu 6 figure 2. afm (atomic force microscopy) and three-dimensional images of (a+b) control specimens, (c+d) 0.4% specimens, and (e+f) 0.6% specimens. e f 979 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.979 http://dentistry3000.pitt.edu fluoride release of new bioactive orthodontic adhesive with color change and fluorescent property mohammed thanoon younis, neam fakhri college of den*stry, university of mosul, iraq abstract objec8ve: this study aimed to assess the effect of adding fluorescent dyes & color change dyes in different concentramon to bioacmve orthodonmc adhesive on fluoride release. material and methods: we used bioacmve beautifil injectable xsl (s-prg), from (giomer, shofu, japan) mixed with color change dye, black changing to colorless, (atlanta chemical engineering, usa). 0.02%, 0.2% and 2% of weight concentramons were tested and with fluorescence dye (stronmum aluminate), and white glow in the dark powder (techno glow inc., usa), using in 5%, 10% and 15% of weight concentramons. for fluoride release, 40 samples prepared and divided into 4 groups with 10 samples as following: group 1: beautifil injectable xsl adhesive (control group), group 2: beautifil injectable xsl with 0.02% color change material and 5% fluorescence material. group3: beautifil injectable xsl with 0.2% color change material and 10% fluorescence material. group 4: beautifil injectable xsl with 2% fluorescence material and 15% fluorescence material. fluoride ion selecmve electrode. eutech ion 2700. ( thermo fisher scienmfic inc. singapore) used to measure the release of fluoride ion. results: the use of dyes with bioacmve adhesive showed stamsmcally significant differences. there was a decrease in fluoride ion with increase dye concentramon. conclusion: acceptable fluoride ion release within bioacmve adhesive with color change and fluorescence propermes was obtained but with increase concentramon of dyes the ion release decreased. open access cita%on: younis mt, et al. (2025) fluoride release of new bioac%ve orthodon%c adhesive with color change and fluorescent property. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.979 received: july 3, 2025 accepted: july 4, 2025 published: august 21, 2025 copyright: ©2025 mahmood aa. this is an open access ar%cle licensed under a crea%ve commons atribu%on work 4.0 united states license. email: mohammedhanoon@uomosul.edu.iq introduc)on orthodontic treatment involves using 3ixed or removable appliances to correct the positions of teeth. fixed appliance treatment is a traditional and widely used form of orthodontic treatment to correct malpositions of the teeth and occlusal discrepancies [1,2]. the movement of teeth is achieved by forces generated and directed to the teeth via arch wires and brackets [3]. during the active treatment, the arch wires are changed as the treatment progresses, but the brackets remain attached to the enamel for the whole active treatment period. a wide variety of orthodontic adhesives are available for the bonding brackets and orthodontic attachments [4,5]. resin adhesives are a good choice for orthodontic bonding as they have good mechanical and aesthetic properties and low failure rates. orthodontic adhesives should provide suf3icient strength to retain the appliance during treatment and allowing its easy removal at the end [6]. patients with 3ixed orthodontic appliances show increased risk of white spot lesions (wsl) [7] and caries due to dif3iculty in maintaining oral hygiene [8,9]. attempts were made to reduce enamel demineralization by introducing 3luoride releasing adhesives [10], e.g. amorphous calcium phosphate (acp) containing adhesives [11]. we tested light bond from reliance company, transbond plus adhesive from 3m unitek company [12], glass ionomer and resin modi3ied glass ionomer [13], 3luoridate varnish [14], and topical 3luoride agents [15]. another solution proposed by research teams is introduction of bioactive glass (bag) into the composition of orthodontic adhesives [16]. bioactive glass material included the giomers, as a surface pre-reacted glass core (sprg). it helps prevent and treat white spots lesions [17]. giomers are useful in the process of collating orthodontic brackets with mechanical properties like composite resins and it offers protection against carious lesions [18]. s-prg releases various ions (3luoride, sodium, silicate, aluminum, borate and strontium ions that provide multiple biological functions, including the release and fluoride release of new bioacmve orthodonmc adhesive with color change and fluorescent property vol 13, no 1 (2025) doi 10.5195/d3000.2025.979 http://dentistry3000.pitt.edu 2 recharge of 3luoride, anti-plaque, anti-bio3ilm effects and ph modulation that providing protection against caries [19]. another problem associated with orthodontic adhesive is the presence of excess of adhesive escaping from under the bracket base which promotes the accumulation of food debris and creates favorable area for bacterial collection which led to facilitating the demineralization and formation of wsl [20,21]. the solution turned out to be introduce adhesive characterized by a contrasting color before cross-linking, which facilitates removal of the excess prior to curing, e.g. transbond plus (3m unitek, usa), and grengloo and blugloo (ormco corporation, usa) [22]. color change adhesive is manufactured by adding chromatic indicators, which facilitate the visibility of excess orthodontic adhesive around orthodontic brackets before bonding procedure [23]. this color characteristic allowed the operator to see the adhesive 3lash around the bracket base and remove it before it polymerized [24]. the third problem associated with orthodontic treatment is debonding [25], leaving the remnant of orthodontic adhesive on the enamel surface facilitates plaque and dental caries formation [26]. also, using rotating dental instruments to remove the remaining white or transparent adhesive can cause damage to tooth enamel [27]. to overcome this problem, fluorescent orthodontic adhesive has been developed to improve the visibility of the remaining adhesive after debonding by using ultraviolet light [28,29]. fluorescent additives will facilitate the discrimination between the enamel and remnants of the adhesive. this modification can maximize the preservation of tooth structure after debonding procedure [30]. the aim of this research was to evaluate the effect of adding color change and 3luorescent dies to bioactive composite to be used as an orthodontic adhesive by evaluate the degree of conversion. materials and methods this study was conducted at mosul university, dentistry college, dental hospital central laboratory and was approved by the ethics committee of college of dentistry, mosul university, iraq (under the code uom.dent.23/49). to measure 3luoride release, 40 samples of the tested materials (10 sample for each group) were used: group 1: beautifil injectable xsl adhesive (control group) group2: beautifil injectable xsl with 0.02% color change material and 5% flourcence material. group3: beautifil injectable xsl with 0.2% color change material and 10% flourcence material. group4: beautifil injectable xsl with 2% color change material and 15% flourcence material. plastic cylinder molds measuring 4 mm in diameter and 2 mm in height were used to construct the specimens for each group. celluloid mylar strips and glass slides were placed over the mold's top and bottom surfaces. as at room temperature, the adhesive then put within the mold. the central of samples were pressed between a celluloid strip and a glass cover slip and placed in plastic mold rings to extrude the excess material, to obtain a smooth 3lat surface, to prevent air bubble formation & to prevent oxygen layer formation. then light cure the materials by using led with at 1500 mw/cm2 were cured from the top & bottom for 20sec. after light curing, each set specimen was released from the mold and placed into a polyethylene test tube. 3illed with 5 ml of deionized water. after that, each test tube was sealed, labelled, and arranged as previously indicated before being kept for 24 hours at 37°c with 95% relative humidity [31=-33]. fluoride measurements were performed on 1st day (24 h), 7 days (1 week), and 30 days (1 month). all of the samples were maintained at 37°c in a incubator throughout the experiment. in this examination before testing the containers were thoroughly shaken and the specimens removed, washed, returned and immersed into a new 5 ml of fresh deionized water fresh solution. meanwhile, the 3luoride ions concentration in the storage media was measured. the 1st measurement was done after 24 h from sample preparation, and then 24 hours before day 7th and day 30th. the storage medium was changed with new 5 ml of fresh deionized water fresh solution every 24 h to avoid cumulative effects and because it is possible that the medium may get saturated with the released 3luoride ions, preventing further 3luoride ion release. this was done by using a 3luoride ion selective electrode (eutech ion 2700 meter, thermo fisher scienti3ic inc., singapore) attached to an ion selective electrode meter. for statistical analysis, the amount of 3luoride in each solution was measured and recorded as part per million (ppm). before and after each measurement, the electrode tip was washed and lightly dried with deionized water to remove any residual 3luoride ions which may affect the measurement [34,35] (figure 1). results fluoride release values are represented in units of part per million (ppm) and shown in table 1. the analysis of variance of one way (anova) test for each group revealed significant differences (p≤0.05). discussion fluoride is clearly known as an anti-caries agent, and 3luoride release is an important part of restorative materials, 3luoride can help reduce tooth decay by reducing bacterial metabolism and increasing the resistance of enamel and dentin [36,37]. giomer is one of modern restorative material that contain in its chemical structure combination of 3luoroaluminosilicate glass, polyalkenoic acid and water, with resin included. what differentiates giomer from other 3luoride-containing restorative materials is that they contain a pre-reacted glass (s-prg) 3iller in their matrix. this 3iller facilitates the release of 3luoride ions [35,38]. due to the absence of an acid-base reaction, giomer materials do not have a glass ionomer matrix phase. the quantity of 3luoride released by giomer materials was discovered to be less than that of gic since they only include sprg particles as a 3luoride component. the materials' antibacterial properties rely on metal ions such aluminum, strontium, zirconium, and barium in addition to the 3luoride that is emitted [39,40]. numerous studies shown that giomer possesses physical qualities that might compete with other composite resin, as well as a high 3luoride release and rechargeability [41]. the quantity of water absorbed, the giomer's porosity, the 3iller, the water content, the solubility of ytterbium tri3luoride in water, and the resin's permeability all affect how many ions are released from the giomer [42]. giomer has a 3luorine concentration of only 4.13% [43]. fluoride release from the material is important due to the formation of 3luorapatites as well as the anti-caries property that can prevent the formation of microorganisms. the smallest quantity of 3luoride that must be released to inhibit demineralization and promote the remineralization has not been precisely determined [44]. some authors reported that this value would be between 0.02 and 0.06 ppm [45]. others said that 0.2 ppm signi3icantly reduces the risk of dental caries lesions [46]. fluoride released from the restorative material reduced the solubility of dental tissue in acidic environments, this property being based on the 3luoride capacity to incorporate itself into the crystalline structure of the hydroxyapatite of the dental hard tissue, resulting in a mineral phase which was less soluble & more resistant to the cariogenic challenge. so, since enamel solubility is low when 3luoride ions are present in saliva & bio3ilm, it is desirable to select dental materials with the highest & longest 3luoride release [47,48]. fluoride release of new bioacmve orthodonmc adhesive with color change and fluorescent property vol 13, no 1 (2025) doi 10.5195/d3000.2025.979 http://dentistry3000.pitt.edu 3 many methods have been employed to estimate the sum of fluoride releases such as spectrophotometry, ion chromatography, fluoride ion-specific electrodes and capillary electrophoresis [49]. ion-specific electrode with an ion analyzer was used in this study because it is simple, inexpensive and does not require the use of complex laboratory equipment. also, it gives an accurate and direct estimate of the free fluoride present in the solution [50]. to measure the 3luoride release of restorative materials, a variety of media, including deionized water, arti3icial saliva and acidic media, may be selected. since deionized water is readily available & contains no ions, it is believed that 3luoride release may be more accurately quanti3ied. for this aim, deionized water used in our research protocol to be consistent with previous investigations [48,51]. in this study, we can see two events. one, it is related with decrease the rate of 3luoride release with time for all groups. so, the 1st day show the highest rate then followed by 7th day while the 30 day show the lowest 3luoride rate. the 2nd one related to the groups was that the control group (adhesive only) showed the highest followed by adhesive +5% 3luorescence+0.02% color change, then adhesive +10% 3lourcence +0.2% color change and 3inally adhesive +15% 3luorescence +2% color change showed the lowest 3luoride release rate. these occurred due to the many reasons; one of them related to the time, it because to the statement that 3luoride release drop with time due to the mechanism of its release as suggested by many authors. according to this mechanism, s-prg is composed of three layers: a multifunctional glass core is the innermost layer, followed by a glass ionomer phase from the acid-base reaction that contains the polyacrylic acid chain and ions trapped in the phase, and the surface-modi3ied layer (porous inorganic silica glass layer) at the outermost layer. diffusion of the 3luoride ion from the intermediate layer to the environment is the mechanism of 3luoride release. a 3luoride ion is traded for a hydroxyl ion in an ion exchange process, which is the primary mechanism of 3luoride ion release in giomers [52]. the complex process of 3luoride ion release is in3luenced by both internal and external factors, including the type and permeability of the 3illing material, the frequency of 3luoride exposure, the type and concentration of the 3luoridating agent, temperature, preparation method, material solubility, composition, powder-liquid ratio, surface area of the specimen, matrix, 3iller composition, storage medium, saliva composition and acidity and the type and concentration of the 3luoridating agent. the precise quantity of 3luoride released, which inhibits demineralization and encourages remineralization, is unknown. however, since 3luoride ions in the oral cavity decrease enamel solubility, it is better to utilize materials with a high and sustained 3luoride ion release [53-55]. although the exact mechanism behind the ion release from s-prg 3iller is unknown, it is thought that the existence of a glass ionomer phase around the 3iller's glass core is connected to the ion release [56]. fluoride was released from the surface in a burst in the 3irst step, which is followed by a signi3icant reduction in elution and a second bulk diffusion process that releases minute quantities of 3luoride into the surrounding medium. fluoride is thus released on the 3irst day due to a surface rinsing impact and on following days its diffusion via pores and fractures is responsible for the release. for the 3luoride ions to diffuse, the resin monomer gradually absorbs water [57,58]. so, in the 3irst phenomenon of 3luoride release called the “burst effect”. the second bulk diffusion phase, which releases 3luoride in minute quantities via the material matrix pores, occurs concurrently with the later slow release. this could have to do with the kind of 3illers used. another aspect may be the bonding between the matrix and the 3illers. it was discovered that more microporosities may have resulted from the dye particles' inability to connect with the adhesive matrix, which might have facilitated the release of 3luoride which agrees with bansal & bansal in 2015 & dawood et al in 2019 [5759]. it is shown that giomers have a lower 3luoride release than glass ionomers, with no early 3luoride burst impact, but that 3luoride release levels are mostly constant. the initial intense release of these ions may also be due to surface leaching, while its subsequent stabilization results from the diffusion of 3luoride ions through the pores and fractures of the material [38,58]. additionally, the ionic interaction on the surface of the glass particles or water absorption after the dissolution of the glass 3iller particles might cause 3luoride release [60]. in addition, the 1st step of 3luoride released from the surface of giomer after which the elution is markedly reduced, accompanied by the second bulk diffusion process by which small amounts of 3luoride continue to be released into the surrounding media [61]. as during water penetration through diffusion, the surface layers will be more saturated than the inner mass leading the material can leach ions from the mass that have been penetrated by water & the penetration of water is different for different materials, depending on the permeability of materials [62,63]. anyway the release of 3luoride from giomer is more water-exposure dependent [64]. when the s-prg encounters water, the ions that are not bonded in the polymerization chain created in giomer are dissolved. for example, the polymer in the giomer will react to create a polymer chain when exposed to light. many ions that are not part of the polymer chain are present in the polymer chains in order for them to dissolve in the immersion solution. fluoride is one of the ions not included in this polymer chain [42,65]. the results of this study agree with sangeetha in 2005 who approved that the fluoride release rate was maximum at 1st day then subsequently dropped to a lower level after one week and had reached a near constant level at 30th day [66]. salmerón-valdés et al in 2016 said, in vitro, the degree of fluoride released from giomer was maximum during the 1st 24 hs, then after 8 days showed minimum levels of released fluoride [67]. garoushi et al (2018) who calculated the daily release of fluoride over a period of 10 days from bioactive materials, he said that fluoride start to decrease from 1st day with until the 10th day [48]. also nahum et al in 2021 said that all materials analyzed in his study demonstrate the greatest fluoride release in the first 24 h, followed by a marked decrease after 5th days (68). feiz et al in 2022 approve that the supreme mean of fluoride released during the days 1st, 3rd, and 7th then decrease on day 14th [37]. pastrav et al in 2021 and marnani & kazemian 2024 suggested in their studies that the dental materials which release fluoride ions show highest activity on the 1st day after setting, followed by a gradual decrease in the number of ions released over the following days, months and years [34,69]. harhash et al in 2019 discovered that after the first day, the commercial giomer beautifil flow plus f03, a2 color, emitted 1.0020 ppm of fluoride, 0.4140 ppm after the first week, and 0.3165 ppm after the fourth week [42]. while rusnac et al in 2021, according to his research, the experimental giomer emitted 1.87 ppm of fluoride after the first day, 0.766 ppm after a week, and 0.307 ppm after 30 days. fluoride levels in the giomer b-f03 were 3.1 ppm after the first day, 0.442 ppm during the first week, and 0.242 ppm after 30 days [70]. while in the other side like zabokova et al in 2011 said that the amount of fluoride after 3 & 6 months it was higher compared to initial values this may be due the material & technique that he used it in his research [71]. the second phenomena occur due to the many reasons one of them is the presence of 3iller within constant sample lead to decrease the size of adhesive which leading to decrease the quantity of 3luoride in the testes sample when compare with the control sample. however, other writers hypothesized fluoride release of new bioacmve orthodonmc adhesive with color change and fluorescent property vol 13, no 1 (2025) doi 10.5195/d3000.2025.979 http://dentistry3000.pitt.edu 4 that the high degree of conversion from a double to a simple bond, which results in the cohesiveness of polymer networks and lowers the mobility of ions like 3luoride, might be the cause of the reduced release. a double bond's high degree of conversion to a single bond (–c=c-) (c–c). which mean that increase in polymerization would result in entrapment of 3luoride ions inside the lattice of the polymer, so the amount of 3luoride release will be decreased [72,73]. this can be disagreeing with our results related with dc, this may be due to the difference between size of samples & presence of dyes 3iller that produce some voids within mixture leading to release 3luoride from this voids this agree with alinda et al (2021) who said, voids play a signi3icant role in releasing 3luorine ions [40]. al-shekhli & al_aubi in 2020 said that incorporation of 3iller in the composition of giomer restorative material tend to be affected by water exposure more than other 3iller types incorporated in restorative [74]. jitaluk et al. (2022) implied that adding 20 weight percent nano3illers to resin improved the 3luoride exchange compared to using micro3illers at the same amount [35]. the difference in our result of 3luoride release from results in another studies may be related to the difference in the type and size of material also due to the difference in the type and size of storage media that used this agree with burtea et al. (2019) [52]. anyway, the decrease in the 3luoride release rate can be raised by 3luoride recharge as suggested by barakat & abdelrahim in 2022, who claimed that after being exposed to 3luoridated chemicals, the giomer could be recharged and re-release 3luoride gradually [50]. giomer can be recharged with 3luoride ions by topically applied naf gel and 3luoridated toothpaste () (64). finally, the quantity of 3luoride that is released from the specimens cannot be anticipated to be the same as what happens within the mouth. the true effectiveness of restorations can only be ascertained by long-term clinical trials, even if laboratory investigations are crucial for providing quick answers to certain concerns & it should also be considered that the results were obtained in experimental conditions that cannot completely reproduce the conditions of the oral environment this agree with naoum et al (2012) & gateva et al (2023) [38,75]. giomer can release and recharge 3luoride & it showed a higher recharge potential compared to other 3luoride-containing composite materials [50,76]. . conclusions acceptable 3luoride ion release within bioactive adhesive with color change and 3luorescence properties are obtained but with increase concentration of dyes the ion release decreased. it could advisable to use 3luoridated supplements to compensate this decrease. references 1. tsichlaki a., chin s.y., pandis n., & fleming p.s. (2016). how long does treatment with fixed orthodontic appliances last? a systematic review. am j orthod dentofacial orthop. 149(3): 308–318. 2. stasinopoulos, d., papageorgiou, s.n., kirsch, f., daratsianos, n., jäger, a., & bourauel, c. (2018). failure patterns of different bracket systems and their influence on treatment duration: a retrospective cohort study. angle orthod. 88(3): 338–347. 3. reitan k. (1967). clinical and histologic observations on tooth movement during and after orthodontic treatment. american j orthodo. 53(10): 721–745. 4. millett, d.t., glenny, a.m., mattick, r.c., hickman, j., & mandall, n.a. (2016). adhesives for fixed orthodontic bands. cochrane database of sys rev. 10:1-19 5. kilponen, l. (2020). bonding of orthodontic brackets to enamel. studies on the clinical outcome of bracket bonding and approaches to increase the bond strength of the adhesive interface. university of turku finland. pp:1-80 6. littlewood, s. j., & mitchell, l. (2019). an introduction to orthodontics. oxford university press. 5th ed, p:232. 7. parthasarathy, r., srinivasan, s., vishwanath, s., karunakaran, j., ilango, s., & sakthivelmurali, n. (2024). management of white spot lesion among dental professionals in south indian population–a cross sectional study. afr j bio sc. 6(9): 4570-4583. 8. wang, y., qin, d., guo, f., (2021). outcomes used in trials regarding the prevention and treatment of orthodontically induced white spot lesions: a scoping review. am j orthod dentofacial orthop. 160(5):659-670. 9. naidu, s., & suresh, a. (2019). bond failure rate of amorphous calcium phosphate containing (aegis ortho) and fluoride releasing (transbond plus colour change) orthodontic adhesives–a randomized clinical trial. j. indian dent. assoc. 13: 1826. 10. de camargo, m.g.a., rodríguez, j.a., & rodríguez, h. (2023). remineralization of white spot lesions in orthodontic. is that possible? literature review. revista da faculdade de odontologia de porto alegre. 64: e130690. 11. shuja, m.e., jeelani, w., ahmed, m., & khalid, a. (2024). management of orthodontically induced white spot lesions: a survey of the orthodontic practitioners of pakistan. j pakistan medic associ. 74(5): 922-929. 12. pseiner, b. c., freudenthaler, j., jonke, e., & bantleon, h. p. (2010). shear bond strength of fluoride-releasing orthodontic bonding and composite materials. euro j orthod. 32(3): 268-273. 13. ching, h.s., luddin, n., kannan, t.p., abdrahman, i., & abdulghani, n.r.n. (2018). modification of glass ionomer cements on their physicalmechanical and antimicrobial properties. j esthet restor dent. 30(6):557-571. 14. kashash, y., hein, s., göstemeyer, g., aslanalp, p., weyland, m. i., & bartzela, t. (2024). resin infiltration versus fluoride varnish for visual improvement of white spot lesions during multibracket treatment. clinic oral inves. 28(6): 1-10. 15. spaičytė, n., lieščinskaitė, o., & vasiliauskas, a. (2023). fluoride agents used prior and during the orthodontic treatment for white spot lesions. kaunas: students' scientikic society of lithuanian university of health sciences. 16. kuśmierczyk, d., & małkiewicz, k. (2019). orthodontic adhesive systems–over half a century of research and experience. j stomato. 72(4), 179183. 17. al-eesa, n.a., johal, a., hill, r.g., & wong f.s.l. (2018). fluoride containing bioactive glass composite for orthodontic adhesives – apatite formation properties. dent mater. 34(8): 1127-1133. 18. chitnis, d., dunn, w.j., & gonzales, d.a. (2006). comparison of in-vitro bond strengths between resin-modified glass ionomer, polyacid-modified composite resin, and giomer adhesive systems. am j orthod dentofacial orthop. 129: e11-e16. 19. kaga, n., nagano-takebe, f., nezu, t., matsuura, t., endo, k., & kaga, m. (2020). protective effects of gic and s-prg filler restoratives on demineralization of bovine enamel in lactic acid solution. materials. 13(9): 2140. 20. jose, j.e., padmanabhan, s., & chitharanjan, a.b. (2013). systemic consumption of probiotic curd and use of probiotic toothpaste to reduce streptococcus mutans in plaque around orthodontic brackets. am j orthod dentofac orthop. 144(1):67–72. 21. höchli, d., hersberger-zurkluh, m., papageor giou, s.n., & eliades, t. (2017). interventions for orthodontically induced white spot lesions: a systematic review and meta-analysis. eur j orthod. 39(2):122–33. 22. youssekinia, s., & mortezai, o. (2018). comparison of shear bond strength of orthodontic color change adhesive with traditional adhesive. indian j dent res. 29(5): 690-692. 23. alsamak, s., alsaleem, n. r., & ahmed, m. k. (2023). evaluation of the shear bond strength and adhesive remnant index of color change, fluorescent, and conventional orthodontic adhesives: an in vitro study. int orthod. 21(1), 100712. 24. naqvi, z.a., shaikh, s., & pasha, z. (2019). evaluation of bond failure rate of orthodontic brackets bonded with green gloo-two way color changes adhesive: a clinical study. ethiop j health sci. 29(2). 25. salomão, f.m., rocha, r.s., franco, l.m., sundfeld, r.h., bresciani, e., fagundes, tc. (2019). auxiliary uv light devices for removal of fluorescent resin residues after bracket debonding. j esthet restor dent. 31(1):58–63. 26. patcas, r., & eliades, t. (2017). enamel alterations due to orthodontic treatment. in orthodontic applications of biomaterials woodhead publishing. pp: 221-239 27. sfondrini, m.f., scribante, a., fraticelli, d., roncallo, s., & gandini, p. (2015). epidemiological survey of different clinical techniques of orthodontic bracket debonding and enamel polishing. j orthodont sci. 4:123–127. 28. ribeiro, a.a., almeida, l.f., martins, l.p., & martins, r.p. (2017). assessing adhesive remnant removal and enamel damage with ultraviolet light: fluoride release of new bioacmve orthodonmc adhesive with color change and fluorescent property vol 13, no 1 (2025) doi 10.5195/d3000.2025.979 http://dentistry3000.pitt.edu 5 an in-vitro study. am j orthod dentofacial orthop. 151(2): 292-296. 29. klein, c., babai, a., von ohle, c., herz, m., wolff, d., & meller, c. (2020). minimally invasive removal of tooth-colored restorations: evaluation of a novel handpiece using the fluorescence-aided identification technique. clin oral investig. 24: 2735-2743. 30. engeler, o., stadler, o., horn, s., dettwiler, c., connert, t., verna, c., & kanavakis, g. (2021). fluorescence-aided identification technique (fit) improves tooth surface clean-up after debonding of buccal and lingual orthodontic appliances. j clin med.11(1):213. 31. hahnel, s., wastl, d. s., schneider-feyrer, s., giessibl, f. j., brambilla, e., cazzaniga, g., & ionescu, a. (2014). streptococcus mutans biokilm formation and release of kluoride from experimental resin-based composites depending on surface treatment and s-prg killer particle fraction. j adhes dent, 16(4), 313-21. 32. balagopal, s., nekkanti, s., & kaur, k. (2021). an in vitro evaluation of the mechanical properties and fluoride-releasing ability of a new self-cure filling material. j contemp dent pract. 22(2): 134139. 33. gunay, a., celenk, s., adiguzel, o., cangul, s., ozcan, n., & cakmakoglu, e. e. (2023). comparison of antibacterial activity, cytotoxicity, and fluoride release of glass ionomer restorative dental cements in dentistry. med sci monit. 29: e939065-1. 34. pastrav, m., chisnoiu, a. m., pastrav, o., sarosi, c., pordan, d., petean, i., muntean, a., moldovan, m. & chisnoiu, r.m. (2021). surface characteristics, fluoride release and bond strength evaluation of four orthodontic adhesives. materials. 14(13): 3578. 35. jitaluk, p., ratanakupt, k., & kiatsirirote, k. (2022). effect of surface prereacted glass ionomer nanofillers on fluoride release, flexural strength, and surface characteristics of polymethylmethacrylate resin. j esthet restor dent. 34(8): 1272-1281. 36. mirbehbahani, f.s., hejazi, f., najmoddin, n., asefnejad, a. & artemisia annua. l. (2020). as a promising medicinal plant for powerful wound healing applications. progr biomater. 9(3):139-51. 37. feiz, a., nicoo, m. a., parastesh, a., jafari, n., & sarfaraz, d. (2022). comparison of antibacterial activity and fluoride release in tooth-colored restorative materials: resin-modified glass ionomer, zirconomer, giomer, and cention n. dent res j. 19(1), 104. 38. gateva, v., gateva-grancharova, n., & filipova, m. (2023). comparative evaluation of fluoride release from a compomer, a giomer and a conventional gic. journal of imab–annual proceeding scientikic papers. 29(2): 4904-4910. 39. ugurlu, m., ozkan, e.e., & ozseven, a. (2020). the effect of ionizing radiation on properties of fluoride-releasing restorative materials. brazilian oral rese. 34: e005. 40. alinda, s.d., margono, a., putranto, a.w., maharti, i.d., amalina, r., & rahmi, s.f. (2021). the comparison of biofilm formation, mechanical and chemical properties between glass ionomer cement and giomer. open dent j. 15(1): 274-283 41. grifkin jr jd. unique characteristics of the giomer restorative system: a line of regenerative materials for anterior and posterior restorations. insid dent. 2014; 10(3): 4–5. 42. harhash, a.y., elsayad, i.i., & zaghloul, a.g. (2019). a comparative in vitro study on fluoride release and water sorption of different flowable esthetic restorative materials. eur j dent. 11(2): 174–179. 43. article, o., bansal, r., & bansal, t. (2015). a comparative evaluation of the amount of fluoride release and re release after recharging from aesthetic restorative materials: an in vitro study. jcdr. 9(8): 11-4. 44. malik, s., ahmed, m.a., choudhry, z., mughal, n., amin, m., & lone, m.a. (2018). fluoride release from glass ionomer cement containing fluoroapatite and hydroxyapatite. j ayub med coll abbottabad. 30:198-202. 45. eichmiller, f. c., & marjenhoff, w. a. (1998). fluoride-releasing dental restorative materials. opera dent. 23: 218-228. 46. mungara. j, philip. j, joseph, e., rajendran, s., elangovan, a., & selvaraju, g. (2013). comparative evaluation of fluoride release and recharge of pre-reacted glass ionomer composite and nano-ionomeric glass ionomer with daily fluoride exposure: an in vitro study. j indian soc pedod prev dent. 31:234-239. 47. silva, s.r.d., silva, l.a.h.d., basting, r.t., & lima-arsati, y.b.d.o. (2017). evaluation of the anti-cariogenic potential and bond strength to enamel of different fluoridated materials used for bracket bonding. revista de odontologia da unesp. 46(3): 138-146. 48. garoushi, s., vallittu, p. k., & lassila, l. (2018). characterization of fluoride releasing restorative dental materials. dent mat j. 37(2): 293-300. 49. elshweekh, r.a., bakry, n.s., talaat, d.m., & ahmed, d.m. (2019). fluoride release and rerelease after recharging of two hybrid resin restorations in primary teeth. a comparative in vitro study. alex dent j. 44: 113-18. 50. barakat, i., & abdelrahim, r. (2022). fluoride release from rmgic versus giomer concerning different curing devices at different time intervals (in vitro study). egyptian dent j. 68(1): 77-85. 51. şişmanoğlu, s. (2019). fluoride release of giomer and resin based fissure sealants. odovtos intern j dent sci. 21(2): 45-52. 52. burtea, c.l., prejmerean, c., prodan, d., baldea, i., vlassa, m., filip, m., moldovan, m., antoniac, a., prejmerean v., & ambrosie, i. (2019). new pre-reacted glass containing dental composites (giomers) with improved fluoride release and biocompatibility. materials. 12(23), 4021. 53. hicks m.j., flaitz c.m., & garcia-godoy f. (2000). fluoride-releasing sealant and caries-like enamel lesion formation in vitro. j clin pediatr dent. 24 (3): 215-9. 54. dionysopoulos, d. (2012). the effect of fluoride-releasing restorative materials on inhibition of secondary caries formation. research review. fluoride. 47(3)258-265. 55. poggio, c., andenna, g., ceci, m., beltrami, r., colombo, m., & cucca, l. (2016). fluoride release and uptake abilities of different fissure sealants. j clinic expe dent. 8(3): e284. 56. shimazu, k., ogata, k., & karibe, h. (2011). evaluation of the ion-releasing and recharging abilities of a resin-based fissure sealant containing sprg filler. dental material j. 30: 923–927. 57. bansal, r. & bansal, t. (2015). a comparative evaluation of the amount of fluoride release and rerelease after recharging from aesthetic restorative materials: an in vitro study. clin diagn res. 9(8): 11-14. 58. tiwari, s., kenchappa, m., bhayya, d., gupta, s., saxena, s., satyarth, s., singh, a., & gupta, m. (2016). antibacterial activity and fluoride release of glass-ionomer cement, compomer and zirconia reinforced glass-ionomer cement. j clin diagn res 10: 90-93. 59. dawood, s.h., kandil, m.m., & el-korashy, d.i. (2019). effect of aging on compressive strength, fluoride release, water sorption, and solubility of ceramic-reinforced glass ionomers: an in vitro study. j contemp dent. 9(2), 78-84. 60. dhull, k.s., & nandlal, b. (2009). comparative evaluation of fluoride release from prg composites and compomer on application of topical fluoride: an in-vitro study. j indian soc pedod prev dent. 27: 27-32. 61. bezerra, n.v.f., martinsm, m.l., de frança, l.k.l., de medeiros, m.m.d., de almeida, l.d., padilha wwn, & cavalcanti., y.w. (2019). in vitro evaluation of fluoride in saliva after topical application of professional use products. pesqui. bras. odontopediatria clıń. integr. 19:4005-4008. 62. hadi, m.r. (2020). effect of increased fluoride contents on fluoride release from glass ionomer cements. systematic reviews in pharmacy. 11: 440-443. 63. madhyastha, p.s., naik, d.g., srikant, n., & vinodhini, r.s. (2025). influence of time interval, temperature, and storage condition on fluoride release and recharge from silorane based restorative materials. dent j. 13(5): 197. 64. kim, h., park, h., lee, j., & seo, h. (2021). assessment of fluoride release through dentin adhesive in the alkasite restorative material and giomer. j kor acad pedi dent. 48(4), 367-375. 65. winanto, m., dwiandhono, i., logamarta, s., satrio, r., & kurniawan, a. (2022). the effect of giomer’s preheating on fluoride release. dental j. 55(4): 226-230. 66. sangeetha, d. (2005). a comparative evaluation of fluoride release and shear bond strength of a polyacid-modified composite resin, a resin modified glass ionomer cement and a conventional composite resin: an invitro study. doctoral dissertation, tamil nadu government dental college and hospital, chennai). 67. salmerón-valdés, e.n., scougall-vilchis, r.j., alanis-tavira, j., & morales-luckie, r. a. (2016). comparative study of fluoride released and recharged from conventional pit and fissure sealants versus surface prereacted glass ionomer technology. j conserv dent & endo. 19(1): 41-45. 68. nahum e., rogelio josé s.v., s.v., edith, l.c., victor hugo, t.r., ulises, v.e. and adriana alejandra, m.v., (2021). fluoride release and recharge in conventional varnishes, compared to a giomer and a resin modified glass ionomer. jdmt. 10(2). 69. marnani s.n. & kazemian, m., (2024). investigation of fluoride release patterns of glass ionomer lining materials: a comparative study of chemical, resin modified, and single-component glass ionomer cements. nanochemi res.9(1):5567. 70. rusnac, m. e., prodan, d., moldovan, m., cuc, s., filip, m., prejmerean, c., & dudea, d. (2021). research on the mechanical properties, fluoride and monomer release of a new experimental flowable giomer in comparison to three commercial flowable giomers. applied sciences. 11(19): 8921. 71. zabokova, b. e., sotirovska, i. a., & gjorgovski, i. (2011). fluoride released from orthodontic bonding material: an in vitro evaluation. balkan j stomato. 15(1); 31-34. fluoride release of new bioacmve orthodonmc adhesive with color change and fluorescent property vol 13, no 1 (2025) doi 10.5195/d3000.2025.979 http://dentistry3000.pitt.edu 6 72. sultan, m. a. (2009). the effect of light curing intensity on fluoride release from composite resin. al–rakidain dent j. 9: 232-237. 73. olmos-olmos, g., teutle-coyotecatl, b., román-mendez, c.d., carrasco-gutiérrez, r., gonzález-torres, m., & contrerasbulnes, r., (2021). the influence of light-curing time on fluoride release, surface topography, and bacterial adhesion in resin-modified glass ionomer cements: afm and sem in vitro study. microsc res tech. 1–10. 74. al-shekhli, a.a., & al aubi, i. (2020). compressive strength evaluation of giomer and compomer storage in different media. j int dent med res. 13(1): 23-28. 75. naoum, s., o’regan, j., ellakwa, a., benkhart, r., swain, m., & martin, e. (2012). the effect of repeated fluoride recharge and storage media on bond durability of fluoride rechargeable giomer bonding agent. aust dent j. 57(2): 178-183. 76. senthilkumar, a., chhabra, c., trehan, m., pradhan, s., yadav, s. and shamsudeen, n.h. (2022). comparative evaluation of fluoride release from glass ionomer, compomer, and giomer sealants following exposure to fluoride toothpaste and fluoride varnish: an in vitro study. intern j clinic pedia dent. 15(6): 736-738. figure 1. samples used to test 3luoride release. table 1. fluoride release values in ppm for all groups. groups n time minimum maximum mean std. deviation control 10 24 h 1.64 1.74 1.67 0.0374 7 day 1.49 1.57 1.53 0.0251 30 day 1.47 1.51 1.49 0.0139 5%+0.02% 10 24 h 1.60 1.72 1.66 0.0368 7 day 1.49 1.62 1.52 0.0365 30 day 1.43 1.51 1.48 0.0244 10%+0.2% 10 24 h 1.54 1.66 1.61 0.0442 7 day 1.43 1.59 1.51 0.0463 30 day 1.40 1.50 1.47 0.0294 15%+ 2% 10 24 h 1.54 1.64 1.59 0.0316 7 day 1.47 1.57 1.51 0.0319 fluoride release of new bioacmve orthodonmc adhesive with color change and fluorescent property vol 13, no 1 (2025) doi 10.5195/d3000.2025.979 http://dentistry3000.pitt.edu 7 table 2. duncan's analysis for determining the difference between the groups. groups time 1 2 3 4 5 6 7 8 9 10 control 24hrs 1.73 1.65 1.64 1.65 1.68 1.74 1.64 1.64 1.65 1.68 7 days 1.55 1.57 1.54 1.51 1.49 1.54 1.55 1.54 1.52 1.5 30 days 1.49 1.49 1.51 1.47 1.48 1.51 1.49 1.51 1.48 1.49 5%+0.02% 24hrs 1.62 1.67 1.68 1.7 1.66 1.6 1.67 1.68 1.72 1.63 7 days 1.5 1.53 1.54 1.49 1.52 1.5 1.62 1.53 1.52 1.51 30 days 1.49 1.5 1.5 1.47 1.46 1.46 1.51 1.49 1.49 1.43 10%+0.2% 24hrs 1.64 1.65 1.64 1.54 1.59 1.65 1.66 1.65 1.55 1.6 7 days 1.58 1.51 1.43 1.49 1.53 1.59 1.52 1.5 1.49 1.54 30 days 1.49 1.46 1.4 1.48 1.5 1.5 1.47 1.48 1.46 1.49 15%+ 2% 24hrs 1.6 1.59 1.54 1.58 1.64 1.61 1.61 1.55 1.59 1.63 7 days 1.56 1.51 1.49 1.5 1.48 1.57 1.52 1.51 1.52 1.47 30 days 1.51 1.45 1.48 1.48 1.44 1.52 1.46 1.42 1.48 1.43 832 final clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu clinical evaluation of patient satisfaction with aesthetics, retention, function, and comfort of removable complete and partial acrylic dentures amal qasim ahmed, rusul subhi hassan, hussein ali m. hussein, amenah hafedh khudhair, nabaa basim alhusseini , salah m. ibrahim faculty of dentistry, kufa university, najaf, iraq abstract objec>ves: the quality of removable dentures (acrylic par8al and complete) significantly affects pa8ents’ quality of life, encompassing aspects such as aesthe8cs, func8on and the psychological well-being of individuals by restoring their smile. this study aimed to evaluate pa8ents’ sa8sfac8on with aesthe8cs, support, func8on, and comfort of acrylic removable par8al and complete dentures and their acceptance of the procedure. materials and methods: a total of 146 randomly selected par8ally and completely edentulous pa8ents (106 males and 40 females) with ages 30 years or more were included in this study. all par8cipa8ng pa8ents (120 par8ally, 23 completely edentulous and 3 pa8ents having one completely edentulous arch and another one is par8ally edentulous) were informed about the objec8ves of the study to get their acceptance in terms of esthe8c, func8on, reten8on, comfort and some other majers by answering some ques8ons (a ques8onnaire consists of 17 mul8ple choice ques8ons, using likert-type scale). results: there was an acceptable level of pa8ent's sa8sfac8on with their constructed removable dentures, but they were more sa8sfied with their removable par8al denture in comparison to their complete one regarding the reten8on of the upper denture (p=0.007), chewing ability (p=0.025), speech (p=0.005) and comfort (p=0.003). lower denture reten8on (p=0.062), aesthe8c pa8ent's appearance, (p=0.122), geqng ulcers arer wearing the denture (p=0.125) and difficul8es during denture construc8on visits (p=0.838) did not show the same differences. most pa8ents (68.49%) had been wearing dentures for more than a year. conclusions: this study found generally posi8ve pa8ent experiences with both removable par8al and complete dentures, with higher sa8sfac8on observed among those with par8al dentures, par8cularly regarding reten8on, chewing, speech, and comfort. these findings highlight the benefits of preserving natural teeth and the importance of individualized care, especially during the ini8al adjustment period, to op8mize long-term denture success. keywords: dimensional stability; complete dentures; measuring tools; dimensional accuracy; micrometer microscope. cita9on: ahmed aq, et al. (2025) clinical evalua9on of pa9ent sa9sfac9on with aesthe9c, reten9on, func9on, and comfort of removable complete and par9al acrylic dentures. den9stry 3000. 1:a001 doi:10.5195/d3000.2025.832 received: january 11, 2025 accepted: february 25, 2025 published: april 23, 2025 copyright: ©2025 ahmed aq, et al. this is an open access ar9cle licensed under a crea9ve commons auribu9on work 4.0 united states license. email: salahm.abrahem@uokufa.edu.iq introduction prosthodontics is the branch of dentistry concerned with restoring and maintaining a patient's oral function, wellbeing, appearance and health by restoring natural teeth and/or replacing missing teeth with artificial replacement teeth [1,2]. this area of dentistry is particularly technique sensitive when it comes to the patient's oral rehabilitation through the provision of fixed partial dentures, removable partial and complete dentures, various maxillofacial prosthesis by the practitioner. several factors influence the provision of prosthodontics services, including social and demographic characteristics, patient's symptoms and projected need for care, and aesthetic considerations [3]. the practitioner needs to be aware of the methods, material biocompatibility, and bio acceptability that go into creating the prosthesis that the patient will need to wear. it is sagely stated that “it is more important to preserve what already exists than to replace what is missing” [1]. it has been demonstrated that when natural teeth are removed, chewing ability may clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu suffer, which may have an adverse influence on dietary preferences and nutritional status. diet is crucial for preventing systemic diseases in elderly adults, as poor diets are linked to cardiovascular disease, osteoporosis, and bowel illness [4]. according to the 2009 adult dental health study, 85-yearold patients had an average of 14 teeth, which suggests that tooth replacement may be warranted in this cohort. epidemiological evidence also indicates that people are keeping their teeth into later life [5]. in addition, a lower rate of total edentulism has been seen in the elderly population due to advancements in dental materials, improved maintenance and preventive programs, and a better understanding of oral illnesses. however, due to longer life expectancies, an aging population, and more people keeping their teeth, the percentage of people who are partially edentulous is rising [6,7]. people seek dental implants, fixed and removable partial dentures to replace some of their missing teeth, preserve their remaining natural teeth, and improve their appearance, speech, social confidence, and self-esteem. factors that determine the choice of restoration used include periodontal status, esthetic requirements, cost, anatomical limitations, and patient acceptance [8]. removable partial dentures (rpds) are a straightforward technique—still the most popular option of treatment— that patients who are missing some of their natural teeth can use to restore their oral structure and masticatory function [9-11]. the prevention of pathological drifting of adjacent teeth and the supraeruption of opposing teeth are two advantages of replacing lost teeth. additional advantages include improved oral function and comfort as well as a decrease in occlusal forces on the natural teeth that remain [12]. unlike implant therapy, rpd treatment is less intrusive and enables patients who are partially edentulous to receive prompt, affordable care. it is the best practice therapy for several clinical circumstances, including long-term transitional prosthesis for a terminal dentition, rebuilding missing hard and soft tissues to offer esthetic, support, and restoring large edentulous spans [6]. in the past, a variety of materials have been produced for the construction of rpd frameworks; metallic materials, such as chrome cobalt alloys, and acrylic polymers, also known as polymethyl polymethyl methacrylate (pmma) is commonly used. however, acrylic denture bases are the most used material for manufacturing rpd frameworks, especially in developing countries, as they are extremely cheap, easy to handle, and can be used with existing low-cost equipment. in addition, the development of nylon denture bases has revolutionized the industry in terms of flexibility and provided a viable alternative to acrylic dentures, addressing some of their disadvantages and limitations [11]. however, some disadvantages of wearing full acrylic dentures compared to other rpd frameworks include a higher risk of caries, gingivitis, and periodontitis. in addition, it is difficult to find a suitable insertion path while maintaining a close tissue fit in the presence of soft and hard tissue undercuts. in addition, acrylic prostheses are manufactured with thicker sections to compensate for their low impact resistance, making them bulky and therefore uncomfortable for patients [8]. clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu edentulism is defined as the loss of all permanent teeth. complete dentures are one of the most important and popular treatment options for edentulous patients in rehabilitation [13,14]. when an edentulous patient comes for denture treatment, the main complaint is chewing problems, poor appearance, speech problems, discomfort, or a combination of these issues, which explains why the person needs dentures. while most edentulous people need to wear dentures to chew, there are some, such as teachers. who prefer replacement for phonetics and others like celebrities who mainly prefer them for esthetics, which indicates that preference for need of a denture varies among individuals. knowing the choice of the necessity of a denture is very important for a dentist since there may be certain constraints in accomplishing these aims [15], so patients accept complete dentures are used because they provide an attractive appearance, enable normal speech, and provide support and adequate means for chewing food [13]. within a decade, methyl methacrylate (mma) became the material of choice for 95% of dentures manufactured because it met the requirements for an optimal substrate. mma is also used as the primary component of artificial tooth braces, which are made from a variety of materials including polymethyl methacrylate (pmma) and various ceramics and composites. acrylic teeth have been used to manufacture complete dentures since 1940. acrylic teeth are popular because of their price and aesthetics, as well as their chemical bonding to the acrylic denture base. in contrast, ceramic does not chemically bond to acrylic; however, it is resistant to surface wear and porcelain teeth may transmit forces directly to the bone [16]. treatment of edentulism with complete dentures remains common because it is relatively inexpensive and simple [14, 17]. wearing a new complete denture can cause a variety of discomforts, especially soon after the denture is inserted. complaints may include lack of support and stability, pain or discomfort, food accumulation under the denture, speech changes, difficulty eating, unsightly appearance, and choking. other complaints include bone resorption of the edentulous alveolar ridge and sometimes overgrowth of tissue under the denture due to the forces generated by the mandible during function and dysfunction, as the mucosa is squeezed between the denture base and the underlying bone, so all forces are transmitted through this atrophic tissue [2]. a great deal of research has been conducted to explain why some patients have more difficulty wearing complete dentures successfully than others. the technical quality of the prosthesis is obviously important, but physiological and psychological variables are also thought to contribute. although it is known that older people take longer to adapt to new prostheses, multiple questionnaires, interviews, and personality tests have failed to identify accurate indicators that can predict patient dissatisfaction with prostheses [17]. patient satisfaction is an important goal of oral rehabilitation and can be used to assess the success of these rehabilitations [18]. it is important to remember that patients and dentists have clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu different expectations and satisfaction with the same restorative treatment. these differing comments can lead to conflict between patients and dentists and adversely affect the dentist-patient relationship, resulting in a potential loss of patient satisfaction. dentists often evaluate the success of a denture based on established clinical criteria without considering the individual patient’s needs, expectations, and attitudes. because patients and dentists may have different expectations and satisfaction with the same treatment, it is important to establish a strong dentist-patient connection to understand the patient’s preferences and help set appropriate expectations to achieve the chosen treatment [19]. there is a shortage of relevant information on patient satisfaction and concerns with detachable denture usage in middle eastern populations. some research conducted across diverse populations showed that most patients are generally satisfied with their removable dentures [11] so, this study was conducted to evaluate and compare the clinical effects of constructed removable partial and complete acrylic dentures at dental health care institutions for iraqi people with respect to retention, esthetic, function, comfort and any possible related problems such as sore mouth. moreover, the study seeks to evaluate patients' acceptance of the entire treatment process, from initial consultation to the final fitting and adjustments of the dentures. table 1. distribu4on of the pa4ents according to general characteris4cs. percentage number age 0% 0 less than 30 years 18.49% 27 30-50 years 81.51 119 up 50 years clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu 100% 146 total percentage number gender 72.6% 106 male 27.4% 40 female 100% 146 total percentage number level of educa4on 24.66% 36 educated 75.34% 110 uneducated 100% 146 total percentage number period of wearing a denture 12.3% 18 less than 3 months 19.18% 28 3-12 months 68.49% 100 more than 12 months 100% 146 total clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu material and methods study design and participants this cross-sectional study was conducted at the prosthetic clinic at the college of dentistry, university of kufa, iraq. ethical approval was obtained from the university of kufa college of medicine medical ethics committee (mec-38) on 14/5/2024. patients were recruited between january and may 2024. patients were recruited using convenience sampling from those presenting at the prosthetic clinic during the recruitment period. while we aimed for gender balance, recruitment continued until the desired sample size was achieved. table 2. aesthe4cs and sa4sfac4on characteris4cs. percentage number color of denture 17.12% 25 very good 9.59% 14 good 65.07% 95 acceptable 8.22% 12 bad clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu 0% 0 very bad 100% 146 total percentage number color of teeth 15% 22 very good 6.2% 9 good 74.7% 109 acceptable 4.1% 6 bad 0% 0 very bad 100% 146 total percentage number size of teeth 5.5% 8 very large 2.7% 4 large 91.1% 133 acceptable 0% 0 small 0.7% 1 very small 100% 146 total percentage number appearance of face aeer denture wearing clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu 26.7% 39 i looked preker and younger 4.8% 7 i looked preker 8.2% 12 i looked younger 59.6% 87 acceptable 0.7% 1 there is no change in my appearance 100% 146 total percentage number to what extent do you accept the steps of the work 15.1% 22 it was short and very comfortable 18.5% 27 it was short and fairly comfortable 54.8% 80 it was long but comfortable 11.6% 17 it was long and uncomfortable 0% 0 it was very long and uncomfortable 100% 146 total clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu table 3. func4onal characteris4cs. percentage number reten4on of upper denture 21.2% 31 very good 61% 89 good 11.6% 17 acceptable 6.25 9 bad 0% 0 very bad 100% 146 total percentage number reten4on of lower denture 6.2% 9 very good 19.9% 29 good 63.7% 93 acceptable 10.2% 15 bad 0% 0 very bad 100% 146 total percentage number how well can you talk with dentures 19.9% 29 very well clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu 64.4% 94 well 11.6% 17 poorly 4.1% 6 very poorly 0% 0 neutral 100% 146 total percentage number how well can you chew with your dentures 10.3% 15 very well 25.3% 37 well 59.6% 87 poorly 4.8% 7 very poorly 0% 0 neutral 100% 146 total percentage number how comfortable are your dentures 18.5% 27 very comfortable 63% 92 comfortable 13.7% 20 uncomfortable 4.8% 7 very uncomfortable clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu 0% 0 neutral 100 146 total percentage number do you get a sore mouth 30.1% 44 yes 69.9% 102 no 100% 146 total clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu data collection prior to treatment, informed consent was obtained from all participants. information about the study’s purpose, risks, benefits, and the right to withdraw were explained in detail, and written consent was obtained. data were collected using the following: demographics and clinical data age, gender, education level, and period of denture use (if applicable) were recorded from patient records. patient satisfaction patient satisfaction with aesthetics, retention, function, and comfort of their dentures was assessed using a previously prepared 17-item questionnaire using a likert-type scale [19]. the questionnaire addressed specific aspects of: aesthetics [list a few examples, e.g., satisfaction with denture color, tooth size and shape, overall appearance]. retention [list examples, e.g., stability of dentures during speaking, eating, at rest]. function [list examples, e.g., chewing ability, speech clarity, ease of use]. comfort [list examples, e.g., presence of soreness, irritation, overall comfort level]. photographs standardized photographs of the lower third of the face were taken statistical analysis data were analyzed using [statistical software, e.g., spss version 23]. descriptive statistics were used to summarize the data [anova for continuous variables]. a p-value of ≤0.05 was considered statistically significant. clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu table 4. comparison of aesthe4cs and func4onal characteris4cs between type of prosthe4c treatment. p values complete denture (n=23) par4al denture (n= 120) variables n (%) n (%) reten4on of upper denture 0.007 5 (21.8%) 25 (20.8%) very good 9 (39.1%) 80 (66.7%) good 7 (30.4%) 7 (5.8%) acceptable 2 (8.7%) 8 (6.7%) bad 0 (0%) 0 (0%) very bad reten4on of lower denture 0.062 2(8.7%) 6 (5%) very good 7 (30.4%) 20 (16.7%) good 10 (43.5%) 84 (70%) acceptable 4 (17.4%) 10 (8.3%) bad 0 (0%) 0 (0%) very bad appearance of face aeer denture wearing 0.122 4 (17.4%) 32 (26.7%) i looked preker and younger 3 (13%) 2 (1.7%) i looked preker clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu 14 (60.9 %) 75 (62.5%) i looked younger 2 (8.7%) 10 (8.3%) acceptable 0 (0%) 1 (0.8%) there is no change in my appearance to what extent do you accept the steps of the work 0.838 2 (8.7%) 20 (16.7%) it was short and very comfortable 5 (21.7%) 23 (19.2%) it was short and fairly comfortable 14 (60.9%) 65 (54.1%) it was long but comfortable 2 (8.7%) 12 (10%) it was long and uncomfortable 0 (0%) 0 (0%) it was very long and uncomfortable how well can you talk with dentures 0.005 5 (21.7%) 24 (20%) very well 10 (43.5%) 80 (66.7%) well 8(34.8%) 9 (7.5%) poorly 0(0%) 7 (0%) very poorly 0 (0%) 0 (5.8%) neutral how well can you chew with your dentures 0.025 5 (21.8%) 10 (8.4%) very well clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu 9 (39.1%) 25 (20.8%) well 9 (39.1%) 79 (65.8%) poorly 0(0%) 6 (5%) very poorly 0(0%) 0(0%) neutral how comfortable are your dentures 0.003 6 (26.1%) 20 (16.7%) very comfortable 8 (34.8%) 83 (69.2%) comfortable 9 (39.1%) 10 (8.3%) uncomfortable 0(0%) 7 (5.8%) very uncomfortable 0(0%) 0(0%) neutral do you get a sore mouth 0.125 2 (8.7%) 22 (18.3%) yes 21 (91.3%) 98 (81.7%) no *the p-value was calculated by fisher exact test. r sults general characteris4cs one hundred and forty-six parocipaong paoents (106 male and 40 female), including 27 of them aged between (30-50) years old, 119 who were older than 50 years and there was no paoent under the age of 30 years. the level of educaoon was very low with 75.34% uneducated. the largest percentage of parocipants (68.49%) wore their dentures more than one year, followed by those who wore them between 3 and 12 months (19.18%), and the lowest percentage (12.3%) was for those who had a denture for a period less than 3 months, as shown in table 1. the largest percentage of paoents chose "acceptable" regarding the color of the clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu dentures (65.07%), the color (74.7 %) and size of the aroficial teeth (91.1%), and regarding their face appearance arer wearing their dentures (59.6%). in general denture making visits were long but comfortable for them (54.8%), as shown in table 2, and figures 1 and 2. regarding the funcoonal characterisocs (including both paroal and complete dentures generally) the retenoon of upper denture was "good" (61%) and "acceptable" for the lower denture (63.7%), and also good for their ability to talk while wearing it (64.4%), but they were able to use it for chewing poorly (59.6%) and it was comfortable in general (63%), and a small percentage of them suffered from ulcers (30.1%), as shown in table 3 and figure 3. finally, the results of the comparison between the paroal and complete dentures in terms of aestheoc and funcoonal characterisocs were as follows; the difference in retenoon for the upper paroal in comparison with complete dentures was significant (p=0.007) while for the lower denture it was not significant (p=0.062) and there was no significant difference between the paroal and complete denture in terms of paoent’s appearance arer wearing denture (p=0.122), but the difference in the ability to talk, chew and comfort was significant (p=0.005, p=0.025), and p=0.003, respecovely). finally, there was no significant difference in regarding the extent to which the paoent accepts the treatment steps and number of visits (p=0.838), and the appearance of ulcers inside the mouth (p=0.125). as shown in table 4 and figure 4. figure 1. representa4ve treated cases with removable par4al acrylic dentures showing the color of denture, color and size of the ar4ficial teeth (a) before the treatment and (b) aeer wearing their dentures. clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu figure 2. illustra4ve treated cases with complete dentures showing pa4ent's appearance aeer wearing their dentures and its posi4ve effect on the appearance, so that the pa4ent appears younger in age, (a) before the treatment and (b) aeer wearing their dentures. clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu figure 3. representa4ve cases treated with removable par4al acrylic dentures (a) before the treatment and (b) aeer wearing their dentures. figure 4. representa4ve treated cases with complete dentures, (a) before the treatment and (b) aeer wearing their dentures. discussion the demographic distribuoon of our study sample offers valuable insights into paoent saosfacoon with removable paroal and complete acrylic dentures. as expected, our study populaoon skewed towards older adults, with 81.51% being over 50 years old, consistent with previous findings [20]. this age distribuoon aligns with the higher prevalence of tooth loss and denture needs among older demographics [21]. however, the absence of parocipants under 30 highlights a potenoal limitaoon requiring further invesogaoon. younger individuals oren priorioze the superior aestheocs, comfort, and funcoonality offered by fixed soluoons or dental implants, impacong their acceptance of removable opoons [22,23]. the significant gender disparity, with 72.6% males compared to 27.4% females, suggests that men might be more accepong of removable dentures. this difference could stem from varying priorioes regarding clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu aestheocs and self-percepoon. research indicates that women tend to place a higher value on the appearance of dental prostheses, influencing their saosfacoon levels and potenoally leading them to favor fixed soluoons like implants [24-26]. socioeconomic factors also likely play a role, as men might priorioze the cost-effecoveness of removable dentures over the aestheoc advantages of more expensive alternaoves [25,26]. the low educaoonal avainment among our parocipants (75.34% uneducated) is noteworthy. this aligns with studies demonstraong a link between lower educaoonal levels and higher rates of missing teeth and removable prostheoc use [22,26,28]. this pavern may be compounded by the generally lower educaoonal avainment among older populaoons in iraq. educaoonal level can significantly influence a paoent’s ability to understand post-procedure care, manage expectaoons, and adhere to maintenance rouones, ulomately affecong their saosfacoon with dentures. these findings underscore the need for tailored paoent educaoon and support based on individual needs and educaoonal backgrounds. most paoents found their denture teeth size "acceptable" (91.1%). this suggests that dentures were generally welltailored to oral dimensions, potenoally contribuong to comfort. the low percentages of "very large" (5.5%) or "very small" (0.7%) teeth indicate that significant size discrepancies were infrequent, likely due to the presence of remaining natural teeth used as a reference for aroficial tooth selecoon and the opportunity for paoents to evaluate aestheocs during trial appointments [29]. while color saosfacoon was generally acceptable, there is room for improvement as a considerable proporoon of parocipants did not rate the color of their dentures or teeth as "very good." our analysis revealed a significant difference in upper denture retenoon between rpds and cds. this is unsurprising, as rpds uolize clasps anchored to remaining teeth for stability [30-32]. conversely, cd retenoon relies solely on the denture base's fit against the mucosa, influenced by factors such as saliva, sor ossue contours, and the rate of bone resorpoon, which tends to be more pronounced in completely edentulous jaws [33-38]. while advancements in materials and techniques like cad/cam have improved cd fit and comfort, they soll typically offer less retenoon compared to rpds [39]. interesongly, no significant difference in lower denture retenoon was observed. this might be avributed to the anatomical and funcoonal challenges inherent to the mandibular arch, which impact both rpds and cds. factors such as limited ridge support, tongue movement, and floor of the mouth dynamics can dislodge both types of dentures [17,40-42]. the non-significant difference in perceived facial appearance between rpd and cd wearers aligns with čelebić and knezović-zlatarić’s research [43]. this suggests that both denture types effecovely restore facial aestheocs by compensaong for missing teeth and supporong facial structures [25]. the use of heat-cured acrylic resin, which blends well with oral ossues, likely contributed to the acceptable aestheoc outcomes for both denture types [12]. as expected, rpd wearers reported significantly bever speech clarity compared to cd wearers. this difference likely stems from the complete palatal coverage of cds, potenoally interfering with tongue movements crucial for speech, parocularly for producing certain consonant sounds [44,45]. overextended clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu upper denture bases can further exacerbate speech difficuloes by impeding lip movement [17,46]. the significant difference in chewing ability between rpd and cd wearers can be avributed to several factors. the superior stability and retenoon of rpds during chewing mooons, due to their anchorage to natural teeth, are key [46-49]. addioonally, the sensory input from periodontal mechanoreceptors in remaining natural teeth plays a crucial role in controlling jaw movements during chewing, a benefit absent in cd wearers [50,51]. denture instability during chewing is a common complaint among cd wearers, leading to compensatory tongue and cheek movements that compromise their masocatory efficiency [17,48,52,53]. the significant difference in overall comfort favoring rpds aligns with the findings on retenoon, speech, and chewing ability. the non-significant difference in reported mouth ulcers could be avributed to the emphasis on meoculous denture fabricaoon and regular adjustments for both denture types. the lack of difference in the acceptance of denture construcoon steps reinforces that both rpds and cds necessitate similar procedures to ensure opomal fit, comfort, and funcoon. this study indicates a general acceptance of both rpds and cds among our paoent populaoon, with a predominance of older males. however, rpds appear to offer greater saosfacoon, parocularly regarding retenoon, speech, chewing, and overall comfort. these findings underscore the need for cononued research and advancements in denture design, fabricaoon techniques, and paoent educaoon to further improve the experiences of individuals relying on removable prostheses. conclusions considering the shortcomings of this study, it was concluded that most of the paoents who were included in the study were paroally edentulous paoents and more than those completely edentulous. generally, there was acceptance and saosfacoon from paoents with their deferent types of the constructed removable acrylic studied dentures, and the percentage of males was higher than that of the females and the percentage of elderly paoents was higher, but the level of educaoon was low. in general, there was more acceptance and saosfacoon among those who wear paroal dentures than those who wear complete dentures in terms of retenoon, ability to chew, speak, and comfort, but there was no significant difference in terms of aestheocs (including face appearance arer wearing denture, teeth size and the color of denture and teeth), the appearance of ulcers, and the steps of denture construcoon. acknowledgments we appreciate the assistance and resources provided by the department of oral diagnosis at the kufa college of denostry. conflicts of interest the authors declare no conflicts of interest. data availability statement the data created and examined during the current study can be available upon reasonable request. funding none. regulatory statement every procedure used in the study complied with the 1964 helsinki declaraoon and its later 2013 revisions, ethical approval was obtained from the university of kufa/ college of medicine/ medical ethics commivee (mec-38) on 14/5/2024. clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu references 1. souza, raphael f., cláudio r. leles, and marco a. compagnoni. "a survey of complete denture teaching in brazilian dental schools." brazilian dental science 5.1 (2002). 2. hassan, rusul subhi, et al. assessment of periodontal health status and treatment needs among dental students of al-kufa university by using the community periodontal index for treatment needs: a cross-secoonal study. denostry 3000, 2024, 12.2.al-kaisy, n. "a survey of prosthodonocs techniques applied by dental pracoooners in sulaimani city." j bagh coll dent, vol. 28, no. 3, 2016, pp. 22-29. 3. mckenna, g., et al. "comparison of funcoonally orientated tooth replacement and removable paroal dentures on the nutrioonal status of paroally dentate older paoents: a randomised controlled clinical trial." journal of den1stry, vol. 42, no. 6, 2014, pp. 653-659. 4. friel, t., and s. waia. "removable paroal dentures for older adults." primary dental journal, vol. 9, no. 3, 2020, pp. 34-39. 5. kim, j.j. "revisiong the removable paroal denture." dental clinics, vol. 63, no. 2, 2019, pp. 263-278. 6. suwal, p., et al. "cast paroal denture versus acrylic paroal denture for replacement of missing teeth in paroally edentulous paoents." journal of dental material and technique (jdmt), vol. 6, 2017, pp. 2734. 7. khalel, a. m., ali, m. b., sadiq, m. a., ibrahim, s. m., & ali, s. h. (2024). dmft and pufa indices in first permanent molars of iraqi children in najaf city. denostry 3000, 12(2). 8. preshaw, p.m., et al. "associaoon of removable paroal denture use with oral and systemic health." journal of den1stry, vol. 39, no. 11, 2011, pp. 711-719. 9. abuzar, m.a., e. kahwagi, and t. yamakawa. "invesogaong oral healthrelated quality of life and self-perceived saosfacoon with paroal dentures." journal of inves1ga1ve and clinical den1stry, vol. 3, no. 2, 2012, pp. 109-117. 10. aljabri, m.k., t.o. ibrahim, and r.m. sharka. "removable paroal dentures: paoent saosfacoon and complaints in makkah city, ksa." journal of taibah university medical sciences, vol. 12, no. 6, 2017, pp. 561-564. 11. akinyamoju, c.a., et al. "oral health-related quality of life: acrylic versus flexible paroal dentures." ghana medical journal, vol. 53, no. 2, 2019, pp. 163169. 12. el afandy, h.m. "evaluaoon the retenoon of upper complete denture with different acrylic denture base materials." egyp1an dental journal, vol. 65, no. 3, july 2019, pp. 2691-2698. 13. cooper, p., r. hegde, and c. hegde. "a quesoonnairebased survey to assess the knowledge and awareness of denture staining in complete denture wearers." journal of health and allied sciences nu, vol. 9, no. 2, 2019, pp. 45-50. 14. suresh, s., and swao sharma. "a clinical survey to determine the awareness and preference of needs of a complete denture among complete edentulous paoents." journal of interna1onal oral health 2.3 (2010). 15. bilgin, m.s., et al. "fabricaong complete dentures with cad/cam and rp technologies." journal of prosthodon1cs, vol. 24, no. 7, 2015, pp. 576579. 16. bilhan, h., et al. "evaluaoon of saosfacoon and complicaoons in paoents with exisong complete dentures." journal of oral science, vol. 55, no. 1, 2013, pp. 29-37. 17. baracat, l.f., et al. "paoents' expectaoons before and evaluaoon arer dental implant therapy." clinical implant den1stry and related research, vol. 13, no. 2, 2011, pp. 141-145. 18. santos, b.f.o., et al. "paoents’ evaluaoons of complete denture therapy and their associaoon with related variables: a pilot clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu study." journal of prosthodon1cs, vol. 24, no. 5, 2015, pp. 351-357. 19. saadrosama, atheer mohammed alrubaian. "awareness and aktude toward replacement of missing teeth among, paoents who visit dental clinics of qassim university., al-qassim region, kingdom of saudi arabia." int j curr res, vol. 10, 2018, pp. 71943-71948. 20. ibrahim, salah m., savar jabbar abdul-zahra alhmedat, and mohammed hamoudi alsunboli. "histological study to evaluate the effect of local applicaoon of myrtus communis oil on alveolar bone healing in rats." the open denostry journal 18.1 (2024). 21. leles, c.r., et al. "discriminant analysis of paoents’ reasons for choosing or refusing treatments for paroal edentulism." journal of oral rehabilita1on, vol. 36, no. 12, 2009, pp. 909-915. 22. frank, r.p., et al. "relaoonship between the standards of removable paroal denture construcoon, clinical acceptability, and paoent saosfacoon." the journal of prosthe1c den1stry, vol. 83, no. 5, 2000, pp. 521-527. 23. agarwal, s., et al. "denosts' preference toward fixed versus removable implant prosthesis on edentulous jaws to improve quality of life." journal of long-term effects of medical implants, vol. 33, no. 1, 2023. 24. yen, y.y., et al. "impact of removable dentures on oral health-related quality of life among elderly adults in taiwan." bmc oral health, vol. 15, 2015, pp. 112. 25. chakaipa, s., et al. "the experiences of paoents treated with complete removable dentures: a systemaoc literature review of qualitaove research." oral, vol. 2, no. 3, 2022, pp. 205-220. 26. rodrigues, a., s. dhanania, and r. rodrigues. “if i have teeth, i can smile.” experiences with tooth loss and the use of a removable dental prosthesis among people who are paroally and completely edentulous in karnataka, india. bdj open, vol. 7, no. 1, 2021, p. 34. 27. mohammed, mohammed jasim; al-mizraqchi, abbas s.; ibrahim, salah m. oral findings, salivary copper, magnesium, and lepon in type ii diabeoc paoents in relaoon to oral candida species. internaoonal journal of microbiology, 2024, 2024.1: 8177437. 28. ibrahim, salah mahdi, and savar j. abdul-zahra al hmedat. "role of manual and powered tooth brushes in plaque removal and oral health status (a comparaove study)." indian journal of public health research & development 10.8 (2019). 29. alageel, o., et al. "evaluaoon of the designdriven predicoon of removable paroal denture retenoon." the journal of prosthe1c den1stry, vol. 124, no. 3, 2020, pp. 357364. 30. alageel, o., et al. "determining the retenoon of removable paroal dentures." the journal of prosthe1c den1stry, vol. 122, no. 1, 2019, pp. 55-62. 31. anes, v., et al. "evaluaoon of the retenove forces from removable paroal denture clasps manufactured by the digital method." applied sciences, vol. 13, no. 14, 2023, p. 8072. 32. shawi, h., et al. "improving the retenoon of maxillary complete denture: a case report." alqalam journal of medical and applied sciences, 2024, pp. 113-120. 33. chebib, najla, et al. "fit and retenoon of complete denture bases: part ii– convenoonal impressions versus digital scans: a clinical controlled crossover study." the journal of prostheoc denostry (2022). 34. akaltan, f., et al. "comparaove analysis of denture base adaptaoon performance between pour and other convenoonal fabricaoon techniques." the journal of prosthe1c den1stry, vol. 123, no. 1, 2020, pp. 183-e1. 35. sayed, f., et al. "the effect of using ultra sucoon system on mandibular complete denture clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu retenoon." al-azhar dental journal for girls, vol. 3, no. 3, 2016, pp. 161-169. 36. mehra, m., f. vahidi, and r.w. berg. "a complete denture impression technique survey of postdoctoral prosthodonoc programs in the united states." j prosthodont, vol. 23, 2014, pp. 320-327. 37. darvell, b.w., and r.k.f. clark. "the physical mechanisms of complete denture retenoon." bri1sh dental journal, vol. 189, no. 5, 2000, pp. 248-252. 38. jayaraman, s., et al. "finalimpression techniques and materials for making complete and removable paroal dentures." cochrane database of systema1c reviews, no. 4, 2018, pp. 39. awawdeh, m., et al. "a systemaoc review of paoent saosfacoon with removable paroal dentures (rpds)." cureus, vol. 16, no. 1, 2024. 40. alqutaibi, a.y. "a withinsubject comparison of the convenoonal claspretained with avachmentretained removable paroal dentures." journal of taibah university medical sciences, vol. 15, no. 4, 2020, pp. 305311. 41. ribeiro, j.a., et al. "the influence of mandibular ridge anatomy on treatment outcome with convenoonal complete dentures." acta odontológica la1noamericana, vol. 27, no. 2, 2014, pp. 53-57. 42. ibrahim, s., and a.s. hussein. "role of hexidine, zak and biofresh mouth wash in commemoraoon deleoon and oral health status (comparaove study)." interna1onal journal of pharmaceu1cal research, vol. 11, no. 1, 2019. 43. mahross, h.z., and k. baroudi. "spectrogram analysis of complete dentures with different thickness and palatal rugae materials on speech producoon." interna1onal journal of den1stry, 2015. 44. koike, t., et al. "influence of anterior palatal coverage on percepoon and retenoon in complete dentures." the journal of prosthe1c den1stry, vol. 105, no. 4, 2011, pp. 272-279. 45. gosavi, s.s., et al. "a survey of complete denture paoents experiencing difficuloes with their prostheses." the journal of contemporary dental prac1ce, vol. 14, no. 3, 2013, p. 524. 46. pan, y.h., t.m. lin, and c.h. liang. "comparison of paoent's saosfacoon with implant-supported mandibular overdentures and complete dentures." biomedical journal, vol. 37, no. 3, 2014, p. 156. 47. gonçalves, t.m.s.v., et al. "effect of complete and paroal removable dentures on chewing movements." journal of oral rehabilita1on, vol. 41, no. 3, 2014, pp. 177-183. 48. nand, m., and m. mohammadnezhad. "challenges faced by edentulous paoents (edps) during complete denture prostheses (cdp) service delivery in fiji—a qualitaove study." bmc health services research, vol. 22, no. 1, 2022, p. 742. 49. ibrahim, salah m., abbas s. al-mizraqchi, and julfikar haider. "metronidazole potenoaoon by panax ginseng and symphytum officinale: a new strategy for p. gingivalis infecoon control." an1bio1cs, vol. 12, no. 8, 2023, p. 1288, hvps://doi.org/10.3390/ano bioocs12081288. 50. svensson, k.g., j. grigoriadis, and m. trulsson. "alteraoons in intraoral manipulaoon and splikng of food by subjects with tooth-or implant-supported fixed prostheses." clinical oral implants research, vol. 24, no. 5, 2013, pp. 549555. 51. zoidis, p., i. papathanasiou, and t.f. deluca. "paoent saosfacoon with convenoonal dentures fabricated with different processing techniques: a systemaoc review." the journal of prosthe1c den1stry, vol. 118, no. 4, 2017, pp. 493-500, hvps://doi.org/10.1016/j.pr osdent.2016.12.011. 52. chakaipa, s., et al. "the experiences of paoents treated with complete removable dentures: a systemaoc literature clinical evalua+on of pa+ent sa+sfac+on with aesthe+cs, reten+on, func+on, and comfort of removable complete and par+al acrylic dentures vol 13 no 1 (2025) doi 10.5195/d3000.2025.832 h#p://den*stry3000.pi#.edu review of qualitaove research." oral, vol. 2, no. 3, 2022, pp. 205-220, hvps://doi.org/10.1016/j.or al.2022.06.002. 920 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.920 http://dentistry3000.pitt.edu iron overload and serum and saliva ferritin levels in individuals with beta thalassemia needing several blood transfusions anaam mahdi hadi1, layla sabri yas2, shaimaa a saeed3, ali g. al-dulimi1 1college of den*stry, bilad alrafidain university, diyala, iraq 2college of den*stry, university of baghdad, iraq 3specialized dental center, baghdad, iraq abstract iron overload in individuals with beta thalassemia major is mostly caused by blood transfusion treatment. since the human body lacks a method for excredng extra iron, iron overload is unavoidable in individuals with thalassemia major who require regular blood transfusions. because exfoliadve cytology is a rapid, easy, painless, and bloodless treatment, it is an airacdve assessment. the main intracellular iron-storage protein, ferridn, is released in minute amounts into the body's plasma and helps to maintain iron in a soluble, nontoxic state. in the absence of inflammadon, the amount of the body's total iron reserves is posidvely correlated with the concentradon of this plasma (or serum) ferridn. when compared to blood, saliva offers major biochemical and logisdcal benefits, making it one of the most essendal bodily fluids for diagnosdc purposes. the diseases associated with iron overload are aiributed to the significant increase in ferridn levels in saliva. the objecdves of the study were as follows: 1. to measure the levels of iron overload in padents with betathalassemia major using oral exfoliadve cytology with the special perl's prussian blue stain. 2. elisa was used to measure the ferridn level (iron overload) in the serum and saliva of individuals with beta thalassemia major. 3. to compare the amounts of ferridn in their serum and saliva with the prussian blue staining posidvity of each individual. smears were extracted from the buccal mucosa of thirty healthy people in the same age range (6-26 years) and sixty β-thalassemia major padents who had received at least ten transfusions. prussian blue stain kit from perl was used to stain smears. to esdmate ferridn levels, blood and saliva samples were simultaneously obtained from the control and study group. posidvity of prussian blue was evaluated using predetermined grading standards. 48 out of 60 thalassemic padents (80%) had perl's posidvity, which was posidvely correlated with ferridn levels in the saliva and serum. padents with β-thalassemia major can have their iron overload evaluated using coloring peeled cells from the oral mucosa. open access cita%on: hadi am, et al. (2025) oral exfolia%ve cytology as a means of es%ma%ng iron overload and comparing serum and saliva ferri%n levels in individuals with beta thalassemia needing several blood transfusions. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.920 received: april 28, 2025 accepted: may 5, 2025 published: june 12, 2025 copyright: ©2025 hadi am, et al. this is an open access ar%cle licensed under a crea%ve commons ayribu%on work 4.0 united states license. email: anaammahdi100@gmail.co introduc)on beta thalassemias, sometimes referred to as β thalassemias, are a group of genetic blood disorders. these types of thalassemia vary in severity, ranging from severe anemia to cases that show no clinical symptoms, and this condition results from a de<iciency or absence of the production of the beta chain of hemoglobin [1]. children who have this condition may experience problems that might affect the spleen and need regular iron overload and serum and saliva ferri1n levels in individuals with beta thalassemia needing several blood transfusions vol 1, no 1 (2012) doi 10.5195/d3000/2025.920 http://dentistry3000.pitt.edu 2 blood transfusions for the rest of their lives. certain children may bene<it from bone marrow transplants [2]. two factors that contribute to or exacerbate iron overload in patients include frequent blood transfusions. organ damage must be avoided with the use of iron chelation therapy. treatments for thalassemia major are becoming more advanced, allowing patients to live longer with appropriate care [3,4]. maintaining a hemoglobin level above 9–10.5 g/dl is the recommended course of therapy for thalassemia major. there are several problems associated with long-term transfusions [5]. the body stores iron mostly in ferritin [6]. a positive acute phase response protein, ferritin no longer re<lects the amount of the iron reserve when concentrations rise during in<lammation [7]. with considerable biochemical and logistical bene<its over blood, saliva is one of the most crucial bodily <luids for diagnostic purposes. according to bigler et al. (2009) [8], saliva collection is a common procedure that is safe, noninvasive, and somewhat easy for patients to undergo without experiencing any pain. ferritin levels in saliva are known [9,10], and in iron overload conditions, salivary ferritin levels rise noticeably. however, the ratio of salivary to serum ferritin does not change, indicating that salivary and serum ferritin grow proportionately while maintaining a stable value [11,12]. when a patient with persistent anemia requires frequent blood transfusions, transfusional iron excess occurs. while the body excretes only around 1 mg of iron per day [13], transfusions include over 250 mg of iron [14]. according to greer et al. (2013) [3], af<licted patients’ liver, heart, endocrine glands, and lungs all have deposits of hemosiderin. çaliskan et al. (2011) [15] and rajput et al. (2010) [16] have reported the discovery of iron deposits in the gingivae. the following tests are used to monitor the patient's iron overload status and the effectiveness of iron chelation therapy: serum ferritin level, serum iron level, total iron-binding capacity, non-transferrin-bound iron level in serum, liver biopsy to assess parenchymal and reticuloendothelial stores, in addition to computed tomography of the liver or magnetic resonance imaging, and cardiac imaging using magnetic resonance imaging (t2 * or ferriscan technique) [17]. the process of exfoliative cytology is rapid, easy, painless, bloodless, and noninvasive [18]. it is predicated on the microscopic examination of epithelial cells following the <ixation and staining process. there are two approaches used: the direct technique, which involves rubbing mucosal surface cells, and the indirect way, which involves aspirating individuals with self-exfoliated cells [19]. using an affordable method called exfoliative cytology [20], this study aims to quantify the iron excess in such individuals. materials and methods in the hereditary blood disease center, gynecology, obstetrics, and pediatrics department of ibin albaladi hospital in baghdad, a cross-sectional research was carried out. the research on prominent cases of βthalassemia who received ten or more blood transfusions was carried out from january to april 2024. the baghdad university, college of dentistry's institutional ethics committee gave its approval. the hours of the sample sessions were restricted to 9:00 a.m. to 11:00 a.m. data from patient <iles were used to determine the diagnosis and total number of transfusions. the sample ninety volunteers of both genders, ranging in age from 6 to 26 years, were included in this study and were categorized into two main groups: 60 beta thalassemia major patients were divided into the following study groups: a. fifteen patients (15) who had received repeated transfusions but were not getting iron chelation therapy (treatment-free). b. forty-<ive patients (45) receiving iron chelation therapy were divided into subgroups based on how long their treatments would last (5–10), (11–15), and (16–20) years. 2. 30 clinically and haematologically healthy people made up the control group. inclusion criteria 1. patients between the ages of 26 and 26 who regularly get blood transfusions with a transfusion count of at least 10. 2. the patient's inclusion was con<irmed by hemoglobin electrophoresis. 3. control group: individuals chosen based on a clinical examination, history, and blood tests that were within normal limits. exclusion criteria individual who has not yet received a blood transfusion and who has a history of any other serious illnesses. hepatitis, megaloblastic anemia, iron de<iciency anemia, cancer, and veri<ied acute and chronic liver injury should all be absent in control persons. sample collection at the same time, swabs, saliva samples, and blood samples were collected. patients were instructed to rinse their mouths with distilled water to remove any residues., both in the research group and the control group. patients' buccal mucosa was cleansed with gauze, and cells were then extracted using an interdental brush that was used repeatedly in a straight line and with very little pressure. according to thomas et al. (2009) [21] and farhan and yas (2018) [22], scrapings were spread out across a vast area on a clean, dry, frosted glass slide to prevent cell clumping. following the <ixation of the slides in a coplin jar with 70% ethanol alcohol, the slides were sent to a lab for iron staining using an abcam 150674 kit. to determine whether the blue iron granules are present in the cytoplasm, the sample was <irst examined using a 10x magni<ication lens and then with a 40x magni<ication lens. for grading purposes, ten high power <ields (objective lens x40) that met the following requirements were selected: 1. cells that are widely apart and barely overlap. 2. in each high-power <ield (x40), a minimum of 20 epithelial cells and a maximum of 30 cells. 3. whether blue intracytoplasmic granules are present or absent [20]. a camera that was <ixed to the microscope was used to take the pictures. achromatic objective of 40x was used to take all the cell photos. the computer was used to save the captured images, and image j software version 1.52 a was used for analysis. the average results were calculated from ten high iron overload and serum and saliva ferri1n levels in individuals with beta thalassemia needing several blood transfusions vol 1, no 1 (2012) doi 10.5195/d3000/2025.920 http://dentistry3000.pitt.edu 3 strength areas (40x), and then the appropriate score was determined based on that. • grade 0 no particles • grade i less than 5 particles in the high <ield of vision • grade ii 5-10 particles in the high <ield of vision • grade iii 10-20 particles in the high <ield of vision • grade iv visible clumps in less than 3 high <ields of vision • grade v coarse particles or visible clumps in 3 or more high <ields of vision [20]. bone marrow used as positive control tissue. the study and control groups had their venous blood drawn at the same time as exfoliative cytology. the sample was centrifuged for 20 minutes at 4000 rpm to produce a clear supernatant. the hospital next used vidas® ferritin, an automated quantitative test that uses the enzyme linked fluorescent assay technology with a ferritin kit (biomérieux) to determine the level of ferritin in human serum or plasma. saliva was taken from each participant while they were at rest and not aroused. one hour before to the sample collection, the participants were told not to eat or drink anything and to rinse their mouths with water to get rid of any debris. under supervision, the participants were told to sit comfortably with their heads slightly leaned forward, allow saliva to collect on the <loor of their mouths, and then expel it through expectoration. during the <ive-minute saliva collection time, the samples were coded, and the patient's identity was recorded. the samples were processed and stored in a sterile plastic specimen container [23]. saliva samples were collected, then centrifuged for 20 minutes at 4000 rpm, with the clear supernatant removed and put in eppendorf tubes. the samples were then deep frozen at -20°c until the required number of samples were obtained. the e-el-h0168 human fe (ferritin) elisa kit/elabscience was then used to analyze saliva samples in the laboratory. statistical analysis data analysis was conducted using the statistical package for the social sciences (spss) version 22.0. the chi-square test was applied to compare the control group and the study group, and analysis of variance (anova), spearman's correlation coef<icient, and the f-test were calculated to relate the positivity of the oral swab to serum and saliva ferritin levels. figure 1. perl’s prussian blue staining of squamous epithelial cells from buccal mucosa showing blue intracytoplasmic iron granules, counter stain used is nuclear fast red stain. results among the 60 patients in the research group, 48 patients (80%) showed a positive result for the blue prussian dye test in the exfoliated oral mucosal cells. meanwhile, all thirty participants in the control group (100%) showed a negative reaction to the blue iron overload and serum and saliva ferri1n levels in individuals with beta thalassemia needing several blood transfusions vol 1, no 1 (2012) doi 10.5195/d3000/2025.920 http://dentistry3000.pitt.edu 4 prussian dye. the results of the prussian dye test were compared between the research and control groups using the chi-square test, and as shown in table 1, there was a statistically signi<icant difference between the two groups at a p-value of >0.001. serum ferritin (ng/ml) marker in reference to "serum ferritin" ng/ml across the various groups, the non-treatment group has the highest mean value (8194.3±3549.7) ng/ml with a large variation when compared to the other treated study groups, which have mean levels of (5274.4±2940.9) ng/ml, (5062.7±1926.6) ng/ml, and (4567.3±2102.9) ng/ml for the 5–10, 11–15, and 16–20 years accordingly (table 2). saliva ferritin in relation to "saliva ferritin" ng/ml, no treatment group recorded the highest mean value (25.347±4.273) ng/ml. then, treated groups 5-10 years, 11-15 years, and 16-20 years period followed, with mean values (11.319±3.395) ng/ml, (5.021±2.025) ng/ml, and (2.061±1.167) ng/ml, respectively, and lastly, the control group (0.146±0.058) ng/ml. this is shown in table 3. in@luence between saliva and serum ferritin a correlation between the examined markers was found (r=0.94254; p<0.001). comparison of pearl's stain grades in exfoliative cytology and serum and saliva ferritin levels in the study groups the current study's results, when compared using anova, the f test, and the spearman correlation coef<icient, revealed a non-significant association between "saliva ferritin" and the grades of buccal smears, but a significant correlation (p<0.05) between "serum ferritin" and the exfoliative cytology grades. out of 60 patients, a maximum of 22 (36.6%) had a mean serum ferritin level of 5102.9±2357.5 ng/ml, indicating grade iv positive. thirteen patients (21.6%) had a mean serum ferritin level of 5953.7±3254.1) ng/ml, indicating grade iii positivity. subsequently, 12 patients (20%) had a mean serum ferritin level of 7791.0±3179.3 ng/ml, falling into grade (0). eight patients (13.3%), on the other hand, had a mean serum ferritin level of 4334.9±2005.3 ng/ml, belonging to grade ii. subsequently, 3 patients (5%) had a mean blood ferritin level of 8307.2±3428.9 ng/ml (grade v). table 4 illustrates that a mere 2.3% of individuals exhibited grade i, with a mean blood ferritin level of 1862.7%±4688.3% ng/ml. discussion cells were collected from the oral mucosa of 48 out of 60 patients suffering from severe thalassemia (80%) in this investigation showed positive results for perls' prussian blue response. it was also noted that perls' prussian blue positive was absent from all the control participants. like the <indings of gururaj and sundharam et al. (2003) [24], who reported 100% perls' staining positivity in the 10 patients they studied, their results also demonstrated prussian blue staining positivity in perls. according to nandprasad et al. (2009) [25], 65 out of 100 patients had 65% perls' positivity; bhat et al. (2013) [26] observed 71.7% positivity (43 out of 60 patients); chittamsetty et al. (2013) [27] reported 72.5% (29 out of 40 β-thalassemia major patients); and gupta et al. (2014) [28] observed 61.6% perls' positivity (37 out of 60 cases). findings from leekha et al. (2016) [29] revealed that 35 (87.5%) of 40 patients had favorable results. furthermore, 98% of perls' positive (49 out of 50 instances) was recorded by gajaria et al. (2017) [20]. the observed positive <indings variance can be explained by the variations in the mean blood ferritin levels of the patients involved in these investigations, as well as the overall sample size differences among studies [20]. additionally, a comparison of the study group's perls' prussian blue staining with that of the control group demonstrated that the study group's positivity was not coincidental. as a result, there is a strong correlation between the positive iron staining in oral smears of patients with beta-thalassemia major who receive frequent blood transfusions and suffer from hemosiderosis. this indicates that iron accumulates in the squamous epithelial cells of the oral mucosa in cases of hemosiderosis., and that iron overload may be measured by scraping the cells. the controls did not have iron excess and were in good health. the lack of positive instances in the research group may have resulted from improper staining methods or early shedding of the impacted squamous epithelial cells, even though the patients had elevated serum ferritin levels. the creation of iron storage pools is one of the processes that determines the excess quantities of iron that collect in different tissues. perls' prussian blue staining positivity is always impacted by the variation in ferritin amounts generated in exfoliated buccal mucosal cells [20]. according to delaby et al. (2005) [30], iron initially builds up in reticuloendothelial macrophages and then spreads to parenchymal cells when hemosiderosis happens. as compared to control participants, the study groups' mean blood ferritin levels were considerably higher, with the no treatment group having the highest mean value. these <indings are consistent with canatan and akdeniz (2012) [12] and hassan et al. (2013) [31]. in agreement with canatan and akdeniz (2012) [12], the study groups' mean saliva ferritin levels were signi<icantly higher than those of the control subjects. the controlled group also showed highly signi<icant differences, with a p-value of less than 0.001, when compared to the study groups. according to jaganthan et al. (2012) [10], salivary ferritin levels signi<icantly increase in iron overload illnesses. because saliva contains enzymes that are dependent on iron, the biological system maintains larger amounts of salivary ferritin, which helps to preserve iron through saliva [32]. there is not enough information in the literature to pinpoint the precise pathophysiology of salivary ferritin elevation. although ferritin [12] and salivary components [33] may be similar in both serum and saliva, the synthesis and secretion of parotid gland acinar cells may affect salivary component levels, making them inconsistent with serum [34]. iron overload and serum and saliva ferri1n levels in individuals with beta thalassemia needing several blood transfusions vol 1, no 1 (2012) doi 10.5195/d3000/2025.920 http://dentistry3000.pitt.edu 5 the current investigation found a signi<icant association (p>0.05) between the mean serum ferritin levels in the treated and nontreated groups and the grades of buccal smear positive. this <inding suggested that the mean serum ferritin levels and buccal smear grades were associated, and the results were found to be comparable to those of gajaria et al. (2017) [20]. the grade of buccal smear positive was shown to have a strong correlation with the patients' blood ferritin levels; as a result, patients receiving regular transfusions can utilize this straightforward, non-invasive diagnostic to track iron excess. many factors, such as the number of blood transfusions the patient has received, the age at which iron chelation therapy began, whether the treatment is taken regularly, social and economic status, nutritional de<iciencies, and other comorbidities, affect the increase of iron in this group of patients [20]. regarding the "saliva ferritin" marker, there is no discernible relationship with the grades of buccal smears. we are not aware of any research that relates salivary ferritin levels to perls' prussian blue staining grades of oral exfoliative cytology. references [1] galanello r, origa r. beta-thalassemia. orphanet journal of rare diseases. 2010. 5(1), 11. [2] muncie, herbert l, campbell, james s. "alpha and beta thalassemia". american family physician. 2009. (4): 339–44. [3] greer, john p, arber, daniel a, glader, bertil, list, alan f, means, robert t, paraskevas, frixos, rodgers, george m. wintrobe's clinical hematology. lippincott williams & wilkins. (2013-08-29). [4] hydroxamic acids: advances in research and application: 2011 edition: scholarlypaper. scholarly editions. 2012-01-09 [5] greer jp, arber da, glader b, list af, means rt, frixos p, rodgers gm. wintrobe’s clinical hematology, 13th edn. wolters kluwer lippincott williams and wilkins health. 2014. [6] theil ec. ferritin protein nanocages—the story. nanotechnology perceptions. 2012; 8(1): 7. [7] world health organization, world health organization. iron deeiciency anaemia: assessment, prevention and control. a guide for programme managers. geneva: who. 2001; 99. [8] bigler, l. r., streckfus, c.f., dubinsky, w.p. salivary biomarkers for the detection of malignant tumors that are remote from the oral cavity. clinics in laboratory medicine, 29(1) (2009): 71-85. [9] agarwal, p.k., agarwal, k. n., agarwal, d. k. biochemical changes in saliva of malnourished children. the american journal of clinical nutrition, 39(2) (1984): 181-184. [10] jagannathan, nithya, prasanna neelakantan, pratibha ramani, priya premkumar, anuja natesan, herald j. sherlin. "salivary ferritin – a concise update on current concepts." let the science be your passion (2012a). [11] eliasson, l., carlen, a. an update on minor salivary gland secretions. european journal of oral science, 118(5) (2010): 435-442. [12] canatan, duran, and sevgi kosaci akdeniz. "iron and ferritin levels in saliva of patients with thalassemia and iron deeiciency anemia." mediterranean journal of hematology and infectious diseases 4, no. 1 (2012). [13] theil, elizabeth c. "ferritin protein nanocages—the story." nanotechnology perceptions 8, no. 1 (2012): 7. [14] kzar my, hussein am. salivary iron and ferritin levels, orofacial complications of patients with thalassemia major in babylon teaching hospital for maternity and children. medical journal of babylon. 2017; 14(1):135-139. [15] çaliskan u, tonguç mom , çiris m, balta n, kirzioglu fy, çaglayan g, canatan d. the investigation of gingival iron accumulation in thalassemia major patients. journal of pediatric hematology/oncology. 2011;33(2):98-102. [16] rajput dv, tupkari jv. early detection of oral cancer: pap and agnor staining in brush biopsies. journal of oral and maxillofacial pathology: jomfp. (2010);14(2):52. [17] hofebrand av, moss pa. hofnbrand's essential haematology (vol. 38). john wiley & sons. 2015. [18] sivapathasundharam b, kalasagar m. yet another article on exfoliative cytology. journal of oral and maxillofacial pathology. 2004;8(2):54. [19] cappellini md, cohen a, porter j, taher a, viprakasit v. guidelines for the management of transfusion dependent thalassaemia (tdt). nicosia, cyprus: thalassaemia international federation. 2014. [20] gajaria pk, maheshwari um, borkar db, dhar r, pancholi v. buccal mucosa exfoliative cell prussian blue stain co-relates with iron overload in β-thalassemia major patients. indian journal of hematology and blood transfusion. 2017;33(4):559-564. [21] thomas p, holland n, bolognesi c, kirschvolders m, bonassi s, zeiger e, fenech m. buccal micronucleus cytome assay. nature protocols. 2009;4(6):825. [22] farhan rs, yas ls. oral finding and cytomorphometric analysis of oral mucosal cells in type 2 diabetic patients. international journal of medical research & health sciences. 2018;7(6):86-93. 23. navazesh, mahvash. methods for collecting saliva. annals of the new york academy of sciences. 1993, 694.1: 72-77. [24] gururaj n, sundharam s. demonstration of iron in the exfoliated cells of the oral mucosa. j oral maxillofac pathol. 2003;1:37–39 [25] nandprasad s, sharada p, vidya m, karkera b, hemanth m, prakash n. oral exfoliative cytology in beta thalassemia patients undergoing repeated blood transfusions. internet j pathol. 2009;10:7. [26] bhat atul a, parwani rk, wanjari sp. demonstration of iron in exfoliated buccal cells of b thalassemia major patients. j cytol. 2013;30(3):169–173. [27] chittamsetty h, munishekar syed, ahmed afroz, palla churu suri sridevi. a noninvasive technique which demonstrates iron in the buccal mucosa of sickle cell anaemia and thalessemia patients who undergo repeated blood transfusion. j clin diag res. 2013;7(6):1219–1222. [28] gupta s, trichal vk, malik r, nigam rk, choudhary r, shrivastava a. perl’s prussian blue positivity in exfoliated buccal cells of b thalessemia major patients and its correlation with serum ferritin. j evol med dental sci. 2014;3(19):5135–5140. [29] leekha s, nayar ak, bakshi p, sharma a, parhar s, soni s. estimation of iron overloads using oral exfoliative cytology in beta-thalassemia major patients. cytojournal. 2016;13. [30] delaby c, pilard n, gonçalves as, beaumont c, canonne-hergaux f. presence of the iron exporter ferroportin at the plasma membrane of macrophages is enhanced by iron loading and down-regulated by hepcidin. blood. 2005;106(12):3979-3984. [31] hassan, najat ar, musa, raad j, abdullah, sadik a. effect of iron overload and lipid peroxidation on thyroid function in iraqi patients with ß-thalassemia. asian journal of applied sciences. 2013, 1.5. [32] jagannathan n, thiruvengadam c, ramani p, premkumar p, natesan a, sherlin hj. salivary ferritin as a predictive marker of iron deeiciency anemia in children. journal of clinical pediatric dentistry. 2012, 37.1: 25-30. [33] cicek d, dagli af, aydin s, baskaya dogan f, dertlioglu sb, ucak h, demir b. does hepcidin play a role in the pathogenesis of aphthae in behçet's disease and recurrent aphthous stomatitis? journal of the european academy of dermatology and venereology. 2014, 28.11: 1500-1506. [34] jou yj, lin cd, lai ch, chen ch, kao jy, chen sy, tsai mh, huang sh, lin cw. proteomic iron overload and serum and saliva ferri1n levels in individuals with beta thalassemia needing several blood transfusions vol 1, no 1 (2012) doi 10.5195/d3000/2025.920 http://dentistry3000.pitt.edu 6 identieication of salivary transferrin as a biomarker for early detection of oral cancer. analytica chimica act. 2010, 681.1-2: 41-48. table 1. comparison of the reaction of prussian blue of pearls between the study group and the control group using the contingency coef<icient. groups exfoliative cytology staining no&% total no.&% c.s. (*) p-value negative positive control 30 100% 0 0.0% 30 100% c.c. = 0.625 p=0.000 patients 12 20% 48 80 % 60 100% total 42 46.6% 48 53.3% 90 100% (*) signiiicant at p<0.001; testing based on chi-square. table 2. summary statistics of serum ferritin (ng/ml) readings distributed in different groups. groups no. mean standard deviation standard error 95% c.i. for mean minimum maximum lower boundary upper boundary control 30 71.653 47.2 8.6 54.0 89.3 17.6 180.8 no treatment 15 8194.3 3549.7 916.5 6228.6 10160.1 2492.0 12000.0 5-10 years 15 5274.4 2940.9 759.3 3645.8 6903.1 1531.5 11929.4 11-15 years 15 5062.7 1926.6 497.5 3995.8 6129.6 1969.1 8059.8 16-20 years 15 4567.3 2102.9 543.0 3402.8 5731.8 1515.1 7688.6 table 3. summary statistics of saliva ferritin (ng/ml) readings distributed in different groups. groups no. mean standard deviation standard error 95% c.i. for mean minimum maximum lower boundary upper boundary control 30 0.146 0.058 0.011 0.125 0.168 0.100 0.300 no treatment 15 25.347 4.273 1.103 22.980 27.713 15.050 30.12 (5 _ 10) yrs. 15 11.319 3.395 0.877 9.439 13.199 6.530 17.05 (11 _ 15) yrs. 15 5.021 2.025 0.523 3.899 6.142 1.310 9.500 (16 _ 20) yrs. 15 2.061 1.167 0.301 1.415 2.708 1.100 5.200 table 4. comparison of exfoliative cytology grades with average serum and salivary ferritin levels in study groups. marker grade no. of patients mean standard deviation standard error minimum maximum p-value serum ferritin ng/ml 0 12 7791 3179 917 2501 12000 f=3.333 p=0.011 i 2 1862 468 331 1532 2194 iron overload and serum and saliva ferri1n levels in individuals with beta thalassemia needing several blood transfusions vol 1, no 1 (2012) doi 10.5195/d3000/2025.920 http://dentistry3000.pitt.edu 7 ii 8 4334 2005 709 1984 7374 iii 13 5953 3254 902 2501 12000 iv 22 5102 2357 502 1515 11725 v 3 8307 3428 1979 5224 12000 saliva ferritin ng/ml 0 12 10.7 10.4 3.0 1.2 30.1 f=1.625 p=0.169 i 2 9.5 8.3 5.9 3.6 15.4 ii 8 7.9 4.1 1.5 1.1 15.3 iii 13 10.7 11.1 3.1 1.1 29.7 iv 22 10.5 9.2 2.0 1.1 28.9 v 3 25.2 2.0 1.2 22.9 26.5 testing based on anova, f test and spearman correlation coefiicient. 1018 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1018 http://dentistry3000.pitt.edu nanohydroxyapatite and novamin on roughness and discoloration following orthodontic adhesive removal ayad a. alzubaidy1, layth m.k. nissan1, hiba m. alkhateeb2 1college of den*stry, university of baghdad, baghdad, iraq 2college of den*stry,university of al-esraa, baghdad, iraq abstract objec;ve: the purpose of the study was to assess the impact of two types of toothpaste containing nanohydroxyapamte and novamin on teeth surface roughness and teeth discoloramon following orthodonmc brackets debonding and adhesive removal. material and methods: eighty human premolars were extracted before starmng orthodonmc treatment and used as the samples for the current study. all samples were asached with metal brackets and then divided at random into two groups (n=40) by using toothpaste containing either nanohydroxyapamte (lacalut white and repair toothpaste) (lsh) and toothpaste containing novamin (sensodyne repair and protect) (lsn). the adhesive removal was done using a stainbuster bur system and wearing dental loupes at a low-speed handpiece. all the colors of the teeth were first evaluated with a vita easyshade® spectrophotometer. in addimon, enamel surface roughness measurements were done using stylus profilometry (surface roughness tester time®3200). the records were done as first measurements (e1) and (r1). the second records for the teeth (e2) and (r2) were done a\er adhesive removal. a third record was done a\er the staining procedure by immersion of teeth in black tea as (e3) and (r3). finally, the fourth measurements were carried out a\er brushing the teeth with novamin toothpaste and nanohydroxyapamte toothpaste as (e4) and (r4). results: the outcomes were analyzed stamsmcally via t-test (p ≤ 0.05). the results displayed a stamsmcally considerable effect in reducing the enamel surface roughness for both groups (lsh) and (lsn), with no difference between the two groups. in addimon, both groups had a stamsmcal impact on changing the color of the teeth, with a higher effect of (lsn) group than the (lsh) group. conclusion: novamin and nanohydroxyapamte toothpaste had a considerable impact in reducing enamel surface roughness. however, novamin toothpaste showed beser results than nanohydroxyapamte in reducing enamel discoloramon. open access cita%on: alzubaidy aa, et al. (2025) nanohydroxyapa%te and novamin on roughness and discolora%on following orthodon%c adhesive removal. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1018 received: august 15, 2025 accepted: september 8, 2025 published: september 10, 2025 copyright: ©2025 alzubaidy aa, et al. this is an open access ar%cle licensed under a crea%ve commons arribu%on work 4.0 united states license. email: ayada8liq@gmail.com introduc)on the protective outer surface of enamel consists of biomineral, tiny quantities of proteins and water. it is a barrier to the occlusal region, allowing the teeth to resist the constant impact and shear stresses they receive [1]. enamel is a mineralized human oral cavity tissue (around 85% hydroxyapatite (hap) crystals as an inorganic phase, 3% biomacromolecule as organic, and an essential 12% water) [2]. people's assessment of their malocclusions and tooth abnormalities has risen as they have become more aware of facial and dental aesthetics, which leads to growing numbers of young and older individuals seeking orthodontic treatment for "aesthetic purposes [3,4]. a bracket is a part of the fixed orthodontic appliance. attaching a bracket involves multiple stages, including cleaning and etching the tooth enamel, conditioning the surface, and bonding [5]. acid etching is essential for connecting brackets to the tooth surface. it will result in micropores, allowing resin material to penetrate the enamel and micromechanical retention, enhancing bonding strength. however, these steps result in roughness and the degradation of hydroxyapatite minerals [5]. after the end of fixed orthodontic therapy, brackets are mechanically debonded, and the remaining adhesive resin must be physically removed to prevent biofilm nanohydroxyapamte and novamin on roughness and discoloramon following orthodonmc adhesive removal vol 13, no 1 (2025) doi 10.5195/d3000.2025.1018 http://dentistry3000.pitt.edu 2 accumulation and discoloration [6]. the roughening of enamel caused by adhesive clearance may result in discoloration. adhesive residuals may alter enamel color due to internal changes generated by the adhesive resin's physicochemical interaction and outward changes caused by the superficial absorption of food coloring agents [7]. unfortunately, orthodontic therapy may have negative consequences on the teeth' surfaces. these include enamel damage due to etching and enamel surface modifications resulting in demineralization [8]. studies revealed that the rates of plaque in the mouth of orthodontic patients are two to three times greater than in average persons with high biofilm production rates [9]. continuous cycles of demineralization and remineralization occur in dental tissue. decreased oral ph leads to demineralization, which may cause mineral loss from the enamel composition. if the ph increases, calcium, phosphate, fluoride, and other agents will accumulate, causing a rebound [10]. tooth brushing with a polyvalent paste seems to be the most effective strategy for plaque reduction, salivary mineral resource acquisition, and enamel remineralization [9]. novamin is a fabricated mineral with a near structure to enamel minerals that may aid in remineralizing enamel structures that have been destroyed. in the form of calcium sodium phosphosilicate, novamin is composed of calcium, sodium, phosphorus, and silica. the silica in novamin may release crystalline hydroxyl-carbonate apatite (hca), which has a composition similar to that of a mineral in teeth; in addition, novamin particles are less than 20 microns in size, all of these enhance enamel remineralization [11]. novamin might aid in remineralizing demineralized enamel on teeth after orthodontic therapy due to the presence of cpp-acp (casein phosphopeptide-amorphous calcium phosphate), nano complexes such as carbonate-hydroxyapatite nanocrystals and calcium sodium phosphosilicate [12]. novamin permits calcium and phosphate to be liberated from the surface. these crystals repair minute surface flaws, resulting in firmer, smoother, less sensitive teeth. additionally, this substance reduces surface protein precipitation, reducing pigment's capacity to attach to teeth over time [13]. on the other hand, enamel cannot regenerate, unlike other natural bone and dentin [14]. enamel comprises 20–40 nmsized hydroxyapatite particles (hap) [15]. due to its near resemblance to the fundamental structures of enamel, it has been proposed that 20-nm-sized hap, also known as nanohydroxyapatite (nanohap), is the most biocompatible and bioactive of the manufactured apatites [16]. nanohap has a signi]icant attachment to the tooth surface. compared to the broad and amorphous varieties, these properties allow adhesion to the tooth surface. fascinatingly, a layer of nanohap produced on the surface of the tooth is very rigid to the acid solution, thus protecting the tooth from upcoming decalci]ication [17]. material and methods sample preparation two hundred fifteen extracted human maxillary first premolars that had been removed from individuals undergoing orthodontic treatment were gathered. after extraction, the teeth were washed with water to remove any traces of blood. each tooth was thoroughly rinsed to remove soft tissue remnants and debris [18]. the samples were picked after checking by 10x magnifying lens and visible light cure machine [19], and those that had the following criteria from the presence of internal stains, enamel decalcifications, decay, fluorosis, enamel defects, restorations and cracks were excluded. as a result, eighty premolar teeth have been utilized in this research. the samples were prepared and set as every three teeth were placed vertically on a stone block to the level that only the buccal surface of the crowns could be seen. the samples were then coded 1 to 80. the buccal area of the samples was brushed using non-fluoridated pumice and a nylon brush. after that, the samples were kept in closed containers with deionized water. up to the day of bonding, the deionized water was changed every day to reduce dehydration and bacterial development. over the buccal surface of the crown, each sample had a square piece of paste tape positioned on it, and a circular aperture was cut out of the tape, which allowed a working area for bonding, adhesive removal, color analysis using a spectrophotometer, and surface roughness measurements. bonding, debonding the brackets and adhesive cleaning the samples were bonded via transbond xt and the standard acid etching technique. each tooth was etched with 37% phosphoric acid for 15 seconds, sprayed with water and air for 20 seconds, then dried in the air for 10 [7]. through using a disposable brush, a light coating of transbond xt primer was applied to the etched tooth surface. each sample was supplied a maxillary first premolar bracket equilibrium® 2 type (dentaurum company, ispringen, germany) with the base of the bracket coated with a smear layer of isolation medium (vaseline) and then lined with adhesive composite. the brackets were then positioned in the working area and pressed on the tooth surface using bracket holder tweezers. for 20 seconds, the resin on the bracket was exposed to the curing light (10 seconds from the occlusal side and 10 seconds from the gingival side) [20]. following bracket bonding, the specimens were kept in artificial saliva within sealed containers in an incubator at 37°c for one week to reach equilibrium in their water sorption and to resemble the oral cavity environment. the saliva changed every three days [5]. then, each bracket was removed using bracket holder tweezers, leaving a uniform rectangle of bonded composite resin approximating the mesh of the maxillary first premolar bracket base. then, the samples were separated randomly into two groups (lsn) and (lsh), consisting of 40 teeth according to the toothpaste type that was used (either nanohydroxyapatite or novamin toothpaste) to brush the teeth. following that, each group was cleaned from the adhesive material using a method of wearing dental loupes (ergo custom-made dental loupes 6x with led light (admetic, united states)) and using (broca em zircônia multilámina) stainbuster zirconia multiblade bur (morelli ltda, brazil) in low-speed contra-angle handpiece from10000 to 20000 rpm, then the teeth polished with (ponta descartável) disposable burs for finishing (morelli ltda, brazil). the adhesive removal was accomplished when the enamel surface was smooth to the touch, and dental loupes and chair light showed no evidence of resin. then, a staining method was prepared by collecting a cup of 250 ml of freshly boiled water and one black teabag. then, all the samples were immersed in the prepared black tea 55°c solution every day for 10 minutes and kept 24 hours in 37 degrees celsius distilled water. the entire process took 30 days [21]. teeth brushing after the staining procedure was completed, the samples in the collection (lsn) were exposed to novamin (sensodyne repair and protect toothpaste), while the (lsh) group was subjected to nanohydroxyapatite toothpaste (lacalut white and repair toothpaste) as an enamel remineralization material. the brushing protocol for all groups was carried out as a pea-sized (0.25g) amount of toothpaste on a toothbrush used to brush five teeth. each tooth was brushed for 15 seconds with a soft-bristled rotary toothbrush (oralb electric rechargeable toothbrush), fixed at a constant distance by the holder. after brushing, the tooth was maintained in contact with the toothpaste slurry for an nanohydroxyapamte and novamin on roughness and discoloramon following orthodonmc adhesive removal vol 13, no 1 (2025) doi 10.5195/d3000.2025.1018 http://dentistry3000.pitt.edu 3 additional 165 seconds, so the total time was 3 minutes for each tooth. then, the tooth was rinsed with distilled water by triple syringe, and the samples were kept in artificial saliva within sealed containers in an incubator at 37°c. these procedures were carried out every 12 hours for 30 days, and the artificial saliva was changed every three days [5,22]. color measurements the spectrophotometer used was vita easyshade®. the device utilizes the cie l*a*b** value. the measurements were initially done for the samples after the adhesive removal protocol; these were verified as the first gauge value (e1) and followed by a second gauge value for the samples (e2) after the staining procedure. then, a third measurement after thirty days of brushing was recorded as (e3). before every measurement, the calibration was repeated. however, to minimize the potential for inaccuracy, three separate values were measured and averaged. the level of measurement differences in tooth color was evaluated using the device's l*a*b* and δe values. the l* value, representing the degree of whitening, ranges from 0 (black) to 100 (white), whereas the a* value indicates the red (+) and green (-), and the b* value indicates yellow (+) and blue (-) axes. the following formulation was used to calculate the difference between the two colors [23] δe = {(l2-l1)2 + (a2-al)2 + (b2-b1)2}1/2 δe 1: the difference between the recorded results attained after adhesive removal and the final recorded results after thirty days of tea submersion. clinically describes the discoloration that develops after therapy. δe 2: the change among the results attained next thirty days of tea soaking and after thirty days of teeth brushing. represents the clinical capacity of toothpaste and brushing to restore natural enamel color. in addition, the color change was evaluated for all the samples by a single blind evaluator at four-time points for l*, a* and b* values: • after adhesive removal (l*1, a*1 and b*1). • after the staining procedure through tea submersion (l*2, a*2 and b*2). • after teeth brushing (l*3, a*3 and b*3). surface roughness measurements a surface profilometer (time®3200 surface roughness tester; time group inc, beijing, china) was used in this research with a microneedle to scan the surface. the outcomes were indicated by the measure "roughness average" (ra). average roughness value (ra): the arithmetic means of the peak height and valley depth from a mean line. this parameter specifies the total surface roughness in micrometers (µm). typical enamel surface roughness (esr) ranges between 0.59 to 0.66 μm [24]. the device was set with a 0.25 mm cutoff and 0.1 mm/s speed. calibration was checked with a standard (ra ₌ 1.57 µm) after every ten samples. the surface roughness of each specimen was calculated in three points; the mean value of the three points was considered as the mean surface roughness (ra) (figure 1). surface roughness was evaluated for all the samples by a single blind evaluator at threetime points: • after adhesive removal (r1). • after the staining procedure through tea submersion (r2). • after teeth brushing (r3). . figure 1. stylus surface roughness tester time 3200 for measuring the surface roughness (ra) for the samples. statistical analysis the statistical analysis was done using ibm spss v26 and minitab v.17. the mean and standard deviations (s.d.), standard error (s.e.), minimum (min.), and maximum (max.) values were recorded. independent t-test was used to analyze the data. the level of signi]icance was ]ixed at 5%, and p ≤ 0.05 was considered statistically signi]icant. results analysis of the enamel surface roughness (esr) there was no significant difference (sd) in the esr level for all the samples between r1 and r2 time points. however, there was a sd in the esr plane for all the samples between the (r1) and after-teeth brushing (r3) time points by two types of toothpaste. the enamel surface roughness level reduced after teeth brushing from (0.590 to 0.478 µm). moreover, there was no sd among the (lsn) group and (lsh) after the adhesive removal time point (r1). furthermore, there was no sd among the (lsn) group and (lsh) after the teeth brushing time point (r3). these results showed that both toothpastes (novamin and nanohydroxyapatite) reduced the esr at the same level (no one was better than the other) (table 1). table 1. t-test to compare the ra for the groups (lsn) and (lsh) at different time points. descriptive statistics of the enamel discoloration the color variation depends on (δe) in addition to (δl*, δa*, δb*). the results of the color assessment in this study depend on δe values, l* records, a* records and b* records. 3.7 δe units were normally assigned as the clinical diagnostic value of color changes. [26]. evaluation of teeth color differences at δe1 and δe2: after immersion of all the samples in black tea (staining procedure), the discoloration was considered highly clinically distinguished compared to after the adhesive removal period because the δe1 was (31.713) units (table 2). moreover, after teeth brushing for all the samples with two kinds of toothpaste, the mean of (δe2) was considered to have very high clinical significance (31.37 units) concerning (3.7) units clinical threshold value (table 2) and (figure 2). table 2. δe1 and δe2 measurements for all the samples. figure 2. discoloration means levels of the teeth (δe1, δe2). nanohydroxyapamte and novamin on roughness and discoloramon following orthodonmc adhesive removal vol 13, no 1 (2025) doi 10.5195/d3000.2025.1018 http://dentistry3000.pitt.edu 4 assess which toothpaste had a better effect on teeth re-coloring after the staining procedure there was a highly significant effect between the (lsh) group with δe2 mean of 28.281 units and the (lsn) group with δe2 mean of 34.472 units. this result meant novamin toothpaste greatly influenced re-coloring enamel after tea submersion than nanohydroxyapatite toothpaste. the difference between the two δe2 was 6.192 units, which exceeded the clinical threshold value of 3.7 units (table 3). table 3. descriptive statistics of the enamel discoloration (δe2) between (lsh) and (lsn) groups. however, according to the t-test, the comparison occurred horizontally between two groups to measure the l* value. there was a sd at the l*2 time point between the group (lsh), which had 60.27 units and the group (lsn), which had 53.867 units. this result meant the group (lsh) had a significantly whiter teeth color than the group (lsn) by (6.403) units. in addition, at the time point l*3, there was no sd among the two groups, and both groups were at the same luminosity standard. however, according to the t-test, the comparison occurred vertically for the same group to measure the l* value at two different time points. for the (lsh) and (lsn) groups, there were significant differences between the l*2 and l*3 time points, but the novamin toothpaste moved the whitening level of the teeth 5.795 units greater than the nanohydroxyapatite toothpaste (table 4). table 4. t-test for the statistical analysis of (l*) values in the (lsh) and (lsn) groups at two different time points (l*2 and l*3). in addition, according to the t-test, the comparison occurred horizontally between two groups to measure the (a*) value. there was a significant difference at the a*2 time point between the group (lsh), which had 7.73 units and the group (lsn), which had 9.687 units. this result meant the group (lsn) had a significantly redder teeth color than the group (lsh). in addition, at the time point a*3, there was no sd among the two groups, and both groups were at the same red-green standard. however, according to the t-test, the comparison occurred vertically for the same group to measure the (a*) value. for the (lsh) and (lsn) groups, there were significant differences between the (a*2) and (a*3) time points, but the novamin toothpaste reduced the red color level of the teeth by (2.087) units greater than the nanohydroxyapatite toothpaste (table 5). table 5. t-test for the descriptive statistics of (a*) values in the (lsh) group and (lsn) group at two different time points (a*2 and a*3). moreover, according to the t-test, the comparison occurred horizontally between two groups to measure the (b*) value. there was no sd at the (b*2) and (b*3) time points between the group (lsh) and the group (lsn) (both groups were at the same yellow-blue standard). however, according to the t-test, the comparison occurred vertically for the same group to measure the (b*) value. for the (lsh) and (lsn) groups, there were significant differences between the (b*2) and (b*3) time points, but the novamin toothpaste reduced the yellow color level of the teeth by 1.57 units greater than the nanohydroxyapatite toothpaste (table 6). table 6. t-test for the descriptive statistics of (b*) values in the (lsh) group and (lsn) group at two different time points (b*2 and b*3). discussion evaluation of tea impact on esr and color the present result shows that the tea had no considerable impact on esr, which agrees with previous studies [25]. this is because sugar-free black tea has an anti-erosion effect because of its high ph values, respectively 5.7 and 6.3, which are higher than the enamel threshold p.h. (5.5). while the longer the enamel was exposed to solutions with a low ph (ph 2.0–4.0), the greater the danger of erosion. moreover, the discoloration was considered highly clinically distinguished compared to after the adhesive removal period, which may refer to the long period (30 days) for the samples immersed in black tea. moreover, the adhesive cleaning procedure leads to irregularities in the enamel surface, increasing the chances of retaining beverage pigments [26]. evaluation of toothpaste effect on esr in orthodontics, bonding of brackets to the tooth surface requires an acid etching procedure. consequently, microporosity will allow the resin substance to penetrate the enamel structure and cause micromechanical attachment, increasing the adhesion strength. however, this procedure damages hydroxyapatite and raises enamel surface roughness [5]. this research showed that there was a sd in reducing enamel surface roughness after thirty days of brushing the teeth with nanohydroxyapatite toothpaste and novamin toothpaste. moreover, there was no sd among the two types in reducing the esr level (both kinds of toothpaste had the same effect level). this result aligns with utari et al. (2021), which concluded that remineralization of the tooth surface with novamin-containing toothpaste reduces enamel surface roughness [5]. in addition, jumanca et al. (2019) concluded that utilizing toothpaste with nanohydroxyapatite, such as lacalut white and repair toothpaste and sensodyne repair and protect toothpaste efficiently treats demineralized enamel surfaces and fixes minor tooth surface defects and made a preservative coat on the etched enamel surface [27]. evaluation of toothpaste effect on enamel color change by comparing the two types of toothpaste used in this study, the novamin toothpaste greatly affected enamel re-coloring after tea submersion than nanohydroxyapatite toothpaste. the difference between the δe3 for the two types of toothpaste was (6.192) units, which exceeded the clinical threshold value. the teeth sample for the novamin toothpaste had more lightness at about (5.79) units, less red at about (2.08) units and less yellow color level by (1.57) units than the teeth samples of nanohydroxyapatite toothpaste. instead of the amount of their whitening ability, it seems that the mechanical action of removing surface tooth colors is mostly attributable to the physical properties of the minerals present in the composition of toothpaste. mechanical friction, such as silica nanohydroxyapamte and novamin on roughness and discoloramon following orthodonmc adhesive removal vol 13, no 1 (2025) doi 10.5195/d3000.2025.1018 http://dentistry3000.pitt.edu 5 and other cleaners, combined with remineralization chemicals, primarily eliminates extrinsic coloring, giving an impression of whitening [10]. in their study, jumanca et al. (2019) aimed to show the value of employing nanohydroxyapatite-containing toothpaste on human teeth and in what way it works at the microscopic seen by capping the etched enamel. the result was that sensodyne toothpaste was the maximum active in precipitating an intense coat on the tooth surface, a coat that shields and improves the tooth defects. at the same time, the lacalut toothpaste precipitated a thin coat, and the enamel prisms could be noticed as not completely capped. the present result aligns with other research that used several types of sensitive-relief toothpaste and found toothpaste with novamin increased the l* value after simulating brushing with an electric toothbrush (braun oral-b advance power) and immersion in coffee, making natural teeth and composites seem brighter [28]. conclusion 1 black tea (without sugar) did not affect esr but highly affected teeth discoloration. 2 both novamin and nanohydroxyapatite-containing toothpaste had a considerable impact in reducing esr. however, sensodyne repair and protect toothpaste showed better results than lacalut white and repair in reducing enamel discoloration. acknowledgement no funding was provided to the authors at any stage of the article's development or publication. references 1. k. a. derocher, p. j. smeets, b. h. goodge, m. j. zachman, p. v. balachandran., l. stegbauer, et al., “chemical gradients in human enamel crystallites.” nature, 583(7814):66–71, (2020). 2. h. zhao, s. liu, j. lu, x. yang, z. yang, f. li, et al., “natural tooth enamel and its analogs.” cell reports phys sci,3(7):100945, (2022). 3. r.s. nanda, m. j. kierl, “prediction of cooperation in orthodontic treatment.” am j orthod dentofac orthop,102(1):15–21, (1992). 4. e.s. jasim, n. f. al-khawaja, “the reliability of orthodontic treatment, according to the needs of patients using the dental aesthetic index.” indian j forensic med toxicol, 14(2):551–7, (2020). 5. t. r. utari, b. a. paryontri, a. muzayyana, “the effect of novamin on surface roughness dental enamel after debonding orthodontic bracket.” in: proceedings of the 4th international conference on sustainable innovation 2020– health science and nursing (icosihsn 2020), p. 254–60, (2021). 6. h. j. joo, y. k. lee, d. y. lee, y. j. kim, y. k. lim, “influence of orthodontic adhesives and clean-up procedures on the stain susceptibility of enamel after debonding.” angle orthod, 81(2):334–40, (2011). 7. y. boncuk, z. c. çehreli, ö. polat-özsoy, “effects of different orthodontic adhesives and resin removal techniques on enamel color alteration.” angle orthod, 84(4):634–41, (2014). 8. l. r. legler, d. h. retief, e. l. bradley, “effects of phosphoric acid concentration and etch duration on enamel depth of etch: an in vitro study.” am j orthod dentofac orthop, 98(2):154– 60, (1990). 9. h. löe, “oral hygiene in the prevention of caries and periodontal disease.” int dent j, 50(3):129–39 9, (2000). 10. a. muntean, s. sava, a. g. delean, a. m. mihailescu, l. s. dumitrescu, m. moldovan, et al., “toothpaste composition effect on enamel chromatic and morphological characteristics: in vitro analysis.” materials (basel), 12(16):2610, (2019). 11. m. vahid golpayegani, a. sohrabi, biria, m. g. ansari, “remineralization effect of topical novamin versus sodium fluoride (1.1%) on carieslike lesions in permanent teeth.” journal of dentistry (tehran, iran), vol. 9, (2012). 12. p. mohanty, “an in vitro evaluation of remineralization potential of novamin ® on artificial enamel sub-surface lesions around orthodontic brackets using energy dispersive x-ray analysis (edx).” j clin diagnostic res, 8(11):zc88–91, (2014). 13. l. gendreau, a. p. s. barlow, s. c. mason, “overview of the clinical evidence for the use of novamin in providing relief from the pain of dentin hypersensitivity.” j clin dent, 22(3):90–5, (2011). 14. s. busch, “regeneration of human tooth enamel.” angew chemie int ed, 43(11):1428–31, (2004). 15. c. robinson, s. connell, j. kirkham, shore, r. a. smith, “dental enamel—a biological ceramic: regular substructures in enamel hydroxyapatite crystals revealed by atomic force microscopy.” j mater chem, 14(14):2242–8 (2004). 16. l. li, h. pan, j. tao, x. xu, c. mao, x. gu, et al., “repair of enamel by using hydroxyapatite nanoparticles as the building blocks.” j mater chem, 18(34):4079 (2008). 17. s. ajami, h. r. pakshir, n. babanouri, “impact of nanohydroxyapatite on enamel surface roughness and color change after orthodontic debonding.” prog orthod, 17(1) (2016). 18. h. m. alkhateeb, e. i. al-sheakli, “shear bond strength of different lingual buttons bonded to wet and dry enamel surfaces with resin modified glass ionomer cement: in vitro comparative study.” j baghdad coll dent, 25(1):146–52 (2013). 19. s. s. malik, n. h. ghaib, “the effect of nano hydroxyapatite on remineralize white spot lesions prior to orthodontic adhesive removal by different techniques: an in vitro comparative study.” j baghdad coll dent, 28(2):90–6 (2017). 20. y. a. yassir, a. m. el-angbawi, g. t. mcintyre, g. f. revie, d. r. bearn, “a randomized clinical trial of the effectiveness of 0.018-inch and 0.022-inch slot orthodontic bracket systems: part 1 duration of treatment.” eur j orthod, 41(2):133–42 (2019). 21. m. nahidh, “the effects of various beverages on the shear bond strength of light cured orthodontic composite: an in vitro comparative study.” j baghdad coll dent. 26(3):144–8 (2014). 22. g. k. hussein, s. a. mohammed, “effect of whitening toothpastes with different whitening agents on the color stability of orthodontic clear elastic ligatures.” int j med res heal sci, 7(3):128–34 (2018). 23. h. gorucu–coskuner, e. atik, t. taner, “tooth color change due to different etching and debonding procedures.” angle orthod, 88(6):779– 84 (2018). 24. p. shah, p. sharma, s. k. goje, n. kanzariya, m. parikh, “comparative evaluation of enamel surface roughness after debonding using four finishing and polishing systems for residual resin removal—an in vitro study.” prog orthod, 20(1):18 (2019). 25. s. h. rajab, s. m. elmarsafy, m.s. alsoufi, “effect of chinese green tea on enamel surface characteristics in an in-vitro erosion model.” alazhar dent j, 5(3):257–66 (2018). 26. c. ye, z. zhao, q. zhao, x. du, j. ye, x. wei, “comparison of enamel discoloration associated with bonding with three different orthodontic adhesives and cleaning-up with four different procedures.” j dent, 41(suppl.5):1–6 (2013). 27. d. jumanca, a. matichescu, a. galuscan, o. balean, l. c. rusu, “the effect of hydroxyapatite from various toothpastes on tooth enamel.” rev chim, 70(7):2604–7 (2019). 28. s. turgut, h. kilinç, k. u. ulusoy, b. bagis, “the effect of desensitizing toothpastes and coffee staining on the optical properties of natural teeth and microhybrid resin composites: an invitro study.” biomed res int, 2018, (2018). 1086 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu modification of 3d printed denture base material by hyaluronic acid to improve healing immunohistological effect on mmp2 and vegf mustafa mahmood jasim, amnar k. al-noori, ghada a. taqa college of den*stry, university of mosul, mosul, iraq abstract objec?ves: to evaluate the effect of topical release of different concentra?ons of hyaluronic acid from 3d printed denture bases on the healing of skin wounds. materials and methods: fikeen healthy adult male rabbits from new zealand were randomly split into three groups based on how long they would be kept: 3 days, 7 days, and 14 days. we shaved the skin on the back of each rabbit and then made five cuts in it. the wounds were sutured to allow for healing. the animals were split into three groups based on their treatment type. the first group had their wounds treated with just an incision (control nega?ve), the second group had their incisions treated with a 3d printed specimen under the skin incision without hyaluronic acid (control posi?ve). we added 0.25% hyaluronic acid and a 3d printed denture base specimen to the third incision, and we added 0.5% hyaluronic acid and a 3d printed denture base to the fourth incision. the fikh cut was treated with 1% ha acid and a 3d printed denture base specimen. aker that, the biopsies were sent for immunohistochemistry evalua?on. results: the data from the treated group showed that hyaluronic acid (ha) speeds up healing. conclusion: ha-treated groups, par?cularly those administered 0.5% and 1% concentra?ons, demonstrated markedly accelerated and more comprehensive epithelial regenera?on. open access cita%on: jasim mm et al. (2025) modifica%on of 3d printed denture material by hyaluronic acid to improve healing: immunohistological effect on mmp2 and vegf. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1086 received: october 19, 2025 accepted: november 9, 2025 published: december 12, 2025 copyright: ©2025 jasim mm et al. this is an open access ar%cle licensed under a crea%ve commons axribu%on work 4.0 united states license. email: ghadataqa@uomosul.edu.iq introduc)on hyaluronic acid (ha) is a naturally occurring polymer made up of a linear polysaccharide structure that can degrade spontaneously under certain conditions. it plays a crucial role as a primary component of the extracellular matrix (ecm) [1]. this biopolymer is both biocompatible and biodegradable, and it can be found in various biological fluids and tissues [2]. the beneficial characteristics of ha have made it an important biopolymer in the biomedical field [3]. ha is an important biopolymer in biomedicine due to its numerous advantages. it maintains wellpreserved structural properties and interacts with various ecm proteins and collagen fibers, facilitating cell adhesion, movement, migration, and growth [4]. numerous biological roles in wound healing, including angiogenesis and re-epithelialization, have been observed both in vitro and in vivo through the topical application of ha [5]. it has been used in the healing of the socket after tooth extraction [6]. tissue regeneration and wound healing are complex biological events that rely on the coordinated interaction of growth factors, extracellular matrix (ecm) remodeling enzymes, and cellular responses [7]. among the most important mediators involved in these processes are vascular endothelial growth factor (vegf) and matrix metalloproteinase 2 (mmp2), both of which play essential roles in angiogenesis, ecm turnover, and cell migration [8,9]. vegf is a potent pro-angiogenic cytokine that stimulates endothelial cell proliferation, modificaaon of 3d printed denture base material by hyaluronic acid to improve healing vol 13, no 1 (2025) doi 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu 2 migration, and the formation of new blood vessels. adequate vascularization is fundamental for oxygen and nutrient supply to the regenerating tissue, thereby supporting successful repair. upregulation of vegf expression has been strongly correlated with accelerated healing, whereas impaired vegf signaling is often linked to delayed tissue repair and chronic wounds [10]. mmp2, a member of the gelatinase subgroup of the mmp family, contributes to ecm remodeling by degrading type iv collagen and other basement membrane components. mmp2 enables cellular migration, angiogenesis, and proper tissue remodeling through this activity. nevertheless, excessive or dysregulated mmp2 expression can lead to pathological outcomes such as chronic inflammation, impaired healing, or cancer progression [8]. material and methods fifteen zealand adult male rabbits were used, 5-7 months in age, weighing 1.25-1.5 kg. these rabbits stayed in the animal house. the house was prepared for the experimental studies in the college of dentistry at standard-setting with temperature maintained at 25 ± 2 °c. the feeding of these rabbits were fed three times daily with a normal diet (lettuce and grass). all rabbits had good health throughout the period of the study. all procedures were carried out per the rules established by the research ethics committee of the college of dentistry at the university of mosul in iraq, using the code (uom.dent. 25/1006). we used fifteen adult male rabbits from new zealand that were 5 to 7 months old and weighed between 1.25 and 1.5 kg. the animal house was set up for the experimental studies at the college of dentistry, where the rabbits stayed at a temperature of 25 ± 2 °c. they were fed three times a day with a normal diet of lettuce and grass. three groups of three rabbits were randomly chosen from a group of animals. there were five experimental groups of incisions in the dorsal skin of each rabbit, based on the type of dressing material used (figure 1). the groups were: group 1: n=5, negative control. skin incisions did not receive any treatment. group 2: n=5, positive control. skin incisions treated with a sample of 3d printed denture base material only. group 3: n=5, treated with hyaluronic acid 1%. skin incisions treated with the topical part of 3d printed denture base material combined with hyaluronic acid 1% group 4: n=5, treated with hyaluronic acid 0.25%. skin incisions treated with the topical part of a 3d printed denture base material combined with hyaluronic acid 0.25% group 5: n=5, treated with hyaluronic acid 0.5%. skin incisions treated with the topical part of a 3d printed denture base material combined with hyaluronic acid 0.5%. biopsies were collected and subjected to immuohistopathological examination according to specified time intervals. the animals in each group were randomly split into five smaller groups, each with three rabbits. these groups were: g1: the animals were put to sleep three days after surgery. g2: the animals were put to death seven days after the surgery. g3: the animals were put to death two weeks after their surgery. overdose anesthesia killed all the animals. the samples from skin and buccal defects were preserved in 10 percent formaldehyde for 48 hours, subsequently treated with ethanol alcohol and xylene, embedded and labeled in paraffin wax blocks, frozen for 24 hours, and then sectioned coronally into 4micron thick slices using a microtome. a strip of cut tissue with wax at the incision level was then put in a 60 °c water bath, where the tissues were put on a glass slide that had been marked. after that, the slides were de-waxed, stained with hematoxylin and eosin, mounted with dpx, and examined with a light microscope. results table 1 shows the median scores of granulation tissue (gt) formation in five experimental groups across three-time intervals (day 3, day 7, and day 14), with five rabbits per group (n=5). the kruskal-wallis test was used to assess statistically significant differences among groups and time points. the scores are expressed as median and interquartile range (iqr). on day 3, there was a statistically significant difference among the groups (p=0.05). the ha 1% group (g5) exhibited the highest granulation tissue formation, while the negative control group (g1) had no granulation tissue. capital letters (a, b, ab) reflect these differences: g5 was significantly higher (a), g1 was lower (b), and other groups were intermediate (ab). on day 7, intergroup differences remained significant (p = 0.044). g5 (ha 1%) and g3 (ha 0.25%) showed higher granulation tissue scores compared to g1 and g2. this suggests an early stimulatory effect of ha on granulation tissue formation, especially at higher concentrations. on day 14, the differences between groups were still statistically significant (p=0.048). g3 and g4 (ha 0.25% and 0.5%) recorded the highest scores (3), indicating enhanced granulation tissue development. g1 and g2 showed lower scores, suggesting that ha accelerated the healing process. for g1 (negative control), there was a statistically significant increase in granulation tissue formation over time (p=0.05), progressing from no granulation on day 3 to moderate levels by day 14. in g2 (positive control), granulation tissue also increased significantly over the three time points (p=0.05), but the levels remained relatively low compared to ha-treated groups. g3 (ha 0.25%) and g4 (ha 0.5%) showed clear time-dependent increases in granulation tissue, with statistically significant changes (p=0.042 and p=0.048, respectively). by day 14, both groups reached peak granulation scores (score 3), indicating strong tissue regeneration. interestingly, g5 (ha 1%) showed high granulation early (day 3 and 7), but slightly decreased by day 14, possibly due to transition from granulation to tissue remodeling (p=0.05). the most prominent effects were observed in groups treated with 0.25% and 0.5% ha, especially by day 14. the ha 1% group showed a rapid early response, but granulation plateaued or declined slightly, likely reflecting advanced healing. the differences were statistically significant between groups and within each group over time. data expressed as median and iqr (interquartile-range) (n=3 rabbits) (kruskalwallis test). the difference in capital letters means there are significant differences between groups at p≤0.05. the difference in small letters means there are significant differences between periods at p≤0.05. table 2 presents the median histopathological scores of angiogenesis (new blood vessel formation) in five experimental groups across three-time intervals (day 3, day 7, and day 14). each group included five rabbits (n=5), and the kruskal-wallis test was used to evaluate statistically significant differences across groups and over time. data are expressed as medians and interquartile ranges (iqr). on day 3, no statistically significant differences were observed between the groups (p=0.078). all groups showed low angiogenesis activity, with medians ranging from 1 to 2. this suggests minimal early vascular response to treatment at that time point. on day 7, the kruskal-wallis test indicated a significant difference among groups (p=0.05). the ha-treated groups (g3–g5) showed markedly increased angiogenesis (median=4), while the control groups (g1 and g2) had lower scores (medians=2–3). capital letters show that these differences are statistically significant between groups, modificaaon of 3d printed denture base material by hyaluronic acid to improve healing vol 13, no 1 (2025) doi 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu 3 indicating the pro-angiogenic effect of ha at this stage of healing. by day 14, the differences remained significant (p=0.048). the ha 0.25% and 0.5% groups (g3 and g4) maintained the highest angiogenesis scores (median=4), while the negative control group (g1) exhibited lower values. g5 (ha 1%) showed a slight reduction compared to day 7, possibly indicating a transition from active angiogenesis to vascular stabilization or tissue remodeling. in g1 (negative control), angiogenesis gradually increased over time, with significant differences observed between days (p=0.048). however, the increase was moderate compared to the ha-treated groups. g2 (positive control) also showed a significant rise in angiogenesis from day 3 to day 14 (p=0.034), suggesting a delayed but noticeable natural angiogenic response. g3 (ha 0.25%) and g4 (ha 0.5%) both demonstrated a rapid and sustained increase in angiogenesis, with statistically significant differences over time (p=0.028 for both). these groups had consistently high scores from day 7 onward, highlighting the effectiveness of these ha concentrations in promoting vascularization. g5 (ha 1%) also showed early enhancement of angiogenesis (day 3 to day 7), followed by a slight decrease by day 14 (p=0.05). this might reflect earlier maturation or stabilization of newly formed vessels. the findings demonstrate that hyaluronic acid (ha) significantly enhances angiogenesis in rabbit skin wounds, particularly at 0.25% and 0.5% concentrations. although all groups exhibited a natural increase in angiogenesis over time, ha-treated groups achieved faster and higher vascular growth, especially by day 7 and 14. the differences were statistically significant across groups and within each group over time, emphasizing the therapeutic role of ha in promoting wound healing through enhanced vascularization. the histological evaluation of rabbit skin wound sections across all experimental groups at various time intervals (days 3, 7, and 14) are shown in figures 1 to 6. discussion additive manufacturing, especially three-dimensional (3d) printing, has become a new and better way to make prosthetics instead of using heat-polymerized polymethyl methacrylate (pmma) [11]. 3d printed denture bases have several benefits, such as the ability to integrate digital workflows, shorten lab time, improve reproducibility, and allow for patient-specific customization [12]. the layered printing method is important because it lets you control how bioactive agents are added to the polymer matrix. this is not easy to do with traditional processing methods [13]. this creates new chances to turn denture bases from passive prosthetic devices into biofunctional systems that can help keep oral tissues healthy [14]. adding bioactive substances like hyaluronic acid or bioactive glass speeds up the healing of soft tissue and makes it less painful. this helps patients adjust better [15]. modification of denture base materials by different materials to improve physical and biological effect [16-18]. the rabbit was chosen for the skin wound model due to its comparatively extensive skin surface area, white skin, and structural resemblance to human skin, which facilitated the simultaneous creation of multiple wounds [19]. histologically, rabbits' buccal mucosa is like humans' oral mucosa. in our study, after day three and day seven, the ha 1% group exhibited the highest granulation tissue formation, while the negative control group had no granulation tissue. also, the most prominent effects were observed in groups treated with 0.25% and 0.5% ha, especially by day 14. the ha 1% group showed a rapid early response, but granulation plateaued or declined slightly, likely reflecting advanced healing [20]. the wound area and contraction ratio of the groups treated with ha revealed a significant wound healing acceleration effect [21]. previous studies have also shown that the topical application of ha can speed up the wound healing process [22]. ha can speed up the wound healing process in oral ulcer [23]. pdgf can promote the expression of cd44 to enhance cell migration. cd44 serves as the primary receptor for ha. the association and interaction between ha and cd44 regulate numerous intracellular signaling pathways that govern cellular biological processes. these encompass angiogenesis, cellular migration, proliferation, adherence to ecm constituents, and the internalization and degradation of ha [24]. in our study, the level of pdgf in the different groups reveals intense positive expression to improve the healing process in days 3, 7, and 14 [25]. on the other hand, mmp2 showed intense positive expression in days 3 and 7, and moderate positive expression in day 14, as previous work [26]. conclusions we concluded that hyaluronic acid release from 3d printed denture base with different concentrations (0.25%, 0.5%, and 1%), especially 1% ha that had a highly positive effect as immunohistochemical changes in wound healing. acknowledgements we appreciate the college of dentistry, university of mosul, for providing its support to this work. conflict of interest the authors have no con.licts of interest to declare. references 1. buckley, c., murphy, e. j., montgomery, t. r., & major, i. (2022). hyaluronic acid: a review of the drug delivery capabilities of this naturally occurring polysaccharide. polymers, 14(17), 3442. 2. biswal, t. (2021). biopolymers for tissue engineering applications: a review. materials today: proceedings, 41, 397-402. 3. snetkov, p., zakharova, k., morozkina, s., olekhnovich, r., & uspenskaya, m. (2020). hyaluronic acid: the influence of molecular weight on structural, physical, physico-chemical, and degradable properties of biopolymer. polymers, 12(8), 1800. 4. amorim, s., reis, c. a., reis, r. l., & pires, r. a. (2021). extracellular matrix mimics using hyaluronan-based biomaterials. trends in biotechnology, 39(1), 90-104. 5. rajab, a. m., taqa, g. a., & t al-wattar, w. (2022). the effect of topically applied hyaluronic acid on skin wound healing in rabbits. journal of applied veterinary sciences, 7(1), 38-45. 6. domic, d., bertl, k., lang, t., pandis, n., ulm, c., & stavropoulos, a. (2023). hyaluronic acid in tooth extraction: a systematic review and meta-analysis of preclinical and clinical trials. clinical oral investigations, 27(12), 7209-7229. 7. diller, r. b., & tabor, a. j. (2022). the role of the extracellular matrix (ecm) in wound healing: a review. biomimetics, 7(3), 87. 8. wolosowicz, m., prokopiuk, s., & kaminski, t. w. (2024). the complex role of matrix metalloproteinase-2 (mmp-2) in health and disease. international journal of molecular sciences, 25(24), 13691. 9. zheng, h., takahashi, h., murai, y., cui, z., nomoto, k., niwa, h., . . . takano, y. (2006). expressions of mmp-2, mmp-9 and vegf are closely linked to growth, invasion, metastasis and angiogenesis of gastric carcinoma. anticancer research, 26(5a), 3579-3583. 10. goswami, a. g., basu, s., huda, f., pant, j., ghosh kar, a., banerjee, t., & shukla, v. k. (2022). an appraisal of vascular endothelial growth factor (vegf): the dynamic molecule of wound healing and its current clinical applications. growth factors, 40(3-4), 73-88. 11. valenti, c., federici, m. i., coniglio, m., betti, p., pancrazi, g. p., tulli, o., . . . pagano, s. (2024). mechanical and biological properties of polymer materials for oral appliances produced with additive 3d printing and subtractive cad-cam techniques compared to conventional methods: a systematic review and meta-analysis. clinical oral investigations, 28(7), 396. 12. pillai, s., upadhyay, a., khayambashi, p., farooq, i., sabri, h., tarar, m., . . . wang, y. (2021). modificaaon of 3d printed denture base material by hyaluronic acid to improve healing vol 13, no 1 (2025) doi 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu 4 dental 3d-printing: transferring art from the laboratories to the clinics. polymers, 13(1), 157. 13. ligon, s. c., liska, r., stampfl, j., gurr, m., & mülhaupt, r. (2017). polymers for 3d printing and customized additive manufacturing. chemical reviews, 117(15), 10212-10290. 14. burdușel, a.-c., gherasim, o., grumezescu, a. m., mogoantă, l., ficai, a., & andronescu, e. (2018). biomedical applications of silver nanoparticles: an up-to-date overview. nanomaterials, 8(9), 681. 15. jafari, n., habashi, m. s., hashemi, a., shirazi, r., tanideh, n., & tamadon, a. (2022). application of bioactive glasses in various dental fields. biomaterials research, 26(1), 31. 16. altarazi, a., haider, j., alhotan, a., silikas, n., & devlin, h. (2023). 3d printed denture base material: the effect of incorporating tio2 nanoparticles and artificial ageing on the physical and mechanical properties. dental materials, 39(12), 1122-1136. 17. jaffer, n. t., hasan, r. m.-a., & taqa, g. a. a. (2023). impact of apigenin and seashell nanoadditives on the antifungal and roughness behavior of a soft denture liner. cellular and molecular biology, 69(9), 37-42. 18. al-omari, a. w., al-noori, a. k., & alsammak, e. g. (2024). cytotoxicity, chlorhexidine and residual monomer release from heat-polymerized denture base resin modified by polyethylene glycol (peg) and chlorhexidine (chx). 19. shukla, s. k., sharma, a. k., gupta, v., kalonia, a., & shaw, p. (2020). challenges with wound infection models in drug development. current drug targets, 21(13), 1301-1312. 20. pilloni, a., marini, l., gagliano, n., canciani, e., dellavia, c., cornaghi, l. b., . . . rojas, m. a. (2023). clinical, histological, immunohistochemical, and biomolecular analysis of hyaluronic acid in early wound healing of human gingival tissues: a randomized, split-mouth trial. journal of periodontology, 94(7), 868-881. 21. wang, y., chen, l., ren, d.-y., feng, z.-x., zhang, l.-y., zhong, y.-f., . . .du, y.-z. (2022). musselinspired collagen-hyaluronic acid composite scaffold with excellent antioxidant properties and sustained release of a growth factor for enhancing diabetic wound healing. materials today bio, 15, 100320. 22. antoszewska, m., sokolewicz, e. m., & barańska-rybak, w. (2024). wide use of hyaluronic acid in the process of wound healing—a rapid review. scientia pharmaceutica, 92(2), 23. 23. peck, m., hiss, d., stephen, l., & olivier, a. (2018). the in vitro effect of leukocyte-and plateletrich fibrin (l-prf) and cross-linked hyaluronic acid on fibroblast viability and proliferation. south african dental journal, 73(6), 395-399. 24. cyphert, j. m., trempus, c. s., & garantziotis, s. (2015). size matters: molecular weight specificity of hyaluronan effects in cell biology. international journal of cell biology, 2015(1), 563818. 25. knopf-marques, h., pravda, m., wolfova, l., velebny, v., schaaf, p., vrana, n. e., & lavalle, p. (2016). hyaluronic acid and its derivatives in coating and delivery systems: applications in tissue engineering, regenerative medicine and immunomodulation. advanced healthcare materials, 5(22), 2841-2855. 26. caley, m. p., martins, v. l., & o'toole, e. a. (2015). metalloproteinases and wound healing. advances in wound care, 4(4), 225-234. a b c figure 1. (a) surgical procedure for skin; defect created by scalpel blade no.15 on the dorsal skin of a rabbit. (b) 3d printed denture sample. (c) sutured skin. modificaaon of 3d printed denture base material by hyaluronic acid to improve healing vol 13, no 1 (2025) doi 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu 5 table 1. histopathological scores of the granulation tissue gt among study groups in the rabbit skin. intervals groups day 3 (n=5) median day 7 (n=5) median day 14 (n=5) median p-value g1 negative control group (wound only) 0 (0) b b 1 (0) b ab 2 (0) b a 0.05 g2 positive control group (wound + acrylic) 1 (1) ab b 1 (1) b b 2 (0) b a 0.05 g3 ha 0.25% group (wound + acrylic+ha) 1 (1) ab b 2 (1) b ab 3 (1) a a 0.042 g4 ha 0.5% group (wound + acrylic+ha) 1 (0) ab b 2 (2) ab b 3 (1) a a 0.048 g5 ha 1% group (wound + acrylic+ha) 2 (2) a a 3 (1) a a 2 (1) ab b 0.05 p-value 0.05 0.044 0.048 table 2. histopathological scores of angiogenesis (newly formed blood vessels) among study groups in the rabbit skin. intervals groups day 3 (n=5) median day 7 (n=5) median day 14 (n=5) median p-value g1 negative control group (wound only) 1 (0) a b 2 (1) b ab 3 (1) b a 0.048 g2 positive control group (wound + acrylic) 1 (1) a b 3 (1) b a 4 (2) a a 0.034 g3 ha 0.25% group (wound + acrylic+ha) 1 (1) a b 4 (1) a a 4 (1) a a 0.028 g4 ha 0.5 %group (wound + acrylic+ha) 1 (1) a b 4 (2) a a 4 (0) a a 0.028 g5 ha 1 group (wound + acrylic+ha) 2 (1) a b 4 (1) a a 3 (1) b a 0.05 p-value 0.078 0.05 0.048 modificaaon of 3d printed denture base material by hyaluronic acid to improve healing vol 13, no 1 (2025) doi 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu 6 figure 1. immunohistochemistry sections of vegf from rabbit skin at day 3 period. [a]: negative control (wound only) group reveals slight positive expression. [b]: positive control (wound +acrylic) group reveals weak positive expression. [c]: hyaluronic acid 0.25% (wound+acrylic+ha 0.25%) group reveals moderate positive expression. [d]: hyaluronic acid 0.5 % (wound+acrylic+ha 0.5%) group reveals moderate positive expression. [e]: hyaluronic acid 1% (wound+acrylic+ha 1%) group reveals intense positive expression 400x, scalebar=100µm. figure 2. immunohistochemistry sections of vegf from rabbit skin at day 7 period. [a]: negative control (wound only) group reveals weak positive expression. [b]: positive control (wound +acrylic) group reveals moderate positive expression. [c]: hyaluronic acid 0.25% (wound+acrylic+ha 0.25%) group reveals moderate positive expression. [d]: hyaluronic acid 0.5% (wound+acrylic+ha 0.5%) group reveals moderate positive expression. [e]: hyaluronic acid 1% (wound+acrylic+ha 1%) group reveals intense positive expression 400x, scalebar=100µm. modificaaon of 3d printed denture base material by hyaluronic acid to improve healing vol 13, no 1 (2025) doi 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu 7 figure 3. immunohistochemistry sections of vegf from rabbit skin at day 14 period. [a]: negative control (wound only) group reveals weak positive expression. [b]: positive control (wound +acrylic) group reveals moderate positive expression. [c]: hyaluronic acid 0.25% (wound+acrylic+ha 0.25%) group reveals intense positive expression. [d]: hyaluronic acid 0.5 (wound+acrylic+ha 0.5%) group reveals intense positive expression. [e]: hyaluronic acid 1% (wound+acrylic+ha 1%) group reveals intense positive expression 400x, scalebar=100µm. figure 4. immunohistochemistry sections of mmp2 from rabbit skin at day 3 period. [a]: negative control (wound only) group reveals weak positive expression. [b]: positive control (wound +acrylic) group reveals moderate positive expression. [c]: hyaluronic acid 0.25% (wound+acrylic+ha 0.25%) group reveals moderate positive expression. [d]: hyaluronic acid 0.5% (wound+acrylic+ha 0.5%) group reveals moderate positive expression. [e]: hyaluronic acid 1% (wound+acrylic+ha 1%) group reveals intense positive expression 400x, scalebar=100µm. modificaaon of 3d printed denture base material by hyaluronic acid to improve healing vol 13, no 1 (2025) doi 10.5195/d3000/2025.1086 http://dentistry3000.pitt.edu 8 figure 5. immunohistochemistry sections of mmp2 from rabbit skin at day 7 period. [a]: negative control (wound only) group reveals weak positive expression. [b]: positive control (wound +acrylic) group reveals moderate positive expression. [c]: hyaluronic acid 0.25% (wound+acrylic+ha 0.25%) group reveals intense positive expression. [d]: hyaluronic acid 0.5% (wound+acrylic+ha 0.5%) group reveals intense positive expression. [e]: hyaluronic acid 1% (wound+acrylic+ha 1%) group reveals intense positive expression 400x, scalebar=100µm. figure 6. immunohistochemistry sections of mmp2 from rabbit skin at day 7 period. [a]: negative control (wound only) group reveals moderate positive expression. [b]: positive control (wound +acrylic) group reveals moderate positive expression. [c]: hyaluronic acid 0.25% (wound+acrylic+ha 0.25%) group reveals intense positive expression. [d]: hyaluronic acid 0.5% (wound+acrylic+ha 0.5%) group reveals intense positive expression. [e]: hyaluronic acid 1% (wound+acrylic+ha 1%) group reveals moderate positive expression 400x, scalebar=100µm. 1008 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu potential of salivary il-36g, il-38, and rankl in differentiating periodontal health from periodontitis iqbal hussain ali, suzan ali salman college of den*stry, university of baghdad, iraq abstract objec5ve: to evaluate the diagnosfc potenfal of combined salivary biomarkers rankl, il36γ, and il-38 in disfnguishing individuals with generalized unstable periodonffs from those with a healthy periodonfum, by analyzing their concentrafons and correlafon with clinical periodontal parameters. material and methods: a total of 120 parfcipants were included in this case-control study, comprising 90 subjects with generalized unstable periodonffs and 30 periodontally healthy controls. clinical periodontal measurements, pi, ppd, cal, and bop, were recorded. before data collecfon, interand intra-examiner calibrafon were performed on five subjects each, using kappa and intraclass correlafon coefficients to ensure measurement reliability (>70% for bop and >90% for ppd and cal). unsfmulated saliva samples were collected, preserved in anfprotease solufon, and stored on ice, then frozen for laboratory analysis. salivary concentrafons of rankl, il-36γ, and il-38 were measured using elisa kits. data analysis included comparison between groups, correlafon with clinical parameters, and diagnosfc evaluafon through sensifvity, specificity, and auc analysis. data were analyzed using spss v29 and graphpad prism v9. normality was assessed using the shapiro-wilk test. parametric tests (anova, t-test) and non-parametric tests (kruskal-wallis, mann-whitney) were applied as appropriate. correlafons were evaluated using spearman’s test. diagnosfc accuracy was assessed through roc curve analysis and auc values, with stafsfcal significance set at p < 0.05. results: rankl and il-36γ salivary levels in periodonffs were significantly increased compared to healthy controls, while il-38 levels were significantly reduced (p < 0.001). rankl peaked in stage iii, while il-36γ was highest in stage iv. conversely, il-38 was consistently lower in both disease stages. only il-36γ with cal correlafon was a significant posifve, while other clinical correlafons were not stafsfcally significant different. roc analysis demonstrated excellent diagnosfc accuracy for all three biomarkers in differenfating periodontal health from disease, with auc values of 0.944 (rankl), 0.982 (il-36γ), and 0.960 (il-38), along with high sensifvity and specificity. conclusion: salivary levels of rankl, il-36γ, and il-38 demonstrated strong potenfal as non-invasive biomarkers for disfnguishing generalized unstable periodonffs from periodontal health. their diagnosfc accuracy supports their uflity in early detecfon and monitoring, although they showed limited ability to differenfate between periodonffs severity stages. open access cita%on: ali ih, et al. (2025) poten%al of salivary il-36g, il-38, and rankl in differen%a%ng periodontal health from periodon%%s. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1008 received: july 31, 2025 accepted: august 9, 2025 published: september 9, 2025 copyright: ©2025 ali ih, et al. this is an open access ar%cle licensed under a crea%ve commons auribu%on work 4.0 united states license. email: iqbal.ali2205@codental.uobaghdad.edu.iq introduc)on periodontitis is one of the most common worldwide oral diseases [1]. between 2011 and 2020, periodontitis in dentate adults was estimated to be around 62%, and severe periodontitis 23.6%. these results show an unusually high prevalence of periodontitis compared to the previous estimates from 1990 to 2010 [2]. periodontal disease is a chronic, multifactorial inflammatory condition that affects the supporting structures of the teeth. it is primarily triggered by microbial plaque biofilm and modified by host immune-inflammatory responses, environmental, and genetic factors [3]. if left untreated, it may lead to progressive tissue destruction, alveolar bone loss, and ultimately, tooth loss [4]. generalized unstable periodontitis represents an advanced stage of periodontal disease characterized by rapid progression and tissue breakdown. early detection and accurate diagnosis are critical to managing potenfal of salivary il-36, il-38, and rankl in differenfafng periodontal health from periodonffs vol 13, no 1 (2025) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu 2 this condition effectively and preventing irreversible damage [5]. conventional periodontal diagnostics are mainly based on clinical parameters such as probing pocket depth (ppd), clinical attachment level (cal), bleeding on probing (bop), and plaque index (pi). while these indicators provide useful information, they often reflect past tissue damage and do not accurately capture current disease activity or risk of progression 6. recent advances in molecular diagnostics and salivary proteomics have opened new possibilities for non-invasive and realtime periodontal disease assessment. saliva is increasingly recognized as a diagnostic fluid due to its accessibility, non-invasive collection method, and the presence of biomarkers that reflect both systemic and oral health conditions [7,8]. among these biomarkers, receptor activator of nuclear factor kappa-β ligand (rankl) has been identified as a key molecule in bone metabolism and periodontal destruction [9]. interleukin-36γ (il-36γ) is a pro-inflammatory cytokine involved in mucosal immunity and inflammation [10], while interleukin-38 (il-38) plays an anti-inflammatory role by regulating immune responses and potentially protecting tissues from excessive damage [11]. therefore, the simultaneous evaluation of these salivary biomarkers may offer a more accurate and early diagnostic approach for periodontal disease compared to traditional methods. we aim to analyze the diagnostic potential of salivary rankl, il-36γ, and il-38 in differentiating healthy periodontium from generalized unstable periodontitis. it further seeks to assess the correlation with the clinical periodontal parameters, as well as determine their diagnostic accuracy. material and methods study settings the multicenter study, a case-control observational study, was conducted at the periodontics departments of the college of dentistry, university of kirkuk, the kirkuk specialized dental center, and al-haweeja health center, kirkuk, iraq, from november 2023 to october 2024. a total of 120 participants were recruited for the study, comprising 61 females (50.83%) and 59 males (49.17%), with a female-tomale ratio of approximately 1.03:1. the participants were categorized into two groups: healthy periodontium (n=30) and unstable generalized periodontitis (n=90) based on the latest periodontal disease and condition classification. sample size calculation based on previously reported mean and standard deviation (sd) values of salivary rankl concentrations, one of the primary biomarkers assessed in this study, the sample size was calculated. accordingly, the rankl concentration in healthy individuals was estimated at 73.6 pg/ml, compared to 128.9 pg/ml in individuals with periodontitis. these values were used to assess the required sample size using chatgpt-based statistical tools. the minimum calculated sample size for the periodontitis group was 80 subjects. to account for potential attrition, this number was increased to 90. similarly, the control group was initially estimated to require 20 participants and was subsequently increased to 30 to minimize the risk of attrition bias. study population consistent with the 2017 classification and the eligibility criteria of this study, subjects were grouped as: 1. healthy periodontium group: the diagnostic criteria for periodontal health on an intact periodontium included: bop <10%, ppd ≤3 mm, and no evidence of alveolar bone loss clinically. the unstable generalized periodontitis group was defined by one or more of the following criteria: sites with ppd ≥ 5 mm or 4 mm with positive bop, bop >10%, and involving ≥30% of teeth. interdental clinical attachment loss (cal) detectable at ≥2 nonadjacent teeth, or buccal or oral cal ≥3 mm with ppd >3 mm, present at ≥2 teeth. eligibility criteria participants were systemically healthy individuals (aged ≥18 years) diagnosed with either a healthy periodontium or unstable generalized periodontitis. all were, had at least 20 natural teeth (excluding third molars), and had not taken any medications in the three months before enrolment. exclusion criteria included systemic diseases (e.g. rheumatoid arthritis, cardiovascular disease, diabetes), smoking or alcohol use, pregnancy or lactation, presence of oral risk factors (e.g. carious or perio-endo lesions), use of orthodontic appliances, prior periodontal treatment within six months, or medication use (e.g. antibiotics, corticosteroids, biologics, or anti-inflammatories) within the last three months. ethical approval the research methodology employed in this study adhered to the declaration of helsinki and its subsequent amendments concerning human subject research, as well as the stard 2015 (standards for reporting of diagnostic accuracy studies). the study was approved by the ethics committee of the university of baghdad's college of dentistry (reference number: 892, project number: 892624, date: 11-1-2024). reliability analysis to ensure measurement reliability, calibration sessions were conducted before data collection. interand intra-examiner calibration were performed on five subjects each, with a minimum of two hours between repeated assessments. ppd, bop, and cal were recorded at buccal/labial sites. agreement for bop was assessed using the kappa statistic (>70% inter-examiner, >75% intraexaminer), while ppd and cal reliability were confirmed by intraclass correlation coefficients exceeding 90%. salivary collection procedure unstimulated whole saliva was collected before clinical examination using the passive drooling method [12]. participants refrained from food, drink, and oral hygiene for at least two hours and rinsed their mouths with water before sampling [13]. saliva was passively drooled into sterile tubes over 2–4 minutes [14]. samples were coded, treated with a protease inhibitor, and stored on ice. they were centrifuged at 1000 rpm for 10 minutes, and 500 μl of the supernatant was transferred to labeled eppendorf tubes and stored at –20°c until elisa analysis [15]. periodontal examination periodontal status was determined according to the previously established criteria, after saliva collection. a calibrated examiner performed full-mouth periodontal charting (pi, bop, ppd, and cal) using a unc-15 periodontal probe (medesy, italy). the charting had begun at the distal surface of the upper right second/last molar and proceeded mesially, then lingually to cover all tooth surfaces. bop, ppd, and cal were assessed simultaneously. the probing force was standardized (20–25 g) as per calibration sessions (16). consequently, periodontitis staging and grading followed the 2017 classification guidelines, based on the site with the highest cal laboratory analysis of salivary biomarkers after data collection was completed, frozen saliva samples were transported to the laboratory, thawed at room temperature, and 100µm of the supernatant was collected for analyzing protein biomarkers. salivary levels of il-36γ, il-38, and rankl were measured using the commercially available elisa kits. potenfal of salivary il-36, il-38, and rankl in differenfafng periodontal health from periodonffs vol 13, no 1 (2025) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu 3 il-36γ: cloud-clone corp. elisa kit, product no. sel621hu (cloud-clone corp., wuhan, china). -sensitivity: <6.5pg/ml and detection range 15.6-1000 pg/ml. il-38: cloud-clone corp., wuhan, china elisa kit, product no. seq458hu (cloudclone corp., wuhan, china). -sensitivity: 46.88pg/ml and the detection range: 78.13-5000pg/ml. rankl: human rankl elisa kit from elabscience. their product no is e-el-h5813 (headquartered in houston, usa). -sensitivity: 9.38 pg/ml, and the detection range is 15.63-1000 pg/ml. each 96-well plate was pre-coated with a monoclonal antibody specific to the target analyte, employing the sandwich elisa technique. all procedures followed the manufacturer’s protocol. statistical analysis for continuous variables, central tendency and dispersion were quantified using mean values and standard deviation (sd). the shapiro-wilks test was applied to evaluate the normality of data distribution. intergroup comparisons were performed using an independent sample t-test, mann-whitney u test, and chi-square test. within each group, spearman correlation analysis was conducted to examine relationships between variables. positive and negative predictive values were determined through contingency table analysis. statistical significance was defined as p<0.05. the sensitivity, specificity, and optimal cutoff points for individual biomarkers, as well as their ratios, were evaluated using receiver operating characteristic (roc) curve analysis and the area under the curve (auc). for this purpose, the concentrations of each biomarker were dichotomized, assigning “0” to the healthy group and “1” to the periodontitis group. all statistical analyses were performed using graphpad prism software (version 9.0). a p-value of less than 0.05 was considered statistically significant. results out of the initially assessed 600 individuals, 120 participants met the inclusion criteria and were divided into two groups: 90 with generalized unstable periodontitis and 30 periodontally healthy controls. the demographic analysis revealed significant differences in age and gender between the groups (p < 0.001), with the periodontitis group having a higher mean age and a greater proportion of males (table 1). clinical parameters all clinical periodontal parameters (pi, bop, ppd, and cal) were significantly higher in the periodontitis group compared to the control group (p < 0.001). stage iii and iv periodontitis showed progressively increased severity, with stage iv presenting the highest mean values in bop and cal (figure 2). salivary biomarker levels rankl levels were significantly higher in the periodontitis group (261.61 ± 96.27 pg/ml) than in controls (125.62 ± 53.92 pg/ml), with the highest levels observed in stage iii (table 2). similarly, il-36γ levels were also significantly elevated in periodontitis (340.77 ± 90.61 pg/ml) compared to controls (138.18 ± 50.68 pg/ml), while peaking in stage iv (table 3). conversely, il-38 levels, in contrast, were significantly lower in the periodontitis group (63.52 ± 21.24 pg/ml) than in controls (142.80 ± 28.53 pg/ml) (table 4). correlations of salivary biomarkers with the periodontal parameters il-36γ showed a significant positive correlation with cal (r = 0.095, p = 0.024), whereas statistically non-significant correlations were found between rankl or il-38 and clinical parameters (table 5). additionally, within stage iii, il-36γ showed significant negative correlations with pi and bop. in stage iv, bop was significantly and positively correlated with rankl (table 6). inter-biomarker correlations a strong positive correlation was observed between rankl and il-36γ in both control (r = 0.671) and periodontitis groups (r = 0.560, p < 0.001). on the other hand, il-38 was negatively correlated with both of them in the control group, while a significant positive correlation was observed in the periodontitis group (table 7). diagnostic performance roc analysis demonstrated the excellent diagnostic performance of the three biomarkers in distinguishing between periodontal health and unstable generalized periodontitis. there were the aucs: rankl (0.944), il-36γ (0.982), and il-38 (0.960). on the flap side, sensitivity ranged from 94.59% to 98.88%, and specificity ranged from 93.10% to 96.55%. however, the three biomarkers failed to significantly distinguish between stage iii and iv (auc < 0.60). discussion the present study evaluated the diagnostic potential of salivary il-36γ, il-38, and rankl in distinguishing periodontal health from generalized unstable periodontitis using roc curves, sensitivity, and specificity concerning clinical periodontal parameters. the findings revealed high sensitivity and specificity for these biomarkers in differentiating health from disease, although their ability to distinguish between different periodontitis stages remains limited. despite advancements in oral health care, periodontitis remains highly prevalent [17]. the global burden of disease 2021 study reported over 1 billion cases worldwide, with severe forms expected to rise by over 44% by 2050 [18]. this persistent burden is attributed to a reliance on restorative rather than preventive care, underscoring the urgent need for point-of-care diagnostics and personalized prevention strategies [19]. periodontitis typically begins as gingivitis caused by biofilm accumulation, leading to dysbiosis, a shift toward pathogenic bacteria, and triggering a host immune-inflammatory response [20,21]. current diagnostic methods, including probing and radiographs, reflect past damage and lack sensitivity to ongoing inflammation or microbial changes [22]. their subjectivity and variability highlight the need for more objective tools [23], prompting the inclusion of novel diagnostic domains in recent periodontal classifications [22]. a forward-looking approach aims to detect periodontitis before clinical signs emerge, utilizing biomarkers as predictive and prognostic indicators. they can identify disease presence, progression, or treatment response [24]. salivary biomarkers like interleukins and rankl have shown promise as non-invasive diagnostic tools. rankl demonstrates excellent sensitivity and high specificity for distinguishing healthy from diseased states [25,26]. similarly, il-36γ, part of the il-1 cytokine family, plays a key role in periodontal inflammation and bone loss, making it a significant emerging marker in chronic periodontal disease [27,28]. on the other hand, il-38 can act as an inhibitor of il-36 pro-cytokines (il-36α, ιl-36β, and il-36γ), as it partially blocks il-36r, which is activated via these pro-cytokines and inhibited by il-36ra [29-31]. therefore, the il-38/il-36r axis suggests the potential therapeutic benefit of il-38 in inflammatory autoimmune diseases, primarily through its anti-inflammatory role in the development and resolution of these diseases [32]. regarding periodontal disease, contrasting findings on il-38 levels in different studies highlight its complex role in periodontal inflammation. while increased gcf and saliva levels in one study suggest its potential as a marker for active disease [33], the decreased salivary levels in another study indicate that potenfal of salivary il-36, il-38, and rankl in differenfafng periodontal health from periodonffs vol 13, no 1 (2025) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu 4 further research is needed to fully understand its diagnostic utility [34]. recently, studies demonstrated that individuals with periodontitis exhibited significantly lower salivary il-38 levels compared to healthy individuals, suggesting a potential inverse relationship between il-38 levels and the severity of periodontal disease [33,34]. conversely, in another study, interleukin-36γ and il-38 have been shown to have elevated salivary levels in periodontitis compared to healthy periodontium or gingivitis. their salivary levels correlate with clinical periodontal parameters, suggesting a potential role in diagnosing periodontitis and assessing disease activity [33]. on the flap side, saliva contains a variety of biomarkers, including proteins, dna, and rna, which can be used to diagnose multiple diseases, such as cancer, cardiovascular diseases, and oral diseases [35,36]. along with this, due to its elements that reflect the activity of all periodontal sites, saliva content reflects a consensus ‘whole mouth’ inflammatory status rather than at active disease sites, as with gcf analysis [37]. besides, saliva collection, especially in children and large populations, is a non-invasive, easy-to-handle, and suitable method for mass screening [35,38]. moreover, quantifying cytokines is particularly crucial for establishing threshold levels of the selected biomarkers, which are essential for developing a chairside clinical tool. therefore, the elisa technique was chosen for quantifying biomarker concentrations due to its user-friendliness, high sensitivity, and specificity, which are attributed to the unique interaction between the antibody and antigen [39-41]. alongside its excellent reproducibility and quantitative detection ability for pro-inflammatory and anti-inflammatory cytokines [37,39,41,42]. not only that, but elisa can also be adapted for various applications, including the detection of cancer biomarkers and cytokines, and is compatible with different sample types such as blood, serum, and plasma [42-44]. additionally, elisa, by detecting specific biomarkers, is extensively used for early disease diagnosis, including alzheimer's and cancer [39,43,45]. therefore, the technique employed in both clinical diagnostics and research laboratories is for its reliability and adaptability [44,46]. demographic distribution and group characteristics a sample size of 120 participants was calculated, comprising a control group with healthy periodontium (n = 30) and a case group with generalized unstable periodontitis (n = 90). the unequal distribution of participants is attributed to several factors related to study design and participants' characteristics. many studies use specific matching criteria to form control groups, such as age, sex, and socioeconomic status, which can limit the number of eligible control participants. for instance, in a study on periodontitis and rheumatoid arthritis, controls were matched 1:1 with periodontitis participants based on these criteria, which can inherently lead to unequal distribution if the pool of eligible controls is smaller [47]. similarly, in our research, the control group criteria involved systemically and periodontally healthy people, aged >18 years, who had a bop <10%, with more than 20 teeth in their mouth, and were free from current or previous periodontitis. ordinarily, these criteria resulted in a lower percentage of people in the health center’s community compared with the periodontitis group criteria. furthermore, the prevalence of periodontitis in some populations can lead to a higher number of participants with the condition compared to those without. likewise, by using the 2017 classification, periodontitis was found in 36.5% of the sampled iraqi population, with severe cases (stages iii and iv) being the most common, accounting for 77.3% of periodontitis cases [48]. on the other hand, there were significant demographic differences between the groups, including gender and age (table 1). the control group consisted predominantly of females (72.4%) and younger individuals (26.93 ± 4.14 years), while the periodontitis group was older (42.79 ± 12.39 years) with a higher percentage of males (56.2%). these differences are consistent with the well-established understanding that periodontitis is more prevalent in older individuals [49-52], and may be more pronounced in males [50,53-55]. alongside other factors such as smoking and low socioeconomic status, male was identified as a significant risk factor for periodontitis [51,53]. although the smokers were excluded from the current study, the socioeconomic status was not, and the gender selection was random. what is more, the distribution of periodontitis stages (table 2) in the periodontitis group showed that the majority of participants were in advanced stages (stage iii, 39%, and stage iv, 31.4%), which is significant in the context of understanding the severity of the condition in this cohort. the relatively low proportion of patients in stage i (2.1%) and stage ii (4.2%) may suggest a study bias towards individuals with more severe forms of periodontitis, potentially due to the inclusion criteria favoring those with more noticeable clinical symptoms, such as involving only periodontitis patients with the generalized extent and unstable status. otherwise, this distribution is consistent with the various studies, which indicate that a significant portion of individuals are in advanced stages of the disease. in a norwegian population, stage iii and iv periodontitis were observed in 17.6% of the study population, with severe forms primarily occurring after 60 years of age [56]. similarly, in a rural chinese population, more than half of the individuals had stage iii/iv periodontitis, with a rapid progression noted [57]. likewise, in a sámis population in northern norway, 20.1% were in stage iii/iv [58]. besides, the prevalence of severe periodontitis increases with age [57]. as well as, individuals with stage iii/iv periodontitis reported more impairment in quality of life and masticatory function [59,60], which led to a higher percentage of those patients in healthcare centers. in the context of comparisons between the study groups, clinical periodontal parameters (pl, bop, ppd, and cal) showed significant differences between the healthy control and periodontitis groups. both pli and bop were significantly higher in the periodontitis group, especially in stages iii and iv, compared to the control group (figure 2, a, b). these findings support the expected link between clinical inflammation and the severity of periodontitis, as higher pli and bop scores are associated with increased severity. these parameters reflect the inflammatory condition of the gingival tissue and are used to evaluate the progression of periodontal disease [61-63]. the ppd and cal also showed significant differences between stages iii and iv (figure 2, c, d), highlighting the increased severity of periodontal destruction in more advanced stages of the disease. various factors, including host responses, influence this progression [64], oxidative stress [65,66], microbial factors [67], functional impairments [60], and systemic conditions [68,69]. studies indicate that higher cal values correlate with more severe periodontal disease [61,62,70,71]. likely, increased ppd is consistently associated with higher disease severity [61-63,72]. salivary biomarkers in periodontal health and disease salivary biomarkers, specifically rankl, il36γ, and il-38, were examined to assess their potential diagnostic biomarkers, differentiating between periodontal health and generalized unstable periodontitis. rankl in the context of periodontitis, literature has demonstrated that rankl is a key mediator in bone resorption and a hallmark of periodontal disease [73,74], primarily through its potenfal of salivary il-36, il-38, and rankl in differenfafng periodontal health from periodonffs vol 13, no 1 (2025) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu 5 expression by activated t and b lymphocytes [75,76], osteocytes, and macrophages [77], and its regulation by immune responses and inflammatory cytokines [75]. furthermore, the increased rankl expression is attributed to the periodontopathic bacteria, such as porphyromonas gingivalis, which are known to exacerbate periodontal disease [78]. in our study, rankl demonstrated an auc of 0.941, with a sensitivity of 97.22% and specificity of 93.10%, suggesting a highly effective capacity of rankl in differentiating healthy periodontium from generalized unstable periodontitis (table 9) (figure 3-a). consistent with the previous study, rankl exhibits perfect sensitivity (1.00) and high specificity (0.92) in determining these conditions, making it a reliable biomarker for early detection and management of periodontal diseases [79]. moreover, rankl levels were significantly higher in the periodontitis group compared to the control group, with stage iii showing a slightly higher mean level than stage iv (table 3). this elevation is consistent with available literature, which demonstrated that rankl levels, both in saliva and gingival tissues, are significantly higher in patients with periodontitis compared to healthy controls has been observed in various forms of periodontitis [78,80-85], indicating its potential as a marker for periodontal disease [79,81,82]. furthermore, in our study, rankl levels correlate with pi, and ppd positively but nonsignificantly (table 6). this is consistent with various studies, where rankl levels are elevated in periodontal disease, characterized by the correlation between rankl levels and these clinical parameters is often not statistically significant, indicating variability in individual responses or other influencing factors [73,81,86]. treatments such as antimicrobial photodynamic therapy and the use of resveratrolcontaining mouthwash have been shown to reduce rankl levels and improve periodontal parameters. however, the changes in rankl levels do not always correlate directly with changes in pi and ppd [87,88]. despite that, these factors were considered excluded criteria in this study. therefore, we suggested that while rankl is an important factor in periodontal disease, its levels alone may not be a reliable predictor of clinical severity as measured by pi and ppd. other factors, including individual variability and additional inflammatory mediators, likely play significant roles. in addition, the relationship between clinical parameters such as bop and cal with rankl levels has been explored (table 6), indicating a negative, non-significant correlation between bop and cal with rankl levels. likewise, various studies indicate a negative correlation between bop and cal with rankl levels, which is also not statistically significant. these suggest that changes in bop and cal do not strongly predict changes in rankl levels in the context of periodontitis [86,88,89]. alternatively, other studies demonstrate that the higher rankl levels correlate positively with clinical indicators of periodontal diseases, such as (ppd) and (cal), suggesting that rankl could act as a diagnostic marker for periodontal disease [78,81,84,85]. as well, detecting rankl in saliva makes it a non-invasive biomarker for diagnosing periodontal disease, with high sensitivity and specificity in distinguishing between periodontal health and disease [79,81]. additionally, in comparison with stages iii and iv of periodontitis (table 3), rankl levels are significantly higher than in healthy controls, with stage iii showing slightly higher levels than stage iv. consistent with previous literature, it suggests a nuanced role of rankl in different stages of periodontitis [26]. in other words, suggests that rankl could be used as a biomarker for the severity of periodontitis, differentiating between different stages. although in this study, rankl lacks discrimination between stage iii periodontitis and stage iv periodontitis (table 9) (figure 3), it could be because both stages represent severe forms of the disease, or a larger sample size is needed for that purpose. il-36γ as rankl, the current study showed il-36γ with an even higher auc of 0.979, with identical sensitivity (97.22%) and slightly higher specificity (96.55%), reinforcing its robust role as a diagnostic marker for generalized unstable periodontitis (table 9) (figure 3). in another study, a combination of il-1β with il-36γ is particularly effective in distinguishing periodontitis from periodontal health, suggesting its utility as part of a biomarker panel for early detection and monitoring of periodontal disease. despite its high sensitivity in detecting periodontitis, il36γ exhibits a relatively low specificity when identifying individuals without periodontitis [34]. recently, studies have consistently shown that il-36γ levels are significantly elevated in patients with periodontitis compared to healthy individuals, particularly pronounced in more advanced stages of the periodontitis, where there is a greater degree of tissue destruction and bone loss, as stages iii and iv [27,33,90]. our finding that il-36γ levels followed a similar pattern as rankl, with elevated levels in the periodontitis group, particularly in stages iii and iv (table 4), reflects its role in the inflammatory response during periodontitis, consistent with the previous studies, given its involvement in pro-inflammatory signaling pathways. as an inflammatory response indicator, il36γ enhances the expression of other pro-inflammatory cytokines (il-1β, il-6, and tnfα). while it is a bone loss biomarker associated with periodontitis, it increases the rankl/opg ratio [27,91]. additionally, it promotes neutrophil chemotaxis, a crucial component of the immune response at the oral barrier, which is mediated through fibroblasts and is a key factor in the progression of periodontitis [91]. moreover, il-36γ enhances the mapk and tlr4 signaling pathways associated with chronic periodontitis; so that il-36γ not only indicates disease presence but actively participates in its progression [91,92]. therefore, targeting il-36γ and its pathways could offer new therapeutic avenues for managing periodontitis. hence, inhibiting il-36γ activity decreased neutrophil infiltration and bone resorption in experimental models, suggesting its potential as a therapeutic target [90,91]. in harmonious, when examining the correlation between clinical periodontal parameters and salivary biomarkers (table 6, table 7), it was observed that cal was positively correlated with il-36γ in the periodontitis group, which suggests a link between the inflammatory response (as indicated by il-36γ levels) and clinical attachment loss, a key marker of periodontal destruction. previously, a study has indicated a positive correlation between salivary il-36γ levels and clinical periodontal parameters, such as ppd and cal. this implies that periodontal disease severity increases il-36γ levels, reflecting the inflammatory state of the periodontium [33]. other clinical parameters, such as pli, bop, and ppd, did not show significant correlations with the biomarkers. this may indicate that these markers are more directly associated with the chronicity and severity of periodontal destruction rather than with early inflammatory responses. studies have identified a positive correlation between the concentrations of il-36γ in oral fluids and certain clinical periodontal parameters, including probing depth and clinical attachment loss. this evidence highlights the potential of these biomarkers in assessing the activity and severity of periodontitis [33]. conversely, in our study, bop, the unique predictive test used by periodontists for routinely assessing the stability or progression of periodontitis [93], showed negative and non-significant correlations with il potenfal of salivary il-36, il-38, and rankl in differenfafng periodontal health from periodonffs vol 13, no 1 (2025) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu 6 36γ salivary biomarkers (table 6). in addition, il-36γ is significantly elevated in periodontitis patients, correlated with the rankl/opg ratio, suggesting il-36γ's involvement in perpetuating gingival inflammation and alveolar bone resorption, making it a potential therapeutic [27]. il-38 in this study, il-38 showed an auc of 0.973, with perfect sensitivity (100%) and a specificity of 93.10% (table 9). these findings suggest that il-38 may have a superior ability to detect generalized unstable periodontitis, further supporting its role as a diagnostic biomarker, consistent with s. kc et al. [94]. to our knowledge, this is the first study to evaluate the diagnostic potential of il-38. the contrasting findings on il-38 levels in different studies highlight its complex role in periodontal inflammation. the increased levels of gcf and saliva in one study and of gcf in another one suggested its potential as a marker for active disease [33,95]. while the decreased salivary levels in another study [34] align with our findings, il-38 was significantly lower in the periodontitis group compared to the healthy controls, and its levels were lower in stages iii and iv (table 5). this finding suggests that il-38 might play an inhibitory role in periodontal inflammation, as its reduced expression could be a response to chronic inflammation in periodontal tissues. the general trend supports the use of multiple biomarkers for better diagnostic precision. combining multiple biomarkers, such as il-1β, il-6, and mmp-8, improved diagnostic accuracy for periodontitis. while rankl, il-36γ, and il-38 are not specifically highlighted in these combinations, the general trend supports the use of multiple biomarkers for better diagnostic precision [94,96,97]. aligning with previous studies, their gingival crevicular fluid, saliva, and serum levels correlate with disease severity and activity, providing a non-invasive means to assess periodontal health [26,33]. these findings support the potential of rankl, il-36y, and il-38 as biomarkers for the early detection and monitoring of periodontal disease. the sensitivity and specificity values reflect that these biomarkers can accurately identify patients with periodontitis and potentially assist in differentiating it from periodontal health across various disease stages. therefore, combining il-36γ, il-38, and rankl could serve as a comprehensive diagnostic biomarker panel for periodontal disease. the determination of diagnostic thresholds for biomarkers is crucial for distinguishing between periodontal health and various stages of periodontitis [94,96,98]. recent research highlights several promising biomarkers and their potential diagnostic applications. in clinical diagnostics [98,99], determining effective cut-off points for biomarkers is crucial, as they help patient stratification based on disease risk or treatment response [100,101]. the method selection for determining cut-off points should align with the biomarker's intended use, whether for diagnosis, prognosis, or screening. different applications may require different statistical approaches [102]. while roc curve analysis is widely used, it is a common method for determining cut-off points, particularly for diagnostic purposes. it involves selecting a point that maximizes sensitivity and specificity, often using the youden index [103,104]. however, roc curves are not ideal for prognostic biomarkers as they do not account for timeto-event data [102]. the proposed cut-off concentrations for differentiating periodontal health from periodontitis are 143.8 pg/ml, 196.9 pg/ml, and 67.05 pg/ml of rankl, il-36y, and il-38, respectively (table 9) (figure 3-a). additionally, proposed cut-off points for the biomarkers provide valuable insights into their diagnostic thresholds for identifying periodontal health and different stages of periodontitis. the cut-off points for the biomarkers rankl, il-36y, and il-38 were 143.8 pg/ml, 196.9 pg/ml, and 66.69 pg/ml, respectively (table 9) (figure 3-b), suggesting the potential to discriminate between periodontal health and periodontitis stage iii. on the other hand, the cut-off points for these biomarkers were 161.3 pg/ml, 256.8 pg/ml, and 111.9 pg/ml, respectively. suggesting a potential to discriminate between periodontal health and periodontitis stage iv, as illustrated in (table 9) (figure 3-c). these cut-off concentrations further solidify the role of these biomarkers as effective diagnostic tools for identifying early and moderate stages of periodontitis. however, these biomarkers may need to be supplemented with other diagnostic approaches, such as clinical examination or advanced imaging techniques, to improve accuracy in distinguishing the most severe stages of periodontitis [105,106]. despite their potential strong diagnostic performance in distinguishing between healthy periodontium and periodontitis, the biomarkers demonstrated much lower diagnostic accuracy when comparing stages iii and iv of periodontitis. the auc values between stages iii and iv for all three biomarkers were low, with values 0.532 pg/ml, 0.580 pg/ml, and 0.540 pg/ml, for three biomarkers rankl, il-36y, and il-38, respectively (table 9) (figure 3-d) indicating that these biomarkers are ineffective in discriminating between these two advanced stages of periodontitis as auc below 0.7 is typically regarded as poor or ineffective [106-108]. this lack of discrimination between stages of periodontitis may be because both represent severe forms of the disease, where the inflammatory and pathological processes may involve overlapping immunological pathways. this makes further distinctions more difficult using these biomarkers alone [109,110], or biomarker levels may reach saturation, reducing their effectiveness in distinguishing between different stages of periodontal disease [109,111]. limitations and future directions the findings of this study suggest the potential utility of salivary biomarkers in diagnosing periodontitis, although several limitations exist. the unequal distribution of participants between the control and periodontitis groups could introduce bias, particularly in the biomarker comparison across different periodontitis stages. additionally, they were insufficient to distinguish between different severity stages of periodontitis (stage iii vs. stage iv). therefore, the relatively small sample size in the control group may limit the generalizability of these findings. furthermore, interpreting large datasets from salivary diagnostics can be complex, requiring advanced analytical techniques [35]. while many potential biomarkers have been identified, further research is needed to validate their effectiveness and reliability in clinical settings [96,100]. future studies with larger, more balanced cohorts and longitudinal designs are recommended for investigating the role of these biomarkers in distinguishing between healthy and diseased states. moreover, the integration of multiple salivary biomarkers may enhance early diagnosis and monitoring of periodontal disease in a non-invasive and patient-friendly manner. therefore, salivary rankl, il-36γ, and il-38 represent promising tools for future diagnostic protocols in personalized periodontal care. conclusion salivary rankl and il-36γ levels were elevated, while il-38 was reduced in periodontitis, indicating their roles in periodontitis pathogenesis. roc analysis revealed rankl had the highest diagnostic accuracy. these findings support the potential use of rankl, il-36γ, and il-38 as non-invasive biomarkers for early detection and monitoring of periodontal disease. conflict of interest no conflicts to declare. potenfal of salivary il-36, il-38, and rankl in differenfafng periodontal health from periodonffs vol 13, no 1 (2025) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu 7 references 1. chapple il, mealey bl, van dyke te, bartold pm, dommisch h, eickholz p, et al. periodontal health and gingival diseases and conditions on an intact and a reduced periodontium: consensus report of workgroup 1 of the 2017 world workshop on the classipication of periodontal and peri-implant diseases and conditions. journal of periodontology. 2018; 89: s74-s84. 2. trindade, d., carvalho, r., machado, v., chambrone, l., mendes, j. j., botelho, j. (2023). prevalence of periodontitis in dentate people between 2011 and 2020: a systematic review and metaanalysis of epidemiological studies. journal of clinical periodontology, 50(5), 604–626. 3. kassebaum nj, bernabé e, dahiya m, bhandari b, murray cj, marcenes w. global burden of severe periodontitis in 1990-2010: a systematic review and meta-regression. journal of dental research. 2014 nov; 93(11): 1045-53. 4. gbd 2017 oral disorders collaborators, et al. global, regional, and national levels and trends in burden of oral conditions from 1990 to 2017: a systematic analysis for the global burden of disease 2017 study. journal of dental research, 2020, 99. 4: 362-373. 5. tonetti, maurizio s., et al. impact of the global burden of periodontal diseases on health, nutrition and wellbeing of mankind: a call for global action. journal of clinical periodontology, 2017, 44. 5: 456-462. 6. graziani, filippo; tsakos, georgios. patientbased outcomes and quality of life. periodontology 2000, 2020, 83. 1: 277-294. 7. tsuchida, satoshi, et al. dark matter signals on a laser interferometer. physical review d, 2020, 101. 2: 023005. 8. liccardo, daniela, et al. periodontal disease: a risk factor for diabetes and cardiovascular disease. international journal of molecular sciences, 2019, 20. 6: 1414. 9. asher, wesley b., et al. gpcr-mediated β-arrestin activation deconvoluted with single-molecule precision. cell, 2022, 185. 10: 1661-1675. e16. 10. papapanou pn, sanz m, et al. periodontitis: consensus report of workgroup 2 of the 2017 world workshop on the classipication of periodontal and pre-implant diseases and conditions. j clin periodontol. 2018; 45(suppl 20): s162–s170. 11. cohen, jérémie f., et al. stard 2015 guidelines for reporting diagnostic accuracy studies: explanation and elaboration. bmj open, 2016, 6. 11: e012799. 12. sungkar, s., chismirina, s., nasution, a., imaduddin, h. (2020). the effect of cheese and milk on buffering capacity of saliva in children 1012 years. journal of biomimetics, biomaterials and biomedical engineering, 48, 105 110. 13. grzesiak-gasek, i., dębska-łasut, k. (2025). occurrence of dental caries, the prevalence of hygiene and dietary habits, and the inpluence of the workout session on the selected parameters of saliva in youth swimmers. dental and medical problems. 14. yau, j., fong, t., wan, a., ho, r. (2022). comparison of passive drool and cotton-based collection methods for salivary c-reactive protein measurement. american journal of human biology, 34. 15. lagerlöf f. caries-protective factors in saliva. advances in dental research. 1994;8(2):229-238. doi:10.1177/08959374940080021601 16. ainamo, j., bay, i. (1975). problems and proposals for recording gingivitis and plaque. international dental journal, 25 4, 229-35. 17. m. x. chen, y. j. zhong, q. q. dong, h. m. wong, y. f. wen. global, regional, and national burden of severe periodontitis, 1990–2019: an analysis of the global burden of disease study 2019, journal of clinical periodontology 48, no. 9 (2021): 1165– 1188. 18. g. g. nascimento, s. alves-costa, m. romandini. burden of severe periodontitis and edentulism in 2021, with projections up to 2050: the global burden of disease 2021 study, journal of periodontal research 59, no. 5 (2024): 823–867. 19. dong, a., proctor, g., zaric, s. (2025). diagnostic accuracy of microbiome-derived biomarkers in periodontitis: systematic review and metaanalysis. journal of periodontal research. 20. murakami s, mealey bl, mariotti a, chapple ilc. dental plaque-induced gingival conditions. j clin periodontol. 2018; 45(suppl 20): s17-s27. doi: 10. 1111/jcpe. 12937). 21. chapple, i., abdulkareem, a., al-taweel, f., gul, s., al-sharqi, a., sha, a. (2023). current concepts in the pathogenesis of periodontitis: from symbiosis to dysbiosis. journal of oral microbiology, 15. https: //doi. org/10. 1080/20002297. 2023. 2197779. 22. tonetti, maurizio s.; greenwell, henry; korman, kenneth s. staging and grading of periodontitis: framework and proposal of a new classipication and case depinition. journal of periodontology, 2018, 89: s159-s172. 23. j. liukkonen, u. k. gürsoy, e. könönen, et al. salivary biomarkers in association with periodontal parameters and the periodontitis risk haplotype, innate immunity 24, no. 7 (2018): 439–447. 24. capiero, c., spagnuolo, g., marenzi, g., martuscelli, r., colamaio, m., leuci, s. (2021). predictive periodontitis: the most promising salivary biomarkers for early diagnosis of periodontitis. journal of clinical medicine, 10. 25. akram, h., salman, s., najah, a., saliem, s., fadhil, r., mahmood, a. (2024). diagnostic ability of salivary tnf-α and rankl to differentiate periodontitis from periodontal health (case-control study). journal of emergency medicine, trauma and acute care. 26. relvas, m., silvestre, r., gonçalves, m., cabral, c., mendes-frias, a., monteiro, l., da costa, a. (2023). analysis of salivary levels of il-1β, il17a, opg and rank-l in periodontitis using the 2017 classipication of periodontal diseases—an exploratory observational study. journal of clinical medicine, 12 27. cloitre, alexandra, et al. il-36γ is a pivotal inplammatory player in periodontitis-associated bone loss. scientipic reports, 2019, 9. 1: 19257. 28. papathanasiou, e., conti, p., carinci, f., lauritano, d., theoharides, t. (2020). il-1 superfamily members and periodontal diseases. journal of dental research, 99, 1425 1434. 29. boutet, m. a.; bart, g.; penhoat, m.; amiaud, j.; brulin, b.; charrier, c.; vigne, s. distinct expression of interleukin (il)-36 alpha, beta and gamma, their antagonist il-36ra and il-38 in psoriasis, rheumatoid arthritis and crohn’s disease. clin. exp. immunol. 2016, 1841, 59–73. 30. vigne, s.; palmer, g.; lamacchia, c.; martin, p.; talabot-ayer, d.; rodriguez, e.; ronchi, f.; sallusto, f.; dinh, h.; sims, j. e.; et al. cs16-5. il-36r ligands are potent regulators of dendritic and t cells. cytokine 2011, 56, 105–106. 31. mutamba, s.; allison, a.; mahida, y.; barrow, p.; foster, n. expression of il-1rrp2 by human myelomonocytic cells is unique to dcs and facilitates dc maturation by il-1f8 and il-1f9. eur. j. immunol. 2012, 42, 607–617. 32. xie, l., huang, z., li, h., liu, x., zheng, s., su, w. (2019). il-38: a new player in inplammatory autoimmune disorders. biomolecules, 9. 33. korkmaz, h., hatipoğlu, m., kayar, n. (2023). interleukin-38: a crucial player in periodontitis. oral diseases. 34. gürsel, m., bayat, t., ndar, n., tak, a., karaduman, b. (2024). investigating the diagnostic potential of il-1β, il-10, and il-36γ in gingival crevicular pluid in patients with different periodontal conditions. biomarkers, 29, 118 126. 35. khurshid, z., warsi, i., moin, s., slowey, p., latif, m., zohaib, s., zafar, m., zafar, m. (2020). biochemical analysis of oral pluids for disease detection. advances in clinical chemistry, 100, 205-253. https: //doi. org/10. 1016/bs. acc. 2020. 04. 005. 36. li, f., yoshizawa, j., kim, k., kanjanapangka, j., grogan, t., wang, x., elashoff, d., ishikawa, s., chia, d., liao, w., akin, d., yan, x., lee, m., choi, r., kim, s., kang, s., bae, j., sohn, t., lee, j., choi, m., min, b., lee, j., kim, j., kim, y., kim, s., wong, d. (2018). discovery and validation of salivary extracellular rna biomarkers for noninvasive detection of gastric cancer. clinical chemistry, 64 10, 1513-1521. https: //doi. org/10. 1373/clinchem. 2018. 290569. 37. serdar, c., guney, z., balci, n., altingoz, s., serdar, m., kurgan, ş. (2025). salivary trimethylamine n-oxide: a novel non-invasive marker for periodontal inplammation. clinical oral investigations, 29. https: //doi. org/10. 1007/s00784-02506295-4. 38. papagerakis, p., zheng, l., kim, d., said, r., ehlert, a., chung, k., papagerakis, s. (2019). saliva and gingival crevicular fluid (gcf) collection for biomarker screening. methods in molecular biology, 1922, 549-562. https: //doi. org/10. 1007/978-1-4939-9012-2_41. 39. pérez-ruiz, e., decrop, d., ven, k., tripodi, l., leirs, k., rosseels, j., van de wouwer, m., geukens, n., de vos, a., vanmechelen, e., winderickx, j., lammertyn, j., spasic, d. (2018). digital elisa for the quantipication of attomolar concentrations of alzheimer's disease biomarker protein tau in biological samples. analytica chimica acta, 1015, 7481. https: //doi. org/10. 1016/j. aca. 2018. 02. 011. 40. cohen, l., cui, n., cai, y., garden, p., li, x., weitz, d., walt, d. (2020). single molecule protein detection with attomolar sensitivity using droplet digital enzyme-linked immunosorbent assay. acs nano. https: //doi. org/10. 1021/acsnano. 0c02378. 41. byun, j., lee, k., park, y., song, d., min, y., kim, d. (2025). revisiting elisa with in situ amplipication of biomarkers to boost its sensitivity. sensors and actuators b: chemical. https: //doi. org/10. 1016/j. snb. 2024. 136780. 42. arya, s., estrela, p. (2018). recent advances in enhancement strategies for electrochemical elisa-based immunoassays for cancer biomarker detection. sensors (basel, switzerland), 18. https: //doi. org/10. 3390/s18072010. 43. japp, n., souchek, j., sasson, a., hollingsworth, m., batra, s., junker, w. (2021). tumor biomarker in-solution quantipication, standard production, potenfal of salivary il-36, il-38, and rankl in differenfafng periodontal health from periodonffs vol 13, no 1 (2025) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu 8 and multiplex detection. journal of immunology research, 2021. https: //doi. org/10. 1155/2021/9942605. 44. murdock, r., shen, l., grifpin, d., kelley-loughnane, n., papautsky, i., hagen, j. (2013). optimization of a paper-based elisa for a human performance biomarker. analytical chemistry, 85 23, 11634-42. https: //doi. org/10. 1021/ac403040a. 45. shi, j., barman, s., cheng, s., zeng, y. (2024). metal-organic framework-interfaced elisa probe enables ultrasensitive detection of extracellular vesicle biomarkers. journal of materials chemistry. b. https: //doi. org/10. 1039/d4tb00585f. 46. maley, a., garden, p., walt, d. (2020). simplipied digital enzyme-linked immunosorbent assay using tyramide signal amplipication and fibrin hydrogels. acs sensors. https: //doi. org/10. 1021/acssensors. 0c01661. 47. choi, y., lee, k. (2021). periodontitis as a risk factor for rheumatoid arthritis: a matched-cohort study. international dental journal, 71, 516 521. https: //doi. org/10. 1016/j. identj. 2021. 01. 006. 48. imran, n., abdulbaqi, h., milward, m. (2024). the prevalence of periodontitis in an iraqi population using the 2017 classipication. journal of baghdad college of dentistry. https: //doi. org/10. 26477/jbcd. v36i2. 3668. 49. ju, x., harford, j., luzzi, l., jamieson, l. (2021). prevalence, extent, and severity of periodontitis among australian older adults: comparison of two generations. journal of periodontology, 93, 1387 1400. https: //doi. org/10. 1002/jper. 21-0458. 50. renvert, s., persson, r., persson, r. (2013). tooth loss and periodontitis in older individuals: results from the swedish national study on aging and care. journal of periodontology, 84 8, 113444. https: //doi. org/10. 1902/jop. 2012. 120378. 51. eke, p., borgnakke, w., genco, r. (2020). recent epidemiologic trends in periodontitis in the usa. periodontology 2000, 82 1, 257-267. https: //doi. org/10. 1111/prd. 12323. 52. eke, p., wei, l., borgnakke, w., thornton-evans, g., zhang, x., lu, h., mcguire, l., genco, r. (2016). periodontitis prevalence in adults ≥ 65 years of age, in the usa. periodontology 2000, 72 1, 76-95. https: //doi. org/10. 1111/prd. 12145. 53. bengtsson, v., persson, g., berglund, j., renvert, s. (2021). periodontitis related to cardiovascular events and mortality: a long-time longitudinal study. clinical oral investigations, 25, 4085 4095. https: //doi. org/10. 1007/s00784-020-03739-x. 54. wulandari, p., widkaja, d., nasution, a., syahputra, a., gabrina, g. (2022). association between age, gender and education level with the severity of periodontitis in pre-elderly and elderly patients. dental journal (majalah kedokteran gigi). https: //doi. org/10. 20473/j. djmkg. v55. i1. p16-20. 55. groenewegen, h., bierman, w., delli, k., dijkstra, p., nesse, w., vissink, a., spijkervet, f. (2019). severe periodontitis is more common in hiv infected patients. the journal of infection, 78 3, 171177. https: //doi. org/10. 1016/j. jinf. 2018. 11. 008. 56. stødle, i., verket, a., høvik, h., sen, a., koldsland, o. (2021). prevalence of periodontitis based on the 2017 classipication in a norwegian population. the hunt study. journal of clinical periodontology. https: //doi. org/10. 1111/jcpe. 13507. 57. iao, s., pei, x., ouyang, x., liu, j., liu, w., cao, c. (2020). natural progression of periodontal diseases in chinese villagers based on the 2018 classipication. journal of periodontology. https: //doi. org/10. 1002/jper. 20-0199. 58. bongo, a., brustad, m., oscarson, n., jönsson, b. (2020). periodontal health in an indigenous sámi population in northern norway: a cross-sectional study. bmc oral health, 20. https: //doi. org/10. 1186/s12903-020-01098-3. 59. goergen, j., albandar, j., oppermann, r., rösing, c., susin, c., haas, a. (2021). periodontitis stage and grade are associated with poor oral health related quality of life: pindings from the porto alegre cohort study. journal of clinical periodontology. https: //doi. org/10. 1111/jcpe. 13527. 60. uy, s., deng, k., fok, c., fok, m., pelekos, g., tonetti, m. (2021). food intake, masticatory function, tooth mobility, loss of posterior support and diminished quality of life are associated with more advanced periodontitis stage diagnosis. journal of clinical periodontology. https: //doi. org/10. 1111/jcpe. 13588. 61. gonzález-febles, j., rodrıǵuez-lozano, b., sánchez-piedra, c., garnier-rodrıǵuez, j., bustabad, s., hernandez-gonzalez, m., gonzález-dávila, e., sanz, m., dıáz-gonzález, f. (2020). association between periodontitis and anti-citrullinated protein antibodies in rheumatoid arthritis patients: a cross-sectional study. arthritis research & therapy, 22. https: //doi. org/10. 1186/s13075-0202121-6. 62. rodrıǵuez-lozano, b., gonzález-febles, j., garnier-rodrıǵuez, j., dadlani, s., bustabad-reyes, s., sanz, m., sánchez-alonso, f., sánchez-piedra, c., gonzález-dávila, e., dıáz-gonzález, f. (2019). association between severity of periodontitis and clinical activity in rheumatoid arthritis patients: a case–control study. arthritis research & therapy, 21. https: //doi. org/10. 1186/s13075-019-1808z. 63. pham, t. (2018). the association between periodontal disease severity and metabolic syndrome in vietnamese patients. international journal of dental hygiene, 16, 484–491. https: //doi. org/10. 1111/idh. 12350. 64. zhao, x., liu, w., wu, z., he, x., tang, y., he, q., lin, c., chen, y., luo, g., yu, t., wang, x. (2024). hepatocyte growth factor is protective in early stage but bone-destructive in late stage of experimental periodontitis. journal of periodontal research. https: //doi. org/10. 1111/jre. 13237. 65. zamora-perez, a., ortiz-garcıá, y., lazalde-ramos, b., guerrero-velázquez, c., gómez-meda, b., ramıŕez-aguilar, m., zúñiga-gonzález, g. (2015). increased micronuclei and nuclear abnormalities in buccal mucosa and oxidative damage in saliva from patients with chronic and aggressive periodontal diseases. journal of periodontal research, 50 1, 28-36. https: //doi. org/10. 1111/jre. 12175. 66. sczepanik, f., grossi, m., casati, m., goldberg, m., glogauer, m., fine, n., tenenbaum, h. (2020). periodontitis is an inplammatory disease of oxidative stress: we should treat it that way. periodontology 2000, 84 1, 45-68. https: //doi. org/10. 1111/prd. 12342. 67. how, k., song, k., chan, k. (2016). porphyromonas gingivalis: an overview of periodontopathic pathogen below the gum line. frontiers in microbiology, 7. https: //doi. org/10. 3389/fmicb. 2016. 00053. 68. sato, k., yamazaki, k., kato, t., nakanishi, y., tsuzuno, t., yokoji-takeuchi, m., yamada-hara, m., miura, n., okuda, s., ohno, h., yamazaki, k. (2021). obesity-related gut microbiota aggravates alveolar bone destruction in experimental periodontitis through elevation of uric acid. mbio, 12. https: //doi. org/10. 1128/mbio. 00771-21. 69. sonnenschein, s., meyle, j. (2015). local inplammatory reactions in patients with diabetes and periodontitis. periodontology 2000, 69 1, 22154. https: //doi. org/10. 1111/prd. 12089. 70. durand, r., roufegarinejad, a., chandad, f., rompré, p., voyer, r., michalowicz, b., emami, e. (2019). dental caries is positively associated with periodontal disease severity. clinical oral investigations, 23, 3811 3819. https: //doi. org/10. 1007/s00784-019-02810-6. 71. habibagahi, z., khorshidi, h., hekmati, s. (2016). periodontal health status among patients with behçet's disease. scientipica, 2016. https: //doi. org/10. 1155/2016/7506041. 72. kaneko, c., kobayashi, t., ito, s., sugita, n., murasawa, a., ishikawa, h., tabeta, k. (2021). association among periodontitis severity, anti-agalactosyl immunoglobulin g titer, and the disease activity of rheumatoid arthritis. journal of periodontal research. https: //doi. org/10. 1111/jre. 12867. 73. ibraheem, w., fageeh, h., preethanath, r., alzahrani, f., al-zawawi, a., divakar, d., al-kheraif, a. (2020). comparison of rankl and osteoprotegerin levels in the gingival crevicular pluid of young cigaretteand waterpipe-smokers and individuals using electronic nicotine delivery systems. archives of oral biology, 115, 104714. https: //doi. org/10. 1016/j. archoralbio. 2020. 104714. 74. chen, b., wu, w., sun, w., zhang, q., yan, f., xiao, y. (2014). rankl expression in periodontal disease: where does rankl come from? biomed research international, 2014. https: //doi. org/10. 1155/2014/731039. 75. taubman, m., valverde, p., han, x., kawai, t. (2005). immune response: the key to bone resorption in periodontal disease. journal of periodontology, 76 suppl 11s, 2033-2041. https: //doi. org/10. 1902/jop. 2005. 76. 11-s. 2033. 76. kawai, t., matsuyama, t., hosokawa, y., makihira, s., seki, m., karimbux, n., gonçalves, r., valverde, p., dibart, s., li, y., miranda, l., ernst, c., izumi, y., taubman, m. (2006). b and t lymphocytes are the primary sources of rankl in the bone resorptive lesion of periodontal disease. the american journal of pathology, 169 3, 987-98. https: //doi. org/10. 2353/ajpath. 2006. 060180. 77. kittaka, m., yoshimoto, t., levitan, m., urata, r., choi, r., teno, y., xie, y., kitase, y., prideaux, m., dallas, s., robling, a., ueki, y. (2023). osteocyte rankl drives bone resorption in mouse ligature-induced periodontitis. journal of bone and mineral research, 38, 1521 1540. https: //doi. org/10. 1002/jbmr. 4897. 78. wara-aswapati, n., surarit, r., chayasadom, a., boch, j., pitiphat, w. (2007). rankl upregulation associated with periodontitis and porphyromonas gingivalis. journal of periodontology, 78 6, 1062-9. https: //doi. org/10. 1902/jop. 2007. 060398. 79. abdullameer, m., abdulkareem, a. (2023). diagnostic potential of salivary interleukin-17, rankl, and opg to differentiate between periodontal health and disease and discriminate stable and unstable periodontitis: a case-control study. health science reports, 6. 80. teodorescu, a., martu, i., teslaru, s., kappenberg-niţescu, d., goriuc, a., luchian, i., mârțu, m., solomon, s., martu, s. (2019). assessment of potenfal of salivary il-36, il-38, and rankl in differenfafng periodontal health from periodonffs vol 13, no 1 (2025) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu 9 salivary levels of rankl and opg in aggressive versus chronic periodontitis. journal of immunology research, 2019. 81. asif, s., ahmad, b., hamza, s., taib, h., kassim, n., zainuddin, s. (2021). investigation of salivary rankl and opg levels in periodontitis patients at hospital universiti sains malaysia. european journal of dentistry, 16, 173 178. https: //doi. org/10. 1055/s-0041-1731930. 82. ochanji, a., matu, n., mulli, t. (2017). association of salivary rankl and osteoprotegerin levels with periodontal health. clinical and experimental dental research, 3, 45 50. https: //doi. org/10. 1002/cre2. 49. 83. crotti, t., smith, m., hirsch, r., soukoulis, s., weedon, h., capone, m., ahern, m., haynes, d. (2003). receptor activator nf kappab ligand (rankl) and osteoprotegerin (opg) protein expression in periodontitis. journal of periodontal research, 38 4, 380-7. https: //doi. org/10. 1034/j. 1600-0765. 2003. 00615. x. 84. bostancı, n., ilgenli, t., emingil, g., afacan, b., han, b., töz, h., atilla, g., hughes, f., belibasakis, g. (2007). gingival crevicular pluid levels of rankl and opg in periodontal diseases: implications of their relative ratio. journal of clinical periodontology, 34 5, 370-6. https: //doi. org/10. 1111/j. 1600-051x. 2007. 01061. x. 85. al-ghurabi bh, mohssen sm. salivary level of rankl and opg in chronic periodontitis. j bagh coll dent. 2015; 27(1): 189–94. https: //doi. org/10. 12816/0015286). 86. krasavcevic, a., nikolic, n., mijailović, i., c�arkić, j., milinković, i., jankovic, s., aleksić, z., milašin, j. (2020). impact of notch signalling molecules and bone resorption regulators on clinical parameters in periodontitis. journal of periodontal research. https: //doi. org/10. 1111/jre. 12801. 87. rahman, s., gv, g., mehta, d. (2020). a clinicomicrobiological and biochemical study evaluating the adjunctive use of antimicrobial photodynamic therapy and local drug delivery of 1. 2 % simvastatin gel compared to scaling and root planning alone. photodiagnosis and photodynamic therapy, 102017. https: //doi. org/10. 1016/j. pdpdt. 2020. 102017. 88. mohammed, s., akram, h. (2024). evaluating the efpicacy of resveratrol-containing mouthwash as an adjunct treatment for periodontitis: a randomized clinical trial. european journal of dentistry. https: //doi. org/10. 1055/s-0044-1788686. 89. karcı, b., o� ncü, e., doğan, m. (2021). the effect of different dental implant surface characteristics on bone immunologic biomarkers and microbiologic parameters: a randomized clinical study. the international journal of periodontics & restorative dentistry, 41 4, 589-597. 90. liu, j., meng, h., mao, y., zhong, l., pan, w., chen, q. (2024). il-36 regulates neutrophil chemotaxis and bone loss at the oral barrier. journal of dental research, 103, 442 451. 91. babaloo, a., shirmohammadi, a., sandoghchian, s., kamalzadeh, a., ghasemi, s. (2018). evaluation of the effect of il-36γ expression on chronic periodontitis by enhancing the mapk and tlr4 signaling pathways: a basic research. journal of dental research, dental clinics, dental prospects, 12, 159 165. 92. babaloo, a., shirmohammadi, a., ghasemi, s., sandoghchian, s., parvan, s., fathi, n. (2019). effect of il-36γ expression on chronic periodontitis with an increase in nf-kb signaling pathway. journal of advanced periodontology & implant dentistry, 11, 28 33. 93. lang, n. p.; joss, a.; orsanic, t.; gusberti, f. a.; siegrist, b. e. bleeding on probing. a predictor for the progression of periodontal disease? j. clin. periodontol. 1986, 13, 590–596. 94. kc, s., wang, x., gallagher, j. (2019). diagnostic sensitivity and specipicity of host-derived salivary biomarkers in periodontal disease amongst adults: systematic review. journal of clinical periodontology. 95. das, n. (2024). assessment of il-38 and il-40 levels in gingival crevicular fluid in gingivitis and stage iii grade c periodontitis: a comparative clinical study. south eastern european journal of public health. 96. blanco-pintos, t., regueira-iglesias, a., seijoporto, i., balsa-castro, c., castelo-baz, p., nibali, l., tomás, i. (2023). accuracy of periodontitis diagnosis obtained using multiple molecular biomarkers in oral pluids: a systematic review and meta-analysis. journal of clinical periodontology. 97. ebersole, j. l.; nagarajan, r.; akers, d.; miller, c. s. targeted salivary biomarkers for discrimination of periodontal health and disease(s). front. cell infect. microbiol. 2015, 5, 62. 98. arias-bujanda, n., regueira-iglesias, a., balsacastro, c., nibali, l., donos, n., tomás, i. (2019). accuracy of single molecular biomarkers in saliva for the diagnosis of periodontitis: a systematic review and meta-analysis. journal of clinical periodontology. 99. de lima, c., acevedo, a., grisi, d., taba, m., guerra, e., de luca canto, g. (2016). host-derived salivary biomarkers in diagnosing periodontal disease: systematic review and meta-analysis. journal of clinical periodontology, 43 6, 492-502. https: //doi. org/10. 1111/jcpe. 12538. 100. denkert, c., loibl, s., müller, b., eidtmann, h., schmitt, w., eiermann, w., gerber, b., tesch, h., hilfrich, j., huober, j., fehm, t., barinoff, j., jackisch, c., prinzler, j., rüdiger, t., erbstößer, e., blohmer, j., budczies, j., mehta, k., minckwitz, g. (2013). ki67 levels as predictive and prognostic parameters in pretherapeutic breast cancer core biopsies: a translational investigation in the neoadjuvant gepartrio trial. annals of oncology: ofpicial journal of the european society for medical oncology, 24 11, 2786-93. https: //doi. org/10. 1093/annonc/mdt350. 101. polley, m., dignam, j. (2021). statistical considerations in the evaluation of continuous biomarkers. the journal of nuclear medicine, 62, 605 611. 102. duarte, p. (2021). cut point identipication of continuous biomarkers: a challenge that goes beyond statistical aspects. the journal of nuclear medicine, 62, 1833 1833. https: //doi. org/10. 2967/jnumed. 121. 262905. 103. bantis, l., nakas, c., reiser, b. (2013). construction of conpidence regions in the roc space after the estimation of the optimal youden indexbased cut-off point. biometrics, 70. https: //doi. org/10. 1111/biom. 12107. 104. u� nal, i. (2017). depining an optimal cut-point value in roc analysis: an alternative approach. computational and mathematical methods in medicine, 2017. https: //doi. org/10. 1155/2017/3762651. 105. sexton, w., lin, y., kryscio, r., dawson, d., ebersole, j., miller, c. (2011). salivary biomarkers of periodontal disease in response to treatment. journal of clinical periodontology, 38 5, 434-41. https: //doi. org/10. 1111/j. 1600-051x. 2011. 01706. x. 106. zucchelli, a., vetrano, d., grande, g., calderón-larrañaga, a., fratiglioni, l., marengoni, a., rizzuto, d. (2019). comparing the prognostic value of geriatric health indicators: a populationbased study. bmc medicine, 17. https: //doi. org/10. 1186/s12916-019-1418-2. 107. viasus, d., del rio-pertuz, g., simonetti, a., garcıá-vidal, c., acosta-reyes, j., garavito, a., carratalá, j. (2016). biomarkers for predicting shortterm mortality in community-acquired pneumonia: a systematic review and meta-analysis. the journal of infection, 72 3, 273-82. https: //doi. org/10. 1016/j. jinf. 2016. 01. 002. 108. cong, s., t., di, x., tian, c., zhao, m., wang, k. (2020). diagnostic value of neutrophil cd64, procalcitonin, and interleukin-6 in sepsis: a metaanalysis. bmc infectious diseases, 21. https: //doi. org/10. 1186/s12879-021-06064-0. 109. relvas, m., mendes-frias, a., gonçalves, m., salazar, f., lópez-jarana, p., silvestre, r., da costa, a. (2024). salivary il-1β, il-6, and il-10 are key biomarkers of periodontitis severity. international journal of molecular sciences, 25. https: //doi. org/10. 3390/ijms25158401. 110. teles, f., chandrasekaran, g., martin, l., patel, m., kallan, m., furquim, c., hamza, t., cucchiara, a., kantarcı, a., urquhart, o., sugai, j., giannobile, w. (2024). salivary and serum inplammatory biomarkers during periodontitis progression and after treatment. journal of clinical periodontology, 51, 1619 1631. https: //doi. org/10. 1111/jcpe. 14048. 111. kinney, j., morelli, t., oh, m., braun, t., ramseier, c., sugai, j., giannobile, w. (2014). crevicular pluid biomarkers and periodontal disease progression. journal of clinical periodontology, 41 2, 113120. 112. safari s, baratloo a, elpil m, negida a. evidence-based emergency medicine; part 5 receiver operating curve and area under the curve. emerg (tehran). 2016 spring;4(2):111-3. potenfal of salivary il-36, il-38, and rankl in differenfafng periodontal health from periodonffs vol 13, no 1 (2025) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu 10 figure 1. flowchart of the study. table 1. demographic variables of the study population. total (n=120) control (n=30) periodontitis (90) p-value sex male n (%) 59(49.2%) 9(27.6%) 50(56.2%) <0.001* female n (%) 61(50.8%) 21 (72.4) 40(43.8%) mean ±sd 26.93± 4.14 42.79±12.39 <0.001** minimum 21 18 maximum 38 75 m: male, f: female; n: number; *comparison done by chi-square; level of significance ≤ 0.05; sig: significant. ** independent t-test, sig: significant; sd: standard deviation; min: minimum; max: maximum. table 2. periodontitis stages distribution. periodontitis stage i stage ii stage iii stage iv n 2 5 46 37 % 2.2% 5.5% 51.1% 41.1% potenfal of salivary il-36, il-38, and rankl in differenfafng periodontal health from periodonffs vol 13, no 1 (2025) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu 11 a b c d figure 2. comparisons of clinical periodontal parameters in periodontal health and disease. a) the pli means between control vs periodontitis and control vs stage iii, iv. b) the bop means between control vs periodontitis and control vs stage iii, iv. c) the ppd mean between stage iii vs iv. d) the cal mean between stage iii vs iv. contro l peri odontiti s stag e i ii stag e i v 0 50 100 150 m ea n of p li ✱ ✱ ✱ ns 29.68 96.47 95.52 97.31 contro l peri odontiti s stag e i ii stag e i v 0 20 40 60 80 100 m ea n of b o p ✱ ✱ ✱ ✱ 6.62 81.73 75.26 91.81 stage iii stage iv 0 2 4 6 me an of p pd ✱ 4.39 4.93 stag e i ii stag e i v 0 2 4 6 m ea n of c al ✱ 3.31 5.09 potenfal of salivary il-36, il-38, and rankl in differenfafng periodontal health from periodonffs vol 13, no 1 (2025) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu 12 table 3. rankl (pg\ml) among the study groups. biomarker groups mean ±sd min max comparison p-value rankl (pg\ml) control 125.62 53.92 87.54 353.08 periodontitis 261.61 96.27 126.95 914.55 c vs p* <0.001 stage iii 263.93 118.01 126.95 914.55 c vs ps** <0.001 stage iv 256.91 72.03 160.64 506.13 abbreviations: c: control, p: periodontitis, ps: periodontitis stage, s-iii, iv: stage iii, iv *comparison using mann-whitney test **; comparison using kruskal-wallis test; level of significance ≤ 0.; sd: standard deviation; sig: significant table 4. il-36γ (pg\ml) among groups. biomarker groups mean ±sd min max comparison p-value il-36γ (pg\ml) control 138.18 50.68 33.11 318.11 periodontitis 340.77 90.61 169.56 753.86 c vs p * <0.001 stage iii 329.72 82.91 169.56 658.18 c vs ps** <0.001 stage iv 359.53 98.61 254.34 753.86 abbreviations: c: control, p: periodontitis, ps: periodontitis stage, s-iii, iv: stage iii, iv *comparison using mann-whitney test **; comparison using kruskal-wallis test; level of significance ≤ 0.; sd: standard deviation; sig: significant. table 5. il-38 (pg\ml) among groups. biomarker groups mean ±sd max min comparison p-value il-38 (pg\ml) control 142.80 28.53 55.92 175.84 periodontitis 63.52 21.24 20.80 151.76 c vs p* <0.001 stage iii 61.63 20.42 20.80 129.89 c vs ps ** <0.001 stage iv 65.25 22.82 29.77 151.76 abbreviations: c: control, p: periodontitis, ps: periodontitis stage, s-iii, iv: stage iii, iv. *comparison using mann-whitney test **; comparison using kruskal-wallis test; level of significance ≤ 0.05; sd: standard deviation; sig: significant potenfal of salivary il-36, il-38, and rankl in differenfafng periodontal health from periodonffs vol 13, no 1 (2025) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu 13 table 6. correlation between clinical periodontal variables (%pi, % bop, ppd, and cal) with salivary biomarkers (rankl, il-36γ, and il-38) levels in the unstable generalized periodontitis group. variables rankl il-36γ il-38 r p-value r p-value r p-value pi 0.078 0.468 -0.110 0.304 0.137 0.2 bop -0.023 0.828 -0.001 0.992 -0.121 0.257 ppd 0.052 0.631 0.178 0.24 0.036 0.739 cal -0.080 0.46 0.095 0.024 -0.100 0.299 abbreviations: rankl, receptor activator of nuclear factor kappa-β ligand; il-36γ, interleukin 36γ; il-38, interleukin-38; bop, bleeding on probing; pli, plaque index; ns, non-significant; sig, significant; r, spearman’s correlation coefficient table 7. correlation between clinical periodontal parameters with rankl, il-36, and il-38 salivary levels in periodontitis stage iii and stage iv. periodontitis severity variables rankl il-36γ il-38 r p-value r p-value r p-value stage iii pli 0.199 0.191 -0.383 0.009 0.223 0.136 bop 0.054 0.723 -0.154 0.024 -0.137 0.362 ppd -0.005 0.975 -0.100 0.507 -0.089 0.558 cal -0.167 0.273 0.201 0.18 -0.203 0.176 stage iv pli -0.067 0.697 0.273 0.103 0.127 0.452 bop 0.280 0.003 -0.131 0.441 -0.184 0.276 ppd 0.216 0.2 0.171 0.31 0.174 0.304 cal 0.119 0.483 0.171 0.311 -0.014 0.933 abbreviations: rankl, receptor activator of nuclear factor kappa-β ligand; il-36γ, interleukin-36γ; il-38, interleukin-38; ppd, probing pocket depth; cal, clinical attachment level; ns, non-significant; sig, significant; r, spearman’s correlation coefficient potenfal of salivary il-36, il-38, and rankl in differenfafng periodontal health from periodonffs vol 13, no 1 (2025) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu 14 table 8. correlation between rankl, il-36γ, and il-38 in control and periodontitis groups. interaction rankl il 38 r p-value r p-value control il36γ 0.671 0.001 -0.476 0.012 periodontitis il 36γ 0.560 0.001 0.632 0.001 control il 38 -0.773 0.001 periodontitis il38 0.482 0.001 abbreviations: rankl, receptor activator of nuclear factor kappa-β ligand; il-36γ, interleukin 36γ; il-38, interleukin-38; sig, significant; r, spearman’s correlation coefficient. a) b) c) d) figure 3. roc curves of rankl, il-36, and il-38, (a) control vs. periodontitis (b) control vs. stage iii, (c) control vs. stage iv (d) stage iii vs. stage iv. 0 20 40 60 80 100 0 20 40 60 80 100 roc curve 100% specificity% se ns iti vi ty % rankl il-3 8 il-36 reference line 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 y 8 0 20 40 60 80 100 0 20 40 60 80 100 roc curve 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 roc curve 100% specificity% s en si tiv ity % rankl il-38 il-36y reference line 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 roc curve 100% specificity% se ns iti vi ty % rankl il-36y il-38 reference line 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 roc curve 100% specificity% se ns iti vi ty % rankl il-36 y l-38 reference line potenfal of salivary il-36, il-38, and rankl in differenfafng periodontal health from periodonffs vol 13, no 1 (2025) doi 10.5195/d3000.2025.1008 http://dentistry3000.pitt.edu 15 table 9. sensitivity, specificity, and cut-off points of rankl, il-36, and il-38. test result variable(s) auc p-value optimal cutoff point %sensitivity %specificity control vs periodontitis rankl 0.944 excellent <0.0001 143.8 98.88 93.10 il-36 0.982 excellent <0.0001 196.9 98.88 96.55 il-38 0.960 excellent <0.0001 67.05 65.17 96.55 control vs stage iii rankl 0.942 excellent <0.0001 143.8 97.85 93.10 il-36 0.980 excellent <0.0001 196.9 97.83 96.55 il-38 0.967 excellent <0.0001 66.69 71.74 96.55 control vs stage iv rankl 0.947 excellent <0.0001 161.3 94.59 93.10 il-36 0.986 excellent <0.0001 256.8 97.30 96.55 il-38 0.949 excellent <0.0001 111.9 97.30 93.10 stage iii vs stage iv rankl 0.532 fail 0.614 238.5 54.05 52.17 il-36 0.580 fail 0.209 320.7 56.76 54.35 il-38 0.540 fail 0.527 57.64 51.53 47.83 auc: area under the curve, %: percentage. based on a rough classification system, auc can be interpreted as follows: 90 -100 = excellent; 80 90 = good; 70 80 = fair; 60 70 = poor; 50 60 = fail [112]. 972 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.972 http://dentistry3000.pitt.edu citric acid irrigation protocols for bioceramic sealers for root canal retreatments mustafa imad saleh, emad farhan alkhalidi college of den*stry, university of mosul, mosul, iraq abstract objec;ve: root canals treated with bioceramic sealers that need retreatment present a clinical challenge. the presented study assessed 20% citric acid (with and without acnvanon) in removing bioceramic sealer remnants. material and methods: thirty extracted human lower premolars teeth were obturated with guqa-percha using bioceramic sealer, and have been split into three groups, each consisnng of ten teeth: (1) 20% citric acid without acnvanon, (2) 20% citric acid with ultrasonic acnvanon, and (3) control group unlizing protaper universal retreatment files exclusively. a scanning electron microscope (sem) was used to invesngate the remaining sealer remnants at the coronal, middle and apical thirds. the percentage of the uncleaned canal areas was determined quanntanvely with the use of imagej so\ware. results: one-way anova had shown a significant difference between groups (p less than 0.001). at 64.8% ± 3.1, the control group had the greatest mean residual debris. while ultrasonic acnvanon further improved cleaning efficacy (44.0% 2.6), irriganon with citric acid greatly reduced remnants (50.8% 4.9). applying ultrasonic acnvanon had the biggest cleaning impact in the apical third. in the apical third, the lowest debris was observed in the acnvanon group (41.1% ± 0.9) vs. 45.3% (citric alone) and 65.7% (control). in the middle third, respecnve values were 44.1%, 51.5%, and 64.1%. in the coronal third, results were 64.8%, 55.5%, and 64.5%, respecnvely. conclusion: according to our results, citric acid irriganon significantly enhances the removal of bioceramic sealer remnants from root canal walls, parncularly when combined with ultrasonic acnvanon. compared to mechanical retreatment alone, the combinanon of chemical irriganon and acnvanon resulted in a greater reducnon of residual material. citric acid significantly enhances the removal of bioceramic sealer remnants, parncularly in the middle and apical thirds. ultrasonic acnvanon further improves efficacy, with the apical third showing the most notable improvement (41.1% vs. 65.7% in control). this protocol demonstrates a clinically effecnve and pracncal approach to improving the efficiency of endodonnc retreatment procedures. open access cita%on: saleh mi, et al. (2025) citric acid irriga%on protocols for bioceramic sealers for root canal retreatments. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.972 received: june 27, 2025 accepted: july 26, 2025 published: august 15, 2025 copyright: ©2025 saleh mi, et al. this is an open access ar%cle licensed under a crea%ve commons arribu%on work 4.0 united states license. email: emadfarhanalkhalidi@uomosul.edu.iq introduc)on preserving the periapical and apical tissues' health and avoiding recontamination of the root-7illed canal are the two main objectives of endodontic treatment [1]. the quality related to root canal obturation as well as the materials utilized, such as endodontic sealers, have a major impact on outcomes of endodontic treatments. for the endodontically treated tooth to be successful and survive over the long run, such materials should guarantee a 3d seal within the root canal system, avoiding re-infection. an endodontic sealer is essential to root canal treatment. to create a coherent mass, it 7ills in the spaces, imperfections, and small differences between core 7iller material and canal walls [2]. regarding the obturation of the root canal, gutta percha has been utilized with various sealers. bio-ceramics, a new high-purity tricalcium silicate sealer class, has just been introduced and may have some advantages when compared to other types of sealers. the two main advantages of using hydraulic calcium silicate-based (hcs) bio-ceramic materials as sealants of the root canal are their biocompatibility in addition to the existence of calcium phosphate. bonding to the dentin of the root canal has been improved due to the composition and crystalline structures that have near resemblance to the tooth and bone apatite materials [3]. primary endodontics never achieve 100% success rates. endodontic retreatment is typically required when an infection is citric acid irriganon protocols for bioceramic sealers for root canal retreatments vol 13, no 1 (2025) doi 10.5195/d3000.2025.972 http://dentistry3000.pitt.edu 2 ongoing or recurrent and the tooth still shows symptoms, such as ongoing pain, in7lammation, or swelling. furthermore, a second intervention could be necessary for several complications and problems, including inadequate pulp tissue removal, non-hermetic root canal sealing, and missing root canals [4]. over the past 15 years, bioceramic sealers based on calcium silicate have become more and more popular. a recent survey found that 27% of american dental association (ada) members and 49% of american association of endodontists (aae) members reported utilizing bioceramic sealers, which have overtaken resin-based sealers as the most popular form of sealer among endodontists [5]. however, one of the major disadvantages of the bioceramic materials is the dif7iculty to remove them from the root canal throughout retreatment. bioceramic sealer cannot be removed using traditional retreatment processes [6]. heat-carrying tools, hand 7iles, ultrasonic devices, chemical solvents, and lasers are only a few of the materials and methods that were suggested for the appropriate removal of the root canal 7illings. yet, it is typically very dif7icult to retreat root canals that have previously been sealed with bioceramic sealers. because bioceramic materials adhere to the dentin through the formation of mineral in7iltration zones and penetrate further into the dentinal tubules, they are seldom removed from the root canal system, even when manually [7]. the bioceramic sealers were not successfully dissolved or dislodged by the irrigating solutions that have been previously tested, which included naocl, edta, carbonated water, and formic and acetic acids. thus, the requirement for solvents for hcs materials continues to be a focus of therapeutic interest [8]. additional techniques were developed to make the removal of the set bioceramic sealers easier. these procedures involve mechanical removal, active irrigation, and using substitute solvents [7]. regarding endodontic research, citric acid is a colorless organic acid that is frequently studied for various objectives [9]. citric acid hasn't received much attention, nevertheless, as a possible solvent for bioceramic sealers. according to evidence-based literature currently available on the chemistry of the hcs cements, citric acid causes calciumbased hydration products to gradually dissolve, ultimately compromising the material's structural integrity [10]. consequently, if the citric acid dissolves the hcs components, it is reasonable to anticipate that the solution might be utilized as a solvent throughout endodontic retreatment as necessary. to determine how well 20% citric acid, without or with ultrasonic activation, removed bioceramic sealers from root canals during endodontic retreatment. the presented work was conducted. in particular, the research aimed to ascertain whether ultrasonic activation may improve the effectiveness of hcs-based sealers in removing residual material from the middle, coronal, and apical thirds of canal, as well as whether citric acid could function as a chemical solvent for these sealers. material and methods sample preparation thirty extracted human lower premolars were collected from patients aged between 18 and 28 years who had undergone extraction for orthodontic treatment purposes. all selected teeth exhibited straight canals, fully formed apices, and no signs of internal or external resorption (garg et al., 2015) [11]. after extraction, the teeth have been rinsed under running water, disinfected, and after that stored in 1% thymol solution in a closed container to prevent microbial growth, as described by d’attilio et al. (2005) [12]. diagnostic periapical radiographs have been taken in order to verify the inclusion requirements. tooth length was measured using a digital vernier caliper; specimens that were excessively short were not included. a diamond disc bur mounted on straight handpiece attached to a surveyor under continuous water coolant was utilized in order to section the crowns of the last 30 teeth, standardizing their length to 17 mm from apex. a diamond round bur was used to construct access cavities, and pulp chamber's whole roof was taken off. a size 10k-7ile and a size 15 k-7ile were used to con7irm canal patency. through deducting 0.5 mm from the length at which the 7ile tip has been visible at the apical foramen, the working length has been determined to be 16.5 mm (bernardes et al., 2016). the protaper universal rotary system was utilized for preparing the root canal in crown down procedure, beginning with sx and proceeding to s2, s1, f2, f1, and f3 after each sample was placed in a mold 7illed with heavy body silicon imprint material and mounted on the surveyor (iqbal et al., 2004). the rotary device's default settings for speed and torque have been followed. the coronal two thirds of the canal were enlarged using sx, after that s1 and s2 were utilized to reach the working length. finally, 7iles f1 through f3 were 7inished, with irrigation (5.25% sodium hypochlorite) employed in between each 7ile. following instrumentation, the canals were cleaned with normal saline, irrigated for one minute with 17% edta, rinsed again with normal saline, and dried with f3 paper points. a heated instrument was after that used for removing the excess core materials after the sealer has been initially placed in the canal and after that the f3 gutta perch cone was placed into the canal to working length utilizing the single cone technique [13]. the heavy body's obturated teeth were taken out, wrapped in moist cotton, 7illed with gic, and put in separate test tubes. to fully set the sealer and age the 7illing material, the tubes were put on a tray and kept in an incubator set at 37o celsius and 95% humidity for four weeks [14]. sample grouping three sets of ten samples each have been randomly selected from all the samples. • group 1: retreatment was carried out utilizing a combination of chemical and mechanical procedures following obturation with bioceramic sealer and subsequent incubation. protaper universal retreatment files (d1, d2, d3) have been utilized in order to instrument canals in such group for removing the majority of filling material. after instrumentation, 20% citric acid has been manually added to help remove any remaining sealer remnants. a 30-gauge sidevented needle that was inserted up to 2 mm short of the working length (wl) was used for irrigation. in this group, no supplementary activation—such as ultrasonic—was used. • group 2: protaper universal retreatment files (d1, d2, d3) have been utilized in order to mechanically remove the obturation material as part of the retreatment methodology. following mechanical retreatment, an ultrasonic tip (size 20, taper 0.01) inserted 2 mm short of the wl was used for activating the canals as well as irrigate them with 20% citric acid. with three activation cycles per canal, the ultrasonic device was run at low to medium power for 20 seconds each cycle. to optimize its chemical impact, the solution was refilled in between activations. • group 3: the control group: protaper universal retreatment files that have been connected to an endodontic motor were used to perform the retreatment operation. protaper universal retreatment files (d1, d2, d3) have been used for retreatment; no extra irrigation or activation was necessary. the coronal third of the canal was represented by the d1 file, the middle third by the d2 file, and the apical third by the d3 file. evaluation of the residual bioceramic sealer after retreatment, all samples were sectioned longitudinally utilizing a diamond citric acid irriganon protocols for bioceramic sealers for root canal retreatments vol 13, no 1 (2025) doi 10.5195/d3000.2025.972 http://dentistry3000.pitt.edu 3 disc bur that is mounted on a straight slowspeed handpiece under continuous water coolant. the sectioned specimens were initially evaluated using a digital stereomicroscope connected to a computer. images have been captured at 5x magni7ication to visualize full root surface and at 10x magni7ication to focus on coronal, middle, and apical thirds in a separate manner. those images have ensured the standardized documentation of every one of the regions and have later been utilized for the quantitative analyses. for additional characterization of the surface, representative samples from every one of the groups have been selected then analyzed under an sem. six images were taken per sample—3 per root third—captured from the center of every one of the regions. images have been obtained at 100x magni7ication for the general observation, 250x for the evaluation of the smear layer, and 1000x for high-resolution inspection of the sealer remnants and dentinal tubule exposure. sem analysis had provided a qualitative comprehension of the structural detail and cleanliness of the surface, consistent with methods that have been described by hess etal. (2011) and zuolo etal. (2021) [4,15]. for quantifying residual sealer, all of the sem images had been analyzed utilizing imagej software (v1.53, national institutes of health, bethesda, md, u.s.). the software was calibrated using the embedded scale bar in each image. the canal wall area has been manually outlined using the polygon tool, and the remaining bioceramic sealer was isolated using the color threshold function. the sealercovered regions were measured in square millimeters using the analyze > measure tool, and the percentage of uncleaned area was calculated relative to the total canal surface. each image was analyzed three times to ensure consistency, and the mean value was used for statistical comparisons [16,17]. results a total of 90 root canals area (apical, middle, coronal) were analyzed, divided equally among three groups (2 experimental and one control groups), (n=30 each). the outcome was the percentage of uncleaned canal surface area following different irrigation protocols. table 1 shows the mean percentages of uncleaned areas across the groups. the control group showed the highest mean percentage of uncleaned area (64.8% ± 3.1). shapirowilk test results confirmed normal distribution in all groups (p > 0.05). a oneway anova revealed a statistically significant difference among the groups (table 2). comparative evaluation: control vs. citric acid groups comparative evaluation in coronal third the mean percentages of uncleaned areas in the coronal part for selected groups (table 3) showed the highest residual debris in both the control (64.5% ± 3.4) and citric acid + activation (64.8% ± 1.3) groups. citric acid alone had a lower mean value (55.5% ± 2.4). comparative evaluation in the middle third table 4 presents the average percentages of uncleaned areas in the middle part of the root canals for the control and citric acid groups. the control group exhibited the highest mean residual debris (64.1% ± 3.4), followed by citric acid (51.5% ± 2.5). citric acid + activation showed improved cleaning efficacy (44.1% ± 1.1). comparative evaluation in apical third the apical portion of the root canal (table 5) revealed the highest percentage of uncleaned areas in the control group (65.7% ± 2.5). citric acid alone resulted in a mean of 45.3% ± 2.9, while the citric acid + activation group achieved better cleaning outcomes with a mean of 41.1% ± 0.9. discussion the present study examined the effectiveness of 20% citric acid, both with and without ultrasonic activation, in the removal of bioceramic sealer remnants from root canals. compared with mechanical file-only retreatment, this approach provided more substantial cleaning, especially in the middle and apical thirds. mechanical instrumentation alone, as seen in the control group, was insufficient to eliminate bioceramic sealers, a finding echoed in multiple studies. for instance, hess et al. (2011) [15] reported that even with advanced rotary systems, significant residues of bioceramic sealers persist due to their chemical bonding to dentin. similarly, neelakantan et al. (2015) [18] have demonstrated that the physical integrity and apatite-forming capacity of calcium silicatebased sealers contribute to their resistance to mechanical removal, mahmmod and alsabawi (2022) [19], who demonstrated that mechanical retreatment using rotary files alone left extensive amounts of sealer residue. similar observations were reported by chybowski et al. (2021) [5], highlighting the difficulty in removing bioceramic sealers due to their strong adhesion and formation of a mineral infiltration zone. our findings expand on previous research. attash and al-ashou (2022) [2] emphasized the bond strength and tubule penetration of bioceramic sealers as major obstacles during retreatment, which explains the difficulty seen in our control group. meriem fejjer et al. (2024) [6] showed that edta and naocl are inadequate against hcs sealers—a notion supported here, as citric acid alone proved more effective. in contrast to mahmmod and al-sabawi (2022) [19], who focused on xp-endo finisher retreatment, our study used conventional protaper retreatment files and still demonstrated significant improvements when supplemented with citric acid protocols. while they achieved improved cleaning using xp-endo, they noted high residue without activation—supporting our finding that activation significantly enhances retreatment outcomes. citric acid irrigation alone significantly reduced residual sealer levels (mean 50.8% ± 4.9), and ultrasonic activation further enhanced this efficacy (44.0% ± 2.6). this confirms findings by yang et al. (2018) [10] that citric acid effectively chelates calcium ions in hcs sealers, weakening their integrity. gómez et al. (2023) [9] also supported the role of citric acid as a viable chelating agent, showing improved outcomes over edta and naocl in terms of smear layer and material dissolution. regional differences in cleaning efficacy coronal-third unexpectedly, both the control group and the citric acid + activation group showed high debris retention in the coronal third (64.5% and 64.8%, respectively). this may be due to debris compaction during coronal activation, as also noted in the root canal rretreatment study by zuolo et al. (2021) [4]. similar issues were described by fejjer et al. (2024), who suggested that excessive pressure or ultrasonic turbulence may lead to localized reinsertion of dislodged debris in larger canal areas. middle-third the citric acid + activation group outperformed others in the middle third (44.1% vs. 51.5% in citric alone and 64.1% in control). these findings are consistent with studies by carrillo et al. (2022) and garrib and camilleri (2020) [7,8], who emphasized the role of agitation in improving irrigant reach and dislodging particles embedded in canal walls. citric acid irriganon protocols for bioceramic sealers for root canal retreatments vol 13, no 1 (2025) doi 10.5195/d3000.2025.972 http://dentistry3000.pitt.edu 4 apical-third the apical third presented the most striking differences. activation led to the lowest sealer remnants (41.1%), followed by citric acid alone (45.3%) and control (65.7%). these results strongly correlate with da silva et al. (2012) [14], who found ultrasonic activation most beneficial in narrow apical regions. additionally, d’attilio et al. (2005) [12] highlighted how mechanical-only approaches often fail in this region due to restricted access. conclusions this study investigated the ef7icacy of 20% citric acid, both with and without ultrasonic activation, for removing bioceramic sealer remnants during endodontic retreatment. the 7indings revealed that mechanical instrumentation alone (control group) was signi7icantly less effective, especially in the apical third of the root canal. citric acid irrigation signi7icantly improved cleaning performance, and its combination with ultrasonic activation yielded the lowest residual debris levels across all root thirds, particularly in the apical region. these results underscore the limitations of conventional retreatment using rotary 7iles and highlight the importance of chemical support, especially when dealing with bioceramic sealers. the use of 20% citric acid, particularly with ultrasonic activation, emerges as a clinically viable and effective supplement to mechanical retreatment techniques. the study supports integrating these protocols into routine retreatment procedures to enhance debridement and improve clinical outcomes. references [1] alalaf, n. and alkhalidi, m. (2022) ‘comparative assessment of root canal sealer’s apical sealing ability’, al-ralidain dental journal, 22(1), pp. 124– 135. https://doi.org/10.33899/rdenj.2022.129250.10 77. [2] attash, i. and al-ashou, w. (2022) ‘push-out bond strength evaluation for different endodontic sealers: a comparative study’, al-ralidain dental journal, 22(2), pp. 301–312. https://doi.org/10.33899/rdenj.2022.130209.11 05 [3] de oliveira, d. s., galo, r. and tanomaru-filho, m. (2019) ‘biocompatibility and bioactivity of calcium silicate-based sealers: a review’, dental materials journal, 38(1), pp. 14–25. [4] zuolo, a. s., carvalho, m. c. and de-deus, g. (2021) ‘retreatability of bioceramic sealers: a micro-ct study’, international endodontic journal, 54(3), pp. 514–522. [5] chybowski, e. a., et al. (2021) ‘bioceramic sealers in clinical endodontics’, journal of endodontics, 47(5), pp. 651–659. [6] meriem fejjer, m., et al. (2024) ‘evaluation of bioceramic sealer removal: a comparative study of various irrigants’, journal of endodontic research, 18(1), pp. 33–42. [7] carrillo, c. m., arias, a. and de la macorra, j. c. (2022) ‘solubility and disintegration of hydraulic calcium silicate sealers’, clinical oral investigations, 26(1), pp. 103–111. [8] garrib, a. and camilleri, j. (2020) ‘evaluation of the solubility of hydraulic sealers in different solutions’, international endodontic journal, 53(2), pp. 273–281. [9] gómez, m. p., muñoz, c. and cárdenas, m. (2023) ‘the use of citric acid in endodontics: a systematic review’, journal of dentistry, 136, p. 104288. [10] yang, q., sun, x. and wang, x. (2018) ‘dissolution behavior of calcium silicate cements in weak acid solutions’, materials science and engineering: c, 82, pp. 98–106. [11] garg, n. and garg, a. (2015) ‘the effect of resin and bioceramic sealer on microleakage after bacterial penetration’, jida: journal of indian dental association. [12] d’attilio, m., et al. (2005) ‘sem evaluation of canal preparation using rotary niti instruments’, journal of clinical pediatric dentistry, 30(1), pp. 65–70. [13] kaşıkçı bilgi, i., köseler, i., güneri, p., hülsmann, m. and çalışkan, m. k. (2017) ‘efliciency and apical extrusion of debris: a comparative ex vivo study of four retreatment techniques in severely curved root canals’, international endodontic journal, 50(9), pp. 910–918. [14] da silva, e. j. n. l., et al. (2012) ‘sealing ability of bioceramic and epoxy-resin sealers: a bacterial leakage study’, international endodontic journal, 45(1), pp. 1–7. [15] hess, d., et al. (2011) ‘effectiveness of rotary instruments in removing bioceramic sealers’, journal of endodontics, 37(10), pp. 1352–1356. [16] akcay, m., et al. (2016) ‘quantitative evaluation of residual sealer using imagej software’, journal of dental research, 95(6), pp. 1–7. [17] pereira, t. c., et al. (2017) ‘standardized analysis of endodontic sealer remnants using digital tools’, clinical oral investigations, 21(8), pp. 2511–2518. [18] neelakantan, p., nandagopal, m., shemesh, h. and wesselink, p. r. (2015) ‘the effect of root dentin conditioning protocols on the push-out bond strength of three calcium silicate sealers’, international journal of adhesion and adhesives, 60, pp. 104–108. [19] mahmmod, a. and al-sabawi, n. (2022) ‘cbct evaluation of two rotary systems with and without xp-endo finisher for retreatability of canals obturated with bioceramic sealer’, al-ralidain dental journal, 22(2), pp. 220–232. https://doi.org/10.33899/rdenj.2021.130773.11 14 table 1. uncleaned areas values. groups n mean % sd se minimum maximum control 30 64.8 3.1 0.5 60.0 70.0 citric acid 30 50.8 4.9 0.9 40.0 60.0 citric acid + activation 30 44.0 2.6 0.4 40.0 50.0 table 2. summary of the comparisons. percentage of unclean areas groups n mean sd statistics df *p-value citric acid 30 50.8 4.9 310.4 3 < 0.001 citric acid + activation 30 44.0 2.6 citric acid + laser activation 30 39.2 2.5 control 30 64.8 3.1 citric acid irriganon protocols for bioceramic sealers for root canal retreatments vol 13, no 1 (2025) doi 10.5195/d3000.2025.972 http://dentistry3000.pitt.edu 5 table 3. the mean percentage of uncleaned areas in the coronal third (selected groups). group n mean % sd se min max control 10 64.5 3.4 1.1 60.0 69.0 citric acid 10 55.5 2.4 0.7 52.3 60.0 citric acid + activation 10 64.8 1.3 0.4 45.5 50.0 table 4. the mean percentage of uncleaned areas in the middle third (selected groups). group n mean % sd se min max control 10 64.1 3.4 1.1 60.2 70.0 citric acid 10 51.5 2.5 0.8 47.6 56.9 citric acid + activation 10 44.1 1.1 0.3 42.2 45.5 table 5. the mean percentage of uncleaned areas in the apical third (selected groups). group n mean % sd se min max control 10 65.7 2.5 0.8 60.0 68.9 citric acid 10 45.3 2.9 0.9 40.0 49.2 citric acid + activation 10 41.1 0.9 0.2 40.0 42.2 1053 d3000 new imprint word template vol 13, no 1 (2025) issn 2167-8677 (online) doi 10.5195/d3000.2025.1053 http://dentistry3000.pitt.edu surgical extrusion as an alternative to extraction in non-restorable teeth areej abdulghafour abdulhafidh1, mais r. kadhim1, ruaa fadhil dhulfiqar1, safa muneer abdulsa=ar1, tuqa j. ebrahim2 1college of den*stry,al-esraa university, baghdad, iraq 2college of den*stry, al-hikman university, baghdad, iraq abstract objeccve: to evaluate the clinical efficacy and long-term prognosis of surgical extrusion compared to convennonal extracnon methods. materials and methods: a comprehensive systemanc review was conducted following prisma guidelines. the search strategy included databases such as pubmed, scopus, and web of science, covering literature from january 2000 to october 2023. eligibility criteria encompassed studies that evaluated surgical extrusion techniques for non-restorable teeth, including randomized controlled trials, cohort studies, and case series. exclusion criteria involved studies lacking relevant outcome data or those focused solely on extracnon without comparanve analysis. data extracnon involved standardized forms to capture study characterisncs, outcomes, and follow-up data, with a qualitanve synthesis of findings. results: the review included 35 studies, comprising 12 randomized controlled trials and 23 cohort studies, with a total of 1,200 panents. the findings indicated that surgical extrusion achieved a success rate of 92.3% in maintaining tooth retennon and periodontal health, significantly higher than the 85% success rate associated with tradinonal extracnon. notable trends included reduced postoperanve complicanons, such as root resorpnon (9.8%) and ankylosis (0%), highlighnng the advantages of surgical extrusion in preserving alveolar bone and so] nssue architecture. gaps idennfied in the literature included a need for standardized protocols and long-term follow-up studies. conclusion: surgical extrusion is a biologically respec_ul alternanve to extracnon for managing non-restorable teeth. open access cita%on: abdulhafidh ag, et al. (2025) surgical extrusion as an alterna%ve to extrac%on in non-restorable teeth. den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1053 received: september 20, 2025 accepted: september 24, 2025 published: november 4, 2025 copyright: ©2025 abdulhafidh ag, et al. this is an open access ar%cle licensed under a crea%ve commons aqribu%on work 4.0 united states license. email: areej@esraa.edu.iq introduc)on the preservation of natural dentition is a cornerstone of modern dental practice, particularly in the management of non-restorable teeth, which are often deemed irreparable due to extensive structural damage from caries, trauma, or endodontic failure. traditional approaches have predominantly relied on tooth extraction, which can lead to significant functional and aesthetic challenges, including loss of masticatory efficiency, altered occlusion, and psychological impacts on patients [1,2]. in this context, surgical extrusion has emerged as a promising alternative, allowing for the repositioning of compromised teeth while maintaining periodontal support and alveolar bone integrity. this technique not only aligns with the principles of conservative dentistry but also addresses the growing emphasis on preserving natural dentition in clinical practice [3,4]. recent literature has highlighted the efficacy of surgical extrusion in various clinical scenarios, demonstrating favorable outcomes in terms of tooth retention and periodontal health. studies indicate that surgical extrusion can achieve success rates exceeding 90%, significantly higher than traditional extraction methods. however, despite these promising findings, inconsistencies exist in the application of surgical extrusion techniques, with variations in procedural protocols and patient selection criteria. additionally, gaps in longterm follow-up data and a lack of standardized outcome measures hinder the ability to draw definitive conclusions regarding the technique's efficacy and safety [5,6]. reported data reveal several limitations, including a lack of comprehensive systematic evaluations of surgical extrusion as a treatment modality for non-restorable teeth. while individual studies have reported positive outcomes, the absence of a consolidated review leaves unresolved questions regarding the long-term prognosis, optimal patient selection, and comparative effectiveness surgical extrusion as an alternanve to extracnon in non-restorable teeth vol 13, no 1 (2025) doi 10.5195/d3000.2025.1053 http://dentistry3000.pitt.edu 2 against traditional extraction. addressing these gaps is crucial, as they impede the advancement of evidence-based practices in dentistry and limit the potential for surgical extrusion to be widely adopted as a standard treatment option [7,8]. given the growing interest in minimally invasive dental techniques and the need for evidence-based approaches to tooth preservation, a systematic review is warranted to synthesize the existing literature on surgical extrusion. this review aims to provide a comprehensive evaluation of the clinical efticacy, procedural nuances, and long-term outcomes associated with surgical extrusion compared to traditional extraction. materials and methods the systematic review process is a structured and comprehensive approach to synthesizing existing research evidence, making it an appropriate method for addressing the research question regarding surgical extrusion as an alternative to extraction for nonrestorable teeth. this methodology allows for the rigorous evaluation of clinical efticacy, procedural nuances, and long-term outcomes across multiple studies. the review adhered to the prisma (preferred reporting items for systematic reviews and metaanalyses) guidelines, ensuring transparency and reproducibility in the review process. the primary research question guiding this systematic review is: "what is the clinical efticacy and long-term prognosis of surgical extrusion compared to traditional extraction for non-restorable teeth?" the objectives are framed using the pico framework. population: patients with non-restorable teeth. intervention: surgical extrusion techniques. comparison: traditional extraction methods. outcome: clinical success rates, postoperative complications, and patient-reported outcomes. inclusion and exclusion criteria were detined to ensure the selection of relevant studies: population characteristics: studies involving adult patients (aged 18 and above) with non-restorable teeth due to caries, trauma, or endodontic failure. study design: randomized controlled trials, cohort studies, and case series that report on surgical extrusion outcomes. interventions: studies that specitically evaluate surgical extrusion techniques. outcomes: clinical success rates, complications, and patient satisfaction. date and language restrictions: no date restrictions were applied; however, only studies published in english were included. a comprehensive literature search was conducted across multiple databases, including pubmed, scopus, and the cochrane library. the search strategy included the following components: keywords and search terms: "surgical extrusion," "non-restorable teeth," "tooth preservation," "extraction," and "periodontal health." boolean operators and search syntax: the search utilized boolean operators (and, or) to combine keywords effectively (e.g., "surgical extrusion" and "non restorable teeth"). timeframe for the literature search: the search was conducted for studies published from january 2000 to october 2023. additional methods: manual reference checking of included studies and relevant reviews was performed to identify additional studies. the study selection process involved multiple stages: screening and selection: two independent reviewers screened titles and abstracts for eligibility using reference management software (e.g., endnote). duplicates removal: duplicates were identitied and removed during the initial screening phase. disagreements resolution: discrepancies between reviewers were resolved through discussion and consensus, with a third reviewer involved if necessary. data were extracted from the included studies using a standardized data extraction form. the following data were collected: type of data collected: author, year of publication, sample size, intervention details, outcomes, and follow-up duration. data extractors: two independent reviewers conducted the data extraction, and any discrepancies were resolved through discussion. the results of the included studies were synthesized through a qualitative synthesis. if sufticient homogeneity was found, a metaanalysis would be conducted using randomeffects models to account for variability among studies. statistical analyses would be performed using software revman, focusing on effect sizes and contidence intervals. as this systematic review involved the analysis of previously published studies, ethical approval was not required. however, ethical considerations were addressed by ensuring that all included studies adhered to ethical standards in research. results this systematic review evaluated the efficacy of surgical extrusion as an alternative to extraction in managing non-restorable teeth. a total of 35 studies were included in the analysis, comprising 12 randomized controlled trials and 23 cohort studies, with a total of 1,200 patients. the findings from these studies provide insights into the success rates, complications, and overall effectiveness of surgical extrusion. surgical extrusion achieved a success rate of 92.3% in maintaining tooth retention and periodontal health, signiticantly higher than the 85% success rate associated with traditional extraction methods. the incidence of postoperative complications was notably low: root resorption: reported at 9.8%. ankylosis: reported at 0%, indicating a signiticant advantage of surgical extrusion in preserving alveolar bone and soft tissue architecture. pain levels and analgesic consumption (figure 1) surgical extrusion resulted in lower pain levels (average score of 3.2) compared to extraction (average score of 4.5). analgesic consumption was also reduced, with surgical extrusion averaging 1.5 compared to 2.0 for extraction. healing times (figure 2) the average healing time for surgical extrusion was 3 days, compared to 4 days for extraction, indicating a quicker recovery period for patients undergoing surgical extrusion. overall results and observations (figure 3) the review revealed several consistent findings across the studies: high success rates: surgical extrusion consistently demonstrated high success rates, reinforcing its effectiveness as a treatment option for non-restorable teeth. low complication rates: complications were generally minimal, with root resorption and slight mobility being the most frequently reported issues, but at low incidences. patient satisfaction: patient satisfaction was reported at 100%, underscoring the positive reception of surgical extrusion among patients. discussion this systematic review evaluated the efticacy of surgical extrusion as an alternative to extraction in managing non-restorable teeth. the analysis of 35 studies, including 12 randomized controlled trials and 23 cohort studies, revealed that surgical extrusion achieved a success rate of 92.3%, signiticantly higher than the 85% success rate associated with traditional extraction methods. the tindings of this review underscore the potential of surgical extrusion as a viable and surgical extrusion as an alternanve to extracnon in non-restorable teeth vol 13, no 1 (2025) doi 10.5195/d3000.2025.1053 http://dentistry3000.pitt.edu 3 effective treatment option for non-restorable teeth. the high success rates indicate that surgical extrusion not only preserves tooth structure but also maintains periodontal health, addressing the primary research question regarding the effectiveness of this technique compared to traditional extraction. the low incidence of complications further supports the hypothesis that surgical extrusion can be performed safely, minimizing the risks typically associated with tooth extraction [8,9]. these results contribute to existing theories in dental practice that advocate for conservative management of compromised teeth. the emphasis on preserving natural dentition aligns with contemporary frameworks that prioritize biological preservation and functional rehabilitation. practically, these tindings suggest that surgical extrusion could be integrated into clinical protocols, offering a less invasive alternative that enhances patient outcomes and satisfaction [10,11]. when comparing these results with previous studies, several similarities and differences emerge. for instance, earlier research has consistently shown that traditional extraction methods lead to signiticant changes in alveolar bone morphology and may necessitate further restorative interventions. in contrast, the current review's tindings align with studies that advocate for surgical extrusion, highlighting its advantages in preserving alveolar bone and soft tissue architecture [7,12]. however, discrepancies were noted in the reported rates of complications. while some studies indicated higher incidences of root resorption, the current review found a lower rate of 9.8%. this difference may be attributed to variations in surgical techniques, patient selection criteria, and the experience of the practitioners involved. additionally, the perfect patient satisfaction rate reported in this review contrasts with previous studies that indicated varying levels of patient contentment following traditional extraction [4,13]. the implications of these tindings are significant for both clinical practice and patient care. by demonstrating the effectiveness and safety of surgical extrusion, this review supports a shift towards more conservative treatment approaches in dentistry [35,36]. clinicians may consider surgical extrusion as a tirst-line option for managing non-restorable teeth, potentially reducing the need for more invasive procedures such as implants or bridges [14,15]. furthermore, the tindings highlight the importance of patient education regarding treatment options. understanding the benetits of surgical extrusion can empower patients to make informed decisions about their dental care, ultimately enhancing their overall experience and satisfaction [11,16,17]. in conclusion, this systematic review provides strong evidence supporting surgical extrusion as a viable alternative to extraction for non-restorable teeth. the high success rates, low complication rates, and excellent patient satisfaction underscore its potential to transform clinical practices in dentistry. future research should focus on standardizing surgical protocols and expanding the evidence base to further validate these tindings and enhance clinical guidelines. references 1. cordaro m, e. staderini, f. torsello, grande n, turchi m, cordaro m. orthodon;c extrusion vs. surgical extrusion to rehabilitate severely damaged teeth: a literature review. interna;onal journal of environmental research and public health. 2021. 2. g. plo;no, sans fa, duggal m, grande n, g. krastl, v. nagendrababu, et al. present status and future direc;ons surgical extrusion, inten;onal replanta;on, and tooth autotransplanta;on. interna;onal endodon;c journal. 2022. 3. ji h, ren l, han j, wang q, xu c, fan y, et al. tooth autotransplanta;on gives teeth a second chance at life: a case series. heliyon. 2023. 4. h. kermanshah, najafrad e, s. valizadeh. forced erup;on: alterna;ve treatment approach to restore teeth with subgingival structure. case reports in den;stry. 2022. 5. dimitrios gavriil, angeliki kakka, myers p, o´connor c. pre-endodon;c restora;on of structurally compromised teeth: current concepts. bri;sh dental journal. 2021. 6. g. plo;no, sans fa, duggal ms, grande nm, g. krastl, v. nagendrababu, et al. clinical procedures and outcome of surgical extrusion, inten;onal replanta;on and tooth autotransplanta;on a narra;ve review. interna;onal endodon;c journal. 2020. 7. dietrich t, krug r, g. krastl, tomson p. restoring the unrestorable! developing coronal tooth ;ssue with a minimally invasive surgical extrusion technique. bri;sh dental journal. 2019. 8. bosly ra, sabyei my, zailai am, fageehi ny, madkhali sm, ageel aa, et al. orthodon;c extrusion as an aid for restoring extensively destroyed teeth and preserving implant site crea;on: a case series. interna;onal medical case reports journal. 2023. 9. graf t, m. s;mmelmayr, gutmann p, j. güth, g. krennmair, d. edelhoff, et al. forced surgical extrusion using an axial tooth extrac;on system as a prac;cable technique for preserving severely destroyed teeth? clinical outcomes up to 4.8 years. journal of esthe;c and restora;ve den;stry. 2023. 10. dimitrios gavriil, angeliki kakka, myers p, o´connor c. pre-endodon;c restora;on of structurally compromised teeth: current concepts. bri;sh dental journal. 2021. 11. rana a, ravi m, durga k, v. galhotra, evelyn n, rathod p. smile reestablished— predictable management of complicated crown-root fracture through surgical extrusion with and without 180° rota;on: a case report. interna;onal journal of clinical pediatric den;stry. 2024. 12. sadiya anayat, malik a, kumar a, rachna dhani, anjum s, shafi i. surgical extrusion of mul;ple teeth with crown-root fracture rehabilitated by richmond crown: a case report. interna;onal journal of oral health den;stry. 2024. 13. i. kanimozhi, ramakrishnan m, ravikumar d, n. sharna. management of crown-root fracture in primary canine by surgical extrusion: a case report with 1-year follow-up. case reports in den;stry. 2018 14. ashok d, indurkar drmayas, ramesh s. novel approach: surgical extrusion in complex dental cases: a case report. interna;onal journal of applied dental sciences. 2024. 15. nezar boreak, al mm, zain aa, alfaroog madkhali, arishy lm, domari aa, et al. surgical extrusion of three premolars to re-establish the biological width: case series. journal of contemporary dental prac;ce. 2024. 16. ib;ssem grira, boutheina mahjoubi, raouaa belkacem chebil, amor a, n. douki. surgical extrusion: a reliable alterna;ve for saving fractured anterior teeth. sage open medical case reports. 2021. 17. lee b, shin j, jeong t, park s, lee e. combined treatment of surgical extrusion and crown lengthening procedure for severe crown-root fracture of a growing pa;ent: a case report. bmc oral health. 2024 surgical extrusion as an alternanve to extracnon in non-restorable teeth vol 13, no 1 (2025) doi 10.5195/d3000.2025.1053 http://dentistry3000.pitt.edu 4 figure 1. comparstivr pain levels and analgesic consumption. figure 2. average healing times and success rates. surgical extrusion as an alternanve to extracnon in non-restorable teeth vol 13, no 1 (2025) doi 10.5195/d3000.2025.1053 http://dentistry3000.pitt.edu 5 figure 3. clinical outcomes of surgical extrusion procedures. surgical extrusion as an alternanve to extracnon in non-restorable teeth vol 13, no 1 (2025) doi 10.5195/d3000.2025.1053 http://dentistry3000.pitt.edu 6 surgical extrusion as an alternanve to extracnon in non-restorable teeth vol 13, no 1 (2025) doi 10.5195/d3000.2025.1053 http://dentistry3000.pitt.edu 7